tjh-2012-3

Review

10.5505/tjh.2012.72681

Fertility-Preserving Treatment Options in Patients with Malignant Hematological Diseases Malin Hematolojik Hastalıklarda Fertilite Koruyucu Tedavi Seçenekleri Mert Küçük1, Ali Zahit Bolaman2, İrfan Yavaşoğlu2, Gürhan Kadıköylü2 Adnan Menderes University, School of Medicine, Department of Obstetrics and Gynecology, Aydın, Turkey Adnan Menderes University, School of Medicine, Department of Internal Medicine, Division of Hematology, Aydın, Turkey

1 2

Abstract The number of patients of reproductive age diagnosed with various malignant hematological diseases increases every year. These patients undergo chemotherapy, radiotherapy, and various other treatments that may have gonadotoxic effects. The life expectancy of these patients is increasing rapidly due to the variety of treatment options. As such, an increasing number of patients—as well as their parents and spouses—express their concerns about the patient’s fertility post treatment. In the present review it was aimed to provide an overview of current fertility-preserving treatment options and the future of fertility preservation.

Key Words: Fertility preservation, Malignant hematological diseases, Ovarian tissue cryopreservation, Embryo cryopreservation

Özet Her yıl üreme çağında çeşitli malin hematolojik hastalıklara yakalanan hasta sayısı artmaktadır. Bu hastalar gonadotoksik etkileri olabilen kemoterapi, radyoterapi ve çeşitli tedaviler almaktadırlar. Hastaların beklenen yaşam süreleri ise çeşitli tedavi seçeneklerinin yardımı ile hızla artmaktadır. Bu nedenle giderek artan sayıda hasta, ebeveynleri ve hastaların eşleri tedavi sonrası hastanın fertilite potansiyeli üzerine endişelerini ifade etmektedirler. Bu derlemede güncel fertilite koruyucu tedavi seçenekleri ve fertilite prezervasyonunun geleceği konusunda bilgi vermek amaçlandı.

Anahtar Sözcükler: Fertilite prezervasyonu, Malin hematolojik hastalıklar, Over doku kryoprezervasyonu, Embriyo kryoprezervasyonu

Introduction It is well known that some treatment options used in patients with malignant hematological diseases negatively affect fertility. The negative effects of various treatments on fertility, the steadily increasing number of cures available, and improvement in 5-year life expectancy have all served to increase the importance of patient quality of life

(QoL). Among patient QoL issues is the desire of patients to become parents post treatment [1].

Schover et al. reported that 76% of childless young cancer survivors reported wanting to become a parent and that they were concerned about the effects of cancer treatment on their fertility [1]. Fertility preservation in cancer patients is becoming a more frequent issue in oncologi-

Address for Correspondence: Mert Küçük, M.D., Adnan Menderes Üniversitesi, Tıp Fakültesi, Kadın Hastalıkları ve Doğum Anabilim Dalı, Aydın, Turkey Phone: +90 256 444 12 56 E-mail: dr.mertkucuk@gmail.com Received/Geliş tarihi : October 13, 2011 Accepted/Kabul tarihi : December 20, 2011

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cal practice. The American Society of Clinical Oncology (ASCO) recently published a clinical guidebook that encourages healthcare professionals to inform patients about and discuss fertility-preserving treatment options [2]. The American Society of Reproductive Medicine (ASRM) Ethics Committee has also expressed its concern for this issue [3].

Today, the fertility-preserving treatment options most commonly offered to patients are cryopreservation of sperm and embryos [2-4]. Pregnancy has also been made possible via oocyte and ovarian tissue cryopreservation [5].

Other fertility preservation methods are still in the experimental stage. This review aimed to provide an overview of currently used fertility-preserving treatment options, those still undergoing experimentation, and a look into the potential future of fertility preservation.

In 1948 it was reported that nitrogen mustard had a toxic effect on the testes [6]. Since then, the adverse effects on reproduction of many treatment procedures have become known.

Chemotherapy can lead to amenorrhea or a reduction in the ovarian reserve [7]. Alkylating agents—particularly cyclophosphamide—are known gonadotoxic agents [7].

Oktay et al. reported that patients treated with alkylating agents had fewer primordial follicles in their ovaries than those that were not treated with these agents [8]. Generally as the dose and duration of treatment increase, so do the negative effects [9]. Younger patients have a larger pool of primordial follicles and are more able to tolerate the chemotherapy. Ovarian follicles are more sensitive to chemotherapy during the proliferative phase of the menstrual cycle [10,11]. Pelvic radiotherapy negatively affects the reproductive system, causing degeneration of primary and primordial follicles [12].

In fact, fertility preservation should be guided by two principles: 1. Use of treatments that are the least gonadotoxic as possible; 2. Use of fertility-preserving treatment options when gonadotoxicity cannot be avoided [13].

The majority of patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin’s lymphoma (HL), and nonHodgkin’s lymphoma (NHL) have reproductive potential prior to treatment, making treatment-related infertility an important issue. Hematopoietic stem cell transplantation (HSCT) is frequently included in the treatment plan of patients with hematologic diseases. During HSCT gonadotoxic agents known to have a very negative effect on future fertility are used [14]. In fact, the potential effect of each

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hematological malignancy and each treatment protocol on future fertility is unique. What follows is a brief explanation of important malignant hematological disorders and HSCT and fertility preservation options [14]. It is important to note that gonadotoxic treatments and/or HSCT are used not only in cases of malignancy, but in patients with precancerous and benign diseases [5].

Lymphomas Among patients with HL and NHL, 5-year survival has increased markedly, approaching approximately greater than 80%. The success of current treatment modalities for malignant hematologic diseases has facilitated an increase in clinicians’ ability to focus on such problems as post-treatment infertility [13]. Semen analysis must be performed in all male patients with lymphomas prior to treatment. The quality of semen is lower in patients with HL and NHL than in healthy male controls. Among male patients diagnosed with HL, 21% and 49% had azoospermia, and moderate or mild semen abnormalities, respectively [14]. The exact cause of these abnormalities has not yet been elucidated. These disorders in HL may be related to fever or pro-inflammatory cytokines, such as interleukin 1 (IL1), IL6, tumor necrosis factor-α, and soluble IL receptors 2 and 6 [14].

A relationship has not been observed between disease stage and quality of semen in HL [15-17]. The folliclestimulating hormone level in male patients was suggested to be a marker of male fertility [18] and inhibin B was suggested as an indirect marker of male fertility [19].

Low levels of inhibin B were associated with impaired spermatogenesis in children and adults receiving chemotherapy [20]. Anti-Müllerian hormone was reported to be an important predictor and marker of gonadal function in women that underwent chemotherapy for HL [21].

The most common cause of gonadal dysfunction in patients with HL is gonadotoxic chemotherapy [15]. Alkylating chemotherapeutic agents, such as procarbazine and/ or cyclophosphamide, cause prolonged azoospermia in 90%-100% of men and premature ovarian failure (POF) in 5%-25% of younger women under 30 years old; however, the risks are low with radiotherapy alone if pelvic radiation or chemotherapy with alkylating agents is not given [22]. Previously the MOPP (mechlorethamine, vincristine, procarbazine, prednisolone), COPP (cyclophosphamide, vincristine, procarbazine, prednisolone), and MOPP-ABV (alternating cycles of mechlorethamine, vincristine, procarbazine, prednisolone, and doxorubicin, bleomycin, vinblastin, and dacarbazine) regimens were frequently

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administered, whereas ABVD (doxorubicin, bleomycin, vinblastin, and dacarbazine) and BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisolone) chemotherapy regimens are currently more popular.

The MOPP, COPP, and MOPP-ABV chemotherapy regimens are more toxic to the gonads than the other mentioned regimens and may cause secondary leukemia. As such, they are not commonly used to treat HL, as they may have greater gonadotoxic effects in HL patients in whom gonadal damage was evident during the pre-treatment period [22]. The ABVD regimen is a non-alkylating regimen that is less gonadotoxic compared to regimens including alkylating agents. Among those treated with the ABVD regimen, only 33% of male patients experienced transient azoospermia, and 8% of female patients developed POF. In comparison to regimens containing alkylating agents, such as BEACOPP, these rates were significantly lower [18]. The gonadotoxic effects of second-line HL chemotherapy regimens, including DHAP (dexamethasone, cytarabine, and cisplatin), ICE (ifosfamide, carboplatin, and etoposide), and MINE (mesna, ifosfamide, mitoxantrone, etoposide), remain unknown [22]. NHL patients often receive the CHOP regimen or CHOP-based regimens; among such patients, the observed rate of gonadal toxicity is low, both in men and women [23].

Leukemias and HSCT Fertility preservation has become an important issue among patients with acute leukemias due to the increase in the success of various chemotherapy protocols. Male leukemia patients had lower pre-treatment semen parameters than healthy controls [24]. Standard chemotherapy regimens used for the treatment of AML and ALL have little toxic effect on reproductive potential [25]. Treatmentrelated infertility in leukemia patients is generally related to HSCT [26].

HSCT, along with its associated gonadotoxic conditioning regimens, has been successfully used for the treatment of both lymphoma and leukemia. HSCT may be performed as autologous or allogeneic transplantation. The gonadotoxic effect of HSCT in leukemia patients is related to whether or not a myeloablative or non-myeloablative conditioning regimen is administered, and whether or not total body irradiation (TBI) is used as a conditioning regimen prior to HSCT [27]. Myeloablative pre-transplant conditioning regimens include alkylating agents and/or TBI [28]. Both alkylating agents and TBI are associated with marked germ cell damage and infertility [29]. Azo-

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ospermia is more frequently encountered in patients that have undergone allogeneic HSCT and received myeloablative conditioning regimens. Additionally, spermatogenesis is frequently impaired in this group of patients; however, spermatogenesis sometimes improves 9-10 years following gonadotoxic therapy [30].

Patients treated with TBI followed by HSCT have a high risk of gonadotoxicity [31]. The gonadotoxic effect of TBI increases in particular when combined with cyclophosphamide [32]. POF is more common when TBI is used for preconditioning. The incidence of POF is lower in cases treated with non-myeloablative conditioning regimens and autologous transplantation [33]. Although rare, recovery of ovarian function can occur years after HSCT in cases in which POF is observed [34]. Radiation can also lead to hypothalamic amenorrhea [35]. One study reported that 68% of patients undergoing radiotherapy of both ovaries developed ovarian failure [36]. Another adverse effect of radiotherapy is a reduction in blood flow to the uterus and a decrease in uterine volume [37]. Pelvic irradiation has been associated with adverse obstetric and neonatal outcomes—among them, spontaneous abortion, low birth weight, and placental anomalies [38-40]. Pelvic irradiation has also been associated with placenta accreta and percreta, as well as uterine rupture [39-44]. Moreover, patients that undergo allogenic HSCT or irradiation, or patients that develop graft-versus-host disease (GVHD) are reported to be at risk for implantation problems [45].

In addition to gonadal damage, these patients may develop vaginal and cervical stenosis, resulting in deterioration of sexual function, with dyspareunia and reproductive failure, or difficult childbirth [45].

Gonadal damage is expected to be extensive in the following patients: patients aged ≥30 years at the time of HSCT, especially those that receive chemotherapy with alkylating agents before HSCT; patients that develop GVHD; patients with a predisposition for infiltration of the gonads; male patients; patients that receive TBI; patients treated with TBI that have undergone allogeneic HSCT; post-pubertal patients [27]. Patients scheduled for HSCT should receive fertility counseling during the pre- and post transplant periods [27].

Fertility Preservation Options Embryo Cryopreservation The ASRM Ethics Committee reported that embryo cryopreservation is the most successful method available today for fertility preservation [3]. Embryo cryopreservation consists of the following 4 steps: 1. Controlled ovarian

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hyperstimulation and induction of multifollicular growth; 2. Retrieval of follicles from the ovaries, generally under transvaginal ultrasound guidance; 3. In vitro fertilization with the partner’s sperm or fertilization via intra-cytoplasmic sperm injection (ICSI); 4. Cryopreservation of the resultant embryos.

Embryo cryopreservation can be performed via traditional slow-freezing or a rapid freezing method referred to as vitrification. Embryo survival after thawing is expected to be around 90% with the vitrification method, versus 75% with the slow freezing method [46,47].

In some of the following cases embryo cryopreservation is unsuitable or difficult to accomplish; 1. The method is not suitable for prepubertal females; 2. In Turkey the patient must be married. Cryopreservation with donor sperm is not legal in Turkey; 3. Supraphysiological estrogen levels during ovarian stimulation are regarded by some clinicians as prohibitive, particularly in patients with hormone-dependent cancers [48]; the use of letrozole, however, is recommended during controlled ovarian hyperstimulation in patients with hormone-dependent cancers [49]; 4. Patients generally need a few weeks to a few months for ovarian stimulation for embryo cryopreservation which sometimes may not be preffered [50].

Sperm Cryopreservation Sperm cryopreservation is a fertility preservation method with a high rate of successful outcomes and is easy to implement. The recommendation is that sperm should be collected three times, each after 48 h of abstinence [51]. In cases in which the underlying pathology is HL, testicular cancer, or leukemia the sperm count and/or quality may be low [52]. With the help of supplementary reproduction techniques and, in particular ICSI, success is possible when even a limited number of sperm are frozen and then thawed [53]. Sperm cryopreservation is possible even if chemotherapy or radiotherapy has already been initiated [54]; however, for maintaining DNA integrity in cases of a low sperm count, it is recommended that sperm be collected before such treatment begins.

Alternatives to obtaining sperm via masturbation include penile vibrator stimulation, testicular aspiration, testicular extraction, electroejaculation [55]. Testicular tissue cryopreservation is another alternative fertility preservation option. Testicular stem cell transplantation and transplantation of frozen-and-thawed testicular cells back to the testes after various gonadotoxic treatments are currently under investigation [13].

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Oocyte Cryopreservation Oocyte cryopreservation is a method in which—similar to embryo cryopreservation—the ovaries are subjected to controlled hyperstimulation, and then the oocytes are retrieved via a minor surgical procedure [56]. This method is unsuitable for pediatric patients. This method has some advantages and disadvantages in comparison with embryo cryopreservation: 1. Oocyte cryopreservation can be used in women without a partner. 2. Oocytes are much more sensitive to freezing and thawing procedures than embryos [57], and as such the success rate of this method is limited; therefore, ASRM still regards this method as experimental [3]; 3. Following freezing and thawing, thickening of the zona pellucida reduces the chances of fertilization of the oocyte [58]. Although ASRM regards this method as experimental, the technology is rapidly advancing [59].

The increase in oocyte survival rates after freezing and thawing following vitrification is promising, but the rate remains low [60]. In short, oocyte cryopreservation is still in the development stage and research is ongoing; nonetheless, results obtained to date show that that technique has great promise.

Ovarian Tissue Cryopreservation Ovarian tissue cryopreservation consists of the following: 1. Surgical—and usually—laparoscopic removal of part of the ovarian tissue; 2. Cryopreservation of the removed ovarian tissue; 3. Thawing and processing of the ovarian tissue when the decision to do so is made, after which time the tissue is subjected to heterotopic or orthotopic implantation; in cases of heterotopic implantation the frontal abdominal wall [61] or the forearm [62] is used, whereas orthotopic sites employed are the ovarian fossa and the pelvic peritoneum. Transplantation can also be made to the cortex of the residual ovary [63,64].

Ovarian tissue cryopreservation appears to be the only choice for prepubertal females and in particular, for postpubertal patients in whom treatment cannot be postponed, even for a short time (cases in which there is no time for ovarian stimulation either for embryo or oocyte cryopreservation) [65]. Another advantage of ovarian tissue cryopreservation is its applicability during any stage of the menstrual cycle. The resistance of primordial follicles to cryotoxicity compared to that of mature oocytes is another advantage of the method [66].

The disadvantages of ovarian tissue cryopreservation are as follows: 1. It requires surgery; 2. It is associated with the risk of thrombosis and hemorrhaging; 3.

Although primordial follicles are more resistant, as there

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is no blood flow at the initial ischemia 66% of oocytes lose their viability [67]; 4. There is a theoretical risk, although low, that with this method malignant cells might find their way back into the patient’s tissues. The ovaries are seldom the site of metastasis, but leukemia, neuroblastoma, and breast cancers have been known to metastasize to the ovaries [68,69]. This is particularly important in cases of BRCA1- and BRCA2-positive mutations [70], in which the removed ovarian tissue must be evaluated histopathologically; 5. The rate of success and clinician experience with this method are quite limited; to date, very few live births have been reported as an outcome of this method [71,72].

In short, ovarian tissue cryopreservation is a new technique with which clinicians have limited experience, and outcome data are in short supply.

Ovarian Transposition, and Intensity-Modulated Radiation Therapy (IMRT) and Gonadal Shielding The aim of ovarian transposition is to move the ovaries of patients with HL, neuroblastoma, Wilms tumor, and other similar conditions to a location in the body that is outside the radiation field [71]. The procedure includes the following: 1. Surgical transposition of the ovaries to an area outside of the field of radiation; 2. Following radiotherapy, the ovaries are returned to their original location.

The success rate for this procedure is reported to be 16%90% [73]. Ovarian transposition can be performed prior to irradiation via laparotomy or laparoscopy [74]. Application of metal clips to the ovaries during the procedure is useful in guiding subsequent X-ray localization. Additionally, a sample of ovarian tissue can be excised during the procedure for cryopreservation. The risks associated with ovarian transposition are postoperative chronic pelvic pain and pelvic adhesions. Although rare, it should also be kept in mind that the ovaries may migrate back to their former locations [75]. Moreover, damage to the ovaries resulting from the combination of chemotherapy and radiotherapy cannot be prevented with this method.

IMRT is a relatively new radiation technique that facilitates delivery of radiation to multiple targets while sparing adjacent tissues. This method is used to minimize the distribution of radiation and the harmful effects of radiation on the ovaries and uterus [76].

Shielding the uterus and ovaries to as great a degree as possible during radiotherapy or dividing the TBI doses may be of benefit to avoid gonadotoxicity [65].

Ovarian Suppression with a GonadotropinReleasing Hormone (GnRH) Analog It is known that the ovaries in prepubertal females are

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more resistant to chemotherapy compared to postpubertal females. GnRH analog treatment is thought to convert the hormonal environment in post-pubertal females to the prepubertal environment, so that the ovaries become more resistant to the toxic effects of chemotherapy. A meta-analysis reported that the use of GnRH analogs prior to chemotherapy was an effective method [77]. Nonetheless, to date the effectiveness of GnRH analog treatment for fertility preservation remains inconclusive [8] and additional large-scale randomized studies are necessary.

Xenotransplantation Xenotransplantation—transplantation of human ovarian tissue into other species—is in the experimental stage [78,79]. Xenotransplantation has been used successfully to create functional oocytes [80]. As xenotransplantation is still in the experimental stage, the procedure raises issues of safety and ethics. The transfer of non-human DNA or contamination with viruses may turn out to be risks associated this procedure [81].

Stem Cells Stem cells are able to differentiate into diverse specialized cell types. Research continues on methods of cloning germ cells from adult somatic cells and the creation of new differentiated cells from stem cells [82,83].

Current Legislation in Turkey According to Turkish regulations concerning assisted reproductive treatment methods and assisted reproductive treatment centers, partners that undergo such treatment may only receive their own reproductive cells. It is illegal to make use of a donor in any way—to harvest an embryo from a donor, transfer an embryo from one assisted reproductive treatment candidate to another via use of eggs or sperm harvested from a candidate, and to use or implant embryos in assisted reproductive treatment candidates that have been harvested from non-candidates [84].

When embryos have been retrieved, these can be cryopreserved with the consent of both partners. If an embryo has been preserved over one year, each year the couple must confirm their desire to continue the preservation by submitting a signed petition. Upon the consent of both partners, in the event of the death of one of the partners or a legally established divorce, or at the end of the determined period, a record is kept by a commission to be appointed by the health directorate of the city and the embryos are destroyed [84].

It is legal for both men and women to preserve reproductive cells and gonad tissue before undergoing treat-

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ment that may damage gonad cells, such as chemotherapy or radiotherapy. To maintain the security of the materials, reproductive cells and gonad tissue are preserved together with a DNA analysis of the donor. In the event that preservation exceeds one year individuals must apply to the authorities with a signed petition confirming the desire to continue the preservation of the tissues/cells. Anytime an individual fails to renew the protocol, or upon his/her request, or in the event of death, a record is kept by a commission to be appointed by the health directorate of the city and the cryopreserved reproductive cells and gonad tissues are destroyed [84]. Reproductive cells and gonad tissues, as well as frozen embryos, may be preserved for a maximum of five years; preservation exceeding five years is subject to the approval of the Ministry of Health [84].

Fertility Preservation Risks and Ethical Considerations Although fertility preservation treatment has undergone significant advancement and continues to generate great interest, and despite the fact that many oncology patients and their families currently seek such treatment, the ethical issues surrounding its use remain unresolved.

At the same time, promising new developments and methods of fertility preservation (e.g. xenotransplantation) complicate the ethics of such treatment. In addition, as each individual and culture has unique psychosocial, cultural, and religious sensitivities and realities, the ethical issues associated with fertility preserving treatment vary accordingly.

An important ethical issue concerns non-adult patients and the informed consent process; in particular, is it ethical for such decisions to be made by the parents of minors.

As such, scientific authorities have stressed the importance of ensuring that children and adolescents are involved in the informed consent process to a degree that is age appropriate [85]. When children or adolescents are concerned, besides age, having the capacity to understand the situation in which the individual is in is more important not age but when participating in the fertility preservation process. It is, however, not always easy to determine whether such capacity exists. A family’s decision about a minor’s future reproductive choice can, for example, result in the rejection of cryopreservation, which in later years becomes an ethical issue if the adult child now wished to become a parent. Additionally, parents that opt for gamete cryopreservation for their child can have the cells destroyed before the patient has reached legal age. Still, another ethical issue concerns the future of cryopreserved embryos or gametes in the event a patient dies, i.e. a healthy partner

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wants to become pregnant with a preserved embryo after her partner has died.

An important ethical consideration is the degree to which patients and their families are informed about experimental fertility preservation options. The information provided to patients should specify that the process is experimental and objective data provided should include information on success rates. Incorrectly raising the hopes of patients and their families when experimental methods are being tested will inevitably bring about many problems. In addition, all oncology patients must be informed about fertility preservation options and their written informed consent must be obtained prior to such treatment. If patients or their legal guardians refuse fertility preservation, clinicians must document their choice.

When a legal issue arises and patients claim that they were not informed about fertility preservation options, or if they admit that they were, but claim to have actually asked for one of the options, in order to resolve a possible dispute, there needs to be a written consent that was signed by the patient and/or the legal guardian before the treatment for cancer. Another issue is the matter of who should obtain a patient’s informed consent—the patient’s attending physician or the reproductive endocrinologist.

It is a possibility that malignant cells may be reintroduced when tissue is re-transplanted during the ovarian tissue cryopreservation procedure [86]. The time needed for oocyte or embryo cryopreservation and sometimes the clinician’s feeling pressed enough to act quickly to start the treatment for cancer or the necessity to do so can also be problematic [87]. The risk of transferring non-human DNA or viruses to humans when employing xenotransplantation must always be a consideration [81], as well as the potential hazards of the interaction of human and animal DNA [88]. Another important ethical issue concerns problems that might arise if a patient becomes pregnant following fertility preservation treatment and then has oncological relapse during the pregnancy [87].

Although there are no studies in this area, it can be assumed that clinicians’ awareness of the matter of fertility preservation may be lacking. The clinicians report that presentation of oncology patients to fertility preservation clinics is not a frequent occurrence. As such, we think that these methods are either underestimated or undervalued by such patients. Hematologists should continue to search objectively for the underlying problems and the reasons for this probable underestimation.

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Conclusion Sperm and embryo cryopreservation are fertility preservation methods proven to be successful. Oocyte cryopreservation and ovarian tissue cryopreservation have also resulted in successful births. Although xenotransplantation, stem cell utilization, and other experimental methods are still under development, they have potential for future success.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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Cancer 1999; 86 (7): 1231-1239 27. Chatterjee R, Kottaridis PD. Treatment of gonadal damage in recipients of allogeneic or autologous transplantation for haematological malignancies. Bone Marrow Transplant 2002; 30 (10): 629-635 28. Chatterjee R, Mills W, Katz M, McGarrigle HH, Goldstone AH. Germ cell failure and Leydig cell insufficiency in postpubertal males after autologous bone marrow transplantation with BEAM for lymphoma. Bone Marrow Transplant 1994; 13 (5): 519-522 29. Kreuser ED, Hetzel WD, Heit W, Hoelzer D, Kurrle E, Xiros N, Heimpel H. Reproductive and endocrine gonadal functions in adults following multidrug chemotherapy for acute lymphoblastic or undifferentiated leukemia. J Clin Oncol 1988; 6 (4): 588-595 30. Anserini P, Chiodi S, Spinelli S, Costa M, Conte N, Copello F, Bacigalupo A. Semen analysis following allogeneic bone marrow transplantation. Additional data for evidence-based counselling. Bone Marrow Transplant 2002; 30 (7): 447451 31. Sklar C. Growth and endocrine disturbances after bone marrow transplantation in childhood. Acta Paediatr Suppl 1995; 411: 57-61

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Blood 1996; 87 (7): 3045-3052 33. Tauchmanovà L, Selleri C, De Rosa G, Esposito M, Orio F Jr, Palomba S, Bifulco G, Nappi C, Lombardi G, Rotoli B, Colao A. Gonadal status in reproductive age women after haematopoietic stem cell transplantation for haematological malignancies. Hum Reprod 2003; 18 (7): 1410-1416 34. Leader A, Lishner M, Michaeli J, Revel A. Fertility considerations and preservation in haemato-oncology patients undergoing treatment. Br J Haematol 2011; 153 (3): 291-308 35. Sklar CA, Constine LS. Chronic neuroendocrinological sequelae of radiation therapy. Int J Radiat Oncol Biol Phys 1995; 31 (5): 1113-1121 36. Stillman RJ, Schinfeld JS, Schiff I, Gelber RD, Greenberger J, Larson M, Jaffe N, Li FP. Ovarian failure in long-term survivors of childhood malignancy. Am J Obstet Gynecol 1981; 139 (1): 62-66 37. Critchley HO, Wallace WH, Shalet SM, Mamtora H, Higginson J, Anderson DC. Abdominal irradiation in childhood; The potential for pregnancy. Br J Obstet Gynaecol 1992; 99 (5): 392-394 38. Green DM, Peabody EM, Nan B, Peterson S, Kalapurakal JA, Breslow NE. Pregnancy outcome after treatment for Wilms tumor: A report from the National Wilms Tumor Study Group. J Clin Oncol 2002; 20 (10): 2506-2513 39. Chiarelli AM, Marrett LD, Darlington GA. Pregnancy outcomes in females after treatment for childhood cancer.

Epidemiology 2000; 11 (2): 161-166 40. Norwitz ER, Stern HM, Grier H, Lee-Parritz A. Placenta percreta and uterine rupture associated with prior whole body radiation therapy. Obstet Gynecol 2001; 98 (5 Pt 2): 929-931 41. Holm K, Nysom K, Brocks V, Hertz H, Jacobsen N, Müller J. Ultrasound B-mode changes in the uterus and ovaries and Doppler changes in the uterus after total body irradiation and allogeneic bone marrow transplantation in childhood.

Bone Marrow Transplant 1999; 23 (3): 259-263 42. Laursen EM, Holm K, Brocks V, Jarden M, Müller J. Doppler assessment of flow velocity in the uterine artery during pubertal maturation. Ultrasound Obstet Gynecol 1996; 8 (5): 341-345 43. Pridjian G, Rich NE, Montag AG. Pregnancy hemoperitoneum and placenta percreta in a patient with previous pelvic irradiation and ovarian failure. Am J Obstet Gynecol 1990; 162: 1205-1206

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44. Boice JD Jr, Tawn EJ, Winther JF, Donaldson SS, Green DM, Mertens AC, Mulvihill JJ, Olsen JH, Robison LL, Stovall M. Genetic effects of radiotherapy for childhood cancer. Health Phys 2003; 85 (1): 65-80 45. Schubert MA, Sullivan KM, Schubert MM, Nims J, Hansen M, Sanders JE, O’Quigley J, Witherspoon RP, Buckner CD, Storb R, Thomas ED. Gynecological abnormalities following allogeneic bone marrow transplantation. Bone Marrow Transplant 1990; 5 (6): 425-430 46. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method. Theriogenology 2007; 67: 73-80 47. Stehlik E, Stehlik J, Katayama KP, Kuwayama M, Jambor V, Brohammer R, Kato O. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts.

Reprod Biomed Online 2005; 11 (1): 53-57 48. Peña JE, Chang PL, Chan LK, Zeitoun K, Thornton MH 2nd, Sauer MV. Supraphysiological estradiol levels do not affect oocyte and embryo quality in oocyte donation cycles. Hum Reprod 2002; 17 (1): 83-87 49. Azim AA, Costantini-Ferrando M, Oktay K. Safety of fertility preservation by ovarian stimulation with letrozole and gonadotropins in patients with breast cancer: A prospective controlled study. J Clin Oncol 2008; 26 (16): 2630-2635 50. Stern CJ, Toledo MG, Gook DA, Seymour JF. Fertility preservation in female oncology patients. Aust N Z J Obstet Gynaecol 2006; 46 (1): 15-23 51. Sanger WG, Olson JH, Sherman JK. Semen cryobanking for men with cancer criteria change. Fertil Steril 1992; 58: 1024-1027 52. Gandini L, Lombardo F, Salacone P, Paoli D, Anselmo AP, Culasso F, Dondero F, Lenzi A. Testicular cancer and Hodgkin’s disease: Evaluation of semen quality. Hum Reprod 2003; 18 (4): 796-801 53. Opsahl MS, Fugger EF, Sherins RJ, Schulman JD. Preservation of reproductive function before therapy for cancer: New options involving sperm and ovary cryopreservation.

Cancer J Sci Am 1997; 3 (4): 189-191 54. Kuczyński W, Dhont M, Grygoruk C, Grochowski D, Wołczyński S, Szamatowicz M. The outcome of intracytoplasmic injection of fresh and cryopreserved ejaculated spermatozoa -- a prospective randomized study.

Hum Reprod 2001; 16: 2109-2113 55. Bahadur G, Ling KL, Hart R, Ralph D, Riley V, Wafa R, Ashraf A, Jaman N, Oyede AW. Semen production in adolescent cancer patients. Hum Reprod 2002; 17 (10): 2654-2656 56. Braude P, Rowell P. Assisted conception. II--in vitro fertilisation and intracytoplasmic sperm injection. BMJ 2003; 327 (7419): 852-855 57. Shaw JM, Oranratnachai A, Trounson AO. Fundamental cryobiology of mammalian oocytes and ovarian tissue.

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58. Matson PL, Graefling J, Junk SM, Yovich JL, Edirisinghe WR. Cryopreservation of oocytes and embryos: Use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization program. Hum Reprod 1997; 12: 1550-1553 59. The Practice Committee of the American Society for Reproductive Medicine: Ovarian tissue and oocyte cryopreservation. Fertil Steril 2004; 82: 993-998 60. Oktay K, Cil AP, Bang H. Efficiency of oocyte cryopreservation: Meta-analysis. Fertil Steril 2006; 86: 70-80 61. Kiran G, Kiran H, Coban YK, Guven AM, Yuksel M. Fresh autologous transplantation of ovarian cortical strips to the anterior abdominal wall at the pfannenstiel incision site.

Fertil Steril 2004; 82: 954-956 62. Oktay K, Economos K, Kan M, Rucinski J, Veeck L, Rosenwaks Z. Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm. JAMA 2001; 286: 1490-1493 63. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B, van Langendonckt A. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004; 364 (9443): 1405-1410 64. Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y, Schiff E, Dor J. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med 2005; 353 (3): 318-321 65. Oktem O, Urman B. Options of fertility preservation in female cancer patients. Obstet Gynecol Surv 2010; 65 (8): 531-542 66. Oktay K, Newton H, Aubard Y, Sahla O, Gosden RG. Cryopreservation of immature human oocytes and ovarian tissue: An emerging technology? Fertil Steril 1998; 69: 1-7 67. Nisolle M, Casanas-Roux F, Qu J, Motta P, Donnez J. Histologic and ultrastructural evaluation of fresh and frozenthawed human ovarian xenografts in nude mice. Fertil Steril 2000; 74: 122-129 68. Curtin JP, Barakat RR, Hoskins WJ. Ovarian disease in women with breast cancer. Obstet Gynecol 1994; 84: 449452 69. Li CI, Anderson BO, Daling JR, Moe RE. Trends in incidence rates of invasive lobular and ductal breast carcinoma. JAMA 2003; 289: 1421-1424 70. Sonmezer M, Oktay K. Fertility preservation in young women undergoing breast cancer therapy. Oncologist 2006; 11: 422-434 71. Gurgan T, Salman C, Demirol A. Pregnancy and assisted reproduction techniques in men and women after cancer treatment. Placenta 2008; 29: 152-159 72. Oktay K, Oktem O. Fertility preservation medicine: A new field in the care of young cancer survivors. Pediatr Blood Cancer 2009; 53 (2): 267-273

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73. Sonmezer M, Oktay K. Fertility preservation in female patients. Hum Reprod Update 2004; 10 (3): 251-266 74. Maltaris T, Koelbl H, Seufert R, Kiesewetter F, Beckmann MW, Mueller A, Dittrich R. Gonadal damage and options for fertility preservation in female and male cancer survivors.

Asian J Androl 2006; 8: 515-533 75. Gabriel DA, Bernard SA, Lambert J, Croom RD 3rd.

Oophoropexy and the management of Hodgkin’s disease. A reevaluation of the risks and benefits. Arch Surg 1986; 121 (9): 1083-1085 76. Wo JY, Viswanathan AN. Impact of radiotherapy on fertility, pregnancy and neonatal outcomes in female cancer patients.

Int J Radiat Oncol Biol Phys 2009; 73 (5): 1304-1312 77. Clowse ME, Behera MA, Anders CK, Copland S, Coffman CJ, Leppert PC, Bastian LA. Ovarian preservation by GnRH agonists during chemotherapy: A meta-analysis. J Womens Health (Larchmt) 2009; 18 (3): 311-319 78. Donnez J, Martinez-Madrid B, Jadoul P, Van Langendonckt A, DemylleD, Dolmans MM. Ovarian tissue cryopreservation and transplantation: A review. Hum Reprod Update 2006; 12: 519-535 79. Snow M, Cox SL, Jenkin G, Trounson A, Shaw J. Generation of live young from xenografted mouse ovaries. Science 2002; 297 (5590): 2227 80. Chao L, Jiang AF, Deng XH, Yu HL, Zhen JH. Capability of oocyte maturation in human cryopreserved ovarian tissue following xenografting. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2008; 30 (5): 583-588

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81. Cozzi E, Bosio E, Seveso M, Vadori M, Ancona E. Xenotransplantation-current status and future perspectives.

Br Med Bull 2006; 75-76: 99-114 82. Pacey AA. Fertility issues in survivors from adolescent cancers. Cancer Treat Rev 2007; 33: 646-655 83. Tsai MC, Takeuchi T, Bedford JM, Reis MM, Rosenwaks Z, Palermo GD. Alternative sources of gametes: Reality or science fiction? Hum Reprod 2000; 15: 988-998 84. http://www.ttb.org.tr/mevzuat/index.php?option=com_con tent&task=view&id=741&Itemid=33 (Accesed 1.09.2011) 85. Committee on Bioethics, American Academy of Pediatrics.

Informed consent, parental permission, and assent in pediatric practice. Pediatrics 1995; 95 (2): 314-317 86. Moffa F, Biacchiardi CP, Fagioli F, Biasin E, Revelli A, Massobrio M, Madon E. Ovarian tissue cryostorage and grafting: An option to preserve fertility in pediatric patients with malignancies. Pediatr Hematol Oncol 2007; 24 (1): 29-44 87. Cohen CB. Ethical issues regarding fertility preservation in adolescents and children. Pediatr Blood Cancer. 2009; 53 (2): 249-253 88. Karpowicz P, Cohen CB, van der Kooy D. It is ethical to transplant human stem cells into nonhuman embryos. Nat Med 2004; 10 (4): 331-335

Research Article

10.5152/tjh.2011.77

Pulmonary Radiological Findings in Patients with Acute Myeloid Leukemia and Their Relationship to Chemotherapy and Prognosis: A Single-Center Retrospective Study Akut Miyeloid Lösemili Hastalarda Akciğer Radyolojik Bulgularının Kemoterapi ve Hastalığın Prognozu ile İlişkisi: Tek Merkezli Retrospektif Çalışma Mehmet S Buğdacı1, Halil Yanardağ1, M. Cem Ar2, Teoman Soysal3, Süleyman Coşkun1, Sabriye Demirci1

İstanbul University, Cerrahpaşa School of Medicine, Department of Pneumophysiology, Division of Internal Medicine, İstanbul, Turkey İstanbul Training and Research Hospital, Department of Hematology, İstanbul, Turkey 3 İstanbul University, Cerrahpaşa School of Medicine, Department of Hematology, Division of Internal Medicine, İstanbul, Turkey 1 2

Abstract Objective: Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Pulmonary are among the most common causes of mortality in AML. This single-center retrospective study aimed to evaluate the relationship between radiological findings of pulmonary at presentation and post chemotherapy on prognosis and clinical outcome in a group of AML patients.

Material and Methods: The study included 278 AML patients. Clinical and radiological findings, laboratory findings, and microbiological culture results were evaluated. Pulmonary complications at presentation and post chemotherapy were compared.

Results: Pulmonary complications were observed in 53 of the patients (19%). Mean age of the patients with and without pulmonary complications was 43.1 ± 15.2 years and 38.8 ± 16.3 years, respectively (P < 0.001). Pulmonary complications were not correlated with gender, AML subtype, or the serum lactate dehydrogenase (LDH) level. The most common cause of pulmonary complications was infection. Pulmonary complications were observed in 29% and 71% of the patients at presentation and post chemotherapy, respectively.

Conclusion: Pulmonary complications were observed more frequently at presentation in neutropenic AML patients of advanced age. The mortality rate was higher among the AML patients that had pulmonary complications at presentation.

Key Words: Acute myeloid leukemia, Pulmonary disease, Radiological findings

Address for Correspondence: Mehmet S BuĞdacı, M.D., İstanbul Üniversitesi, Cerrahpaşa Tıp Fakültesi, Fatih, İstanbul, Turkey Phone: +90 212 414 34 14 E-mail: msbugdaci@gmail.com Received/Geliş tarihi : Marh 9, 2011 Accepted/Kabul tarihi : June 13, 2011

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Özet Amaç: Akut miyeloid lösemi (AML) erişkinde en sık görülen akut lösemidir. AML’de mortaliteyi yol açan en önemli sebeplerden biri pulmoner olaylardır. Bu retrospektif tek merkezli çalışmanın amacı tanı anında ve kemoterapi sonrası gelişen radyolojik akciğer bulgularının hastalığın klinik gidişi ve prognozu üzerine etkisinin araştırılmasıdır.

Gereç ve Yöntemler: Çalışmaya 278 AML hastası dahil edildi. değerlendirildi. Tanı anındaki ve kemoterapi sonrası pulmoner bulgular, hastalara ait klinik ve radyolojik bulgular, laboratuvar verileri ve mikrobiyolojik kültür sonuçları ile değerlendirildi.

Bulgular: Pulmoner olaylar hastaların 53’ünde (%19) görüldü. Pulmoner bulguları olan ve olmayan hastaların ortalama yaşları sırasıyla 43.1±15.2 ve 38.8±16.3 olarak bulundu (p<0,001). Akciğer bulguları ile cinsiyet, AML alt tipi ve LDH düzeyleri arasında ilişki saptanmadı. En sık rastlanılan pulmoner olay enfeksiyondu. Pulmoner olaylar hastaların %29’unda tanı anında, %71’inde tedavi sonrası gözlendi.

Sonuç: Pulmoner olaylar ileri yaştaki, nötropenik hastalarda tanı anında daha sık gözlendi. Tanı anında pulmoner komplikasyonu bulunan AML’li hastalarda mortalite daha yüksek bulundu.

Anahtar Sözcükler: Akut miyeloid lösemi, Pulmoner hastalık, Radyolojik bulgular Introduction Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by malignant proliferation of myeloid precursor cells. It is the most common form of acute leukemia in adults.[1,2,3] The prognosis of acute leukemias is poor, despite improvements in supportive and definitive therapies.[4] Severe pulmonary complications, including acute pulmonary insufficiency and infectious pneumonia, frequently occur in patients with acute leukemia.[5,6] Such pulmonary complications may be of infectious or non-infectious origin. Pulmonary infections are the most important cause of morbidity and mortality in patients with hematological malignancies undergoing chemotherapy.[7] Furthermore, high mortality rates nearly 60% were reported in leukemia patients with radiologically evident pulmonary infiltration.[8] The bulk of data on pulmonary findings in patients with AML consists primarily of chemotherapy-related complications.[6,7,9-11] Data are lacking on other aspects of pulmonary findings in AML, such as the association between AML subtypes and pulmonary complications, and the correlation between pulmonary complications at presentation and prognosis. As such, the aim of the present single-center retrospective study was to evaluate the relationship between radiological findings of pulmonary complications at presentation and post chemotherapy on prognosis and clinical outcome in patients with AML. Materials and Methods Presentation and follow-up records of 1066 patients with hematological malignancies that presented to University of Istanbul, Cerrahpaşa School of Medicine,

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Hematology Department, between 1995 and 2006 were retrospectively reviewed. There were 475 patients diagnosed as acute leukemia, of which 278 with AML were included in the study. AML was diagnosed based on morphology and immunophenotyping. The patients were classified according to French-American-British (FAB) criteria. Flow cytometry was used for immunophenotyping.

Methodology of radiographic studies Thoracic high-resolution computed tomography (HRCT) was performed using a Siemens Somatom Plus (Siemens, Erlangen, Germany) scanner during breathholding at full inspiration, according to Gevenois et al [12]. At the beginning of the procedure radiographic fields were marked on digital topograms. Using a cross sectional thickness of 1 mm and a cross sectional interval of 10 mm, deep inspiration images of the whole lung parenchyma— from apex to base—were obtained using the high-spatialfrequency (bone) reconstruction algorithm. Every section over the diaphragm for each lung was evaluated separately.

Definitions Consolidation was considered homogeneous amorphous opacities observed via air bronchogram to obscure many of the underlying vessels. Ground-glass appearance was characterized as a patchy or diffuse hazy increase in lung opacity that did not obscure the underlying vessels.

Radiologically, nodules are focal, rounded opacities of varying size, with either well- or ill-defined borders. In the present study, rounded opacities without any specific features were simply defined as non-specific nodular opacity.

Air-containing nodules were considered cavitary nodules.

Thoracic findings were classified as pleural, parenchymal,

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Bugdacı MS, et al: Pulmonary Radiological Findings in AML

and pleuroparenchymal, according to radiological appearance. Lesions were considered parenchymal if they involved the pulmonary tissue and bronchial tree. Pleural thickening was associated with fibrosis, pleural effusion, or pleural involvement.

ated using the chi-square or Pearson’s correlation tests, as appropriate. Student’s t test was used for comparison of parametric data. A P value less then 0.05 was considered to be statistically significant. SPSS v.12.0 for Windows was used for statistical evaluation.

Radiological, clinical, and laboratory findings were evaluated together, so as to determine their etiology.

Consolidation and abscesses were considered to be due to bacterial agents initially if supportive clinical findings were present. Nodules without specific characteristics, such as a centrilobular nodule or acinar nodule (± halo finding), were considered to be of fungal origin. Cavitary lesions were diagnosed as tuberculosis if confirmed via microbiological culture results. Radiological diagnosis of pulmonary embolism was based on spiral CT findings or high-probability ventilation/perfusion scintigraphy.

Invasive pulmonary aspergillosis was diagnosed according to EORTC/MSG consensus criteria.[13] Leukostasis was considered a leukocyte count >100 x 109 L–1 and the presence of pulmonary symptoms (dyspnea and diffuse interstitial infiltrates chest radiographs), only after having excluded other plausible causes. The diagnosis of bacterial pneumonia was based on the presence of a pathogen concentration >103 CFU mL–1 in culture. Respiratory findings were grouped as those that were observed at presentation and those that occurred post chemotherapy.

Results

Drugs Cytosine arabinoside (100-200 mg m–2 on d 1-7) and daunorubicin (45 mg m–2 on d 1-3), idarubicin (12 mg m–2 on d 1-3), or mitoxantrone (12 mg m–2 on d 1-3) were administered as induction treatment for AML. High-dose cytosine arabinoside (1.5-3 g m–2 q12h on d 1, 3, and 5 [6 doses]) was administered for consolidation purposes following remission. Regimens containing all-transretinoic acid (ATRA) and idarubicin were used for remission induction and consolidation in patients with acute promyelocytic leukemia.

Broad-spectrum beta-lactam antibiotics and carbapenems with or without aminoglycoside were administered in febrile patients with neutropenia (<500 mm–3). The addition of glycopeptide antibiotics and/or antifungal agents, as well as re-evaluation of initial antibiotic therapy, were usually carried out according to IDSA guidelines.[14,15] The study protocol was approved by the Istanbul University, Cerrahpaşa School of Medicine Ethics Committee.

Statistics Parametric variables are presented as mean ± standard deviation (mean ± SD). Non-parametric data were evalu-

The study included 278 AML patients. In all, 169 (60.7%) were male (mean age: 39.2 ± 16 years) and 109 (39.3%) were female (mean age: 40.3 ± 16.5 years).

Among the patients, 241 (86.7%) had de novo AML and 37 (13.3%) had AML secondary to chronic myeloid leukemia, myelodysplastic syndrome, sideroblastic anemia, aplastic anemia, or chemotherapy. In total, 47 (16.9%) patients were aged ≥65 years.

Pulmonary findings were observed in 53 (19.1%) of the 278 AML cases (Table 1 and the Figure). Mean age of the patients with and without radiologically confirmed pulmonary complications was 43.1 ± 15.2 years and 38.8 Table 1: The etiology of radiologically confirmed pulmonary complications and the frequency of Aspergillus infection in the AML patients (n = 53), according to EORTC/MSG consensus criteria.

Cause Infection Bacterial* Bacterial** Mycobaterium tuberculosis*** Aspergillus fumigatus Proven Probable Possible Non-infectious causes Malignant infiltration**** Pulmonary embolism Pneumothorax Cardiac disease Undetermined Cure with AB and/or AF treatment

n (%) 30 (56.6) 8 (15.0) 7 (13.3) 1 (2.0) 2 (3.8) 3 (5.7) 9 (17.0) 13 (24.5) 2 (3.8) 4 (7.5) 2 (3.8) 5 (9.4) 10 (18.8) 4 (7.5)

AB: Antibiotic; AF: antifungal treatment. *Definitive cause confirmed via microbiological cultures. **Possible bacterial etiology supported by radiological and clinical findings. ***Definitive diagnosis based on direct observation of resistant mycobacteria in the sputum and positive cultures. **** The 2 cases with leukemic infiltration had the M5 and M4/5 subtypes.

Diagnoses were based on radiological and clinical findings.

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achieved in 4 of the patients with pulmonary complications of undetermined origin.

Pulmonary complications were observed at initial presentation in 16 (28.9%) of the patients, whereas in 37 (71.1%) patients they were observed post chemotherapy.

Mortality was 43.8% in the patients with pulmonary complications at presentation and 24.6% in those that developed pulmonary complications post chemotherapy. Table 2 summarizes the characteristics of the AML patients with pulmonary complications at presentation and post chemotherapy.

Figure: Leukemia cases included in the study and the distribution of AML patients with pulmonary complications.

± 16.3 years, respectively; the difference was significant (P < 0.001). Pulmonary complications and gender were not statistically correlated (P = 0.96). Radiologically confirmed pulmonary complications occurred at a higher frequency in the patients with the M4 subtype (32.6%), though the difference was not significant (P = 0.55). Leukemic infiltration of the lungs was observed in 2 patients with AML M5 (n = 1) and AML M4/5 (n = 1). There wasn’t a significant difference in the serum lactate dehydrogenase (LDH) level between the patients with and without pulmonary complications (P = 0.290).

In all, 81% of the patients had parenchymal involvement, 15.1% had pleural involvement, and 3.8% had pleuropulmonary involvement. CT findings in the patients with pulmonary lesions were as follows: ground-glass appearance (n = 18); nodular opacity (n = 15); pneumonic consolidation (n = 9); non-homogenous opacity (n = 8); fungus ball (n = 1); nodule with cavity (n = 1); abscess (n = 1). Etiological distribution of the pulmonary complications showed that in 56.6% of the cases the underlying cause was infection. Non-infectious and undetermined causes were responsible for the pulmonary complications in 24.5% and 18.8% of the cases, respectively. Clinical response to antibiotic and/or antifungal treatment was

Discussion Pulmonary infections are the most common cause of pulmonary radiological findings and are among the most common causes of mortality in AML.[7] Chemotherapy and allogeneic stem cell transplantation are the treatment options in patients in good general condition. The cure rate with chemotherapy alone is low and is usually not durable. Pulmonary complications are the primary cause of mortality, both before and after chemotherapy; therefore, detection of probable pulmonary involvement before commencing chemotherapy could help prevent negative outcomes that might occur during the post-treatment pancytopenic period. As such, predicting the risk of pulmonary complications in AML might play a crucial role in the success of chemotherapy by reducing complicationrelated morbidity and mortality.

Pulmonary complications were observed in 19% of the present study’s patients, which is a lower rate than previously reported. For instance, Ewig et al.[16] observed respiratory findings in 30% of patients and Chaoui et al.[4] reported a rate of 46%. Unlike the present study, Chaoui et al.’s included patients with a higher median age (median: 55 years; range: 17-85 years) that had de novo, and secondary acute lymphoid and myeloid leukemias, which might explain why the rate of pulmonary complications was higher in their study. The most frequent cause of pulmonary complications in the present study was infec-

Table 2: Characteristics of the AML patients with pulmonary complications at presentation and post chemotherapy.

Age (years) Neutrophil leukocyte count (mL–1) Hospitalization period (months) AML: Acute myeloid leukemia; F: female; M: male.

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At presentation (n = 16) 50 ± 15.5 373 ± 423 3.5 ± 2.7

Post chemotherapy (n = 37) 32 ± 11.0 702 ± 457 10 ± 4.5

P 0.001 0.001 0.001

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tions (30%), including those of bacterial and fungal origin; similar findings have been reported.[4,16,17] Rano et al. reported that pulmonary complications were most frequently caused by bacterial infections, followed by fungal infections.[18] Proven or probable invasive pulmonary aspergillosis was observed in 95% of patients with pulmonary complications in the present study, whereas rates reported in the literature are 5% in acute leukemias[19] and 6%-8.3% in AML[20].

AML is a rare cause of leukemia in individuals aged <15 years;[21] it is a disease of late adulthood and the median age of newly diagnosed AML patients is 65 years.

[22] Deschler and Lübbert reported an incidence of 1.8/100,000 in patients aged <65 years and an incidence of 17/100,000 in patients aged >65 years.[23] Mean age of the male and female patients in the present study was 39.2 ± 16 years and 40.3 ± 16.5 years, respectively.

Pulmonary leukemic infiltration was noted in 2 of the present study’s AML patients; 1 with the M5 subtype and 1 with the M4/5 subtype. This finding in accordance with the current knowledge. Autopsy studies reported that pulmonary infiltration occurs at a rate of 24%-64% in AML M5 cases, versus 5% for clinically and radiologically diagnosed leukemic lung infiltration.[24,25] No correlation was observed between the serum LDH level and pulmonary complications in the present study, which is agreement with Chaoui et al.,[4] who reported that there wasn’t a difference in LDH levels between patients with and without pulmonary complications.

In the present pulmonary complications occurred more frequently in the patients with advanced age particularly > 65-year-old and there was a statistically significant correlation between age and pulmonary complications (P = 0.001), which is similar to the findings reported by Rossini et al.[6] Unexplained causes of pulmonary complications were observed in 18.8% of the present study’s patients.

Similar findings were reported by Chaoui et al. and Rano et al., who reported that pulmonary complications of unexplained origin occurred in 25% and 19% of immunocompromised patients, respectively.[4,18] Pulmonary complications of unidentified origin could be associated with the absence of sufficient diagnostic work-up due to poor general status or severe cytopenias.

In the present study 33% of the non-infectious causes of pulmonary complications were cardiac pathologies.

Cardiac failure-related pulmonary complications occurred primarily during the first week of induction chemotherapy, suggesting that the pulmonary complications were re-

Bugdacı MS, et al: Pulmonary Radiological Findings in AML

lated to chemotherapy or hydration. Cardiovascular dysfunction was reported to occur easily in anemic patients receiving high-volume fluid replacement, and combination chemotherapy consisting of anthracycline and cytosine arabinoside.[26] The present study’s most important findings are as follows: 1. The frequency of pulmonary complications did not differ significantly between AML subtypes; 2. Pulmonary complications at presentation occurred more often in patients of advanced aged with a low absolute neutrophil leukocyte count. Consequently, the baseline absolute neutrophil leukocyte count is an important prognostic factor in patients with advanced age; 3. Ground-glass appearance was the most common nonspecific radiological finding and should be considered as infection in AML patients until proven otherwise; 4. The serum LDH level was observed to be a prognostic factor in the AML patients; however, it is a non-specific marker of pulmonary complications.

In conclusion, the most common pulmonary complication in the present study was infection. We think that the presence of pulmonary complications at diagnosis is a predictor of poor prognosis in AML patients.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Cheson BD, Cassileth PA, Head DR, Schiffer CA, Bennett JM, Bloomfield CD, Brunning R, Gale RP, Grever MR, Keating MJ. Report of the NCI-sponsored workshop on definitions of diagnosis and response in acute myeloid leukemia. J Clin Oncol 1990; 8: 813-819 2. Pizzo PA. Fever in immucompromised patients. N Engl J Med 1999; 341: 893-900 3. Wade JC. Management of infection in patients with acute leukemia. Hematol Oncol Clin North Am 1993; 7: 293-315 4. Chaoui D, Legrand O, Roche N, Cornet M, Lefebvre A, Peffault de Latour R, Sanhes L, Huchon G, Marie JP, Rabbat A. Incidence and prognostic value of respiratory events in acute leukemia. Leukemia 2004; 18: 670-675

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Bugdacı MS, et al: Pulmonary Radiological Findings in AML

5. Krowka MJ, Rosenow EC, Hoagland HC. Pulmonary of bone marrow transplantation. Chest 1985; 87: 237-246 6. Rossini F, Verga M, Pioltelli P, Giltri G, Sancassani V, Pogliani EM, Corneo G. Incidence and outcome of pneumonia in patients with acute leukemia receiving first induction therapy with antracyclines-containing regimens.

Haematologica 2000; 85: 1255-1260 7. Demirkazik FB, Akin A, Uzun O, Akpinar MG, Ariyürek MO. CT findings in immunocomprimised patients with pulmonary infections. Diagn Interv Radiol 2008; 14: 75-82 8. Wardman AG, Milligan DW, Child JA, Delamore IW, Cooke NJ. Pulmonary infiltrates and adult acute leukaemia: Empirical treatment and survival related to the extent of pulmonary radiological disease. Thorax 1984; 39: 568-571 9. Sverzellati N, Zompatori M, Poletti V, Geddes DM, Hansell DM. Small chronic pneumothoraces and pulmonary parenchymal abnormalities after bone marrow transplantation. J Thorac Imaging 2007; 22: 230-234 10. Bruno C, Minniti S, Vassanelli A, Pozzi-Mucelli R. Comparison of CT features of aspergillus and bacterial pneumonia in severely neutropenic patients. J Thorac Imaging 2007; 22: 160-165 11. Marchiori E, Escuissato DL, Gasparetto TD, Considera DP, Franquet T. “Crazy-Paving” patterns on high resolution CT scans in patients with pulmonary after hematopoietic stem cell transplantation. Korean J Radiol 2009; 10: 21-24 12. Gevenois PA, de Maertelaer V, De Vuyst P, Zanen J, Yernault JC. Comparison of computed density and macroscopic morphomtery in pulmonary emphysema. Am J Respir Crit Care Med 1995; 152: 653-657 13. Subirà M, Martino R, Rovira M, Vazquez L, Serrano D, De La Cámara R. Clinical applicability of the new EORTC/MSG classification for invasive pulmonary aspergillosis in patients with hematological malignancies and autopsy-confirmed invasive aspegillosis. Ann Hematol 2003; 82: 80-82 14. Hughes WT, Armstrong D, Bodey GP, Brown AE, Edwards JE, Feld R, Pizzo P, Rolston KV, Shenep JL, Young LS. 1997 guidelines for the use of antimicrobial agents in neutropenic patients with unexplained fever. Infectious Diseases Society of America. Clin Infect Dis 1997; 25: 551-573

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Turk J Hematol 2012; 29: 217-222

15. Hughes WT, Armstrong D, Bodey GP, Bow EJ, Brown AE, Calandra T, Feld R, Pizzo PA, Rolston KV, Shenep JL, Young LS. 2002 guidelines for the use of antimicrobial agents in neutropenic patients with cancer. Clin Infect Dis 2002; 34: 730-751 16. Ewig S, Glasmacher A, Ulrich B, Wilhelm K, Schäfer H, Nachtsheim KH. Pulmonary infiltrates in neutropenic patients with acute leukemia during chemotherapy, outcome and prognostic factors. Chest 1998; 114: 444-451 17. Rosenow EC 3rd, Wilson WR, Cockerill FR 3rd. Pulmonary disease in immunocompromised host (second of two parts).

Mayo Clin Proc 1985; 60: 473-487 18. Rañó A, Agustí C, Jimenez P, Angrill J, Benito N, Danés C, González J, Rovira M, Pumarola T, Moreno A, Torres A. Pulmonary infiltrates in non-HIV immunocompromised patients: a diagnostic approach using non-invasive and bronchoscopic procedures. Thorax. 2001 May;56(5): 379387 19. Segal BH. Aspergillosis. N Engl J Med 2009; 360: 1870-1884 20. Cornet M, Fleury L, Maslo C, Bernard JF, Brücker G. Invasive Aspergillosis Surveillance Network of the Assistance Publique-Hôpitaux de Paris: Epidemiology of invasive aspergillosis in France: A six-year multicentric survey in the greater Paris area. J Hosp Infect 2002; 51: 288-296 21. Aquino VM. Acute myelogenous leukemia. Curr Probl Pediatr Adolesc Health Care 2002; 32: 50-58 22. Forman D, Stockton D, Møller H, Quinn M, Babb P, De Angelis R, Micheli A. Cancer pravalence in UK: Results from the EUROPREVAL study. Ann Oncol 2003; 14: 648-654 23. Deschler B, Lübbert M. Acute myeloid leukemia: Epidemiology and etiology. Cancer 2006; 107 (9): 2099-2107 24. Vlad O, Peddinghaus ME, Rubin R. Acute monocytic leukemia presenting as pulmonary infiltration. Commun Oncol 2008; 5: 328-330 25. Darmon M, Azoulay E. Prognosis of Acute Monocytic Leukemia (French-American-British Classification M5). J Clin Oncol 2005; 23: 1327 26. Briasoulis E, Pavlidis N. Noncardiogenic pulmonary edema: unusual and serious complication of anticancer therapy.

Oncologist 2001; 6: 153-161

Commentary

10.5505/tjh.2012.16013

Pulmonary Complications in Acute Myeloid Leukemia Selami K. Toprak Baskent University, School of Medicine, Department of Hematology, Ankara, Turkey

In this issue of the journal, Bugdaci et al. reports on the pulmonary radiological findings in patients with acute myeloblastic leukemia (AML) and their relation to chemotherapy and prognosis [1]. Respiratory events occurred in 19% of the 278 patients, especially in advanced aged patients with low absolute neutrophil leukocyte counts at initial presentation of disease. Pulmonary findings were due to infection in 56.6% of cases and to another cause in 24.5%. These results and the rate of pulmonary events with undetermined etiologies were consistent with similar studies from different groups such as Chaoui et al. and Rano et al. [2, 3].

In the present study, Bugdaci et al. reported infection as the most common pulmonary complication in AML. Although most cases represent infectious pneumonia, with bacteria (28.3%) and fungi (26.5%) the leading pathogens, noninfectious etiologies such as leukemic infiltration (3.8%), pulmonary embolism (7.5%), pneumothorax (3.8%), and cardiac disease (9.4%) also must be taken into account.

Both pulmonary leukostasis and malignant infiltration of the lung have been described in patients with AML [4].

In contrast to leukostasis, leukemic pulmonary infiltration has been defined in AML patients without hyperleukocytosis, suggesting that the type of blasts and their affinity for the pulmonary endothelium may be involved in the development of ARF. Bugdaci et al. reported two cases of AML M5 and M4/5 with pulmonary leukemic infiltration at initial presentation of disease. Azoulay et al. showed that adding dexamethasone (10 mg every 6 hours, until leukopenia occurred) to the chemotherapy regimen in AML M5 with acute respiratory failure from leukemiarelated pulmonary involvement significantly diminished mortality [5].

Pulmonary manifestations including leukemia-related respiratory involvement and infectious process such as

hypoxemic acute respiratory failure (ARF) and pneumonia, constitute diagnostic and therapeutic emergencies, and contribute to the morbidity and mortality during the course of AML [6]. In patients with AML and pulmonary infiltrates, infection must be suspected and treated empirically, because ARF, the leading reason for intensive care unit admission, still carries a 50% overall mortality rate despite improvements in supportive therapy. However, clinically defined respiratory events may be directly due to the leukemia-related involvement consist of leukostasis, leukemic infiltrates and lysis pneumopathy [5]. In addition to rapid cytoreduction by hydration and chemotherapy, anti-inflammatory therapy appears to be effective in improving outcomes in these patients. For many years, glucocorticoids (GCs) are often clinically used to treat lymphoid leukemia and lymphoma. The induction of apoptosis is thought to be a main mechanism mediating the therapeutic effect of GCs [7]. In contrast to acute lymphoblastic leukemia, AML cells have usually been resistant to these agents [8]. Nevertheless, steroid therapy has been used in AML with pulmonary disease of infectious or noninfectious origin, to prevent acute respiratory distress syndrome through various mechanism, including decrease cytokine and oxidant release, blast adhesion to endothelial cells and blast degeneration within the interstitium [5].

In conclusion, irrespective of their etiology, respiratory events are of a major prognostic significance [2]. Moreover, leukemic pulmonary involvement should be considered in AML patients with noninfectious lung infiltration.

References 1. Bugdaci MS, Yanardag H, Ar MC, Soysal T, Coskun S, Demirci S. Pulmonary radiological findings in patients with acute myeloid leukemia and their relation to chemotherapy and prognosis: a single center retrospective study. Turk J Hematol. 2012;29:217-222.

Address for Correspondence: Selami K. Toprak, M.D., Başkent Üniversitesi, Tıp Fakültesi, Hematoloji Bilim Dalı, Ankara, Turkey Phone: +90 312 212 29 12 E-mail: sktoprak@yahoo.com

223

Toprak SK,: Pulmonary Complications in Acute Myeloid Leukemia

2. Chaoui D, Legrand O, Roche N, Cornet M, Lefebvre A, Peffault de Latour R, Sanhes L, Huchon G, Marie JP, Rabbat A. Incidence and prognostic value of respiratory events in acute leukemia. Leukemia. 2004;18(4):670-675.

3. Rañó A, Agustí C, Jimenez P, Angrill J, Benito N, Danés C, González J, Rovira M, Pumarola T, Moreno A, Torres A. Pulmonary infiltrates in non-HIV immunocompromised patients: a diagnostic approach using non-invasive and bronchoscopic procedures. Thorax. 2001;56(5):379-387.

4. Azoulay E, Fieux F, Moreau D, Thiery G, Rousselot P, Parrot A, Le Gall JR, Dombret H, Schlemmer B. Acute monocytic leukemia presenting as acute respiratory failure. Am J Respir Crit Care Med. 2003;167(10):1329-1333.

5. Azoulay É, Canet E, Raffoux E, Lengliné E, Lemiale V, Vincent F, de Labarthe A, Seguin A, Boissel N, Dombret H, Schlemmer B. Dexamethasone in patients with acute lung injury from acute monocytic leukaemia. Eur Respir J. 2012;39(3):648-653.

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Turk J Hematol 2012; 29: 223-224

6. Uzunhan Y, Cadranel J, Boissel N, Gardin C, Arnulf B, Bergeron A. The specific pulmonary manifestations of acute myeloid leukaemia. Rev Mal Respir. 2010;27(6):589-598.

7. Kanatani Y, Kasukabe T, Okabe-Kado J, Hayashi S, Yamamoto-Yamaguchi Y, Motoyoshi K, Nagata N, Honma Y. Transforming growth factor beta and dexamethasone cooperatively enhance c-jun gene expression and inhibit the growth of human monocytoid leukemia cells. Cell Growth Differ. 1996;7(2):187-196.

8. Hicsonmez G. A novel approach to treatment in childhood acute myeloblastic leukemia and myelodysplastic syndrome with high-dose methylprednisolone as a differentiation -and apoptosis- inducing agent of myeloid leukemic cells. Turk J Hematol. 2010;27:1-7.

Research Article

10.5505/tjh.2012.24392

Analysis of Chromosomal Aberrations and FLT3 gene Mutations in Childhood Acute Myelogenous Leukemia Patients Çocukluk Çağı Akut Myeloid Lösemi Hastalarında Kromozomal Değişiklikler ve FLT3 Geni Mutasyonlarının Araştırılması Ender Coşkunpınar1, Sema Anak2, Leyla Ağaoğlu2, Ayşegül Ünüvar2, Ömer Devecioğlu2, Gönül Aydoğan3, Çetin Timur4, Ahmet Faik Öner5, Yıldız Yıldırmak6, Tiraje Celkan7, İnci Yıldız7, Nazan Sarper8, Uğur Özbek1 İstanbul University, Institute of Experimental Medical Research, Department of Genetics, İstanbul, Turkey İstanbul University, School of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey 3 Bakırköy Maternity and Children’s Hospital, İstanbul, Turkey 4 Göztepe Education and Research Hospital, Department of Pediatric Hematology, İstanbul, Turkey 5 Yüzüncü Yıl University, School of Medicine, Department of Pediatrics, Van, Turkey 6 Şişli Etfal Education and Research Hospital, Department of Pediatric Hematology, İstanbul, Turkey 7 İstanbul University, Cerrahpaşa School of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey 8 Kocaeli University, School of Medicine, Department of Children’s Health and Diseases, Kocaeli, Turkey 1 2

Abstract Objective: To identify the well-known common translocations and FLT3 mutations in childhood acute myelogenous leukemia (AML) patients in Turkey.

Material and Methods: The study included 50 newly diagnosed patients in which t(15;17), t(8;21), and inv(16) chromosomal translocations were identified using real-time PCR and FLT3 gene mutations were identified via direct PCR amplification PCR-RE analysis.

Results: In all, t(15;17) chromosomal aberrations were observed in 4 patients (8.0%), t(8;21) chromosomal aberrations were observed in 12 patients (24.0%), inv(16) chromosomal aberrations were observed in 3 patients (6.0%), and FLT3ITD mutations were observed in 2 patients (4.0%); FLT3-D835 point mutation heterozygosity was observed in only 1 patient (2.0%) patient.

Conclusion: Despite of the known literature, a patient with FLT3-ITD and FLT3-D835 double mutation shows a better survival and this might be due to the complementation effect of the t(15;17) translocation. The reportedmutation rate in this article (4%) of FLT3 gene seems to be one of the first results for Turkish population.

Key Words: Childhood AML, FLT3 gene mutations, ITD, D835 mutations, Chromosomal translocations

Address for Correspondence: Uğur Özbek, M.D., İstanbul Üniversitesi DETAE Genetik AD, Vakıf Gureba Cad. Capa, Fatih 34093 İstanbul, Turkey Phone: +90 212 414 20 00 (33312) E-mail: uozbek@istanbul.edu.tr Received/Geliş tarihi : December 28, 2010 Accepted/Kabul tarihi : February 21, 2012

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Coşkunpınar E, et al: Chromosomal Aberrations and FLT3 Mutations in AML

Turk J Hematol 2012; 29: 225-232

Özet Amaç: Bu çalışmada Türk çocukluk çağı AML hastalarında sık görülen kromozomal translokasyonların ve FLT3 mutasyonlarının belirlenmesi amaçlandı.

Gereç ve Yöntemler: Yeni tanı almış 50 hastada real time PCR yöntemi ile t(15;17), t(8;21), inv(16) kromozomal translokasyonlarının varlığı ve PCR yöntemi ile FLT3 geni ITD tipi mutasyonlar ve D835 nokta mutasyonları varlığı araştırıldı.

Bulgular: Olguların 4’ünde t(15;17) (8.0%), 12’sinde t(8;21) (24%), 3’ünde inv(16) (6.0%) kromozomal düzensizlikleri, 2 olguda FLT3-ITD mutasyonu (4.0%), 1 olguda FLT3-D835 nokta mutasyonu (2,0%) varlığı tespit edildi. t(15;17) pozitif AML M3’lü bir olguda hem FLT3-ITD hem de FLT3-D835 mutasyonları bakımından heterozigotluk tespit edildi.

Sonuç: Literatürden farklı olarak, bir hastamızın daha uzun sağkalımının t(15;17) translokasyonunun mutasyonlu bireylerde iyi yönde düzeltici etkisinin neden olabileceği düşünülmektedir. FLT3 geninde görülen bu mutasyon oranı (4%) Türk toplumu için ilk sonuçlardan biridir.

Anahtar Sözcükler: Çocukluk çağı akut myeloid lösemi, FLT3 gen mutasyonları, İnternal tandem duplikasyon-ITD, D835 mutasyonları, Kromozomal translokasyonlar

Materials and Methods

Introduction Acute myelogenous leukemia (AML) is a malignant disease of myeloid stem cells linked to oncogenic fusion proteins, which is due to chromosome translocations and inversions. Numerous translocations have been described in AML, of which the most common are t(8;21), t(15;17), and inv(16). These recurring translocations are currently used as the basis for classification of AML [1]. As such, AML-associated fusion proteins function as aberrant transcriptional regulators, with the potential to interfere with normal myeloid cell differentiation [1-3]. FMS-like tyrosine kinase 3 (FLT3)—a new member of the receptor tyrosine kinase (RTK) III subfamily—was originally identified in hematopoietic stem/progenitor cells and is important for normal lymphohematopoietic stem cell function [4].

FLT3 is aberrantly expressed in the most of AML patients.

The FLT3 gene is located on chromosome 13 (13q12) [5].

To date, 2 distinct types of FLT3 gene mutations have been identified in AML cases: 1. Internal tandem duplication (ITD) mutations, which occur within the juxtamembrane region of the gene; 2. Point mutations that occur at codon 835 (D835) within the kinase domain. Both types of mutations constitutively activate FLT3 tyrosine kinase activity [6]. FLT3 gene mutations are strongly associated with leukocytosis and poor prognosis in AML patients [5,7,8].

Patients with either of these mutations have a higher risk of recurrence and a lower survival rate [8]. It was recently reported that the FLT3 gene mutant/normal ratio can be used as a marker for the selection of therapy [5-8]. The present study aimed to indentify the well-known common AML translocations and FLT3 mutations in childhood AML patients in Turkey.

226

Patients The study included 50 newly diagnosed childhood AML patients (28 male and 22 female) that presented for molecular diagnosis to Istanbul University, Institute of Experimental Medicine, Istanbul, Turkey, between October 2007 and July 2008. The Istanbul University, School of Medicine Ethics Committee approved the study protocol (project No. 1850/2007) and informed consent was provided by the patients’ parents. Diagnostic bone marrow samples were divided into 2 parts; 2 x 107 cells were preserved in RTL buffer (cat. No. 79216, Qiagen, Germany) at –80 °C until RNA isolation and the remainder of the samples were used for DNA isolation, according to the manufacturer’s instructions (cat. No. 11796828001 Roche Applied Sciences, Germany).

Determination of t(15:17), t(8:21) and inv(16) chromosome abnormalities Total RNA was extracted from bone marrow specimens using a QIAamp RNA Blood Mini Kit (cat. No. 52304, Qiagen, GmbH, D-40724 Hilden, Germany), according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA, as previously described [7]. The quality of the obtained cDNA was evaluated via ß-globin PCR performed using the following primers: forward: 5’ GAA GAG CCA AGG ACA GGT AC 3’; reverse: 5’ CAA CTT CAT CCA CGT TCA CC 3’. Chromosomal abnormalities [t(15; 17), t(8; 21), and inv(16)] were identified via real-time PCR, using the LightMix primer/probe set (cat. No. 40-013516 cat. No. 40-0196-16 cat. No. 40-0229-16 TIB Molbiol GmbH, Berlin, Germany), and the Light Cycler FastStart

Turk J Hematol 2012; 29: 225-232

Coşkunpınar E, et al: Chromosomal Aberrations and FLT3 Mutations in AML

DNA Master Hyprobe Kit (cat. No. 03515575001, Roche Diagnostics, GmbH, Mannheim, Germany).

Detection of FLT3-ITD mutations FLT3-ITD mutations were indentified via PCR. The forward primer was in exon 14 (14F 5’-GCAATTTAGGTATGAAAGCCAGC-3’) and the reverse primer was in exon 15 (15R 5’-CTTTCAGCATTTTGACGGCAACC-‘3), as described by Wang et al. [4]. Amplification was performed in a reaction volume of 50 µL with 100 ng µL–1 of DNA, 10 pmol of each primer, 10 mmol dNTP, and 2.5 U of Ex-Taq DNA polymerase (cat. No. RR001A Takara, Japan) in the buffer (10 mmol L–1 of TrispHCl [pH 8.3], 50 mmol L–1 of KCl, and 1.5 mmol L–1 of MgCl2). The PCR conditions were as follows: initial denaturation at 95 °C for 5 min, 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s for 30 cycles, and elongation for 10 min at 72 °C. Amplification products were analyzed on 3% agarose gel stained with ethidium bromide and samples with the specific PCR products (329bp) were considered as positive for FLT3-ITD mutations. The specific amplicons were purified using the QIAEX II Gel Extraction Kit (cat. No. 20021, Qiagen, Hilden, Germany), according to the manufacturer’s instructions, and directly sequenced for confirmation of PCR. Detection of FLT3-D835 mutations FLY3-D835 mutations were identified using the PCRRFLP method. The primers employed were 20F 5’-CGC-

a

b

c

d

CAGGAACGTGCTTG-3’ and 20R 5’-GCAGCCTCACATTGCCCC-3’, as described by Wang et al. [4]. At codon 835 an aspartate amino acid is encoded, providing a recognition site for restriction enzyme EcoRV; as such, mutants can be detected via the loss of this enzyme restriction site.

The PCR setup was as described above. The specific products were detected on agarose gel, followed by EcoRV (cat.

No. 50-720-3590 Takara, Japan) digestion at 37 °C for 4 h. Restriction products were detected on a 3.5% agarose gels and undigested PCR product indicated the presence of the mutation (Figure). The results were confirmed via direct sequencing.

Statistical analysis Clinical and laboratory characteristics at diagnosis were statistically correlated (age, sex, WBC count, hemoglobin level, PLT count, blast cells rate) with t(15;17), t(8;21), inv(16) chromosomal aberrations, and FLT3 mutations (Table 1). Fisher’s exact test and Pearson’s chi-square test were performed using SPSS v.12.0. P values less than 0.05 were considered statistically significant.

Results Diagnoses—based on French-American-British (FAB) classification—were as follows: M0 (n = 3); M1 (n = 9); M2 (n = 15); M3 (n = 12); M4 (n = 3); M5 (n = 5); M7 (n = 1). Additionally, 1 patient was lacking clinical data and could not be classified, and another patient that died following BMT and couldn’t be classified was thought to

Figure. a. PCR amplification of the FLT3-ITD region (lane M: size marker; lanes 1-10: normal samples). b. PCR amplification of the FLT3-ITD region (lane M: size marker; lanes 1-3: normal samples; lane 4: negative control; lane 5: FLT3/ITD-positive case). c. PCR amplification of FLT3-D835 (lane M: size marker; lanes 1-6: normal samples; lane 7: negative control). d. D835 mutation detection (lane M: size marker; lane 1; FLT3-D835-positive case; lane 2: wild type; lane 3: EcoRV undigested sample).

227

228

M

M

M

F

F

F

F

F

M

M

M

M

M

M

M

F

M

F

F

F

M

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

144

72

54

84

14

168

204

180

24

120

84

144

96

60

132

180

120

120

132

1

20

AGE (months)

3.730

27.000

1.800

11.900

74.600

5.500

1.400

1.300

5.450

9.800

23.700

10.710

12.100

8.500

5.200

3.600

1.100

4.100

1.200

132.000

15.920

11.50

8.30

8.20

7.20

8.10

3.60

12.80

8.90

7.70

7.40

10.70

10.20

7.40

11.00

6.90

11.20

7.90

7.00

5.90

NA

8.90

24.000

38.000

17.000

45.000

79.000

6.000

25.000

291.000

61.000

28.000

86.000

29.000

22.000

71.000

43.000

104.000

31.000

99.000

32.000

NA

15.000

73

48

94

49

80

48

51

81

37

61

72

47

48

53

45

22

100

40

60

NA

44 Positive

Negative

ITD

Negative

Positive

Negative

Negative Positive Negative Negative

Negative Positive Negative Negative

Positive Negative Negative Negative

Negative Positive Negative Negative

Negative Negative Positive

Negative Positive Negative Negative

Negative Positive Negative Negative

Negative Positive Negative Negative

Negative Positive Negative Negative

Negative Positive Negative Negative

Positive Negative Negative

Negative Positive Negative Negative

Negative Positive Negative Negative

Negative Positive Negative Negative

Negative Positive Negative Negative

Negative Positive Negative Negative

Positive Negative Negative Negative

Negative Negative Positive

Positive Negative Negative Negative

Negative Negative Negative

Negative Negative Positive

BM PLT WBC Hb Blast T(15;17) T(8;21) Inv(16) count count (g dL–1) 9 –1 9 –1 (x10 L ) (%) (x10 L )

WBC: White blood cell, Hb: hemoglobin, PLT: platelet, BM: bone marrow, FAB: French-American-British; WT: wild type; HET: heterozygous; NA: not available.

SEX

Case No.

Table 1: Characteristics of childhood AML patients carrying FLT3 gene mutations and/or chromosomal aberrations.

WT

WT

WT

WT

WT

WT

WT

WT

WT

WT

HET

WT

WT

WT

WT

WT

WT

WT

WT

WT

WT

D835

NA

48.8

1.73

NA

31.7

58.6

70.7

NA

1.76

86.5

8.81

69.5

44.2

15.9

33.8

45.2

4.0

1.38

NA

NA

47.4

M2

M2

M3

M2

M5

M2

M2

M1

M1

M2

M3

M2

M2

M2

M2

M2

M3

M1

M3

NA

M0

CD34 FAB (%) Classification

Coşkunpınar E, et al: Chromosomal Aberrations and FLT3 Mutations in AML Turk J Hematol 2012; 29: 225-232

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Coşkunpınar E, et al: Chromosomal Aberrations and FLT3 Mutations in AML

Table 2: Patient mutation status and clinical features.

Characteristics

FLT3 Negative Patients (n) 48

Age (years) 0-5 5-10 10-18 Sex Male Female WBC (x109 L–1) ≤50 >50 Unknown Hemoglobin (g dL–1) ≤10 >10 Unknown Platelet (x109 L–1) ≤50 >50 Unknown Blast cells (%) ≤80 >80 Unknown

FLT3 Positive Patients (n) 2

15 11 22

2 -

26 22

2 -

38 8 2

1 1 -

31 16 1

1 1

21 25 2

1 1

30 13 5

1 1

be M0 or M7. Median age of the patients was 8.42 ± 5.24 years (range: 0-18 years). The median white blood cell (WBC) count was 30,394.17 ± 57,255.86 mL–1 (range: 890-260,000 mL–1), the median platelet (PLT) count was 83,851.06 ± 76,349.87 L–1 (range: 2 x 109-291 x 109 L–1), the median hemoglobin level was 3.4 ± 2.67 g dL–1 (range: 3.4-14.3 g dL–1), and the median bone marrow blast rate was 60.86% ± 22.9% (range: 12%-100%).

FAB classification and clinical features of the 50 childhood AML patients are summarized in Table 2. In all, 4 patients were positive for t(15;17), 12 were positive for t(8; 21), and 3 were positive for inv(16) (Table 3). The 4 t(15;17)-positive patients were classified as M3, and 9 of the 12 t(8;21)-positive patients were M2, 2 were M1, and 1 was M4. Among the inv(16)-positive patients, 1 was

Table 3: The frequency of translocations in the childhood AML patients.

Chromosome translocation breakpoints

Positive (n = 50)

Rate (%)

t(8;21)(q22;q22)

13

26

inv(16)(p13;q22)

3

6

t(15;17)(q22;q21)

4

8

M0, 1 was M1, and 1 was M5. None of the patients were classified as M6; therefore, statistical evaluation of the FAB M6 patients was excluded. In total, 2 patients had FLT3 gene mutations, 1 of which was classified as AML-M3 and interestingly the FLT3-D835 mutation was not a deletion, but a point mutation (g.IVS20 +49 A>G) that also changed the EcoRV restriction site. Both of these changes were previously described and are known to increase expression of FLT3 [1,5,6].

The hemoglobin level in the patients with FLT3-ITD mutations was significantly lower than in the patients without the mutation. In the present study there was a correlation between FAB M2 classification and t(8;21) positivity (P = 0.005), and between FAB M3 classification and t(15;17) positivity (P = 0.009), which is in agreement with previous reports. In addition inv(16) was positive in the FAB M0, M1, and M5 patients with P values of P = 0.001, P = 0.003, P = 0.002 respectively.None of the FAB M4 patients were positive. Bone marrow blast rates below and higher than 80% were compared with t(8; 21) positivity and found that patients with t(8;21) had higher blast rates than non translocated ones (P = 0.049).

Discussion In addition to observation of the standard clinical features and laboratory analysis, the diagnosis of AML requires additional procedures, including pathological examination, immunophenotyping, cytogenetics examination, and molecular diagnostics. Identification of the specific cytogenetic abnormality is important for selection of appropriate therapy and prognostic analysis [1,9]. Numerous translocations have been described in AML, of which the most frequent are t(15;17), t(8;21), and inv(16), accounting for 20%-30% of all chromosomal aberrations [1,1012].These aberrations, depending on their structure, lead to expression of a chimeric protein with new functions [8,13].Prognosis is considered goodin cases oft(15;17)

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associated with AML-M3, t(8;21), inv(16) associated with AML-M2, and inv(16) associated with AML-M4 [3,14,15].

It is known that the blast level in AML-M2 patients is 30%90% [16-18].

In the present study the bone marrow blast rate in 11 of the 12 FAB M2 patients with t(8;21) translocation was over 80%, which shows that the bone marrow blast level in childhood AML patients classified as FAB M2 is high.

Additionally, inv(16) was strongly correlated with FAB classification (P = 0.043). The 3 inv(16)-positive AML patients were classified as follows: M0 (n = 1); M1 (n = 1); M5 (n = 1). According to the literature, inv(16) occurs more frequently in patients classified as FAB M4 [19] and AML-M4 occurs more frequently in patients classified as FAB M4, primarily in patients aged ≥50 years [17,18]. The presence of inv(16) is indicative of a good prognosis in FAB M4 patients, but data concerning the relationship between inv(16) and other AML FAB classifications are lacking. In the present study inv(16) was not observed in any of the FAB M4 patients. The present findings are in agreement with those reported by Dash et al. and the American National Cancer Institute AML guideline [14,20].The FLT3 gene is expressed primarily in hematopoietic stem cells [5,21,22].

Moreover, human leukemia and lymphoma cell lines also express FLT3 protein [22]. The 2 most common mutations of the FLT3 gene are FLT3-ITD and FLT3-D835. Among the 50 childhood AML patents in the present study, only 2 had FLT3-ITD mutations, of which 1 also had FLT3D835 point mutation. The incidence of FTL3-ITD mutation in childhood AML patients (5-16%) is lower than that in adult AML patients (20-25%) [22,25]. Meshinchi et al. reported that the incidence of FLT3-TKD (tyrosine kinase domain) mutations in childhood AML patients was 6.7% [27], versus 7% reported by Karabacak et al. [23]. The known FLT3 gene mutation rate in adult AML patients is 20%-25% [22,25]; however, Liang et al. [24] reported a mutation rate in childhood AML patients of 11.3%, and Kondo et al. [22], Iwai et al. [25], and Krstovski et al. (26) reported rates of 5.3%-16.5%.

Our data is one of the first results FLT3-ITD mutation evaluation in Turkish pediatric AML patients and the mutation rate is 4.0%, which is much lower than the other study groups [23,27]. In the present study there wasn’t a correlation between FLT3 gene mutations, and age, gender, the WBCcount, blast cell rate, or FAB classification. Although it was reported that the FLT3-ITD mutation rate increases with age Meshinchi et al. [27], Kondo et al. [22] and Wang et al. [4] and was not observed a significant difference between the mutation rate and the age in our results.

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In the present study there wasn’t a significant difference between FLT3-ITD mutations and the WBC count. The WBC count in 95% of the present study’s patients was less than 50 x 109 L–1, which is in agreement with the findings reported by Moreno et al. [28]. The most common feature of AML is chronic, severe anemia, which damages bone marrow. The present results show that there was a relationship between a low hemoglobin level and FLT3-ITD mutations, but not between a low hemoglobin level and FLT3-D835 mutation, which might have been due to the small number of patients withFLT3-D835 point mutation as well as the small study population. Pre-clinical studies highlighted the potential use of inhibitors against FLT3 kinase activity. Most of patients carrying FLT3 gene mutation have a 50% decrease in the peripheral blast count, along with inhibition of receptor autophosphorylation.

Herein we described the development of anemia in patients carrying FLT3 gene mutation. It is also known that FLT3 mutations are poor prognostic markers of AML. A 7-year-old male patient in the present study classified as AML-M3 had double FLT3 mutations and t(15;17) translocation. He was in remission for 1 year, and died after 19 months of diagnosis. While FLT3 mutation causes continuous tyrosine kinase activity, t(15;17) translocation deregulates RARα transcription factor and cell differentiation stops. Thus, AML occurs due to the combined effects of FLT3 gene mutations and t(15;17) translocation. To the best of our knowledge our 7-year-old male patient is the first reported case to have been in remission for approximately 1 year with double FLT3 mutation and translocation. This is also the first case with double FLT3 mutation and t(15;17) translocation that is reported inTurkish population. Most FLT3 gene mutations are reported as individual mutations and only a few cases of double mutation have been reported [5,22,29,30]; Moreno et al. reported 4 cases in 2003 [28] and Wang et al. reported 2 cases in 2005 [4]. All the reported double mutation patients died after induction therapy or relapsed in first months after diagnosis [28]. These results indicate that FLT3-ITD and FLT3-D835 mutations are markers of poor prognosis. In contrast to previous reports our 7-year-old patient survived longer, which might have been due to the combined effect of t(15;17) translocation (31,32); however, the quality of clinical response to FLT3 inhibitors has been minor, with many patients transiently responding with a decreased blast count(31,32). This finding needs to be confirmed by in vitro studies in which patients are followed-up for longer periods of time. It is not clear if FLT3 double mutation causes upregulation in tyrosine kinase activity or increases cell survival. AML occurs as a result of excessive prolifera-

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tion and differentiation of myelogenous blasts. The present study is the first to perform detailed molecular analysis of Turkish childhood AML patients. The low FLT3 gene mutation rate (4%) seems to be unique to this study’s population. This result and the effects of double mutations need to be evaluated in larger patient groups.

We wish to thank the Research Council of Istanbul University for supporting this study.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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Philadelphia: JB Lippincott, 3rd ed. 1999 19. Colovic M, Jurisic V, Pavlovic S, Terzic T, Colovic N. FLT3/D835 mutation and inversion of chromosome 16 in leukemic transformation of myelofibrosis. Eur J Intern Med 2006; 17 (6): 434-435 20. Dash A, Gilliland DG. Molecular genetics of acute myeloid leukaemia. Best Pract Res Clin Haematol 2001; 14 (1): 49-64 21. Drexler HG. Expression of FLT3 receptor and response to FLT3 ligand by leukemic cells. Leukemia 1996; 10 (4): 588-599

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Research Article

10.5505/tjh.2012.94547

Comparison of Stored Umbilical Cord Blood and Adult Donor Blood: Transfusion Feasibility Saklanan Kordon Kanının Erişkin Bağışçı Kanıyla Karşılaştırılması: Transfüzyon için Kullanılabilirlik Çalışması Rola Sahyoun-Tokan1, Saadet Arsan2, Ömer Erdeve2, Nuri Solaz6, Aslıhan Avcı4, Serenay Elgün-Ülkar4, Elif Gülyapar7, Zeynep Üstünyurt7, Zeynep Bıyıklı5, Sabri Kemahlı3,6 Ankara University, Faculty of Medicine, Department of Pediatrics, Ankara, Turkey Ankara University, Faculty of Medicine, Department of Pediatrics, Division of Neonatology, Ankara, Turkey 3 Ankara University, Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Ankara, Turkey 4 Ankara University, Faculty of Medicine, Department of Biochemistry, Ankara, Turkey 5 Ankara University, Faculty of Medicine, Department of Biostatistics, Ankara, Turkey 6 Ankara University, Faculty of Medicine, Serpil Akdağ Blood Center, Ankara, Turkey 7 Dr. Zekai Tahir Burak Women’s Hospital, Ankara, Turkey 1 2

Abstract Objective: This study aimed to compare the storage properties of red blood cell (RBC) concentrates of umbilical cord blood (UCB) and adult donor blood (ADB), and to evaluate the feasibility of UCB-RBC concentrate as an autologous source for blood transfusion in very low birth weight (VLBW) preterm neonates.

Material and Methods: In all, 30 newborn (10 preterm, 20 full term) UCB and 31 ADB units were collected.

RBC concentrates were stored and compared with regard to pH, potassium (K+), 2,3-biphosphoglycerate (2-3-BPG), adenosine tri-phosphate (ATP), plasma Hb, and bacterial contamination on d 1, 21, and 35 of storage.

Results: The K+ level increased with time and differed significantly between storage d 1 and 21, and between storage d 1 and 35 in both the UCB and ADB units. Initial and d 21 K+ levels were higher in the UCB units than in the ADB units. The 2,3-BPG level did not differ significantly between the UCB-PRC and ADB-PRC samples. After 35 d of storage both UCB-PRC and ADB-PRC samples exhibited significant differences from the initial free Hb, intracellular ATP, and pH values. Significant differences in intracellular ATP and pH were also observed between the UCB-PRC and ADB-PRC samples.

Conclusion: The volume of harvested and prepared UCB-PRC can be used for some of the blood transfusions required during the neonatal period and thus may decrease the number of allogeneic transfusions, especially in preterm newborns.

The hematological and biochemical changes that occurred in UCB during storage were comparable with those observed in ADB, and do not pose a risk to the immature metabolism of neonates. UCB-RPC prepared and stored under standard conditions can be a safe alternative RBC source for transfusions in VLBW newborns.

Key Words: Transfusion, Newborn, VLBW preterm, Umbilical cord blood, Blood storage

Address for Correspondence: Sabri Kemahlı, M.D., Ankara Üniversitesi, Tıp Fakültesi, Çocuk Sağlığı ve Hastalıkları Anabilim Dalı, Cebeci 06100 Ankara, Turkey Phone: +90 312 595 66 35 E-mail: kemahli@medicine.ankara.edu.tr Received/Geliş tarihi : February 3, 2011 Accepted/Kabul tarihi : January 30, 2012

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Özet Amaç: Çalışmada kordon kanı ile erişkin bağışçı kanlarından elde edilen eritrosit süspansiyonlarının özelliklerini ve saklama sırasında oluşan değişiklikleri izleyerek, kordon kanından elde edilen eritrosit süspansiyonlarının çok düşük doğum ağırlıklı yenidoğanlarda otolog bir transfüzyon kaynağı olarak kullanılabilirliğini değerlendirmek amaçlanmıştır.

Gereç ve Yöntemler: 30 yenidoğan (20 preterm, 10 term) kordon kanı ile 31 erişkin bağışçı kanı toplandı. Hazırlanan eritrosit süspansiyonları standard koşullarda saklanarak 1., 21. ve 35. günlerde pH, potasyum (K+), 2,3-BPG, adenozin trifosfat (ATP), plazma Hb ve bakteriyel kontaminasyon bakımından incelendi.

Bulgular: Potasyum değerlerinin zamanla artarak, hem kordon kanı hem de erişkin kanlarında 1.-21. ve 1-35. günler arasında anlamlı farklar saptandı. İlk ve 21. Gün değerlerinin kordon kanında erişkin kanlarından daha yüksek olduğu saptandı. 2,3-BPG değerleri kordon kanı ve erişkin kanlarında anlamlı fark göstermedi.Saklamanın 35. gününde hem kordon kanı hem de erişkin kanlarında serbest Hb, ATP ve pH değerlerinin ilk değerlere gore anlamlı fark gösterdiği belirlendi.Bu parametreler açısından kordon kanı ve erişkin kanı grupları arasında da fark olduğu görüldü.

Sonuç: Toplanan ve hazırlanan kordon kanı eritrosit süspansiyonları yenidoğan dönemindeki transfüzyon gereksinimini kısmen karşılayabilir ve böylece allogeneik transfüzyonları azaltabilir. Saklama sırasında oluşan hematolojik ve biyokimyasal değişiklikler erişkin bağışçı kanlarındakilere benzerlik göstermekte olup yenidoğan metabolizması için bir risk oluşturacak düzeyde değildir. Kordon kanından hazırlanıp standart koşullarda saklanan eritrosit süspansiyonları çok düşük doğum ağırlıklı yenidoğanlar için bir seçenek olabilir.

Anahtar Sözcükler: Transfüzyon, Yenidoğan, Çok düşük doğum ağırlıklı preterm, Kordon kanı, Kan saklama Introduction The majority of neonates usually have uncomplicated hematological adaptation to their physiological circumstances following delivery; however, very premature neonates are prone to suffer from mild to severe anemia, which must be corrected. At present, adult donor blood (ADB) is used for neonates that require transfusion [1,2,3].

Although the risk of transfusion-associated infections has been significantly reduced due to changes in blood collection, testing, processing, and storage, the risks associated with blood transfusion have not been reduced to zero and these risks are well documented [4,5].

Umbilical cord blood (UCB) is emerging as an option for autologous red cell transfusion in low-birth-weight (LBW) and preterm infants. Its autologous nature negates the risks inherent in transfusion from adult donors; however, few studies have compared the properties of red blood cell (RBC) concentrates from UCB and ADB, and the changes that occur in each during storage. The aim of the present study was to compare the storage properties of UBC and ADB RBC concentrates, and to evaluate the feasibility of UBC RBC concentrates as an autologous source of blood transfusion in very low birth weight (VLBW) preterm neonates.

Material and Methods Participants ADB samples were collected at Ankara University, Ser-

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pil Akdağ Blood Center, Ankara, Turkey, from 31 healthy male and female donors that met the standard blood donor criteria and provided informed consent to participate in the study. The donors were tested to rule out hepatitis B, hepatitis C, HIV, and syphilis infection. UCB was collected at Zekai Tahir Burak Women’s and Maternity Hospital, Ankara, Turkey, from 20 full-term and 10 preterm infants (gestational age <32 weeks) that were delivered via caesarean section; informed consent was obtained from their parents before delivery.

Exclusion criteria for the collection of UCB were maternal viral or bacterial infections, including suspected chorioamnionitis (defined by the presence of purulent amniotic fluid, C-reactive protein [CRP] >10 mg L–1, or an immature to total white blood cell (I:T) ratio in the mother >0.15). None of the blood units tested in this study were used for transfusion.

The study protocol was approved by the ethics committees of Ankara University, School of Medicine and Zekai Tahir Burak Women’s and Maternity Hospital, and was performed in accordance with the Declaration of Helsinki.

Blood collection Standard units (450 ± 45 mL) of blood were drawn from each adult donor into a main blood bag that contained 63 mL of citrate-phosphate-dextrose (CPD). Pediatric blood bag sets (Kansuk Laboratuarı, Istanbul Turkey) were used for UCB collection. As standard blood bags con-

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tain CPD solution at a ratio of 1:7 for 450 mL of whole blood, the volume of CPD for use in UCB collection bags was calculated using the same ratio and an estimated maximum UCB volume of 140 mL; thus, UCB was collected into bags containing 21 mL of CPD. Placental blood collection was performed using a specifically modified placental blood collection system. Before placental blood collection the blood collecting system’s primary bag was placed approximately 1 m below the level of the placenta. Placental blood was collected immediately after birth, with the placenta in utero. Cord clamping was performed immediately after the baby was born and then the puncture site was identified approximately 10 cm from the placenta-cord interface. The umbilical cord vein was then punctured with a cannula attached at the longer collection tube of the collection system. The collecting tube was clamped before the blood clotted and the blood flow stopped. Collected blood was immediately transported to the blood bank where it was separated into blood components, as RBCs and plasma.

Separation and RBC unit preparation Collected UCB was stored under standard storage conditions (2-6 °C) and processed within 24 h of collection.

In all, 30 newborn (10 preterm and 20 full-term) UCB and 31 ADB units were collected. UCB and ADB units were centrifuged at 2600 g for 5.5 min. Whole blood was then separated into plasma and buffy coat depleted RBCs using a blood component separation device. The total collected volume of UBC packed red cell concentrate (UCB-PRC) and 31 ADB-PRC samples were recorded. Then, 8 mL of blood from the main bag was transferred to each of 2 accessory bags. Blood samples for testing were withdrawn from the accessory bags to avoid contamination of the main bag on d 21 and d 35. All blood units were stored in the same blood bank refrigerator at 4 °C. Biochemical tests Biochemical (2,3-biphosphoglycerate [2,3-BPG], adenosine triphosphate [ATP], free Hb, K+, and pH) parameters were measured in all UCB-PRC samples on d 1, 21, and 35 of storage. Data were compared with data obtained from 31 ADB-PRC samples stored under the same conditions. Microbiological testing was performed on all samples via blood cultures using the Bactec system. The recommended blood volume (2 mL) was injected into 50-mL Bactec NR 660 aerobic and anaerobic pediatric blood culture bottles (Becton Dickinson, Franklin Lakes, NJ, USA).

Inoculation of the samples was performed every week.

Cultures were assessed daily for the presence of bacterial

Tokan RS, et al: Comparison of Cord Blood and Adult Blood

growth for 1 week after inoculation. Serum K+ was measured via ion-selective electrode (ISE Olympus AU 600, Hamburg, Germany) ATP Measurement Intracellular ATP was measured via bioluminescence assay. The test principle is based on luciferase from Photinus pyralis (American firefly) catalyzing the following reaction: ATP + D-luciferin + O2 → oxyluciferin + PPi + AMP + CO2 + light.

The methods specified in the commercial kit (ATP Bioluminescence Assay Kit CLS II, Roche Cat. No. 1699695) were used.

2,3-BPG measurement 2,3-BPG was measured spectrophotometrically based on the change in absorbency at 340 nm caused by oxidation from NADH to NAD due to glycerol-3-phosphate dehydrogenase (GDH), according to the methods specified in the commercial kit (2,3-biphosphoglycerate Kit, Roche, Cat. No.10148334001).

Free Hb measurement Free Hb was measured spectrophotometrically, as described previously [6].

Statistical analysis Data analysis was performed using SPSS v.11.5. As the variables 2,3-BPG, ATP, K+, and pH were not normally distributed, Friedman’s ANOVA followed by Wilcoxon test with Bonferroni correction was used to determine the differences within groups, based on time. The differences of measured variables between UCB and ADB was analyzed using the non-parametric Mann-Whitney U test with Bonferoni correction [7].

In that case 3 different tests were performed to reduce the error made by Bonferroni correction and thus, P < 0.05/3 = P < 0.017 was considered statistically significant; when analyzing differences between 2 different time points, this level was accepted as P < 0.05/2 = P < 0.025, as 2 different tests were performed.

Results Characteristics of the collected and processed blood The volume of UCB collected ranged between 43 and 94 mL, depending on gestational age. The harvested UCB volume was 43-77 mL (mean: 57.1 ± 10.5 mL) for pre-

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On d 35 of storage free Hb (Table 5), pH (Table 6), and intracellular ATP values (Table 7) differed significantly from the initial values in both UCB and ADB RBCs. Significant differences in these parameters were also observed between UCB-PRC and ADB-PRC.

term newborns and 62-94 mL (mean: 77.8 ± 10.5 mL) for full-term newborns. Birth weight was associated with the volume of collected UCB; approximately 24.8 mL of UCB kg–1 of bodyweight was collected, irrespective of gestational age. Mean RBC volume, on the other hand, was 14.6 ± 2.4 mL kg–1 for the preterm newborns and 12.6 ± 1.5 mL kg–1 for the full-term newborns.

Discussion The present study aimed to evaluate changes in UCBPRC and ADB-PRC in an additive storage medium (CPD) during storage. This preclinical study was considered to be essential for determining the feasibility of the clinical use of UCB. No transfusions were performed with the blood units collected during this study; however, the use of UCB for transfusion has been studied previously. Tamayo [8] reported the successful transfusion of 12 unfractionated UCB specimens (volume: 25-150 mL) in 1966 and suggested that UCB could be used instead of allogeneic transfusion. Ballin et al. published a case report describing how transfusion of 2 units of autologous UCB in a preterm baby did not cause any side effects [9]. In addition to the publication by Anderson et al. [10], Strauss published a warning on the clinical use of UCB, in which he suggested that the relative risks due to infections, changes during

Microbial contamination Bacterial contamination was not observed in any of the UCB or ADB units during storage.

Changes during storage Storage parameters for the 30 UCB-PRC and 31 ADBPRC samples are shown in Table 2. The K+ level increased with time in both groups, with significant differences between d 1 and 21, and between d 1 and 35. Initial and d 21 levels were higher in the UCB samples than in the ADB samples (Table 3). The 2,3-BPG level did not differ significantly between UCB-PRC and ADB-PRC samples (P > 0.017); however, there was a significant decrease (P < 0.01) on d 21 and d 35—as compared to d 1—in both groups (Table 4).

Table 1: Characteristics of the neonates and UCB volume.

Birth weight

n

Gestational age

Mean UCB

Mean RBC

(weeks)

Volume (mL)

Volume (mL)

(g) 1500-1999

4

31.4 ± 0.4

49.0 ± 7.6

25.2 ± 3.9

2000-2499

6

31.9 ± 0.2

62.6 ± 8.5

32.9 ± 5.7

2500-2999

4

39.4 ± 0.7

74.3 ± 7.

40.1 ± 6.0

3000-3999

15

38.9 ± 1.0

77.9 ± 10.9

41.7 ± 3.4

> 4000

1

39

92

52

Table 2: Mean hematological and biochemical parameters.

Day 1 K (mmol L ) 2,3-BPG (mmol L–1) Free Hb (g dL–1) Intracellular ATP (mmol L–1) pH Bacterial Contamination +

236

–1

Day 21

Day 35

UCB

ADB

UCB

ADB

UCB

ADB

6.5 ± 2.2 1.2 ± 0.4 20.8 ± 2.7 1 ± 0.3 x 10–7 6.5 ± 0.2

3.50 ± 0.2 1.1 ± 1.0 21.1 ± 2.2 1.3 ± 0.4 x 10–7 7.0 ± 0.0

35.9 ± 3.7 0.2 ± 0.1 28.0 ± 2.3 0.2 ± 0.7 x 10–7 6.3 ± 0.1

33.1 ± 4.0 0.2 ± 0.2 22.3 ± 1.8 0.5 ± 0.5 x 10–7 6.7 ± 0.0

38.3 ± 5.0 0.2 ± 0.1 28.0 ± 2.2 0.0 ± 0.02 x 10–7 6.2 ± 0.1

40.5 ± 3.1 0.2 ± 0.3 30 ± 2.5 0.3 ± 0.1 x 10–7 6.6 ± 0.1

Ø

Ø

Ø

Ø

Ø

Ø

Turk J Hematol 2012; 29: 233-241

Tokan RS, et al: Comparison of Cord Blood and Adult Blood

Table 3: Comparison of K+ values in UCB and ADB samples over time.

Day 1

Day 21

Day 35

UCB (mmol L–1)

6.5 ± 2.2 (3.2-10.6)

35.9 ± 3.7 (27.5- 40.1)

38.3 ± 5.0 (34.6-54.2)

ADB (mmol L–1)

3.5 ± 0.2 (2.9-3.7)

33.1 ± 4.0 (24.6-37)

40.5 ± 3.1 (33.3-46.6)

P Values in each group Days 1-21 P < 0.001 Days 1-35 P < 0.001 Days 21-35 P > 0.05 Days 1-21 P < 0.05 Days 1-35 P < 0.001 Days 21-35 P > 0.05

P Values Between Groups

Day 1 P < 0.001 Day 21 P = 0.01 Day 35 P = 0.1

Table 4: Comparison of 2,3,-BPG values in UCB and ADB samples over time.

Day 1

Day 21

Day 35

UCB (mmol L–1)

1.2 ± 0.4 (0.5-2.1)

0.2 ± 0.1 (0.1-0.6)

0.2 ± 0.1 (0.1-0.6)

ADB (mmol L–1)

1.1 ± 1.0 (0.7-3.9)

0.2 ± 0.2 (0.0-0.8)

0.2 ± 0.3 (0.1-1.9)

storage, and efficacy—as compared to allogeneic transfusions—should be clarified before the start of clinical studies [11].

Eichler et al. studied VLBW infants (body weight <1000 g) and collected a net mean UCB volume of only 37 mL; RBC preparation was successful only in exceptional cases. They did conclude that the preparation of autologous RBCs from UCB collected from preterm infants was technically possible [12]. One of the concerns of UCB has been the small volume of blood collected. Researchers have tried to show the correlation between the volume of collected blood and birth weight, type of delivery (vaginal

P Values in each group

P Values Between Groups

Days 1-21 P < 0.001 Days 1-35 P < 0.001 Days 21-35 P> 0.05 Days 1-21 P < 0.001 Days 1-35 P < 0.001 Days 21-35 P > 0.05

Day 1 P = 0.026 Day 21 P = 0.038 Day 35 P = 0.035

or caesarean section), and collection method (ex utero versus in utero) [12-19]. Despite some inconsistent results, Surbek et al. [14,15], Pafumi et al. [16,17], and Solves et al. [19] reported that in utero collection after caesarean delivery yielded higher collection volume. As such, in utero collection was employed in the present study.

Some studies examined the correlation between the volume of collected blood, and birth weight and gestational age of newborns. Garritsen et al. and Brune et al. harvested approximately 20 mL kg–1 of bodyweight, independent of birth weight, using both in utero and ex utero collection [20,21]. Eichler et al., on the other hand, reported

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Table 5: Comparison of free Hb values in UCB and ADB samples over time.

Day 1

Day 21

Day 35

UCB (g dL–1)

20.8 ± 2.7 (15-25)

28.0 ± 2.3 (22.8-31.7)

28.0 ± 2.0 (23.3-31.3)

ADB (g dL–1)

21.2 ± 2.2 (16.9-23.6)

22.3 ± 1.8 (18.6-27)

30.0 ± 2.5 (25.4-33.8)

P Values in each group Days 1-21 P < 0.001 Days 1-35 P < 0.001 Days 21-35 P > 0.05 Days 1-21 P > 0.05 Days 1-35 P < 0.001 Days 21-35 P < 0.001

P Values Between Groups

Day 1 P = 0.61 Day 21 P < 0.001 Day 35 P = 0.017

Table 6: Comparison of pH values in UCB and ADB samples over time.

UCB

ADB

Day 1

Day 21

Day 35

6.5 ± 0.2 (6.2-7.0)

6.3 ± 0.1 (6.1-6.4)

6.2 ± 0.1 (6.0-6.3)

7.0 ± 0.0 (7.0-7.1)

6.7 ± 0.0 (6.7-6.8)

6.6 ± 0.1 (6.5-6.7)

P Values in each group Days 1-21 P < 0.001 Days 1-35 P < 0.001 Days 21-35 P > 0.05 Days 1-21 P < 0.01 Days 1-35 P < 0.001 Days 21-35 P > 0.05

P Values Between Groups

Days 1, 21 and 35 P < 0.001

Table 7: Comparison of ATP values in UCB and ADB samples over time.

UCB (mmol L–1)

ADB (mmol L–1)

238

Day 1

Day 21

Day 35

1.0 ± 0.3 x 10–7 (0.3 -1.6 x 10–7)

0.2 ± 0.7 x 10–7 (0.0 - 2.1 x 10–7)

0.0 ± 0.2 x 10–7 (0.0- 0.5 x 10–7)

1.3 ± 0.4 x 10–7 (0.5-2.1 x 10–7)

0.5 ± 0.5 x 10–7 (0.3 -2.1 x 10–7)

0.3 ± 0.1 x 10–7 (0.0-0.5 x 10–7)

P Values in each group Days 1-21 P > 0.05 Days 1-35 P < 0.001 Days 21-35 P < 0.001 Days 1-21 P > 0.05 Days 1-35 P < 0.001 Days 21-35 P < 0.001

P Values Between Groups

Day 1 P = 0.029 Day 21 P = 0.021 Day 35 P < 0.001

Turk J Hematol 2012; 29: 233-241

that there was an inverse correlation between the volume of collected blood, and gestational age and birth weight.

They harvested 43 mL kg–1 of body weight from preterm infants [12]. Surbek et al. collected a median volume of 21 mL (range: 8-38 mL) following delivery of infants with a gestational of 22-32 weeks and a median volume of 49 mL (range: 21-103 mL) after delivery of infants with a gestational age of 33-36 weeks; birth weight and the volume of collected blood were not correlated [14,15]. Jansen et al. reported a UCB collection volume of 23 mL kg–1 in infants with a gestational age <32 weeks, but did not note a correlation between the volume of harvested UCB, and gestational age, birth weight, placental weight, or method of delivery [22]. Khodabux et al. observed a correlation between the collected blood volume and gestational age.

The collected volume of UCB was 32 ± 7.7 mL for infants with a gestational age of 24-28 weeks, 44 ± 27.4 mL for those with a gestational age 28-30 weeks, and 33 ± 13.3 mL for infants with gestational age of 30-32 weeks. The volume of harvested UCB was 16 ± 15 mL kg–1 in infants with a birth weight <1000 g, 18 ± 18 mL kg–1 for those weighing 1000-1250 g, and 20 ± 23 mL kg–1 for those with a birth weight >1250 g; however, birth weight and the volume of harvested blood were not correlated [23].

Mean volume of harvested UCB in the present study was 57.11 ± 10.46 mL in the preterm infants and 77.84 ± 10.54 mL in the full-term infants, and was 24.87 mL kg–1 of bodyweight, irrespective of gestational age. Mean RBC volume, on the other hand, was 14.63 ± 2.43 mL kg–1 in the preterm infants and 12.55 ± 1.46 mL in the full-term newborns, which is similar to previous reports.

Mean UCB volume kg–1 of birth weight was higher in the preterm infants; however, correlation analysis between the collected volume, and birth weight and gestational age was not performed.

Numerous studies reported that K+, 2,3-BPG, ATP, and pH values in stored blood and blood components change with time, and research has been conducted in an effort to increase the quality of stored blood [24-30]. Studies on the biochemical and hematological properties of stored UCB, however, are less common. Garritsen et al. studied whether or not UCB-PRC could be used as an alternative to ADB-PRC. They developed a system for collecting and preparing UCB-PRC, and measured standard storage parameters during 35 d of storage in extended storage medium (SAG mannitol). The initial laboratory UCB-PRC parameters were similar to those of ADB-PRC. After 35 d of storage UCB-PRC had a hemolysis rate 1% higher than that of ADB-PRC and a significant decrease in ATP. UCB-

Tokan RS, et al: Comparison of Cord Blood and Adult Blood

PRC met the same quality criteria as ADB-PRC after 35 d of storage [20].

Bifano et al. studied ‘placental whole blood’ stored in CPDA for 28 d and reported that the K+ level on d 28 was comparable to that in stored ADB. In the UCB ATP, 2,3-BPG, and pH decreased, and some morphological changes, an increase in osmotic fragility, and a minimal increase in hemolysis were noted. They also reported that fetal erythrocytes were preserved better in citrate-phospohate-adenine-1 solution (CPDA-1) than in CPD [31].

The hemolysis rate, however, was higher in the studies

by Brune et al. [32], Garritsen et al. [20], and Widing et al. [33]. In a recent review Khodabux and Brand reported that the preservative solution used, not the manipulation of UCB, might contribute to storage damage. This has been suggested by some other studies that reported better results using the storage solution PAGGSM that contained additional phosphate and guanosine than those obtained using SAG-M [34].

In the present study the 2,3-BPG level did not differ significantly between UCB-PRC and ADB-PRC (P > 0.017). After 35 d of storage, free Hb, intracellular ATP, and pH values were significantly different than initial values in both UCB RBCs and ADB RBCs. Additionally, it was observed that 2,3-BPG, ATP, and pH values decreased in both blood groups, whereas K+ increased. These significant differences in both blood groups were thought to have been due to an increase in hemolysis and decrease in intracellular ATP. The higher K+ value on d 1 of storage reached a similar level in ADB on d 35. Bacterial contamination has been one of the concerns of the groups dealing working with UCB. In the present study bacterial contamination was not observed in any of the stored UCB-PRC samples. Eichler et al. had raised concerns with regard to the high rate of bacterial contamination [12]. Garritsen et al. [20] reported a bacterial contamination rate of 1.84% in 390 collected UCB units, and Jansen et al. reported there was bacterial contamination in 6 of 91 (7%) collected UCB units stored for 30 days [22].

In conclusion, the volume of harvested and prepared UCB-PRC is similar to previous studies and can supply for some of the blood transfusions required in neonatal period and thus, its use may decrease number of allogeneic transfusions, especially in premature newborns. Hematological and biochemical changes that occur to UCB during storage are comparable to those observed in ADB and do not pose a risk to the relatively immature metabolism of neonates [35-37]. The lack of any observable bacterial contami-

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nation in the present study indicates that the collection method employed was reliable. The present findings show that the collection, separation, and storage of UBC-PRC, and the changes that occur during 35 d of storage can yield a product that is feasible for use under clinical conditions.

In addition, autologous transfusion is safer than allogeneic transfusion, in terms of immunohematology and infection risk. The use and outcomes of UCB however, remain to be determined by further clinical studies.

Abbreviations: VLBW: Very low birth weight, UCB: Umbilical cord blood, ADB: Adult donor blood, RBC: Red Blood Cell, K+: Potassium, 2,3-BPG: 2,3-biphosphoglycerate, ATP: Adenosine tri-phosphate, UCB-PRC: Umbilical cord blood- packed red blood cell concentrate, A-PRC: Adult packed red blood cell concentrate, CPD: Citrate-phosphatedextrose additive solution This study has been supported by Ankara University Scientific Research Projects Fund References 1. Holland BM, Jones JG, Wardrop CA. Lessons from the anaemia of prematurity. Hematol Oncol Clin North Am 1987;1 (3): 355-366 2. Shannon KM. Anaemia of Prematurity : Progress and prospects. Am J Pediatr Hematol Oncol 1990; 12: 14-20 3. Ramasethu J, Luban NL. Red Blood Cell transfusion in the newborn. Seminars in Neonatology 1999; 4: 5-16 4. Nathanson N. Emergence of new viral infections: Implications for the blood supply. Biologicals 1998; 26 (2): 77-84 5. Chamberland ME. Surveillance for blood borne infections.

Thromb Haemost 1999: 82: 494-499 6. Van Kampen E, Zijlstra WG. Standardization of hemoglobinometry: II. The hemiglobincyanide method.

Clin Chim Acta 1961; 6: 538–544 7. Siegel S, Castellan Jr. NJ. Nonparametric Statistics For The Behavioral Sciences. New York: McGraw-Hill Inc.,1998 8. Tamayo JG. The use of residual placental blood for transfusion. J Philipp Med Assoc 1966; 42 (7): 399-406 9. Ballin A, Arbel E, Kenet G, Berar M, Kohelet D, Tanay A, Zakut H, Meytes D. Autologous umbilical cord blood transfusion. Arch Dis Child Fetal Neonatal Ed 1995: 73: 181-183 10. Anderson S, Fangman J, Wager G, Uden D. Retrieval of placental blood from umbilical vein to determine volume, sterility and presence of clot formation. Am J Dis Child 1992; 146 (1): 36-39

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11. Strauss RG. Autologous transfusions for neonates using placental blood. A cautionary note. Am J Dis Child 1992; 146 (1): 21-22 12. Eichler H, Schaible T, Richter E., Zeiger W, Voller K, Leveringhaus A, Goldmann SF. Cord blood as a source of autologous RBC’s for transfusion to preterm infants.

Transfusion 2000; 40: 1111-1117 13. Reboredo N, Diaz A, Castro A, Villaescusa RG. Collection, processing and cryopreservation of umbilical cord blood for unrelated transplantation. Bone Marrow Transplant 2000; 26: 1263–1270 14. Surbek DV, Glanzmann R, Senn HP, Hoesli I, Holzgreve W. Can cord blood be used for autologous transfusion in preterm neonates? Eur J Pediatr 2000; 159 (10): 790-791 15. Surbek DV, Holzgreve W, Steinmann C, Hahn S, Gratwohl A, Wodnar-Filipowicz A, Tichelli A. Preterm birth and the availability of cord blood for HPC transplantation.

Transfusion 2000; 40: 817–820 16. Pafumi C, Farina M, Bandiera S, Cavallaro A, Pernicone G, Russo A, Iemmola A, Chiarenza M, Leonardi I, Calogero AE, Calcagno A, Cianci A. Differences in umbilical cord blood units collected during cesarean section, before or after the delivery of the placenta. Gynecol Obstet Invest 2002; 54: 73–77 17. Pafumi C, Milone G, Maggi I, Mancari R, Farina M, Russo A, Pernicone G, Bandiera S, Giardina P, Franceschini A, Calogero AE, Cianci A. Umbilical cord blood collection in Cesarean section: A comparison before and after placental delivery. Arch Gynecol Obstet 2002; 266: 193–194 18. Sparrow RL, Cauchi JA, Ramadi LT, Waugh CM, Kirkland MA. Influence of mode of birth and collection on WBC yields of umbilical cord blood units. Transfusion 2002; 42: 210–215 19. Solves P, Mirabet V, Larrea L, Moraga R, Planelles D, Saucedo E, Uberos FC, Planells T, Guillen M, Andres A, Monleon J, Soler MA, Franco E. Comparison between two cord blood collection strategies. Acta Obstet Gynecol Scand 2003; 82: 439-442 20. Garritsen HS, Brune T, Louwen F, Wüllenweber J, Ahlke C, Cassens U, Witteler R, Sibrowski W. Autologous red cells derived from cord: collection, preparation, storage and quality controls with optimal additive storage medium (Sagmannitol). Transfus Med 2003; 13: 303-310 21. Brune T, Garritsen H, Hentschel R, Louwen F, Harms E, Jorch G. Efficacy, recovery and safety of RBCs from autologous placental blood: Clinical experience in 52 newborns. Transfusion 2003; 43: 1210-1216 22. Jansen M, Brand A, von Lindern JS, Scherjon S, Walther FJ. Potential use of autologous umbilical cord blood red cells for early transfusion needs of premature infants. Transfusion 2006; 46: 1049-1056

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23. Khodabux CM, von Lindern JS, van Hilten JA, Scherjon S, Walther FJ, Brand A. A clinical study on the feasiblility of autologous cord blood transfusion for anemia of prematurity.

Transfusion 2008; 48: 1634-1643 24. Wallas CH. Sodium and potassium changes in blood bank stored human erythrocytes. Transfusion 1979; 19: 210-215 25. Moroff G, Dende D. Characterization of biochemical changes occuring during storage of red cells. Comparative studies with CPD and CPDA-1 anticoagulant-preservative solutions. Transfusion 1983; 23: 484-489 26. Hess JR, Greenwalt TG. Storage of blood cells: New approaches. Transfus Med Rev 2002; 16: 283-295 27. Sezdi M, Bayık M, Ülgen Y. Storage effect on the Cole-Cole parameters of erythrocyte suspensions. Physiol Meas 2006; 27: 623-635 28. Högman CF, Löfh H, Meryman HT. Storage of Red Blood cells with improved maintenance of 2,3- Bisphosphoglycerate.

Transfusion 2006; 46: 1543-1552 29. Yoshida T, AuBuchon JP, Dumont LJ, Gorham JD, Gifford SC, Foster KY, Bitensky MW. The effects of additive solution pH and metabolic rejuvenation on anaerobic storage of red cells. Transfusion 2008; 48: 2096-2105 30. de Korte D, Kleine M, Korsten HG, Verhoeven AJ. Prolonged maintenance of 2,3-diphopsphoglycerate acid and adenosine triphosphate in red blood cells during storage. Transfusion 2008; 48: 1081-1089 31. Bifano EM, Dracker RA, Lorah K, Palit A. Collection and 28-day storage of human placental blood. Pediatr Res 1994; 36: 90-94

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32. Brune T, Garritsen H, Witteler R, Schlake A, Wüllenweber J, Louwen F, Jorch G, Harms E. Autologous placental blood transfusion for the therapy of anaemic neonates. Biol Neonate 2002: 81: 236–243 33. Widing L, Bechensteen AG, Mirlashari MR, Vetlesen A, Kjeldsen-Kragh J. Evaluation of nonleukoreduced red blood cell transfusion units collected at the delivery from the placenta. Transfusion 2007; 47: 1481–1487 34. Khodabux CM, Brand A. The use of cord blood for transfusion purposes: Current status. Vox Sang 2009; 97: 281-293 35. Parshuram CS, Joffe AR. Prospective study of potassiumassociated acute transfusion events in pediatric intensive care. Pediatr Crit Care Med 2003; 4: 65-68 36. Vohra HA, Adluri K, Willets R, Horsburgh A, Barron DJ, Brawn WJ. Changes in potassium concentration and hematocrit associated with cardiopulmonary bypass in paediatric cardiac surgery. Perfusion 2007; 22: 87-92 37. Dani C, Perugi S, Benuzzi A, Corsini I, Bertini G, Pratesi S, Rubaltelli FF. Effects of red blood cell transfusions during the first week of life on acid-base, glucose and electrolytes in preterm neonates. Transfusion 2008; 48: 2302-2307

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Research Article

10.5505/tjh.2012.94547

Comparison of Stored Umbilical Cord Blood and Adult Donor Blood: Transfusion Feasibility Saklanan Kordon Kanının Erişkin Bağışçı Kanıyla Karşılaştırılması: Transfüzyon için Kullanılabilirlik Çalışması Rola Sahyoun-Tokan1, Saadet Arsan2, Ömer Erdeve2, Nuri Solaz6, Aslıhan Avcı4, Serenay Elgün-Ülkar4, Elif Gülyapar7, Zeynep Üstünyurt7, Zeynep Bıyıklı5, Sabri Kemahlı3,6 Ankara University, Faculty of Medicine, Department of Pediatrics, Ankara, Turkey Ankara University, Faculty of Medicine, Department of Pediatrics, Division of Neonatology, Ankara, Turkey 3 Ankara University, Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Ankara, Turkey 4 Ankara University, Faculty of Medicine, Department of Biochemistry, Ankara, Turkey 5 Ankara University, Faculty of Medicine, Department of Biostatistics, Ankara, Turkey 6 Ankara University, Faculty of Medicine, Serpil Akdağ Blood Center, Ankara, Turkey 7 Dr. Zekai Tahir Burak Women’s Hospital, Ankara, Turkey 1 2

Abstract Objective: This study aimed to compare the storage properties of red blood cell (RBC) concentrates of umbilical cord blood (UCB) and adult donor blood (ADB), and to evaluate the feasibility of UCB-RBC concentrate as an autologous source for blood transfusion in very low birth weight (VLBW) preterm neonates.

Material and Methods: In all, 30 newborn (10 preterm, 20 full term) UCB and 31 ADB units were collected.

RBC concentrates were stored and compared with regard to pH, potassium (K+), 2,3-biphosphoglycerate (2-3-BPG), adenosine tri-phosphate (ATP), plasma Hb, and bacterial contamination on d 1, 21, and 35 of storage.

Results: The K+ level increased with time and differed significantly between storage d 1 and 21, and between storage d 1 and 35 in both the UCB and ADB units. Initial and d 21 K+ levels were higher in the UCB units than in the ADB units. The 2,3-BPG level did not differ significantly between the UCB-PRC and ADB-PRC samples. After 35 d of storage both UCB-PRC and ADB-PRC samples exhibited significant differences from the initial free Hb, intracellular ATP, and pH values. Significant differences in intracellular ATP and pH were also observed between the UCB-PRC and ADB-PRC samples.

Conclusion: The volume of harvested and prepared UCB-PRC can be used for some of the blood transfusions required during the neonatal period and thus may decrease the number of allogeneic transfusions, especially in preterm newborns.

The hematological and biochemical changes that occurred in UCB during storage were comparable with those observed in ADB, and do not pose a risk to the immature metabolism of neonates. UCB-RPC prepared and stored under standard conditions can be a safe alternative RBC source for transfusions in VLBW newborns.

Key Words: Transfusion, Newborn, VLBW preterm, Umbilical cord blood, Blood storage

Address for Correspondence: Sabri Kemahlı, M.D., Ankara Üniversitesi, Tıp Fakültesi, Çocuk Sağlığı ve Hastalıkları Anabilim Dalı, Cebeci 06100 Ankara, Turkey Phone: +90 312 595 66 35 E-mail: kemahli@medicine.ankara.edu.tr Received/Geliş tarihi : February 3, 2011 Accepted/Kabul tarihi : January 30, 2012

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Özet Amaç: Çalışmada kordon kanı ile erişkin bağışçı kanlarından elde edilen eritrosit süspansiyonlarının özelliklerini ve saklama sırasında oluşan değişiklikleri izleyerek, kordon kanından elde edilen eritrosit süspansiyonlarının çok düşük doğum ağırlıklı yenidoğanlarda otolog bir transfüzyon kaynağı olarak kullanılabilirliğini değerlendirmek amaçlanmıştır.

Gereç ve Yöntemler: 30 yenidoğan (20 preterm, 10 term) kordon kanı ile 31 erişkin bağışçı kanı toplandı. Hazırlanan eritrosit süspansiyonları standard koşullarda saklanarak 1., 21. ve 35. günlerde pH, potasyum (K+), 2,3-BPG, adenozin trifosfat (ATP), plazma Hb ve bakteriyel kontaminasyon bakımından incelendi.

Bulgular: Potasyum değerlerinin zamanla artarak, hem kordon kanı hem de erişkin kanlarında 1.-21. ve 1-35. günler arasında anlamlı farklar saptandı. İlk ve 21. Gün değerlerinin kordon kanında erişkin kanlarından daha yüksek olduğu saptandı. 2,3-BPG değerleri kordon kanı ve erişkin kanlarında anlamlı fark göstermedi.Saklamanın 35. gününde hem kordon kanı hem de erişkin kanlarında serbest Hb, ATP ve pH değerlerinin ilk değerlere gore anlamlı fark gösterdiği belirlendi.Bu parametreler açısından kordon kanı ve erişkin kanı grupları arasında da fark olduğu görüldü.

Sonuç: Toplanan ve hazırlanan kordon kanı eritrosit süspansiyonları yenidoğan dönemindeki transfüzyon gereksinimini kısmen karşılayabilir ve böylece allogeneik transfüzyonları azaltabilir. Saklama sırasında oluşan hematolojik ve biyokimyasal değişiklikler erişkin bağışçı kanlarındakilere benzerlik göstermekte olup yenidoğan metabolizması için bir risk oluşturacak düzeyde değildir. Kordon kanından hazırlanıp standart koşullarda saklanan eritrosit süspansiyonları çok düşük doğum ağırlıklı yenidoğanlar için bir seçenek olabilir.

Anahtar Sözcükler: Transfüzyon, Yenidoğan, Çok düşük doğum ağırlıklı preterm, Kordon kanı, Kan saklama Introduction The majority of neonates usually have uncomplicated hematological adaptation to their physiological circumstances following delivery; however, very premature neonates are prone to suffer from mild to severe anemia, which must be corrected. At present, adult donor blood (ADB) is used for neonates that require transfusion [1,2,3].

Although the risk of transfusion-associated infections has been significantly reduced due to changes in blood collection, testing, processing, and storage, the risks associated with blood transfusion have not been reduced to zero and these risks are well documented [4,5].

Umbilical cord blood (UCB) is emerging as an option for autologous red cell transfusion in low-birth-weight (LBW) and preterm infants. Its autologous nature negates the risks inherent in transfusion from adult donors; however, few studies have compared the properties of red blood cell (RBC) concentrates from UCB and ADB, and the changes that occur in each during storage. The aim of the present study was to compare the storage properties of UBC and ADB RBC concentrates, and to evaluate the feasibility of UBC RBC concentrates as an autologous source of blood transfusion in very low birth weight (VLBW) preterm neonates.

Material and Methods Participants ADB samples were collected at Ankara University, Ser-

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pil Akdağ Blood Center, Ankara, Turkey, from 31 healthy male and female donors that met the standard blood donor criteria and provided informed consent to participate in the study. The donors were tested to rule out hepatitis B, hepatitis C, HIV, and syphilis infection. UCB was collected at Zekai Tahir Burak Women’s and Maternity Hospital, Ankara, Turkey, from 20 full-term and 10 preterm infants (gestational age <32 weeks) that were delivered via caesarean section; informed consent was obtained from their parents before delivery.

Exclusion criteria for the collection of UCB were maternal viral or bacterial infections, including suspected chorioamnionitis (defined by the presence of purulent amniotic fluid, C-reactive protein [CRP] >10 mg L–1, or an immature to total white blood cell (I:T) ratio in the mother >0.15). None of the blood units tested in this study were used for transfusion.

The study protocol was approved by the ethics committees of Ankara University, School of Medicine and Zekai Tahir Burak Women’s and Maternity Hospital, and was performed in accordance with the Declaration of Helsinki.

Blood collection Standard units (450 ± 45 mL) of blood were drawn from each adult donor into a main blood bag that contained 63 mL of citrate-phosphate-dextrose (CPD). Pediatric blood bag sets (Kansuk Laboratuarı, Istanbul Turkey) were used for UCB collection. As standard blood bags con-

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tain CPD solution at a ratio of 1:7 for 450 mL of whole blood, the volume of CPD for use in UCB collection bags was calculated using the same ratio and an estimated maximum UCB volume of 140 mL; thus, UCB was collected into bags containing 21 mL of CPD. Placental blood collection was performed using a specifically modified placental blood collection system. Before placental blood collection the blood collecting system’s primary bag was placed approximately 1 m below the level of the placenta. Placental blood was collected immediately after birth, with the placenta in utero. Cord clamping was performed immediately after the baby was born and then the puncture site was identified approximately 10 cm from the placenta-cord interface. The umbilical cord vein was then punctured with a cannula attached at the longer collection tube of the collection system. The collecting tube was clamped before the blood clotted and the blood flow stopped. Collected blood was immediately transported to the blood bank where it was separated into blood components, as RBCs and plasma.

Separation and RBC unit preparation Collected UCB was stored under standard storage conditions (2-6 °C) and processed within 24 h of collection.

In all, 30 newborn (10 preterm and 20 full-term) UCB and 31 ADB units were collected. UCB and ADB units were centrifuged at 2600 g for 5.5 min. Whole blood was then separated into plasma and buffy coat depleted RBCs using a blood component separation device. The total collected volume of UBC packed red cell concentrate (UCB-PRC) and 31 ADB-PRC samples were recorded. Then, 8 mL of blood from the main bag was transferred to each of 2 accessory bags. Blood samples for testing were withdrawn from the accessory bags to avoid contamination of the main bag on d 21 and d 35. All blood units were stored in the same blood bank refrigerator at 4 °C. Biochemical tests Biochemical (2,3-biphosphoglycerate [2,3-BPG], adenosine triphosphate [ATP], free Hb, K+, and pH) parameters were measured in all UCB-PRC samples on d 1, 21, and 35 of storage. Data were compared with data obtained from 31 ADB-PRC samples stored under the same conditions. Microbiological testing was performed on all samples via blood cultures using the Bactec system. The recommended blood volume (2 mL) was injected into 50-mL Bactec NR 660 aerobic and anaerobic pediatric blood culture bottles (Becton Dickinson, Franklin Lakes, NJ, USA).

Inoculation of the samples was performed every week.

Cultures were assessed daily for the presence of bacterial

Tokan RS, et al: Comparison of Cord Blood and Adult Blood

growth for 1 week after inoculation. Serum K+ was measured via ion-selective electrode (ISE Olympus AU 600, Hamburg, Germany) ATP Measurement Intracellular ATP was measured via bioluminescence assay. The test principle is based on luciferase from Photinus pyralis (American firefly) catalyzing the following reaction: ATP + D-luciferin + O2 → oxyluciferin + PPi + AMP + CO2 + light.

The methods specified in the commercial kit (ATP Bioluminescence Assay Kit CLS II, Roche Cat. No. 1699695) were used.

2,3-BPG measurement 2,3-BPG was measured spectrophotometrically based on the change in absorbency at 340 nm caused by oxidation from NADH to NAD due to glycerol-3-phosphate dehydrogenase (GDH), according to the methods specified in the commercial kit (2,3-biphosphoglycerate Kit, Roche, Cat. No.10148334001).

Free Hb measurement Free Hb was measured spectrophotometrically, as described previously [6].

Statistical analysis Data analysis was performed using SPSS v.11.5. As the variables 2,3-BPG, ATP, K+, and pH were not normally distributed, Friedman’s ANOVA followed by Wilcoxon test with Bonferroni correction was used to determine the differences within groups, based on time. The differences of measured variables between UCB and ADB was analyzed using the non-parametric Mann-Whitney U test with Bonferoni correction [7].

In that case 3 different tests were performed to reduce the error made by Bonferroni correction and thus, P < 0.05/3 = P < 0.017 was considered statistically significant; when analyzing differences between 2 different time points, this level was accepted as P < 0.05/2 = P < 0.025, as 2 different tests were performed.

Results Characteristics of the collected and processed blood The volume of UCB collected ranged between 43 and 94 mL, depending on gestational age. The harvested UCB volume was 43-77 mL (mean: 57.1 ± 10.5 mL) for pre-

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On d 35 of storage free Hb (Table 5), pH (Table 6), and intracellular ATP values (Table 7) differed significantly from the initial values in both UCB and ADB RBCs. Significant differences in these parameters were also observed between UCB-PRC and ADB-PRC.

term newborns and 62-94 mL (mean: 77.8 ± 10.5 mL) for full-term newborns. Birth weight was associated with the volume of collected UCB; approximately 24.8 mL of UCB kg–1 of bodyweight was collected, irrespective of gestational age. Mean RBC volume, on the other hand, was 14.6 ± 2.4 mL kg–1 for the preterm newborns and 12.6 ± 1.5 mL kg–1 for the full-term newborns.

Discussion The present study aimed to evaluate changes in UCBPRC and ADB-PRC in an additive storage medium (CPD) during storage. This preclinical study was considered to be essential for determining the feasibility of the clinical use of UCB. No transfusions were performed with the blood units collected during this study; however, the use of UCB for transfusion has been studied previously. Tamayo [8] reported the successful transfusion of 12 unfractionated UCB specimens (volume: 25-150 mL) in 1966 and suggested that UCB could be used instead of allogeneic transfusion. Ballin et al. published a case report describing how transfusion of 2 units of autologous UCB in a preterm baby did not cause any side effects [9]. In addition to the publication by Anderson et al. [10], Strauss published a warning on the clinical use of UCB, in which he suggested that the relative risks due to infections, changes during

Microbial contamination Bacterial contamination was not observed in any of the UCB or ADB units during storage.

Changes during storage Storage parameters for the 30 UCB-PRC and 31 ADBPRC samples are shown in Table 2. The K+ level increased with time in both groups, with significant differences between d 1 and 21, and between d 1 and 35. Initial and d 21 levels were higher in the UCB samples than in the ADB samples (Table 3). The 2,3-BPG level did not differ significantly between UCB-PRC and ADB-PRC samples (P > 0.017); however, there was a significant decrease (P < 0.01) on d 21 and d 35—as compared to d 1—in both groups (Table 4).

Table 1: Characteristics of the neonates and UCB volume.

Birth weight

n

Gestational age

Mean UCB

Mean RBC

(weeks)

Volume (mL)

Volume (mL)

(g) 1500-1999

4

31.4 ± 0.4

49.0 ± 7.6

25.2 ± 3.9

2000-2499

6

31.9 ± 0.2

62.6 ± 8.5

32.9 ± 5.7

2500-2999

4

39.4 ± 0.7

74.3 ± 7.

40.1 ± 6.0

3000-3999

15

38.9 ± 1.0

77.9 ± 10.9

41.7 ± 3.4

> 4000

1

39

92

52

Table 2: Mean hematological and biochemical parameters.

Day 1 K (mmol L ) 2,3-BPG (mmol L–1) Free Hb (g dL–1) Intracellular ATP (mmol L–1) pH Bacterial Contamination +

236

–1

Day 21

Day 35

UCB

ADB

UCB

ADB

UCB

ADB

6.5 ± 2.2 1.2 ± 0.4 20.8 ± 2.7 1 ± 0.3 x 10–7 6.5 ± 0.2

3.50 ± 0.2 1.1 ± 1.0 21.1 ± 2.2 1.3 ± 0.4 x 10–7 7.0 ± 0.0

35.9 ± 3.7 0.2 ± 0.1 28.0 ± 2.3 0.2 ± 0.7 x 10–7 6.3 ± 0.1

33.1 ± 4.0 0.2 ± 0.2 22.3 ± 1.8 0.5 ± 0.5 x 10–7 6.7 ± 0.0

38.3 ± 5.0 0.2 ± 0.1 28.0 ± 2.2 0.0 ± 0.02 x 10–7 6.2 ± 0.1

40.5 ± 3.1 0.2 ± 0.3 30 ± 2.5 0.3 ± 0.1 x 10–7 6.6 ± 0.1

Ø

Ø

Ø

Ø

Ø

Ø

Turk J Hematol 2012; 29: 233-241

Tokan RS, et al: Comparison of Cord Blood and Adult Blood

Table 3: Comparison of K+ values in UCB and ADB samples over time.

Day 1

Day 21

Day 35

UCB (mmol L–1)

6.5 ± 2.2 (3.2-10.6)

35.9 ± 3.7 (27.5- 40.1)

38.3 ± 5.0 (34.6-54.2)

ADB (mmol L–1)

3.5 ± 0.2 (2.9-3.7)

33.1 ± 4.0 (24.6-37)

40.5 ± 3.1 (33.3-46.6)

P Values in each group Days 1-21 P < 0.001 Days 1-35 P < 0.001 Days 21-35 P > 0.05 Days 1-21 P < 0.05 Days 1-35 P < 0.001 Days 21-35 P > 0.05

P Values Between Groups

Day 1 P < 0.001 Day 21 P = 0.01 Day 35 P = 0.1

Table 4: Comparison of 2,3,-BPG values in UCB and ADB samples over time.

Day 1

Day 21

Day 35

UCB (mmol L–1)

1.2 ± 0.4 (0.5-2.1)

0.2 ± 0.1 (0.1-0.6)

0.2 ± 0.1 (0.1-0.6)

ADB (mmol L–1)

1.1 ± 1.0 (0.7-3.9)

0.2 ± 0.2 (0.0-0.8)

0.2 ± 0.3 (0.1-1.9)

storage, and efficacy—as compared to allogeneic transfusions—should be clarified before the start of clinical studies [11].

Eichler et al. studied VLBW infants (body weight <1000 g) and collected a net mean UCB volume of only 37 mL; RBC preparation was successful only in exceptional cases. They did conclude that the preparation of autologous RBCs from UCB collected from preterm infants was technically possible [12]. One of the concerns of UCB has been the small volume of blood collected. Researchers have tried to show the correlation between the volume of collected blood and birth weight, type of delivery (vaginal

P Values in each group

P Values Between Groups

Days 1-21 P < 0.001 Days 1-35 P < 0.001 Days 21-35 P> 0.05 Days 1-21 P < 0.001 Days 1-35 P < 0.001 Days 21-35 P > 0.05

Day 1 P = 0.026 Day 21 P = 0.038 Day 35 P = 0.035

or caesarean section), and collection method (ex utero versus in utero) [12-19]. Despite some inconsistent results, Surbek et al. [14,15], Pafumi et al. [16,17], and Solves et al. [19] reported that in utero collection after caesarean delivery yielded higher collection volume. As such, in utero collection was employed in the present study.

Some studies examined the correlation between the volume of collected blood, and birth weight and gestational age of newborns. Garritsen et al. and Brune et al. harvested approximately 20 mL kg–1 of bodyweight, independent of birth weight, using both in utero and ex utero collection [20,21]. Eichler et al., on the other hand, reported

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Turk J Hematol 2012; 29: 233-241

Table 5: Comparison of free Hb values in UCB and ADB samples over time.

Day 1

Day 21

Day 35

UCB (g dL–1)

20.8 ± 2.7 (15-25)

28.0 ± 2.3 (22.8-31.7)

28.0 ± 2.0 (23.3-31.3)

ADB (g dL–1)

21.2 ± 2.2 (16.9-23.6)

22.3 ± 1.8 (18.6-27)

30.0 ± 2.5 (25.4-33.8)

P Values in each group Days 1-21 P < 0.001 Days 1-35 P < 0.001 Days 21-35 P > 0.05 Days 1-21 P > 0.05 Days 1-35 P < 0.001 Days 21-35 P < 0.001

P Values Between Groups

Day 1 P = 0.61 Day 21 P < 0.001 Day 35 P = 0.017

Table 6: Comparison of pH values in UCB and ADB samples over time.

UCB

ADB

Day 1

Day 21

Day 35

6.5 ± 0.2 (6.2-7.0)

6.3 ± 0.1 (6.1-6.4)

6.2 ± 0.1 (6.0-6.3)

7.0 ± 0.0 (7.0-7.1)

6.7 ± 0.0 (6.7-6.8)

6.6 ± 0.1 (6.5-6.7)

P Values in each group Days 1-21 P < 0.001 Days 1-35 P < 0.001 Days 21-35 P > 0.05 Days 1-21 P < 0.01 Days 1-35 P < 0.001 Days 21-35 P > 0.05

P Values Between Groups

Days 1, 21 and 35 P < 0.001

Table 7: Comparison of ATP values in UCB and ADB samples over time.

UCB (mmol L–1)

ADB (mmol L–1)

238

Day 1

Day 21

Day 35

1.0 ± 0.3 x 10–7 (0.3 -1.6 x 10–7)

0.2 ± 0.7 x 10–7 (0.0 - 2.1 x 10–7)

0.0 ± 0.2 x 10–7 (0.0- 0.5 x 10–7)

1.3 ± 0.4 x 10–7 (0.5-2.1 x 10–7)

0.5 ± 0.5 x 10–7 (0.3 -2.1 x 10–7)

0.3 ± 0.1 x 10–7 (0.0-0.5 x 10–7)

P Values in each group Days 1-21 P > 0.05 Days 1-35 P < 0.001 Days 21-35 P < 0.001 Days 1-21 P > 0.05 Days 1-35 P < 0.001 Days 21-35 P < 0.001

P Values Between Groups

Day 1 P = 0.029 Day 21 P = 0.021 Day 35 P < 0.001

Turk J Hematol 2012; 29: 233-241

that there was an inverse correlation between the volume of collected blood, and gestational age and birth weight.

They harvested 43 mL kg–1 of body weight from preterm infants [12]. Surbek et al. collected a median volume of 21 mL (range: 8-38 mL) following delivery of infants with a gestational of 22-32 weeks and a median volume of 49 mL (range: 21-103 mL) after delivery of infants with a gestational age of 33-36 weeks; birth weight and the volume of collected blood were not correlated [14,15]. Jansen et al. reported a UCB collection volume of 23 mL kg–1 in infants with a gestational age <32 weeks, but did not note a correlation between the volume of harvested UCB, and gestational age, birth weight, placental weight, or method of delivery [22]. Khodabux et al. observed a correlation between the collected blood volume and gestational age.

The collected volume of UCB was 32 ± 7.7 mL for infants with a gestational age of 24-28 weeks, 44 ± 27.4 mL for those with a gestational age 28-30 weeks, and 33 ± 13.3 mL for infants with gestational age of 30-32 weeks. The volume of harvested UCB was 16 ± 15 mL kg–1 in infants with a birth weight <1000 g, 18 ± 18 mL kg–1 for those weighing 1000-1250 g, and 20 ± 23 mL kg–1 for those with a birth weight >1250 g; however, birth weight and the volume of harvested blood were not correlated [23].

Mean volume of harvested UCB in the present study was 57.11 ± 10.46 mL in the preterm infants and 77.84 ± 10.54 mL in the full-term infants, and was 24.87 mL kg–1 of bodyweight, irrespective of gestational age. Mean RBC volume, on the other hand, was 14.63 ± 2.43 mL kg–1 in the preterm infants and 12.55 ± 1.46 mL in the full-term newborns, which is similar to previous reports.

Mean UCB volume kg–1 of birth weight was higher in the preterm infants; however, correlation analysis between the collected volume, and birth weight and gestational age was not performed.

Numerous studies reported that K+, 2,3-BPG, ATP, and pH values in stored blood and blood components change with time, and research has been conducted in an effort to increase the quality of stored blood [24-30]. Studies on the biochemical and hematological properties of stored UCB, however, are less common. Garritsen et al. studied whether or not UCB-PRC could be used as an alternative to ADB-PRC. They developed a system for collecting and preparing UCB-PRC, and measured standard storage parameters during 35 d of storage in extended storage medium (SAG mannitol). The initial laboratory UCB-PRC parameters were similar to those of ADB-PRC. After 35 d of storage UCB-PRC had a hemolysis rate 1% higher than that of ADB-PRC and a significant decrease in ATP. UCB-

Tokan RS, et al: Comparison of Cord Blood and Adult Blood

PRC met the same quality criteria as ADB-PRC after 35 d of storage [20].

Bifano et al. studied ‘placental whole blood’ stored in CPDA for 28 d and reported that the K+ level on d 28 was comparable to that in stored ADB. In the UCB ATP, 2,3-BPG, and pH decreased, and some morphological changes, an increase in osmotic fragility, and a minimal increase in hemolysis were noted. They also reported that fetal erythrocytes were preserved better in citrate-phospohate-adenine-1 solution (CPDA-1) than in CPD [31].

The hemolysis rate, however, was higher in the studies

by Brune et al. [32], Garritsen et al. [20], and Widing et al. [33]. In a recent review Khodabux and Brand reported that the preservative solution used, not the manipulation of UCB, might contribute to storage damage. This has been suggested by some other studies that reported better results using the storage solution PAGGSM that contained additional phosphate and guanosine than those obtained using SAG-M [34].

In the present study the 2,3-BPG level did not differ significantly between UCB-PRC and ADB-PRC (P > 0.017). After 35 d of storage, free Hb, intracellular ATP, and pH values were significantly different than initial values in both UCB RBCs and ADB RBCs. Additionally, it was observed that 2,3-BPG, ATP, and pH values decreased in both blood groups, whereas K+ increased. These significant differences in both blood groups were thought to have been due to an increase in hemolysis and decrease in intracellular ATP. The higher K+ value on d 1 of storage reached a similar level in ADB on d 35. Bacterial contamination has been one of the concerns of the groups dealing working with UCB. In the present study bacterial contamination was not observed in any of the stored UCB-PRC samples. Eichler et al. had raised concerns with regard to the high rate of bacterial contamination [12]. Garritsen et al. [20] reported a bacterial contamination rate of 1.84% in 390 collected UCB units, and Jansen et al. reported there was bacterial contamination in 6 of 91 (7%) collected UCB units stored for 30 days [22].

In conclusion, the volume of harvested and prepared UCB-PRC is similar to previous studies and can supply for some of the blood transfusions required in neonatal period and thus, its use may decrease number of allogeneic transfusions, especially in premature newborns. Hematological and biochemical changes that occur to UCB during storage are comparable to those observed in ADB and do not pose a risk to the relatively immature metabolism of neonates [35-37]. The lack of any observable bacterial contami-

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nation in the present study indicates that the collection method employed was reliable. The present findings show that the collection, separation, and storage of UBC-PRC, and the changes that occur during 35 d of storage can yield a product that is feasible for use under clinical conditions.

In addition, autologous transfusion is safer than allogeneic transfusion, in terms of immunohematology and infection risk. The use and outcomes of UCB however, remain to be determined by further clinical studies.

Abbreviations: VLBW: Very low birth weight, UCB: Umbilical cord blood, ADB: Adult donor blood, RBC: Red Blood Cell, K+: Potassium, 2,3-BPG: 2,3-biphosphoglycerate, ATP: Adenosine tri-phosphate, UCB-PRC: Umbilical cord blood- packed red blood cell concentrate, A-PRC: Adult packed red blood cell concentrate, CPD: Citrate-phosphatedextrose additive solution This study has been supported by Ankara University Scientific Research Projects Fund References 1. Holland BM, Jones JG, Wardrop CA. Lessons from the anaemia of prematurity. Hematol Oncol Clin North Am 1987;1 (3): 355-366 2. Shannon KM. Anaemia of Prematurity : Progress and prospects. Am J Pediatr Hematol Oncol 1990; 12: 14-20 3. Ramasethu J, Luban NL. Red Blood Cell transfusion in the newborn. Seminars in Neonatology 1999; 4: 5-16 4. Nathanson N. Emergence of new viral infections: Implications for the blood supply. Biologicals 1998; 26 (2): 77-84 5. Chamberland ME. Surveillance for blood borne infections.

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Research Article

10.5505/tjh.2012.58751

The Association Between JAK2V617F Mutation and Bone Marrow Fibrosis at Diagnosis in Patients with Philadelphia-Negative Chronic Myeloproliferative Neoplasms Philadelphia Negatif Kronik Miyeloproliferatif Neoplazili Hastaların Tanısında JAK2V617F Mutasyonu ile Kemik İliğindeki Fibrozun İlişkisi M. Cem Ar1, Deram Büyüktaş2, A. Emre Eşkazan3, Şeniz Öngören Aydın4, Eda Tanrıkulu2, Zafer Başlar4, A. Nur Buyru5, Burhan Ferhanoğlu4, Yıldız Aydın4, Nükhet Tüzüner6, Teoman Soysal4 İstanbul Training and Research Hospital, Department of Hematology, İstanbul, Turkey İstanbul University, Cerrahpaşa Medical School, Department of Internal Medicine, İstanbul, Turkey 3 Diyarbakır Training and Research Hospital, Department of Hematology, Diyarbakır, Turkey 4 İstanbul University, Cerrahpaşa Medical School, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey 5 İstanbul University, Cerrahpaşa Medical School, Department of Medical Biology, İstanbul, Turkey 6 İstanbul University, Cerrahpaşa Medical School, Department of Pathology, İstanbul, Turkey 1 2

Abstract Objective: Bone marrow fibrosis is the second most common complication that causes morbidity and mortality in patients with Philadelphia-negative myeloproliferative neoplasms (MPNs). The aim of this study was to investigate the association between JAK2V617F mutation and bone marrow fibrosis at diagnosis in patients with MPNs.

Material and Methods: In total, 149 patients with MPNs were retrospectively evaluated to determine if there was an association between the histological grade of bone marrow fibrosis and JAK2V617F mutation.

Results: In all, 67.7% of the patients carried the mutated JAK2 gene. The presence of JAK2V617F mutation was not associated with the occurrence of bone marrow fibrosis (P = 0.55) or its grade at diagnosis (P = 0.65).

Conclusion: Molecular mechanisms or genetic defects other than JAK2V617F may underlie the occurrence of bone marrow fibrosis in patients with MPNs.

Key Words: JAK2V617F, Myeloproliferative disease, Bone marrow fibrosis

Özet Amaç: Kemik iliği fibrozu, Philadelphia negatif miyeloproliferatif neoplazili (MPN) hastalarda morbidite ve mortaliteye yol açan ikinci en sık komplikasyondur. Bu çalışmanın amacı MPN’li hastalarda tanı anında kemik iliğinde görülen fibroz ile JAK2V617F mutasyonu arasındaki ilişkinin araştırılmasıdır.

Address for Correspondence: M. Cem Ar, M.D., İstanbul Eğitim ve Araştırma Hastanesi, Kasap İlyas Mah. Org. Abdurrahman Nafiz Gürman Cd. 34098 İstanbul, Turkey Phone: +90 212 459 67 79 E-mail: muhcar@superonline.com Received/Geliş tarihi : June 6, 2011 Accepted/Kabul tarihi : October 19, 2011

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Ar MC, et al: JAK2V617F and Marrow Fibrosis in MPN

Gereç ve Yöntemler: Çalışmaya retrospektif olarak dahil edilen 149 MPN tanılı hasta JAK2V617F mutasyonu ile kemik iliğindeki fibroz arasındaki ilişki açısından değerlendirilmiştir.

Bulgular: Çalışmaya alınan hastaların %67.7’sinde JAK2V617F gen mutasyonuna rastlandı. Ancak JAK2V617Fmutasyonu ile kemik iliğindeki fibroz varlığı ve derecesi arasında ilişki saptanmadı (sırasıyla, P = 0.55 ve P = 0.65).

Sonuç: MPN’de görülen kemik iliği fibrozunun altında JAK2V617F mutasyonu dışında bazı moleküler mekanizma veya genetik bozuklukların yattığı sonucuna varılmıştır.

Anahtar Sözcükler: JAK2V617F, Miyeloproliferatif hastalık, Kemik iliği fibrozu Introduction Polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocythemia (ET) are the 3 classical Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) that are characterized by clonal proliferation of multipotent hematopoietic progenitor cells. New discoveries concerning the molecular pathogenesis of MPNs have changed the nature of their classification and diagnosis.[1] Janus kinase 2V617F (JAK2V617F) point mutation is a recently identified acquired genetic defect that is present in 95% and 50% of patients with PV and ET/PMF, respectively.[2-4] The mutation encodes an inducible tyrosine kinase of the intracellular signaling pathway that promotes myeloid proliferation and differentiation.

Bone marrow fibrosis occurs either as a primary disease (PMF) or as a late complication of PV and ET. Fibrosis contributes to morbidity and mortality in patients with MPNs together with additional risk factors.[5,6] Patients usually suffer from fatigue, malaise, weight loss, bone pain, and abdominal distension. The severity of symptoms is associated with the extent of anemia and splenomegaly. Unfortunately, treatment has been palliative and mostly disappointing until recently, consisting mainly of transfusion and other modalities that reduce the size of the spleen. Discovery of JAK2V617F mutation and current progress in the molecule targeted treatment technologies lead to the development of JAK2 inhibitors which seem to

yield promising results in selected groups of patients with myelofibrosis and created a certain enthusiasm. But further evidence from randomized studies should be awaited before drawing firm conclusions on the role of JAK2 inhibitors in the management of MPN associated bone marrow fibrosis.

It has been reported that there is a possible relationship between the homozygosity of JAK2V617F mutation and the extent of bone marrow fibrosis in patients with PV.[7,8] Tefferi et al. reported that PV patients with homozygous JAK2V617F mutation were more likely to transform into myelofibrosis (23% vs. 2% of patients with homozygous and heterozygous JAK2V617F mutation, respectively).

Passamonti et al. also suggested that PV patients homozygous for JAK2V617F are more prone to transformation into myelofibrosis with myeloid metaplasia.[9] The invention of potent JAK2-inhibitor drugs seem to open a new era in the treatment of classic MPNs. The impact of JAK2 inhibitors on bone marrow fibrosis is of clinical importance. As such, the present study aimed to investigate the association between JAK2V617F mutation and bone marrow fibrosis at diagnosis in patients with classic MPNs. Materials and Methods Patients We retrospectively reviewed the records of 149 patients with MPNs that were diagnosed and treated between 1996 and 2009 at our institution. Patients were diagnosed

Table 1: Bone marrow fibrosis.

Bone marrow fibrosis, as rearranged and defined in the present study no fibrosis minimal moderate marked

Corresponding Grading Systems for Fibrosis I II (Grades from 0 to 3) (Grades from 0 to 4) 0 0 1 1 2 2, 3 3 4

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Ar MC, et al: JAK2V617F and Marrow Fibrosis in MPN

Table 2: Patient characteristics.

Patients (n) Male:female ratio Median age at diagnosis (years) [range] Median blood counts at diagnosis [range] Hemoglobin (g dL–1) Hematocrit (%) Leukocyte (x109 L-1) Platelet (x109 L-1) JAKV617F status, n (%) Positive Negative Bone marrow fibrosis, n (%) Present Absent MPNs, n (%) PV ET PMF uMPN

149 77:72 61 [17-86]

13.2 [6.2-19.4] 42.0 [18.1-61.9] 9.01 [2.0 - 933.5] 452 [40 - 1391] 101 (67.7) 52 (32.3) 107 (71.8) 42 (28.2) 80 (53.7) 37 (24.8) 18 (12.1) 14 (9.4)

BM: Bone marrow; ET: essential thrombocythemia; MPNs: myeloproliferative neoplasms; PMF: primary myelofibrosis; PV: polycythemia vera; uMPN: unclassifiable myeloproliferative neoplasm.

according to the international criteria for MPNs that were valid at the time they were diagnosed.[10,11] Bone marrow fibrosis at diagnosis was assessed by an expert hematopathologist and scored according to one of the two grading scales that was in use at the time of pathologic examination as reported in detail elsewhere.[11,12] For practical purposes we merged these two grading scales by rearranging the patients into 4 subgroups with ‘no’, ‘minimal’, ‘moderate’ and ‘marked’ fibrosis. The 2 pathological grading scales and the corresponding 4 subgroups are given in Table 1. JAK2V617F testing has been available at our hospital since 2006; therefore, patients in the study that were diagnosed before 2006 underwent JAK2V617F testing upon entering the study and all the other patients were tested during their initial evaluation. The study protocol was approved by the Ethics Committee of Cerrahpaşa Medical Faculty and was conducted according to the prin-

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ciples of the Declaration of Helsinki. All the participants provided written informed consent to participate in the study.

Methods JAK2V617F mutation testing was performed via allelespecific polymerase chain reaction (PCR) in all the patients.

Into ethylene diamine tetra-acetic acid (EDTA)-containing sample tubes (Greiner Bio-One GmBH, Kremsmünster, Austria), 2 mL of venous blood was collected from each patient via peripheral venipuncture. Using standard techniques, as previously described,[13] DNA was extracted from the blood samples and stored at –70 °C. PCR amplification for the detection of JAK2V617F and visualization of the PCR products were carried out using a commercial assay (Seeplex JAK2 Genotyping Kit).

Statistics Pearson’s chi-square test and the odds ratios were used to compare and describe the fibrosis occurrence rate between JAK2V617F-positive and negative groups. Statistical analysis was performed using SPSS v.15.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was set at P < 0.05.

Results Among the 149 patients included, 80 had PV, 37 had ET, and 18 had PMF. Despite exhibiting the classical laboratory and clinical features of MPNs, 14 patients could not be assigned to a specific subgroup and were diagnosed as unclassifiable MPN (uMPN). Patient characteristics are shown in Table 2. In all, 67.7% of the patients carried the mutated JAK2 gene. The frequency of JAK2V617F mutation was highest in the patients with uMPN (85.7%), followed those with PV (80%), PMF (50%), and ET (43.2%).

Bone marrow fibrosis (varying in grade) was observed in 107 of the 149 patients at the time of diagnosis (Table 3).

Bone marrow fibrosis at diagnosis was not associated with the JAK2V617F mutation (P = 0.55). The odds ratio for bone marrow fibrosis between the JAK2V617F-positive and JAK2V617F-negative patients was 1.27 (95% CI: 0.58-2.77), i.e. occurrence rate of fibrosis in bone marrow was not increased in JAK2V617F-positive subjects (Figure 1). Analysis of the relation of JAK2V617F to bone marrow fibrosis in PV, ET, PMF, and uMPN subgroups separately did not yield a significant association between fibrosis and the JAK2V617F mutation (Table 4). The grade of bone marrow fibrosis at diagnosis did not differ significantly between patients with and without JAK2V617F mutation (P = 0.65) (Figure 2).

Turk J Hematol 2012; 29: 242-247

Ar MC, et al: JAK2V617F and Marrow Fibrosis in MPN

Figure 1: JAK2V617F-positivity and bone marrow fibrosis

Figure 2: JAK2V617F-positivity according to different grades of bone marrow fibrosis

Table 3: Distribution of MPN patients with and without bone marrow fibrosis, according to JAKV617F status.

BM fibrosis grade

Patients (n = 149)

JAK status

with fibrosis

without fibrosis

mild

moderate

marked

Total

PV (n = 80)

positive negative positive negative positive negative positive negative

44 9 9 16 9 9 9 2 107

20 7 7 5 0 0 3 0 42

27 8 7 10 1 0 6 2 63

10 1 2 5 1 3 2 0 22

7 0 0 1 7 6 1 0 22

64 16 16 21 9 9 12 2

ET (n = 37) PMF (n = 18) uMPN (n = 14) Total

BM: Bone marrow; ET: essential thrombocythemia; uMPN: unclassifiable myeloproliferative neoplasm; PMF: primary myelofibrosis; PV: polycythemia vera.

Discussion The classic MPNs are considered clonal disorders that occur due to some mutations in hematopoietic progenitor cells. Despite new discoveries concerning the molecular mechanism of MPNs, their genetic background is not fully known and requires further investigation. JAK2V617F is an acquired gain-of-function mutation that has been recently described in patients with Philadelphia chromosome-negative MPNs, including primarily PV and ET. The mutated gene encodes the tyrosine kinase called JAK2, which, with

other mediators, plays an important role in induction of myeloid cell proliferation and differentiation. JAK2 is an essential component of the intracellular signaling system associated with certain cytokines such as interleukin (IL)3, IL-5, and colony stimulating factors and with growth factors like thrombopoietin, and erythropoietin.[14] Fibrosis is an essential descriptive component of PMF, and occurs in 5-14% of patients with PV and 15-20% of patients with ET at diagnosis.[11,15,16] In patients with fully developed disease bone marrow is replaced by fibrous

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Turk J Hematol 2012; 29: 242-247

Table 4: The association between JAK2V617F mutation and bone marrow fibrosis at diagnosis in the MPN subgroups

Patients (n) Subgroup PV ET PMF uMPN Total

JAKV617F mutation Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative

without BM fibrosis

with BM fibrosis

20 7 7 5 0 0 3 0 30 12

44 9 9 16 9 9 9 2 71 36

P

OR (95%CI)

0.35

1.71 (0.56-5.25)

0.21

0.04 (0.09-1.64)

*

*

*

*

0.55

1.27 (0.58-2.77)

BM: Bone marrow; CI: confidence interval; ET: essential thrombocythemia; uMPN: unclassifiable myeloproliferative neoplasm; PMF: primary myelofibrosis; PV: polycythemia vera. *Statistical analysis could not be performed due to the small number of patients.

connective tissue, causing extramedullary hematopoiesis.

Myelofibrosis is the second most common complication in patients with classic MPNs, which leads to cytopenias, splenomegaly, poor quality of life, and reduced survival.

[5,17] Intramedullary fibrosis in patients with MPNs occurs through induction of stromal elements in bone marrow via a set of mechanisms. It has been postulated that the mutant JAK2 causes increased proliferation of granulocytes and platelets which in turn, leads to bone marrow fibrosis by producing large quantities of stimulatory cytokines, such as transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF), and fibroblast growth factor-beta (FGF-β).[3,7,9] The exact role of JAK2V617F in bone marrow fibrosis remains to be determined.

JAK2V617F has been reported to occur in 40-91% of patients with MPN-associated myelofibrosis[7]; in the present study 71 of the 107 patients (66%) with bone marrow fibrosis at diagnosis carried the mutation. Considering that the prevalence of JAK2V617F in the MPN patients without bone marrow fibrosis (71%) did not differ significantly from those with fibrosis, we think that there should be other genetic and environmental factors—apart from JAK2V617F that contribute to the deposition of fibrous tissue in the marrow. Passamonti et al. reported that JAK2V617F mutation activates granulocytes and mobilizes CD34 cells, and that the transition of JAK2V617F from heterozygosity to homozygosity could play a role in the

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progression of PV to post-PV myelofibrosis;[9] however, this has not yet been confirmed.

The discovery of the association between JAK2V617F and MPNs has facilitated the invention of new targeted treatment strategies and resulted in the development of JAK2 inhibitor drugs for patients with myelofibrosis. A limited number of studies on the use of JAK inhibitors in patients with myelofibrosis reported a dramatic decrease in the size of splenomegaly, but no significant improvement in leukocyte counts, anemia, number of platelets, and bone marrow fibrosis has been observed.[18,19,20] This incomplete resolution of signs and symptoms with JAK2 inhibition supports our view, i.e. JAK2V617F is not the only actor of the scene, there should be other molecular mechanisms and/or mutations which contribute to the development of fibrosis in the bone marrow of patients with MPNs. Although limited by its retrospective design, the present study clearly shows that the JAK2V617F mutation was not associated with the occurrence of bone marrow fibrosis or its grade at diagnosis. Additional large-scale multi-center studies with MPN patients are required to further delineate the impact of JAK2 and/or other additional factors on the development of bone marrow fibrosis.

Studies on newly discovered target-oriented drugs—apart from their contribution to treatment success—may help in uncovering the probable molecular mechanisms of MPNs.

Turk J Hematol 2012; 29: 242-247

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Tefferi A, Thiele J, Orazi A, Kvasnicka HM, Barbui T, Hanson CA, Barosi G, Vestovsek S, Birgegard G, Mesa R, Reilly JT, Gisslinger H, Vannucchi AM, Cervantes F, Finazzi G, Hoffman R, Gilliland DG, Bloomfield CD, Vardiman JW. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia and primary myelofibrosis: Recommendations from an ad hoc international expert panel. Blood 2007; 110 (4): 1092-1097 2. James C, Ugo V, Le Couédic JP, Staerk J, Delhommeau F, Lacout C, Garçon L, Raslova H, Berger R, BennaceurGriscelli A, Villeval JL, Constantinescu SN, Casadevall N, Vainchenker W. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434: 1144-1148 3. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli A, Cazzola M, Skoda RC. A gain-offunction mutation of JAK2 in myeloproliferative disorders.

N Engl J Med 2005; 352: 1779-1790 4. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, Vassiliou GS, Bench AJ, Boyd EM, Curtin N, Scott MA, Erber WN, Green AR. Cancer Genome Project: Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054-1061 5. Tefferi A. Myelofibrosis with myeloid metaplasia. N Engl J Med 2000; 342: 1255-1265 6. Cervantes F, Passamonti F, Barosi G. Life expectancy and prognostic factors in the classic BCR/ABL-negative myeloproliferative disorders. Leukemia 2008; 22: 905-914 7. Tefferi A, Lasho TL, Schwager SM, Steensma DP, Mesa RA, Li CY, Wadleigh M, Gary Gilliland D. The JAK2V617F tyrosine kinase mutation in myelofibrosis with myeloid metaplasia: Lineage specificity and clinical correlates. Br J Haematol 2005; 131: 320-328 8. Tefferi A, Lasho TL, Schwager SM, Strand JS, Elliott M, Mesa R, Wadleigh M, Lee SJ, Gilliland DG. The clinical phenotype of wild-type, heterozygous, and homozygous JAK2V617F in polycythemia vera. Cancer 2006; 106 (3): 631-635 9. Passamonti F, Rumi E, Pietra D, Della Porta MG, Boveri E, Pascutto C, Vanelli L, Arcaini L, Burcheri S, Malcovati L, Lazzarino M, Cazzola M. Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders. Blood 2006; 107: 3676-3682

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10. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Eds. Steven H. Swerdlow, Elias Campo, Nancy Lee Haris, Elaine S. Jaffe, Stefano A. Pileri, Harald Stein, Jürgen Thiele, James W. Varidman. WHO, OMS, International Agency for Research on Cancer, 4th ed. Lyon, 2001 11. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Eds. Steven H. Swerdlow, Elias Campo, Nancy Lee Haris, Elaine S. Jaffe, Stefano A. Pileri, Harald Stein, Jürgen Thiele, James W. Varidman. WHO, OMS, International Agency for Research on Cancer, 4th ed. Lyon, 2008 12. Bauermeister DE. Quantitation of bone marrow reticulin: A normal range. Am J Clin Pathol 1971; 56: 24-31 13. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells.

Nucleic Acids Res 1988; 16 (3): 1215 14. Kralovics R, Teo SS, Buser AS, Brutsche M, Tiedt R, TichElli A, Passamonti F, Pietra D, Cazolla M, Skoda RC. Altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of Jak2.

Blood 2005; 106: 3374-3376 15. Barbui T, Thiele J, Passamonti F, Rumi E, Boveri E, Randi ML, Bertozzi I, Marino F, Vannucchi AM, Pieri L, Rotunno G, Gisslinger H, Gisslinger B, Müllauer L, Finazzi G, Carobbio A, Gianatti A, Ruggeri M, Nichele I, D’Amore E, Rambaldi A, Tefferi A. Initial bone marrow reticulin fibrosis in polycythemia vera exerts an impact on clinical outcome.

Blood 2012; 119 (10): 2239-2241 16. Kreft A, Büsche G, Ghalibafian M, Buhr T, Fischer T, Kirkpatrick CJ. The Incidence of Myelofibrosis in Essential Thrombocythaemia, Polycythaemia vera and Chronic Idiopathic Myelofibrosis: A Retrospective Evaluation of Sequential Bone Marrow Biopsies. Acta Haematol 2005; 113:137-143 17. Campbell PJ, Bareford D, Erber WN, Wilkins BS, Wright P, Buck G, Wheatley K, Harrison CN, Green AR. Reticulin accumulation in essential thrombocythemia: Prognostic significance and relationship to therapy. J Clin Oncol 2009; 27 (18): 2991-2999 18. Tefferi A. New insights into the pathogenesis and drug treatment of myelofibrosis. Curr Opin Hematol 2006; 13: 87-92 19. Mesa R, Gale RP. Hypothesis: How do JAK2-inhibitors work in myelofibrosis? Leuk Res 2009; 33: 1156-1157 20. Verstovsek S, Kantarjian H, Mesa RA, Pardanani AD, Cortes-Franco J, Thomas DA, Estrov Z, Fridman JS, Bradley EC, Erickson-Viitanen, Vadi K, Levy R, Tefferi A. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med 2010; 363: 1117-1127

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Research Article

10.5505/tjh.2012.13008

Does Reactive Thrombocytosis Observed in Iron Deficiency Anemia Affect Plasma Viscosity?

Demir Eksikliği Anemisinde Görülen Reaktif Trombositoz Plazma Viskozitesini Etkiler mi?

Selami K. Toprak1, İbrahim Tek2, Sema Karakuş1, Nihat Gök1, Nazmiye Kurşun3 Baskent University, School of Medicine, Department of Hematology, Ankara, Turkey Medicana International Ankara Hospital Cancer Center, Ankara, Turkey 3 Ankara University, School of Medicine, Department of Biostatistics, Ankara, Turkey 1 2

Abstract Objective: The accompanying thrombocytosis is referred to as the major factor associated with thromboembolism in iron deficiency anemia (IDA). Increased viscosity may increase the risk of thrombosis. We hypothesized that increased platelet count -with reactive thrombocytosis- might also affect plasma viscosity. We planned to evaluate the influence of normal and high platelet count on plasma viscosity in IDA patients.

Material and Methods: The patient population consisted of fifty-three newly diagnosed and untreated women aged between 18 and 62 years with IDA. Group 1 consisted of 33 patients, platelet levels below 400 x 109/L. Group 2 consisted of 20 patients, platelet levels above 400 x 109/L. Measurements of plasma viscosity were performed using Brookfield viscometer.

Results: Mean plasma viscosity was found as 1.05 ± 0.08 mPa.s. in Group 1, and 1.03 ± 0.06 mPa.s. in Group 2. Mean plasma viscosity was not statistically different. White blood cell count was significantly higher in Group 2. Vitamin B12 levels were significantly higher in Group 2, while folic acid levels were higher in Group 1 (p=0.011 and p=0.033). Plasma viscosity was correlated with erythrocyte sedimentation rate (r=0.512 p=0.002) in Group 1 and inversely correlated with vitamin B12 (r=−0.480 p=0.032) in Group 2.

Conclusion: Despite the significant difference between groups in terms of platelet count, no significant difference was detected in plasma viscosity and this finding could be explained as the following; 1-These platelets were not thrombocythemic platelets; 2-Similar to the theory about leukocytes, higher platelet counts – even non-thrombocythemic – may increase plasma viscosity; 3-Evaluating platelet count alone is not sufficient and the associating red-cell deformability should also be taken into account; and 4-Although other diseases that could affect viscosity are excluded, some definitely proven literature criteria such as fibrinogen, hyperlipidemia, and the inflammatory process should also be evaluated by laboratory and clinical measures.

Key Words: Iron deficiency anemia, Reactive thrombocytosis, Thrombocythemia, Plasma viscosity

Özet Amaç: Demir eksikliği anemisine eşlik eden trombositoz, gelişebilecek tromboembolizm için en önemli etken olarak görülmektedir. Artmış viskozite, tromboz riskini de arttırabilir. Buradan hareketle, trombosit sayısındaki artışın da Address for Correspondence: Selami K. Toprak, M.D., Başkent Üniversitesi, Tıp Fakültesi, Hematoloji Bilim Dalı, Ankara, Turkey Phone: +90 312 212 29 12 E-mail: sktoprak@yahoo.com Received/Geliş tarihi : January 20, 2012 Accepted/Kabul tarihi : February 21, 2012

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Toprak SK, et al: Reactive Thrombocytosis and Plasma Viscosity

-reaktif trombositoz- plazma viskozitesini etkileyebileceği varsayımında bulunduk. Demir eksikliği anemisi bulunan hastalarda, normal ve yüksek trombosit sayısının, plazma viskozitesi üzerindeki etkisini değerlendirmeyi amaçladık.

Gereç ve Yöntemler: Yakın zamanda demir eksikliği anemisi tanısı alıp, daha herhangi bir tedavi başlanmayan ve yaşları 18 ile 62 arasında değişen 53 kadın hasta çalışmaya dahil edilmiştir. Grup 1, trombosit sayısı < 400 x 109/L olan 33 hastadan oluşmaktadır. Grup 2, trombosit sayısı > 400 x 109/L olan 20 hastadan oluşmaktadır. Plazma viskozite ölçümleri, Brookfield viscometer kullanılarak yapılmıştır.

Bulgular: Ortalama plazma viskozitesi, Grup 1’de 1.05 ± 0.08 mPa.s. ve Grup 2’de ise 1.03 ± 0.06 mPa.s. olarak bulunmuştur. İki grup arasında, ortalama plazma viskozitesinde istatistiksel bir fark yoktur. Grup 2’de lökosit sayısı anlamlı olarak yüksek bulunmuştur. Grup 1’de folik asid ve Grup 2’de ise vitamin B12 düzeyleri, diğer gruba göre anlamlı olarak yüksek saptanmıştır (p=0.011 ve p=0.033). Plazma viskozitesi, Grup 1’de eritrosit sedimentasyon hızıyla doğru (r=0.512 p=0.002) ve Grup 2’de ise vitamin B12 düzeyi ile ters korelasyon göstermektedir (r=−0.480 p=0.032).

Sonuç: Her iki grup arasında trombosit sayısı bakımından anlamlı bir fark olmasına karşın, plazma viskozitesinde istatistiksel bir fark bulunamamıştır. Bunun nedenleri olarak şunlar sayılabilir: 1- Bu trombositler, “trombositemik” özellik taşımamaktadırlar. 2- Lökositlerle alakalı teoriye benzer şekilde, belki de ancak çok yüksek trombosit sayısı -trombositemik olmasalar bile- plazma viskozitesini arttırabilmektedir. 3- Sadece trombosit sayısını değerlendirmek yeterli olmamakta, ek olarak eritrositlerin yapısal özellikleri de hesaba katılmalıdır. 4- Viskoziteyi etkileyebilecek başka hastalıkların dışlanmasına karşın, fibrinojen, hiperlipidemi ve enflamatuvar sürecin de laboratuvar ve klinik olarak değerlendirilmesi uygun olacaktır.

Anahtar Sözcükler: Demir eksikliği anemisi, Reaktif trombositoz, Trombositemi, Plazma viskozitesi Introduction Thrombocytosis is frequently encountered as a coincidental laboratory finding. The causes of thrombocytosis, in which the platelet count exceeds the upper limit can be categorized as 1- reactive or secondary due to infections, trauma, surgery, iron deficiency (ID), or occult malignancy; 2- clonal, including essential thrombocythemia (ET) and other myeloproliferative disorders; and 3- familial [1].

The clinical features of secondary thrombocytosis are almost always a result of the underlying disorder provoking the reaction. Even though thrombocytosis is benign and self-limiting in most cases and virtually absent in reactive thrombocytosis -unless provoked by other features-, this disorder can result in hemorrhage or thrombosis [2, 3].

The accompanying thrombocytosis is frequently referred to as the major factor associated with thrombembolism in iron deficiency anemia (IDA) [4].

Plasma viscosity is known to have a close relationship with blood flow. Increased viscosity may increase the risk of thrombosis or thromboembolic events [5]. Many factors can affect plasma (and/or blood) viscosity, such as white blood cell and platelet count, hematocrit, immunoglobulins, and fibrinogen.

We hypothesized that increased platelet count -with reactive thrombocytosis- might also affect plasma viscos-

ity. In the present study, we planned to evaluate the influence of normal and high platelet count on plasma viscosity in IDA patients.

Materials and Methods Study Population The patient population consisted of fifty-three newly diagnosed and untreated women aged between 18 and 62 years with a demonstrable cause of IDA. There were two groups. Group 1 consisted of 33 patients with IDA and platelet levels below 400 x 109/L. Group 2 consisted of 20 patients with IDA and platelet levels above 400 x 109/L. Patient characteristics are summarized in table 1.

Table 1: Patient characteristics

Patients Age, years Median Range Etiology Menorrhagia Peptic ulcer Hemorrhoid

Group 1 33

Group 2 20

34 18-55

42.5 29-62

25 2 6

14 3 3

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Menorrhagia was the most frequent cause of IDA in both groups. Patients possessing chronic renal failure, hypertension, coronary vascular disease, diabetes mellitus, cigarette addiction, hyperlipidemia, coagulopathy, acute blood loss, infectious disease, connective tissue disorders, anemia of chronic disease, and cancer were excluded from the study. Group 2 patients are evaluated for primary thrombocytosis and other causes of reactive thrombocytosis and those with other disorders causing reactive thrombocytosis were also excluded.

The study protocol was approved by the local ethics committee and written and signed informed consent was provided by all participants.

Methods IDA was diagnosed based on hemoglobin (Hb), red blood cell indexes, serum iron, serum iron binding capacity, serum ferritin, and transferrin saturation test results, as previously reported [6, 7, 8]. Thrombocytosis was defined as platelet count ≥ 400 x 109/L in at least two blood samples.

To measure plasma viscosity, 5 cc of blood samples were collected from patients into anticoagulated tubes. All

samples were frozen because immediate measurement of plasma viscosity was not possible and the results of fresh frozen samples and freshly studied blood samples were the same. By this method, all samples were studied at the same time and errors that could be due to calibration of the test machine were minimized. Blood samples taken into anticoagulated tubes were first centrifuged at 3000 rpm for 5 min and then the separated plasma was frozen at −40°C. On the day of measurement, all samples were melted and recentrifuged and then measured at 37°C in a Brookfield DV– II + Cone Plate Viscometer (Brookfield, Stoughton, MA, USA) machine, which was calibrated with distilled water. Each sample was measured four times and the average of the measurements was taken. Some sources indicate that the normal value of plasma viscosity is between 1.3 and 1.65 mPa.s., while others state that it is 1.10-1.30 mPa.s. at 37°C and independent of age and gender [9, 10].

Statistical Analysis Variables are presented as mean ± SD. Analyses of variance was performed to test the difference between two groups with respect to viscosity and the other laboratory measurements according to their platelet count using Ttest and Mann-Whitney U test. The Pearson’s Correlation

Table 2: Mean of different variables in two groups

Viscosity Platelet count (x 109/L) MPV (fL) Hemoglobin (g/dL) White blood cell count (x 109/L) MCH (pg) MCV (fL) Serum iron (µg/mL) Serum iron binding capacity (µg/dL) Ferritin (ng/mL) Transferrin saturation (%) C-reactive protein (mg/L) TSH (uIU/mL) Vitamin B12 (pg/mL) Folic acid (ng/mL) ESR (mm/h)

Group 1 1.05 ± 0.08 277.90 ± 54.16 9.82 ± 2.31 10.48 ± 1.25 6.63 ± 1.66 23.98 ± 3.63 73.62 ± 8.92 22.42 ± 11.20 416.39 ± 58.23 7.85 ± 4.13 7.84 ± 3.97 3.76 ± 2.68 1.81 ± 0.90 307.09 ± 105.10 8.45 ± 2.75 16.90 ± 5.32

Group 2 1.03 ± 0.06 507.45 ± 67.70 9.60 ± 3.74 8.77 ± 1.66 8.04 ± 1.95 20.29 ± 7.01 60.30 ± 12.63 20.90 ± 12.73 434.15 ± 59.56 8.94 ± 4.52 9.40 ± 4.56 3.73 ± 2.90 1.76 ± 0.94 397.30 ± 144.20 6.79 ± 2.54 19.15 ± 4.78

P 0.421 p<0.001 0.795 0.069 0.007 0.015 p<0.001 0.650 0.291 0.473 0.198 0.956 0.838 0.011 0.033 0.129

MPV: Mean platelet volume; MCH: Mean corpuscular hemoglobin; MCV: Mean corpuscular volume; TSH: Thyroid stimulating hormone; ESR: Erythrocyte sedimentation rate.

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Figure 1: Correlation of plasma viscosity with erythrocyte sedimentation rate in Group 1

Figure 2: Correlation of plasma viscosity with vitamin B12 in Group 2

was used to describe a correlation between independent parameters. A P values less than 0.05 were considered to be statistically significant. All statistical analyses were conducted using SPSS v.15.0 software (SPSS, Inc., Chicago, IL, USA).

2 (Figure 1 and 2). Platelet count, mean platelet volume (MPV), WBC count, Hb level, red blood cell indexes, iron indexes, and the others variables such as thyroid stimulating hormone (TSH), CRP and folic acid levels were not associated with plasma viscosity in both groups.

Results

Discussion

The mean platelet count in Group 1 was 277.90 x 10 /L (range: 155-368 x 109/L). The mean platelet count in Group 2 was 507.45 x 109/L (range: 415-645 x 109/L) (p<0.001).

Markers of platelet activation and haemorrheological indices have been demonstrated to play a role in the pathophysiology of atherosclerosis and cardiovascular events through thrombosis [11]. Compared to primary thrombocytosis such as that caused by essential thrombocythemia, reactive thrombocytosis is generally regarded as benign [3]. Nevertheless, reactive thrombocytosis has been reported to cause severe and even lethal complications [3, 12]. The mechanism of this possible association between reactive thrombocytosis-iron deficiency and thrombosis is unknown. Thus we also wanted to investigate whether or not the reactive thrombocytosis, the process of triggering thrombosis occurs by a possible increase in plasma viscosity. We defined the lower limit of thrombocytosis as 400 x 109/L and when we compared the plasma viscosity of the 20 patients in the group with thrombocytosis with the other group, no significant difference could be detected.

In a study consisting of 113 patients with diagnosed iron deficiency anemia, pancytopenia, polycythemia vera, essential thrombocythemia, idiopathic thrombocytopenic purpura, myelodysplastic syndrome, aplastic anemia, and thalassemia, thrombocytosis –along with many associating variables – had a positive effect on blood viscosity while not affecting plasma viscosity in parallel with our study [5]. Similarly, in another study comparing 20 cases with splenectomy (15 trauma related, 4 idiopathic thrombocytopenic purpura, 1 splenic cyst) to healthy controls, although the difference in thrombocyte count between groups was statistically significant, no significant difference could be detected in plasma viscosity [13]. In this

9

Mean plasma viscosity was found as 1.05 ± 0.08 mPa.s. in Group 1, and 1.03 ± 0.06 mPa.s. in Group 2, (normal 1.39 ± 0.08) [9]. The mean plasma viscosity was not statistically different. Table 2 shows the mean of different variables in 53 patients.

Although there was no significant difference between the two groups in terms of hemoglobin (Hb) and even serum iron, serum iron binding capacity, serum ferritin, and transferrin saturation, levels of mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) were significantly lower in Group 2 (p=0.015 and p<0.001, respectively). In terms of excluding any associating acute or chronic condition that could affect viscosity, it is important that the C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) levels were within the normal range in both groups (Table 2). However, the white blood cell (WBC) count, which is known to affect viscosity, was significantly higher in Group 2.

Vitamin B12 levels were significantly higher in Group 2, while folic acid levels were significantly higher in Group 1 (p=0.011 and p=0.033, respectively).

Plasma viscosity was significantly positively correlated with ESR (r=0.512 p=0.002) in Group 1 and inversely correlated with vitamin B12 (r=−0.480 p=0.032) in Group

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same study, apart from the absence of a significant correlation between viscosity and thrombocyte count, it was reported that the rapidly increasing thrombocyte count following splenectomy returned to normal in the following weeks while the increase in plasma viscosity persisted.

The researchers stated that the most important factor here was the decreased red-cell deformability following splenectomy. In the study of Rozenberg et al., patients with history of myocardial infarct were compared with healthy controls and no significant correlation could be detected between blood viscosity and thrombocyte count [14].

The concentration of red cells is the major factor determining the viscosity of normal blood [15]. Leukocytes have little effect because of their relatively small numbers.

When the leukocyte count is greatly elevated, changes in viscosity of blood have been noted [15]. WBC count was increased in Group 2, however, it is meaningful that this alone did not affect plasma viscosity. In Ho’s study, similar to increased thrombocyte count, increased WBC count influenced blood viscosity while no significant relation could be detected in plasma viscosity [5]. Some authors suggest that for the leukocyte count be able to affect both the whole blood and plasma viscosity, the granulocyte component should be increased, along with the total WBC count reaching almost 50 x 109/L [15].

There was not a correlation between MCH, MCV and plasma viscosity in both groups; but levels of MCH and MCV were significantly lower in Group 2. The lower levels of these markers in the group with higher platelet count could be explained by a longer and deeper iron depletion in this group.

Folic acid and vitamin B12 levels were within the normal levels, however, the folic acid level in Group 1 and the vitamin B12 level in Group 2 were significantly higher than the other group and this showed that they did not affect plasma viscosity. Nonetheless, serum vitamin levels in Group 2 were inversely correlated with plasma viscosity (Figure 2). Remacha et al. compared 326 patients with thrombosis to 351 control cases and reported that serum vitamin B12 levels were remarkably decreased in the group with thrombosis [16]. The researchers stated that this was related to the hyperhomocysteinemia, which was significantly different in the patient group. It was demonstrated that high plasma homocystein levels were positively correlated with viscosity both in patients with coronary arterial disease and controls [17]. Thus, although homocystein levels were not measured in the present study, the relation between low B12 levels and high viscosity is meaningful.

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In Group 1, the correlation between ESR and plasma viscosity was an expected result [10]. However, it is doubtful why this same tendency could not be observed in Group 2. It was reported that ESR was positively correlated with plasma viscosity in case groups with pulmonary tuberculosis and malignant lymphomas [18]. In this same study, no correlation could be detected between ESR and plasma viscosity in the sickle cell anemia group and healthy controls and furthermore, ESR was remarkably lower in patients with sickle cell anemia when compared with controls. When the more pronounced effect of red cell deformability on plasma viscosity is taken into account and the fact that this parameter is not tested in the patient groups is considered, the non-correlation between ESR and viscosity in Group 2, in which the platelet count is higher than Group 1, can be explained.

As a result; in contrast to normal platelets in healthy individuals, the circulating thrombocythemic platelets (e.g. chronic myeloproliferative disorders) spontaneously activate and secrete their products, thus forming aggregates that transiently plug the microcirculation or result in occlusive platelet thrombi in vessels [19]. In patients with thrombocythemia associated with chronic myeloproliferative disorders, increased hematocrit and viscosity aggravate the platelet-mediated microvascular ischemic and thrombotic syndrome of thrombocythemia to major arterial and venous thrombotic complications. In the present study, despite the significant difference between groups in terms of platelet count, no significant difference was detected in plasma viscosity and this finding could be explained as the following; 1-these platelets were not thrombocythemic platelets; 2-similar to the theory about leukocytes, much higher platelet counts (e.g. > 1.000 x 109/L) than observed in Group 2 (mean: 507.45 x 109/L) – even non-thrombocythemic– may increase plasma viscosity; 3-evaluating platelet count alone is not sufficient and the associating red-cell deformability should also be taken into account; and 4-although other diseases that could affect viscosity are excluded, some definitely proven literature criteria such as fibrinogen, hyperlipidemia, and the inflammatory process should also be evaluated by laboratory and clinical measures. We hope that further studies performed on larger number of patients -reactive thrombocytosis- and controls, additionally including thrombocythemic disorders, with the possibility of encountering higher platelet counts and analyzing additional criteria that could influence viscosity can illuminate the dark spots of current clinical practice in reactive thrombocytosis which seems innocent at the present.

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References 1. Kaushansky K. Reactive Thrombocytosis. In: Kaushansky K, Lichtman MA, Beutler E, Kipps TJ, Seligsohn U, Prchal JT, eds. Williams Hematology. 8th ed. New York: McGraw Hill, 2010: 1929-1932 2. Khan PN, Nair RJ, Olivares J, Tingle LE, Li Z. Postsplenectomy reactive thrombocytosis. Proc (Bayl Univ Med Cent) 2009; 22 (1): 9-12 3. Keung YK, Owen J. Iron deficiency and thrombosis: Literature review. Clin Appl Thromb Hemost 2004; 10 (4): 387-391 4. Stolz E, Valdueza JM, Grebe M, Schlachetzki F, Schmitt E, Madlener K, Rahimi A, Kempkes-Matthes B, Blaes F, Gerriets T, Kaps M. Anemia as a risk factor for cerebral venous thrombosis? An old hypothesis revisited. Results of a prospective study. J Neurol 2007; 254 (6): 729-734 5. Ho CH. White blood cell and platelet counts could affect whole blood viscosity. J Chin Med Assoc 2004; 67 (8): 394-397 6. Means RT Jr, Glader B. Anemia. General considerations.

In: Greer JP, Foerster J, Rodgers GM, Paraskevas F, Glader B, Arber DA, Means RT Jr, eds. Wintrobe’s Clinical Hematology. 12th ed. Philadelphia: Lippincott Williams and Wilkins 2009, pp. 779-809 7. Andrews NC. Iron deficiency and related disorders. In: Greer JP, Foerster J, Rodgers GM, Paraskevas F, Glader B, Arber DA, Means RT Jr, eds. Wintrobe’s Clinical Hematology.

12th ed. Philadelphia: Lippincott Williams and Wilkins 2009, pp. 810-34 8. Goddard AF, James MW, McIntyre AS, Scott BB. British Society of Gastroenterology: Guidelines for the management of iron deficiency anaemia. Gut 2011; 60 (10): 1309-1316 9. Rosenson RS, McCormick A, Uretz EF. Distribution of blood viscosity values and biochemical correlates in healthy adults. Clin Chem 1996; 42 (8 Pt 1): 1189-1195 10. Késmárky G, Kenyeres P, Rábai M, Tóth K. Plasma viscosity: A forgotten variable. Clin Hemorheol Microcirc 2008; 39 (1-4): 243-246

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11. Senen K, Topal E, Kilinc E, ten Cate H, Tek I, Karakoc Y, Yetkin E. Plasma viscosity and mean platelet volume in patients undergoing coronary angiography. Clin Hemorheol Microcirc 2010; 44 (1): 35-41 12. Crowley JJ, Hannigan M, Daly K. Reactive thrombocytosis and stroke following cardiopulmonary bypass surgery: Case report on three patients. Eur Heart J 1994; 15: 1144 13. Robertson DA, Simpson FG, Losowsky MS. Blood viscosity after splenectomy. Br Med J (Clin Res Ed) 1981; 283 (6291): 573-575 14. Rozenberg MC. Blood viscosity and thrombosis in vitro in patients with previous myocardial infarction. Angiology 1968; 19 (9): 527-533 15. Steinberg MH, Charm SE. Effect of high concentrations of leukocytes on whole blood viscosity. Blood 1971; 38 (3): 299-301 16. Remacha AF, Souto JC, Piñana JL, Sardà MP, Queraltó JM, Martí-Fabregas J, García-Moll X, Férnandez C, Rodriguez A, Cuesta J. Vitamin B12 deficiency, hyperhomocysteinemia and thrombosis: A case and control study. Int J Hematol 2011; 93 (4): 458-464 17. von Eckardstein A, Malinow MR, Upson B, Heinrich J, Schulte H, Schönfeld R, Köhler E, Assmann G. Effects of age, lipoproteins, and hemostatic parameters on the role of homocyst(e)inemia as a cardiovascular risk factor in men.

Arterioscler Thromb 1994; 14 (3): 460-464 18. Salawu L, Durosinmi MA. Erythrocyte rate and plasma viscosity in health and disease. Niger J Med 2001; 10 (1): 11-13 19. Michiels JJ, Berneman Z, Schroyens W, Koudstaal PJ, Lindemans J, Neumann HA, van Vliet HH. Plateletmediated erythromelalgic, cerebral, ocular and coronary microvascular ischemic and thrombotic manifestations in patients with essential thrombocythemia and polycythemia vera: A distinct aspirin-responsive and coumadin-resistant arterial thrombophilia. Platelets 2006; 17 (8): 528-544

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Research Article

10.5152/tjh.2011.75

Secondary Infections in Cancer Patients with Febrile Neutropenia Febril Nötropenik Hastalarda Gelişen Sekonder İnfeksiyonların Değerlendirilmesi Alpay Azap1, Gülden Yılmaz Bozkurt1, Meltem Kurt Yüksel2, Hakan Kutlu1, Pervin Topçuoğlu3, Adalet Aypak4, Hamdi Akan3 Ankara University, School of Medicine, Department of Infectious Diseases and Clinical Microbiology, Ankara, Turkey Medicana International Ankara Hospital, Ankara, Turkey 3 Ankara University, School of Medicine, Department of Hematology, Ankara, Turkey 4 Ankara Numune Education and Research Hospital, Department of First Infectious Diseases and Clinical Microbiology, Ankara Turkey 1 2

Abstract Objective: Patients with neutropenia due to cancer chemotherapy are prone to severe infections. Cancer patients can experience >1 infectious episode during the same period of neutropenia. This study aimed to determine the etiological and clinical characteristics of secondary infectious episodes in cancer patients with febrile neutropenia and to identify the factors associated with the risk of secondary infectious episodes.

Material and Methods: All cancer patients that received antineoplastic chemotherapy at Ankara University, School of Medicine, Department of Hematology between May 2004 and May 2005 and developed neutropenia were included in the study. Data were collected using survey forms that were completed during routine infectious diseases consultation visits.

Categorical data were analyzed using the chi-square test, whereas Student’s t-test was used for continuous variables.

Multivariate logistic regression analysis was performed to identify independent predictors of secondary infections (SIs).

Results: SIs were observed during 138 (53%) of 259 febrile neutropenic episodes. Of the 138 episodes, 89 (64.5%) occurred in male patients with a mean age of 40.9 years (range: 17-76 years). In total, 80% of the SIs were clinically or microbiologically documented. Factors on d 4 of the initial febrile episode were analyzed via a logistic regression model.

The presence of a central intravenous catheter (OR: 3.01; P < 0.001), acute myeloid leukemia (AML) as the underlying disease (OR: 2.12; P = 0.008), diarrhea (OR: 4.59; P = 0.005), and invasive aspergillosis (IA) during the initial febrile episode (OR: 3.96; P = 0.009) were statistically significant risk factors for SIs.

Conclusion: Among the cancer patients with neutropenia in the present study, AML as the underlying disease, the presence of a central venous catheter, diarrhea, and IA during the initial febrile episode were risk factors for the development of SIs. Key Words: Hematologic malignancy, Febrile neutropenia, Secondary infection

Özet Amaç: Antineoplastik kemoterapi uygulanan kanser hastalarının hemen tamamında nötropeni gelişmektedir.

Nötropenik dönemde birden fazla febril atak ortaya çıkabilmektedir. Bu çalışmada, nötropenik hastalarda ilk febril atak Address for Correspondence: Alpay Azap, M.D., Ankara Üniversitesi Tıp Fakültesi, İnfeksiyon Hastalıkları ve Klinik Mikrobiyoloji Anabilim Dalı, Ankara, Turkey Phone: +90 312 310 33 33 E-mail: Alpay.Azap@medicine.ankara.edu.tr Received/Geliş tarihi : March 31, 2011 Accepted/Kabul tarihi : May 17, 2011

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sonrasında gelişen ikincil atakların etiyolojik ve klinik özellikleri ile birlikte sekonder infeksiyon için risk faktörlerinin belirlenmesi amaçlanmıştır.

Gereç ve Yöntemler: Ankara Üniversitesi Tıp Fakültesi Hematoloji AD’da çeşitli maligniteler nedeniyle 2004 Mayıs ve 2005 Mayıs tarihleri arasında kemoterapi uygulanan ve febril atak/ataklar gelişen hastalar çalışmaya dahil edilmiş ve bu hastaların kayıtları geriye dönük olarak taranmıştır. Kategorik değişkenler ki-kare testi, devamlı değişkenler ise Student’s test uygulanarak değerlendirilmiştir. İkincil febril atak için risk faktörlerini belirlemede lojistik regresyon analizi uygulanmıştır.

Bulgular: Çalışma periyodunda gelişen 259 febril nötropenik atağın 138’inde (%53) sekonder infeksiyon gözlemlenmiştir. Sekonder infeksiyon gelişen hastaların %64.5’i erkek iken ortalama yaş 40,9’dur (17-76). Gelişen sekonder infeksiyonların yaklaşık %80’i klinik ya da mikrobiyolojik dokümante infeksiyonlardır. İlk ateş atağının 4. gününde incelenen faktörler için lojistik regresyon analizi uygulandığında; sekonder infeksiyon gelişimi için risk faktörleri olarak sanral venöz kateter bulunması (odds oranı [OR], 3.01; P < 0.001), altta yatan hastalık olarak Akut Myeloid Lösemi (AML) olması (OR, 2.12; P = 0.008), diare olması (OR, 4.59; P = 0.005) ve ilk atakta invazif aspergilloz (İA) tanısı bulunması (OR, 3.96; P = 0.009) belirlenmiştir.

Sonuç: Santral venöz kateter bulunması, altta yatan hastalığın AML olması, diare görülmesi ve ilk atakta İA gelişmiş olması nötropenik hastalarda sekonder infeksiyon riskini arttırmaktadır.

Anahtar Sözcükler: Hematolojik malignite, Febril nötropeni, Sekonder infeksiyon Introduction During the last 2 decades major developments in the treatment of malignant diseases have been realized, including bone marrow transplantation, aggressive antineoplastic treatment modalities, and the use of intravascular catheters. Nonetheless, all these developments are associated with longer periods of neutropenia, severe mucositis, and widespread use of antimicrobial therapy or prophylaxis, which probably lead to the emergence of drug-resistant microorganisms. Consequently, patients undergoing such treatment are prone to severe infections, and cancer patients can experience multiple infectious episodes during the same period of neutropenia [1,2].

Recently, consideration of the level of risk of severe infection in patients treated for febrile neutropenia has been strongly recommended. To date, several studies have sought to identify severe infection risk factors in febrile neutropenia patients, and mortality rates in febrile neutropenia patients have been reported; however, only a few studies examined the factors associated with the risk of secondary infections (SIs) [1-5].

As such, the aim of the present study was to determine the etiological and clinical characteristics of SIs in cancer patients with neutropenia, and to identify the factors associated with the risk of SIs. Methods and Materials All cancer patients that received antineoplastic chemotherapy at Ankara University, School of Medicine, Depart-

ment of Hematology between May 2004 and May 2005 and developed neutropenia were included in the study.

The local ethics committee approved the study. Fever was defined as ≥2 axillary temperature measurements of ≥38 °C within a 12-h period or 1 measurement of ≥38.5 °C. Patients were considered to be neutropenic if their neutrophil count was <500 cells mm–3, or <1000 cells mm–3 and expected to decrease to <500 cells mm–3 within 24-48 h.

SI was defined as follows: i) any clinical or microbiologically documented infection that did not exist at the time of initial evaluation, but developed during empirical therapy or within 1 week after discontinuation of therapy, and ii) fever that responded to empirical therapy, but recurred after an afebrile period of 48 h during empirical therapy [1,6].

Each primary infection (PI) and SI was classified as a microbiologically documented infection (MDI), clinically documented infection (CDI) (objective and detectable signs of infection with a lack of microbiological documentation), or fever of unknown origin (FUO). Variables assessed on d 4 of the PI were age, sex, underlying hematological disease, comorbid diseases, receipt of a bone marrow transplant, severity of neutropenia, presence of a central venous catheter (CVC), history of fungal infection prior to the study, presence of oral mucositis, presence of diarrhea (>3 times d–1), presence of a catheter-related infection, administration of antibacterial, antiviral, and antifungal prophylaxis, antimicrobials used during the treatment of PIs, presence of invasive fungal infections, type of infection documentation (MDI, CDI, or FUO), and, if

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MDI, the infectious microorganism. Episodes of SI were evaluated in terms of the type of infection documentation and the infectious microorganisms.

Data were collected using structured survey forms that were completed during routine infectious diseases consultation visits. Categorical data were analyzed via the chisquare test, whereas Student’s t-test was used for continuous variables. Multivariate logistic regression analysis was performed to identify independent predictors of SIs. Data were analyzed using Stata v.8.0 statistical software (Stata Corp., Texas, USA) and the level of statistical significance was set at P < 0.05, using two-sided comparisons.

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(54%, n = 35), gram-negative bacilli (43%, n = 28), and fungi (3%, n = 2). Possible and probable invasive aspergillosis (IA) was diagnosed in 35 (14%) of the patients, based on European Organization for Research and Treatment of Cancer (EORTC) criteria [7]. Among the episodes of SI, 81 (59%) of the 138 episodes were CDI, 27 (20%) were MDI, and 30 (21%) were FUO. In total, 27 bacterial pathogens were isolated, of which 12 (44%) were gram-positive bacteria and 15 (56%) were gram-negative bacilli. No fungi were isolated.

SIs were observed during 138 (53%) of 259 febrile neutropenic episodes. Of the 138 episodes, 89 (64.5%) occurred in male patients with a mean age was 40.9 years (range: 17-76 years). Acute myeloid leukemia (AML) (64.5%) was the most common underlying disease, followed by acute leukemoid leukemia (16.5%), lymphoma (11.8%), and others (multiple myeloma, myelodysplastic syndrome, chronic myeloid leukemia, and aplastic anemia) (7.2%). In all, 14% of the patients had undergone bone marrow transplantation and only 1 patient had a comorbid disease (diabetes mellitus).

The baseline characteristics of the febrile neutropenic patients and SI episode rate according to these characteristics are presented in Table 1. Univariate analysis was performed in order to identify the risk factors for SI and included such variables as gender, age, diarrhea, neutrophil count, grade 3-4 mucositis, IA during the PI, CVC, CDI, or MDI during the episode of PI, and AML as the underlying disease. Among these factors, univariate analysis showed that diarrhea (P < 0.001), IA during the episode of PI (P < 0.001), CVC (P < 0.001), CDI or MDI during the episode of PI (P = 0.009), and AML as the underlying disease (P = 0.001) were strongly correlated with SIs. Neutropenia and severe neutropenia (<100 mm–3) were not associated with SIs.

Among the episodes of PI, 121 (47%) were FUO, 73 (28%) were MDI, and 65 (25%) were CDI. The microbial agents responsible for PIs were gram-positive bacteria

The baseline risk factors during PI were analyzed using a logistic regression model of multivariate analysis.

The presence of a CVC (OR: 3.01; P < 0.001), AML as the

Results

Table 1: Characteristics of the febrile neutropenic patients and the SI rate according to these characteristics.

Variable Gender, n (%) Male Mean age in years (range) Diarrhea (%) Neutrophil count <100 mm–3 (%) Grade III-IV oral mucositis IA during PI Central venous catheter CDI or MDI during PI AML as underlying disease

Neutropenic Episodes (n = 259)

SIs

P* 0.173

157 (60.6) 40.3 (18-76) 30 (11.6) 140 (54) 35 (13.5) 35 (13.5) 160 (61.8) 138 (53.3) 143 (55.2)

89 (56.7) 40.9 (17-76) 25 (83.3) 81 (57.9) 23 (65.7) 30 (85.7) 102 (63.7) 84 (60.9) 89 (62.2)

0.736a <0.001 0.109 0.113 <0.001 <0.001 0.009 0.001

IA: Invasive aspergillosis; AML: acute myeloid leukemia, FEN: febrile neutropenia; CDI: clinically documented infection; MDI: microbiologically documented infection. *Predicted by chi-square test; aStudent’s t-test.

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Table 2: Multivariate analysis via logistic regression modeling of the factors associated with SIs.

Variable IA during PI AML as the underlying disease Diarrhea CVC

OR 3.96

95% CI 1.40-11.18

P 0.009

2.12

1.22-3.70

0.008

4.59 3.01

1.56-13.46 1.69-5.34

0.005 <0.001

underlying disease (OR: 2.12; P = 0.008), diarrhea (OR: 4.59; P = 0.005), and IA during PI (OR: 3.96; P = 0.009) were statistically significant risk factors for SIs (Table 2).

Discussion Identification of the risk factors for infection is a logical approach in the treatment of febrile neutropenic patients.

Although various studies have reported these risk factors, SIs were considered in only a few [1-5, 8-11]. The frequency of SIs in adults varies between 12% and 24%. Recently Paganini et al. reported an SI rate of 17% in children [4]. In the present study the SI incidence rate was much higher—56%. This may have been related to underlying disease and the chemotherapeutic regimen, as AML was the underlying disease in 64.5% of the patients and 14% patients had undergone bone marrow transplantation. At the time of the study prophylactic use of such antibacterial agents as quinolones was not practiced at our institution, which may also be a factor related to the high incidence of SIs in the present study.

AML as the underlying disease, presence of a CVC, diarrhea, and IA during PI were associated with the development of SIs in the present study. The presence of a CVC was previously reported to be a marker of SIs [1,2]. CVCs may promote skin and soft tissue infections, and are associated with bacteremia, especially due to gram-positive pathogens [12,13]. As empirical treatment regimens used at the onset of an episode of febrile neutropenia do not usually target gram-positive bacteria, CVC is a reliable risk factor for SI; however, Serra et al. reported that there wasn’t a relationship between the presence of CVC and the development of SIs [14].

The other independent factor associated SIs in the present study was AML as the underlying disease. Patients with AML as the underlying disease were administered more intensive chemotherapeutic regimens than the other patients in the study and therefore were more prone to SIs. The risk of SIs increases significantly in the presence

of diarrhea; the integrity of the gastrointestinal mucosa is compromised and bacterial translocation occurs more easily in patients with diarrhea.

Serra et al. reported that the duration and severity of neutropenia are risk factors for developing SIs [14]. The duration of severe neutropenia (<100 neutrophils mm–3) was strongly associated with SIs in a study by Nucci et al.

[2]. Nevertheless, neutropenia (<500 neutrophils mm–3) and severe neutropenia were not associated with SIs in the present study. With respect to the present findings, Feld et al. reported that the duration of neutropenia had no effect on the development of SIs [15]. Akova et al. reported that the neutrophil count on d 0 was not a predictive factor for SIs, whereas persistently and severely neutropenic patients were prone to SIs [1].

IA during PI was also a risk factor for SIs in the present study. SIs occurred in 36 of the 49 patients with a history of IA. Such an association has not been previously reported, and might be due to the fact that IA usually occurs in high-risk patients with profound neutropenia for a long time (>10 days) [16]. On the other hand, the duration (>10 days) and severity (<100/mm-3) of neutropenia were not observed to be independent risk factors for SIs based on statistical analysis of the present study’s data. The effect of the duration and severity of neutropenia on SI might have been obscured by the high incidence of IA among the patients included in the present study.

The present study has some limitations. First, stage of the underlying disease and chemotherapeutic regimens (remission induction or consolidation) were not included in the statistical analysis. The stage of underlying disease and chemotherapeutic regimens have major impact on the duration and severity of neutropenia, both of which were not associated with SIs in the study. As such, the stage of underlying disease was not considered to be an important factor. Second, as the study group consisted of adults with a mean age of 40.3 years, only 1 patient had comorbid disease (diabetes mellitus). As such, it was not possible to assess the association between comorbidities and the development of SIs. In conclusion, although SIs in neutropenic patients are common, they are rarely studied. The present study analyzed SIs in febrile neutropenic patients to identify the risk factors for SIs. AML as the underlying disease, presence of a CVC, diarrhea, and IA during PI are factors that may be considered as risk factors for SIs in neutropenic patients.

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Azap A, et al: Secondary Infections in Cancer Patients with Febrile Neutropenia

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Akova M, Paesmans M, Calandra T, Viscoli C. International Antimicrobial Therapy Group of the European Organization for Research and Treatment of Cancer: A European Organization for Research and Treatment of CancerInternational Antimicrobial Therapy Group Study of secondary infections in febrile neutropenic patients with cancer. Clin Infect Dis 2005; 40: 239-245 2. Nucci M, Spector N, Bueno AP, Solza C, Perecmanis T, Bacha PC, Pulcheri W. Risk factors and attributable mortality associated with superinfections in neutropenic patients with cancer. Clin Infect Dis 1997; 24: 575-579 3. Freifeld AG, Bow EJ, Sepkowitz KA, Boeckh MJ, Ito JI, Mullen CA, Raad II, Rolston KV, Young JA, Wingard JR. Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 2011; 52: 56-93 4. Paganini H, Caccavo J, Aguirre C, Gómez S, Zubizarreta P. A scoring system to predict superinfections in high risk febrile neutropenic children with cancer. Bol Med Hosp Infant Mex 2011; 68: 40-47 5. Paesmans M. Risk factors assessment in febrile neutropenia.

Int J Antimicrob Agents 2000; 16: 107-111 6. Hughes WT, Armstrong D, Bodey GP, Bow EJ, Brown AE, Calandra T, Feld R, Pizzo PA, Rolston KV, Shenep JL, Young LS. 2002 Guidelines for the use of antimicrobial agents in neutropenic patients with cancer. Clin Infect Dis 2002; 34: 730-751 7. Ascioglu S, Rex JH, de Pauw B, Bennett JE, Bille J, Crokaert F, Denning DW, Donnelly JP, Edwards JE, Erjavec Z, Fiere D, Lortholary O, Maertens J, Meis JF, Patterson TF, Ritter J, Selleslag D, Shah PM, Stevens DA, Walsh TJ. Invasive Fungal Infections Cooperative Group of the European Organization for Research and Treatment of Cancer; Mycoses Study Group of the National Institute of Allergy and Infectious Diseases: Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: An international consensus. Clin Infect Dis 2002; 34: 7-14

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8. Talcott JA, Siegel RD, Finberg R, Goldman L. Risk assessment in cancer patients with fever and neutropenia: A prospective, two-center validation of a prediction rule. J Clin Oncol 1992; 10: 316-322 9. Klastersky J, Paesmans M, Rubenstein EB, Boyer M, Elting L, Feld R, Gallagher J, Herrstedt J, Rapoport B, Rolston K, Talcott J. The Multinational Association for Supportive Care in Cancer risk index: A multinational scoring system for identfying low risk febrile neutropenic cancer patients. J Clin Oncol 2000; 18: 3038-3051 10. Guiguet M, Blot F, Escudier B, Antoun S, Leclercq B, Nitenberg G. Severity of illness scores for neutropenic cancer patients in an intensive care unit: Which is the best predictor? Do multiple assessment times improve the predictive value? Crit Care Med 1998; 26: 488-493 11. Paganini HR, Aguirre C, Puppa G, Garbini C, Javier RG, Ensinck G, Vrátnica C, Flynn L, Iacono M, Zubizarreta P. Febrile Neutropenia Study Group: A prospective, multicentric scoring system to predict mortality in febrile neutropenic children with cancer. Cancer 2007; 109: 2572-2579 12. Pizzo PA, Ladisch S, Robichaud K. Treatment of gram positive septicemia in cancer patients. Cancer 1980; 45: 206-207 13. Raad II, Bodey GP. Infectious complications of indwelling vascular catheters. Clin Infect Dis 1992; 15: 197-208 14. Serra P, Santini C, Venditti M, Mandelli F, Martino P. Superinfections during antimicrobial treatment with betalactam-aminoglycoside combinations in neutropenic patients with hematologic malignancies. Infection 1985; 13: 115-122 15. Feld R, Goodman PJ, Higgins B, De Pauw BE, Deresinski S, Donnelly JP. Prognostic factors fort he development of superinfection in febrile neutropenic cancer patients (abstract no 1695). In: Program and abstracts of the 32th Interscience Conference on Antimicrobial Agents and Chemotherapy. Washington, DC: American Society for Microbiology, 1992 16. Mühlemann K, Wenger C, Zenhäusern R, Täuber MG. Risk factors for invasive aspergillosis in neutropenic patients with hematologic malignancies. Leukemia 2005; 19: 545-550

Research Article

10.5505/tjh.2012.15986

Psychiatric Problems in Children and Adolescents with Sickle Cell Disease, Based on Parent and Teacher Reports Orak Hücre Anemili Çocuk ve Ergenlerde Ebeveyn ve Öğretmen Bildirilerine Dayalı Psikiyatrik Sorunlar Özalp Ekinci1, Tanju Çelik2, Şule Ünal3, Cahit Özer4 Ozalp Ekinci Child and Adolescent Psychiatry Clinic, Hatay, Turkey Mustafa Kemal University, School of Medicine, Department of Pediatrics, Hatay, Turkey 3 Antakya State Hospital, Pediatric Hematology Clinic, Hatay, Turkey 4 Mustafa Kemal University, School of Medicine, Department of Family Medicine, Hatay, Turkey 1 2

Abstract Objective: This study aimed to investigate the occurrence of psychiatric problems in children and adolescents with sickle cell disease (SCD).

Material and Methods: The Child Behavior Checklist for ages 4-18 years (CBCL/4-18), Conners’ Parent Rating Scale (CPRS), Conners’ Teacher Rating Scale (CTRS-R), and The Turgay DSM-IV Based Child and Adolescent Behavior Disorders Screening and Rating Scale, clinician and parent forms (T-DSM-IV-S) were given to the caregivers and teachers of 31 children with SCD aged between 7-18 years and the caregivers and teachers of 34 age matched controls with irondeficiency anemia.

Results: The SCD patients had higher scores on all 4 of scales. Among the subscales, internalizing problems, and attention problems were more prominent in the SCD patients.

Conclusion: Children and adolescents with SCD appear to have an increased risk for psychiatric problems. Regular psychological evaluation and referral to child and adolescent psychiatry clinics may facilitate timely diagnosis and effective treatment of at-risk children and adolescents.

Key Words: Sickle cell disease, Psychiatric problems, Children, Adolescents

Özet Amaç: Bu çalışma Orak Hücre Anemisi (OHA) tanılı çocuk ve ergenlerde psikiyatrik sorunların varlığını incelemeyi amaçlamıştır.

Gereç ve Yöntemler: 4-18 yaş arası için Çocuk Davranış Kontrol Listesi (ÇDKL/4-18), Conners Ebeveyn Derecelendirme Ölçeği (CEDÖ), Conners Öğretmen Derecelendirme Ölçeği (CÖDÖ-R) ve Turgay DSM-IV’e dayalı Çocuk ve Ergen Davranış

Address for Correspondence: Özalp Ekinci, M.D., Özalp Ekinci Çocuk ve Yetişkin Psikiyatri Kliniği, Antakya, Hatay, Turkey Phone: +90 326 215 85 75 E-mail: ozalpekinci@yahoo.com Received/Geliş tarihi : January 12, 2012 Accepted/Kabul tarihi : February 6, 2012

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Bozuklukları Tarama ve Derecelendirme Ölçeği, klinisyen ve ebeveyn formları (T-DSM-IV-Ö) 7-18 yaş arası OHA tanılı 31 çocuğun ve 34 yaş olarak eşlenmiş demir eksikliği anemisi tanılı kontrol grubunun bakım vericileri ve öğretmenlerine verildi.

Bulgular: OHA tanılı çocuk ve ergenler her 4 ölçekte de daha yüksek puanlar aldılar. Ölçek alt puanlarında içe atım sorunları ve dikkat sorunları öne çıkıyordu.

Sonuç: OHA tanılı çocuk ve ergenlerin psikiyatrik sorunlar açısından yüksek riskte olduğu görünmektedir. Olgulara düzenli psikolojik değerlendirme yapılması ve çocuk ve ergen psikiyatri kliniklerine refere edilmesi risk altında olan olguların zamanında tanısı ve etkin tedavisine katkı sağlayabilir.

Anahtar Sözcükler: Orak hücre anemisi, Psikiyatrik sorunlar, Çocuk, Ergen Introduction Sickle cell disease (SCD) is a genetic hemoglobin disorder characterized by a chronic course and disabling complications. SCD patients experience episodic complications affecting multiple organ systems and an increased risk of infections. Recurrent, acute pain that often requires emergency management is the hallmark of SCD and negatively affects quality of life. Children and adolescents with SCD face many challenges associated with living with a chronic condition that requires lifelong medical management. Treatment and hospitalization of SCD patients of school age can further disrupt everyday life due to absence from school [1,2]. As observed in children and adolescents with other chronic illnesses, the current literature suggests that those with SCD may be at risk for adjustment problems and impaired psychosocial functioning [2-4] A number of studies have shown that the frequency of neuropsychiatric problems in children and adolescents with SCD is higher than that in normal controls [5].

Among the neuropsychiatric problems, depression, anxiety disorders, learning problems, and attention deficit are commonly reported [6-8]. The present study aimed to investigate the frequency of psychiatric problems, with a special emphasis on attention-deficit/hyperactivity disorder (ADHD) symptoms, in children and adolescents with SCD. Materials and Methods Study group The study included SCD patients aged between 7-18 years that presented to the Antakya Thalassemia Center, Hatay, Turkey, between May 2010 and May 2011. The study protocol was approved by the Mustafa Kemal University, School of Medicine Ethics Committee. Prior to administration, the study questionnaires were explained to the participants by a child and adolescent psychiatry specialist, and informed consent was provided by the

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adolescent patients and the parents’ of pediatric patients.

Patients with documented mental retardation (MR) were excluded from the study, as were those diagnosed with MR at the time of the study process. From among the 35 SCD patients that initially gave consent, 31 fulfilled the study requirements and participated in the study. The control group included 34 children and adolescents with iron deficiency anemia that presented to the Antakya Mustafa Kemal University Hospital Pediatric Clinic.

Tools Conners’ Parent Rating Scale (CPRS) Conners’ Parent Rating Scale (CPRS) is a 48-item Likert-type scale used to assess problematic behaviors in children [9]. In addition to a total score, there are 5 subscale scores: Conduct problems; impulsivity and hyperactivity; learning problems; anxiety; psychosomatic problems.

Dereboy et al. reported that the CPRS-Turkish Version is valid and reliable for use in the Turkish population [10].

Conners’ Teacher Rating Scale-Revised (CTRS-R) Conners’ Teacher Rating Scale (CTRS-R) is a commonly used teacher rating scale for diagnosing behavioral problems in children [11]. The 28-item CTRS-R provides a total score and 3 subscale scores: Attention deficit; hyperactivity; conduct problems. Şener et al. reported that the CTRS-R-Turkish Version is valid and reliable for use in the Turkish population [12].

The Turgay DSM-IV Based Child and Adolescent Behavior Disorders Screening and Rating Scale, clinician and parent forms (T-DSM-IV-S) The Turgay DSM-IV Based Child and Adolescent Behavior Disorders Screening and Rating Scale, clinician and parent forms (T-DSM-IV-S) was developed by Turgay [13]) and consists of 42 items that measure attention-deficit, hyperactivity, impulsivity, and disruptive behaviors.

In the present study a shorter version of the scale was used

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Özalp E, et al: Psychiatric Problems in Children with Sickle Cell Disease

that included 9 attention-deficit items, 6 hyperactivity items, and 3 impulsivity items. Ercan et al. reported that the T-DSM-IV-S Scale-Turkish Version is valid and reliable for use in the Turkish population [14].

Child Behavior Checklist for Age 4-18 years (CBCL/4-18):

Statistical analysis All study data were analyzed using SPSS v.13.0 software. Student’s t test was used to compare patient group and control group means. The level of statistical significance was set at P = 0.05.

Results

The Child Behavior Checklist for age 4-18 years (CBCL/4-18) was developed by Achenbach and Edelbrock to evaluate adequate and problematic behavior in children, based on parental reporting [15]. The 112-item CBCL/418 provides a total score, internalizing and externalizing scores, and somatic complaints, anxiety/depression, social problems, thought problems, attention problems, delinquent behaviors, and aggressive behaviors subscale scores.

In the present study the items’ T scores were calculated with Cross Informant Program V 4.0 [16] and entered into the study database. Erol and Şimşek reported that the CBCL/4-18-Turkish Version is valid and reliable for use in the Turkish population [17].

Mean age in the SCD group (n=31) was 12.6 ± 3.2 years, versus 12.0 ± 2.9 years in the control group (n=34).

In all, 19 (61%) SCD patients and 16 (47%) controls were male. Table 1 shows the CBCL/4-18 total and subscale scores in the 2 groups. Mean CBCL/4-18 total score was significantly higher in the SCD group (P = 0.02), as was the internalizing subscale score (P = 0.01), withdrawn subscale score (P = 0.001), somatic complaints subscale score (P = 0.05), anxiety/depressive symptoms subscale score (P = 0.03), thought problems subscale score (P = 0.01), and attention problems subscale score (P = 0.01).

CPRS scores in the SCD and control groups are shown in Table 2. CPRS total score (P = 0.001), conduct prob-

Table 1: CBCL/4-18 scores in the SCD and control groups.

CBCL/4-18 Scores Total Internalizing subscale Externalizing subscale Withdrawn subscale Somatic complaints subscale Anxiety/depressive symptoms subscale Social problems subscale Thought problems subscale Attention problems subscale Delinquent behaviors subscale Aggressive behaviors subscale

SCD Mean ± SD 65.0 ± 10.0 68.4 ± 9.9 56.1 ± 10.3 65.0 ± 11.3 69.2± 11.4 65.1 ± 8.4 59.6 ± 8.3 64.0 ± 7.3 64.8 ± 8.3 57.3 ± 7.4 58.1 ± 7.0

Controls Mean ± SD 58.4 ± 11.4 61.6 ± 10.4 52.4 ± 10.2 58.2 ± 8.3 62.8 ± 12.7 60.6 ± 8.2 57.8 ± 8.3 57.7 ± 7.8 59.0 ± 9.4 54.7 ± 7.1 55.3 ± 7.8

P 0.02 0.01 0.15 0.01 0.05 0.03 0.38 0.01 0.01 0.15 0.13

Table 2: CPRS scores in the SCD and control groups.

CPRS Scores Total Conduct problems subscale Impulsivity/hyperactivity subscale Learning problems subscale Anxiety subscale Psychosomatic problems subscale

SCD Mean ± SD 41.1 ± 18.1 7.7 ± 5.5 4.4 ± 2.8 4.7 ± 3.0 11.1 ± 3.2 7.0 ± 3.0

Controls Mean ± SD 20.4 ± 8.2 5.2 ± 2.8 4.3 ± 2.1 3.9 ± 2.1 3.2 ± 2.5 1.7 ± 1.7

P 0.001 0.002 0.91 0.24 0.001 0.001

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Table 3: CTRS-R scores in the SCD and control groups.

CTRS-R Scores

SCD Mean ± SD

Controls Mean ± SD

P

Total

21.8 ± 13.0

18.3 ± 8.8

0.20

Attention deficit subscale

7.6 ± 4.6

4.4 ± 3.2

0.002

Hyperactivity subscale

6.7 ± 4.7

7.0 ± 3.5

0.83

Conduct problems subscale

3.4 ± 3.2

3.1 ± 2.8

0.78

Table 4: T-DSM-IV-S scores in the SCD and control groups.

T-DSM-IV-S Scores

SCD Mean ± SD

Controls Mean ± SD

P

ADHD total

19.7 ± 12.0

10.2 ± 7.9

0.001

Inattentiveness subscale

9.5 ± 6.7

4.9 ± 4.4

0.002

Hyperactivity subscale

10.2 ± 6.9

5.3 ± 4.3

0.001

lems subscale score (P = 0.002), anxiety subscale score (P = 0.001), and psychosomatic problems subscale score (P = 0.001) were significantly higher in the SCD group. CTRSR scores in the SCD and control groups are shown in Table 3. The CTRS-R attention deficit subscale score was significantly higher in the SCD group (P = 0.002). T-DSM-IV-S scores in the SCD and control groups are shown in Table 4. T-DSM-IV-S total score (P = 0.001), inattentiveness subscale score (P = 0.002), and hyperactivity/impulsivity subscale score (P = 0.001) were significantly higher in the SCD group.

Discussion Hemoglobinopathies are among the most common genetic diseases in Turkey. The incidence of the SCD trait is 10.0% in the Çukurova region of southern Turkey [18].

In the present study the frequency of psychiatric problems in children and adolescents with SCD treated at a single center in this region was investigated. As compared to the control group, the SCD patients had higher psychiatric problem scores on all 4 psychiatric scales administered.

The SCD patients had significantly higher CBCL/4-18 subscale scores. Among the CBCL/4-18 subscale scores, internalizing, including somatic complaints, anxiety/ depressive symptoms, and thought problems scores were higher in the SCD group. Internalizing problems and emotional symptoms (that meet or do not meet DSM-IV criteria) commonly occur in children with chronic medical diseases [19]. Previous studies on children and adolescents with SCD reported high frequencies of internalizing

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problems, including symptoms of depression and anxiety [6,20-22]. These symptoms may be attributed to be born with a chronic illness, the unpredictable painful nature of the disease and the feelings of frustration or a sense of injustice that the children have been afflicted [2]. Somatic symptoms are important in the differential diagnosis of children with SCD. Children with SCD may experience chronic pain directly related to SCD or in the form of additional somatic psychiatric symptoms. Somatic complaints may also underlie unrecognized depression or anxiety; therefore, psychiatric consultation should be considered for children and adolescents with SCD that have chronic somatic complaints.

In the present study the parent-rated CPRS total score and conduct problems subscale, anxiety subscale, and psychosomatic problems subscale scores were higher in the SCD group, whereas the teacher-rated CTRS-R scores showed that the number of children and adolescents with attention deficit was higher in the SCD group. This discrepancy between parent- and teacher-rated scores could have been due to numerous factors. Children with chronic medical diseases like SCD usually experience social withdrawal and isolation at school [4,20,23-25]. For instance, children with SCD are usually limited from physical exercise. Frequent hospitalization and absence from school, and related academic failure may also contribute to the social isolation of children and adolescents with SCD [26,27]. As such, SCD patients are usually observed as silent and withdrawn at school [2,4,5,27]. In contrast to school where authority and rules are strict, at home SCD patients may feel free to

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express their feelings and behavioral characteristics. Parents’ fears about their children with SCD may also lead to a parenting style characterized by low-level authority at home and unresponsiveness to problematic behaviors.

Taken together, the excessive control and/or suppressed behavioral problems of children and adolescents with SCD at school may be more apparent at home.

In the present study the SCD patients had higher T-DSM-IV-S total, inattentiveness subscale, and hyperactivity/impulsivity subscale scores. CTRS-R and CBCL/4-18 attention-deficit subscale scores in the SCD group were also higher than in the control group. The present findings are in accordance with those of previous studies that reported children with SCD have an increased risk of concentration problems [8,20,28]— especially sustained attention [8]. Although not definitively proven, deficits in attention are often related to the presence and severity of cerebral injury [28-30]. Frontal lobe abnormalities, especially anterior infarcts, appear to play a major role in attention deficit [8,31]. Although there is an available literature on attention deficit in SCD, to the best of our knowledge the present study is the first to link SCD with ADHD. It must be noted that the parent-rated CPRS used in the present study does not indicate a clinical diagnosis of ADHD, which must be based on a structured clinical interview; however, the possible comorbidity of ADHD in SCD may not be considered surprising. Available data show that the 2 conditions share some neurocognitive features. Attention deficit, executive functions, and working memory are the well-known basic cognitive components of ADHD [32], and have been shown to be impaired in children with SCD [8,33,34].

As the present study used a cross-sectional design, the direct causal relationship of the variables could not be evaluated. The high frequency of psychiatric problems in the SCD group may have, at least partly, been due to some endogenous factors, including personality characteristics.

Investigation of personality characteristics in SCD and control groups in a prospective study might yield interesting findings. In addition, whether a high frequency of psychiatric problems is specific to SCD or is also valid for other hemoglobinopathies remains unknown. Multicenter studies with larger patient groups, including children with other hemoglobinopathies, are necessary to more fully elucidate the relationship between SCD and psychiatric problems in children and adolescents.

The present study has several limitations, including a small sample size, use of subjective questionnaires for data collection, and lack of a structured psychiatric interview.

Özalp E, et al: Psychiatric Problems in Children with Sickle Cell Disease

In addition, the prevalence of silent cerebral infarcts was not examined in the present study, which is also considered a limitation. As such, the extent to which the presence of cerebral infarcts was responsible for the frequency and types of psychiatric problems observed in the present study is unknown. In contrast, the major strength of the present study is its use of both parent- and teacher-rated data. Previously, most relevant studies used only parent-rated data.

As school is a major domain in a child’s life, teachers’ objective information may be considered valuable.

Conclusion Children and adolescents with SCD appear to be at increased risk for psychiatric problems. According to the present findings, internalizing symptoms and attention problems were the most common psychiatric symptoms in the SCD patients. Thus, early identification and frequent monitoring of psychiatric problems in children with SCD is critical. Regular psychological evaluation and referral of at-risk children and adolescents to child and adolescent psychiatry clinics may facilitate timely diagnosis and effective treatment.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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21. Benton TD, Boyd R, Ifeagwu J, Feldtmose E, Smith-Whitley K. Psychiatric diagnosis in adolescents with sickle cell disease: a preliminary report. Curr Psychiatry Rep 2011; 13: 111-115 22. Alao AO, Dewan MJ, Jindal S, Effron M. Psychopathology in sickle cell disease. West Afr J Med 2003; 22: 334-337 23. Tunde-Ayinmode MF, Adelekan ML. Psychosocial impact of sickle cell disease in children seen at University of Ilorin Teaching Hospital, Ilorin, Nigeria. East Afr Med J 2005; 82: 73-78 24. Barlow JH, Ellard DR. The psychosocial well-being of children with chronic disease, their parents and siblings: An overview of the research evidence base. Child Care Health Dev 2006; 32: 19-31 25. Dale JC, Cochran CJ, Roy L, Jernigan E, Buchanan GR. Health-related quality of life in children and adolescents with sickle cell disease. J Pediatr Health Care 2011; 25: 208-215 26. Schatz J. Brief report: Academic attainment in children with sickle cell disease. J Pediatr Psychol 2004; 29: 627-633 27. Devine D, Brown RT, Lambert R, Donegan JE, Eckman J. Predictors of psychosocial and cognitive adaptation in children with sickle cell syndromes. Journal of Clinical Psychology in Medical Settings 1998; 5: 295-313 28. Nabors NA, Freymuth AK. Attention deficits in children with sickle cell disease. Percept Mot Skills 2002; 95: 57-67 29. Schatz J, Brown RT, Pascual JM, Hsu L, DeBaun MR. Poor school and cognitive functioning with silent cerebral infarcts and sickle cell disease. Neurology 2001; 56: 1109-1111 30. Brown RT, Davis PC, Lambert R, Hsu L, Hopkins K, Eckman J. Neurocognitive functioning and magnetic resonance imaging in children with sickle cell disease. J Pediatr Psychol 2000; 25: 503-513 31. Craft S, Schatz J, Glauser TA, Lee B, DeBaun MR. Neuropsychologic effects of stroke in children with sickle cell anemia. J Pediatr 1993; 123: 712-717 32. Barkley RA. Behavioral inhibition, sustained attention, and executive functions: constructing a unifying theory of ADHD. Psychol Bull 1997; 121: 65-94 33. Wang W, Enos L, Gallagher D, Thompson R, Guarini L, Vichinsky E, Wright E, Zimmerman R, Armstrong FD. Neuropsychologic performance in school-age children with sickle cell disease: A report from the Cooperative Study of Sickle Cell Disease. J Pediatr 2001; 139: 391-397 34. Brandling-Bennett EM, White DA, Armstrong MM, Christ SE, DeBaun M. Patterns of verbal long-term and working memory performance reveal deficits in strategic processing in children with frontal infarcts related to sickle cell disease.

Developmental Neuropsychology 2003; 24: 423-434

Case Report

10.5505/tjh.2012.62134

Familial Hemophagocytic Lymphohistiocytosis with A665G Perforin Gene Mutation: A Case Report A665G Perforin Gen Mutasyonu Olan Ailesel Hemofagositik Lenfohistiyositozis: Bir Olgu Sunumu İdil Yenicesu1, Geneviève De Saint Basile2, Hamdi Cihan Emeksiz1, Buket Dalgıç1 1 2

Gazi University, School of Medicine, Department of Pediatrics, Ankara, Turkey Hôpital Necker-Enfants Malades, Paris, France

Abstract Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disease. Presentation of the disease such as primarily fever, hepatosplenomegaly, and cytopenia, which are the results of functional degradation in cytotoxic T-lymphocytes and natural killer cells, activation of macrophages and T-lymphocytes, over production of proinflammatory cytokines, and hemophagocytosis. In all, 5 genetic loci have been identified in FHL, and all known affected genes encode critical components of the granule exocytosis pathway, which is essential for the release of cytotoxic granules and proteases that are necessary for targeted cell death. Herein we present an FHL patient with a severe clinical course and a very rare perforin gene mutation. The patient was homozygous for A665G mutation. However, the child died in a short period of time. Prenatal diagnosis was performed in the family and the fetus was found to be heterozygous for the mutation.

Key Words: Familial hemophagocytic lymphohistiocytosis, Perforin gene, A665G homozygous mutation, Prenatal diagnosis, Turkey

Özet Ailesel Hemofagositik Lenfohistiyositozis(AHL), genetik olarak oldukça heterojen bir hastalıktır. Sıklıkla ateş, hepatosplenomegali, sitopeni gibi bulgular görülebilir. Bu bulgular doğal katil hücreler ve sitotoksik T lenfositlerde fonksiyonel bozulma, makrofaj ve T lenfositlerde aktivasyon, proinflamatuar sitokinlerde artış ve hemofagositoz sonucu oluşur. AHL’de 5 ayrı genetik bölge tanımlanmıştır. Tüm bu bölgelerin ortak özelliği granul ekzositoz yolağı için kritik görev gören maddelerin yapımında rol oynamalarıdır. Bu makalede çok nadir perforin mutasyonuna sahip ve ağır klinik tabloyla erken dönemde kaybedilen bir olgu sunulmuştur. Hastada homozigot A665G mutasyonu bulundu. Hasta kısa sure sonra kaybedildi. Sonraki gebelikte prenatal tanı konuldu. Fetusda aynı mutasyonun heterozigotluğu gösterildi.

Anahtar Sözcükler: Ailesel hemofagositik lenfohistiyositozis, Perforin geni, A665G homozigot mutasyonu, Prenatal tanı, Türkiye

Address for Correspondence: İdil Yenicesu, M.D., Gazi Üniversitesi, Tıp Fakültesi, Pediatrik Hematoloji Bilim Dalı, Beşevler, 06500, Ankara, Turkey Phone: +90 312 202 50 09 E-mail: iyenicesu@gazi.edu.tr Received/Geliş tarihi : June 2, 2011 Accepted/Kabul tarihi : February 13, 2012

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Introduction Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disease. The clinical presentation of FHL includes primarily fever, hepatomegaly, and cytopenia, which are the results of functional degradation in cytotoxic T-lymphocytes and natural killer (NK)-cells, activation of macrophages and T-lymphocytes, excessive production of proinflammatory cytokines, and hemophagocytosis [1-3]. According to a study from Sweden, the incidence of FHL is 1.2 in 1,000,000 [4], but as intermarriage is common in Turkey the incidence of FLH in Turkey is probably higher. A study from Turkey reported that the frequency of primary hemophagocytic lymphohistiocytosis (HLH) is 7.5 in 10,000 among hospitalized patients [5].

Five loci causing FHL have been identified, as well as the underlying genetic defect for 4 of them. FHL1 is a locus first described in 1999 following linkage analysis in 2 Pakistani families, although the gene remains unknown.

FHL2 encodes mutations in the perforin (PFR1) gene.

Mutations in the UNC13D gene (FHL3) interfere with the role of the encoded protein Munc 13-4 in cytolytic granule exocytosis and FHL4 states the mutations in the STX11 gene and production of syntaxin 11, which also plays a role in cytotoxic granule release. Recently, mutations in STXB2, which encode syntaxin binding protein 2 (Munc 18-2) and cause impaired protein expression and impaired NK-cell cytotoxic granule exocytosis, were also described (FHL5) [6-12]. Perforin gene mutations are not uncommon in Turkey. Herein we present an FHL patient with a severe clinical course and a very rare homozygous perforin gene mutation.

Case Report A 3.5-month-old male infant presented with abdominal protuberance, fever, and cough, and was admitted to hospital. One week earlier he developed a fever with cough, was diagnosed as lower respiratory tract infection, and was given antibiotic treatment at another hospital.

The parents are first-degree cousins. The initial physical examination performed at our hospital showed the following signs: jaundice, grunting, intercostal retraction, and marked hepatosplenomegaly. No one else in the patient’s family had a similar history.

Complete blood count findings were as follows: white blood cell count: 4.7 x 109 L–1; absolute neutrophil count: 752 mm–3; hemoglobin: 4.8 g dL–1; platelet count: 15 x 109 L–1. Further laboratory test results include elevated transaminases (ALT: 4202 u L–1; AST: 1689 u L–1), elevated

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lactate dehydrogenase (1984 u L–1), and elevated serum ferritin (40,000 µg mL–1). Serum triglycerides was 207 mg mL–1, serum total bilirubin was 6.14 mg dL–1, serum direct bilirubin was 4.78 mg dL–1, serum uric acid was 30.9 mg dL–1, and fibrinogen was 50 mg dL–1. Arterial blood gas analysis was consistent with metabolic acidosis. Due to respiratory depression the patient was mechanically ventilated with multi-component transfusion support. He was given antibiotic treatment (teicoplanin, ceftriaxone, and fluconazole) during his hospitalization.

This patient underwent bone marrow aspiration, which showed profuse hemophagocytosis. The clinical and laboratory findings, and consanguineous parents strongly suggested FLH which lead to the commencement of the HLH 2004 protocol [13]. The patient’s elevated uric acid level and acidosis were treatment resistant, which led to the decision to perform peritoneal dialysis. Peritoneal dialysis does not have a clinically significant effect on cyclosporine, etoposide, or dexamethasone plasma clearance. As such, supplemental doses were not given during the dialysis procedure. Despite all treatment efforts, the patient died 30 h after admission due to multi-organ failure.

Postmortem splenic examination showed profuse hemophagocytosis. While alive the patient was in extremely poor clinical condition and was intubated; therefore, investigation of the central nervous system could not be performed, but postmortem cerebrospinal fluid examination showed mononuclear cell infiltration.

Written informed consent for abdominal autopsy was obtained from the patient’s family. The bone marrow core biopsy showed normal cellular components and abundant hemophagocytic macrophage infiltration based on CD68 immunohistochemical staining. Liver smear showed extramedullary hematopoiesis and microvesicular steatosis, as well as massive hemophagocytic macrophage infiltration via CD68, particularly in portal regions and in some sinusoidal regions. An increase in sinusoidal macrophages was noted in lymph nodes dissected from the peripancreatic region and mesocolon. Adrenal medulla showed massive CD68-positive macrophage infiltration. Focal histiocytic cellular groups were observed in kidney specimens.

Post-mortem genetic testing showed homozygosity for A665G PRF 1 mutation. The patient was the parents’ first child—the result of the mother’s first pregnancy. As the parents were consanguineous, the family was provided genetic counseling about subsequent pregnancies. One year later the mother was again pregnant and the fetus was determined to be a carrier for the same mutation; however, spontaneous abortion terminated the pregnancy.

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Discussion When the immune system is triggered by an infectious agent histiocytes (macrophages and dendritic cells), NKcells, and cytotoxic T-lymphocytes (CTLs) are activated, mutually stimulating each other via receptor interaction, as well as secretion of a variety of inflammatory cytokines and chemokines. In immune-competent individuals this concerted action leads to of the death of infected cells, removal of antigen, and termination of the immune response. In cases of inherited or acquired NK-cell and CTL defects this process is impaired, infected cells do not undergo apoptosis, and persistently high cytokine levels lead to the clinical picture of HLH [2].

The cytotoxic function of NK-cells and CTLs is mediated by release of cytolytic granules, which contain perforin, granzymes, and other serine-specific proteases, into the immunological synapse that forms between the effector and the target cell. Cytolytic granules must travel to the contact site, dock and fuse with the plasma membrane, and release their contents [2]. All known affected genes encode critical components of the granule exocytosis pathway, which is essential for the release of cytotoxic granules and proteases necessary for targeted cell death in HLH [1].

The most common mutations involve the perforin 1 gene, as in the presented case [6,14]. Perforin is expressed in the granules of T-cells and NK-cells, and creates pores in the cytoplasm of targeted cells, thereby allowing granzymes and proteases to enter cells and ultimately induce apoptosis after entering the cell nucleus [1]; however, the perforin gene mutation may not lead to the development of the clinical features of FHL until a patient comes into contact with viral infectious agents and environmental agents [12,15].

Lee et al. previously described a case where one allel has the same mutation as our case while the mutation of the other allel could not be found [16]. Recently, the same mutation in homozygous state was described in a family from Turkey [17]. To the best of our knowledge the presented case is the second homozygous case with this mutation in the current literature. Lee et al. showed that this mutation occurs in a region of the perforin protein that exhibits amphipathic conformation in order to interact with and traverse the membrane lipid bilayer, and exhibits the highest degree of homology to the putative lipid-binding domain of the complement components. The absence of NK-cell function in their patient suggests that mutation in this region interferes with the function of the perforin protein [16].

Yenicesu Ä°, et al: A665G Perforin Gene Mutation

In addition to the diagnosis of FHL, an understanding of its genetic or acquired nature is also of great value, especially in families with consanguineous marriage and a history of early onset of FHL in their offspring. The family of the presented case had the same characteristics. Prenatal diagnosis was of special importance to the presented caseâ&#x20AC;&#x2122;s family because he was their first child and they planned to have another baby. Currently, the most important problem is variation in FHL mutations according to countryâ&#x20AC;&#x201D; almost 50% of which have not yet been determined. This was reported to be as 50% in Turkey and 70% in Germany [13].

Due to the fact that FHL is quite common in Turkey, identification of the molecular pathologies in FHL patients is critical. Okur reported that 9 of 37 Turkish FHL patients (24%) were FHL type-2 based on linkage analysis. Genomic sequencing of the entire coding region of the perforin gene led to the identification of 5 different pathologic changes in these patients. In all, 3 patients (33%) carried the same nonsense W374X mutation in exon 3, and the 6 remaining patients (67%) had 4 different missense variations (G149S, V50M, A91V, and novel A523D).

Among these missense changes, G149S S was detected in 2 patients, V50M in 1 patient, and A91V in 2 patients [18]. In contrast, 2 earlier studies reported that 30% and 44% of Turkish FHL patients had perforin mutations, of which, 67% and 86%, respectively, had W374X mutation [10,14]. These 3 studies indicate that the incidence of perforin mutation is similar, ranging from 24%-44% and that W374X mutation is the most common type of perforin mutation in the Turkish population [10,14,18].

In the Turkish population perforin gene mutation is the most commonly detected mutation in the 4 genes known to be responsible for HLH. Among perforin gene mutations, W374X is known as the Turkish mutation and is common in Turkey; this mutation, which has a severe clinical outcome, is thought to have first emerged in Southeast Anatolia [19]. Clinical symptoms appear at birth and infants are usually symptomatic until 6 months of age.

The A665G (H222R) mutation observed in the presented case is rare and associated with a severe disease course. It may occasionally lead to hydrops fetalis or become symptomatic during the first months of life [19,20]. Risma et al. classified perforin gene mutations into 3 groups, based on flow cytometry, immunohistochemistry, and immunoblotting methods: class 1 are missense mutations with partial maturation of perforin; class 2 are missense mutations with no apparent proteolytic maturation of perforin; class 3 are missense mutations with no recognizable forms

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of perforin. Among these 3 classes, class 3 missense mutations are associated with the most severe disease course [21]. The A665G mutation in the presented case was in class 3 and was associated with severe clinical manifestations, resulting in mortality at age 3 months.

The aim of this case presentation was to increase pediatricians’ awareness of HLH. HLH is a life-threatening disease that may be difficult to distinguish from severe sepsis.

A simple clinical approach may be helpful in diagnosing HLH. As such, autopsy studies suggest that HLH may be under recognized in intensive care unit patients [22-24].

Several diagnostic criteria protocols have recently been developed for HLH. The most current (HLH-2004 protocol) suggests that HLH can be diagnosed if 5 of 8 diagnostic criteria are met [13]. In recent years elevated levels of ferritin have become more important in diagnosis, especially levels >10,000 mg dL–1 [25]. The presented case had an extremely high ferritin level (40,000 mg dL–1). HLH should be ruled out in any previously healthy child that abruptly develops fever, hepatosplenomegaly, cytopenia, and jaundice.

Chemo-immunotherapy based on the HLH-2004 protocol includes corticosteroids, epipodophyllotoxins, and cyclosporine, and succeeds in controlling the disease in the majority of patients; however, cure is rarely achieved.

Most patients suffer an early death unless they undergo hematopoietic stem cell transplantation (HSCT), which is the only curative approach available to date [13]. In conclusion, mutations like V50M in HLH patients become symptomatic later in life [18] and are associated with a slow clinical course, whereas mutations like W374X and A665G are associated with early onset of clinical symptoms, a rapid and severe disease course, and mortality shortly afterward due to multiple organ failure, as in the presented case. As such, the clinical outcome in HLH patients often depends on how rapidly and precisely diagnosis is established. In the near future it is expected that other candidate genes will be identified that will facilitate the establishment of appropriate individualized therapies, including chemotherapy, immunotherapy, HSCT, and perhaps gene therapy. Until that time, as practicing clinicians our task is to acquire sufficient knowledge for timely diagnosis and treatment of HLH. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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References 1. Verbsky JW, Grossman WJ. Hemophagocytic lymphohistiocytosis: Diagnosis, pathophysiology, treatment and future perspectives. Ann Med 2006; 38: 20-31 2. Janka GE. Familial and acquired hemophagocytic lymphohistiocytosis. Eur J Pediatr 2007; 166: 95-109 3. Janka GE. Hemophagocytic syndromes. Blood Reviews 2007; 21: 245-253 4. Henter JI, Elinder G, Soder O, Ost A. Incidence in Sweden and clinical features of familial hemophagocytic lymphohistiocytosis. Acta Paediatr Scan 1991; 80: 428-435 5. Gürgey A, Göğüş S, Ozyürek E, Aslan D, Gümrük F, Cetin M, Yüce A, Ceyhan M, Seçmeer G, Yetgin S, Hiçsönmez G. Primary hemophagocytic lymphohistiocytosis in Turkish children. Pediatr Hematol Oncol 2003; 20: 367–371 6. Stepp SE, Dufourcq-Lagelouse R, Le Deist F, Bhawan S, Certain S, Mathew PA, Henter JI, Bennett M, Fischer A, de Saint Basile G, Kumar V. Perforin gene defects in familial hemophagocytic lymphohistiocytosis. Science 1999; 286: 1957-1959 7. Ohadi M, Lalloz MR, Sham P, Zhao J, Dearlove AM, Shiach C, Kinsey S, Rhodes M, Layton DM. Localization of a gene for familial hemophagocytic lymphohistiocytosis at chromosome 9q21.3-22 by homozygosity mapping. Am J Hum Genet 1999; 64: 165-171 8. Feldmann J, Callebaut I, Rapuoso G, Certain S, Bacq D, Dumont C, Lambert N, Ouachee-Chardin M, Chedeville G, Tamary H, Minard-Colin V, Vilmer E, Blanche S, Le Deist F, Fischer A, de Saint Basile G. Munc 13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis(FLH3). Cell 2003; 115: 461-473 9. Zur Stadt U, Schmidt S, Kasper B, Beutel K, Diler AS, Henter JI, Kabisch H, Schneppenheim R, Nurnberg P, Janka G, Hennies HC. Linkage of familial hemophagocytic lymphohistiocytosis (FHL) type-4 to chromosome 6q24 and identification of mutations in syntaxin 11. Hum Mol Genet 2005; 14: 827-834 10. Zur Stadt U, Beutel K, Kolberg S, Schneppenheim R, Kabisch H, Janka G, Hennies HC. Mutation spectrum in children with primary hemophagocytic lymphohistiocytosis: Molecular and functional analyses of PRF1, UNC13D, STX11 and RAB27A. Hum Mutat 2006; 27: 62-68 11. Côte M, Ménager MM, Burgess A, Mahlaoui N, Picard C, Schaffner C, Al-Manjomi F, Al-Harbi M, Alangari A, Le Deist F, Gennery AR, Prince N, Cariou A, Nitschke P, Blank U, El-Ghazali G, Ménasché G, Latour S, Fischer A, de Saint Basile G. Munc18-2 deficiency causes familial hemophagocytic lymphohistiocytosis type 5 and impairs cytotoxic granule exocytosis in patient NK cells. J Clin Invest 2009; 119: 3765-3773

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12. Freeman HR, Ramanan AV. Review of haemophagocytic lymphohistiocytosis. Arch Dis Child 2011; 96: 688-693 13. Henter JI, Horne AC, Arico M, Egeler RM, Filipovich AH, Imashuku S, Ladish S, McClain K, Webb D, Winiarski J, Janka G. HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007; 48: 124-131 14. Göransdotter E, Fadeel B, Nilsson-Ardnor S, Söderhäll C, Samuelsson A, Janka G, Schneider M, Gurgey A, Yalman N, Révész T, Egeler R, Jahnukainen K, Storm-Mathiesen I, Haraldsson A, Poole J, de Saint Basile G, Nordenskjöld M, Henter J. Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis. Am J Hum Genet 2001; 68: 590-597 15. Albayrak M, Kaya Z, Yılmaz-Keskin E, Stadt UZ, Koçak U, Gürsel T. Fatal Epstein-Barr virus infection in a case familial hemophagocytic lymphohistiocytosis with syntaxin-11 mutation. Turk J Pediatr 2009; 51 (4): 371–374 16. Lee SM, Villanueva J, Sumegi J, Zhang K, Kogawa K, Davis J, Filipovich AH. Characterisation of diverse PRF1 mutations leading to decreased natural killer cell activity in North American families with haemophagocytic lymphohistiocytosis. J Med Genet 2004; 41: 137-144 17. Aslan D. A665G mutation in PRF1 in a Turkish infant with familial hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2011; 56: 319–320 18. Okur H, Balta G, Akarsu N, Oner A, Patiroglu T, Bay A, Sayli T, Unal S, Gurgey A. Clinical and molecular aspects of Turkish familial hemophagocytic lymphohistiocytosis patients with perforin mutations. Leukemia Research 2008; 32: 972-975

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19. Balta G, Okur H, Unal S, Yarali N, Gunes AM, Unal S, Turker M, Guler E, Ertem M, Albayrak M, Patiroglu T, Gurgey A. Assessment of clinical and laboratory presentations of familial hemophagocytic lymphohistiocytosis patients with homozygous W374X mutation. Leuk Res 2010; 34: 1012– 1017 20. Lipton JM, Weastra S, Hawerty CE, Roberts D, Harris NL. Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 28-2004. Newborn twins with thrombocytopenia, coagulation defects and hepatosplenomegaly. N Eng J Med 2004; 351: 1120-1130 21. Risma KA, Frayer RW, Filibovich AH, Sumegi J. Aberant maturation of mutant Perforin underlies the clinical diversity of hemophagocytic lymphohistiocytosis. J Clin Invest 2006; 116: 182-192 22. Gauvin F, Toledano B, Champagne J, Lacroix J. Reactive hemophagocytic syndrome presenting as a component of multiple organ dysfunction syndrome. Crit Care Med 2000; 28: 3341-3345 23. Strauss R, Neureiter D, Westenburger B, Wehler M, Kirchner T, Hahn EG. Multifactorial risk analysis of bone marrow histiocytic hyperplasia with hemophagocytosis: A postmortem clinicopathologic analysis. Crit Care Med 2004; 32: 1316-1321 24. Nahum E, Ben-Ari J, Stain J, Schonfeld T. Hemophagocytic lymphohistiocytic syndrome: Unrecognized cause of multiple organ failure. Pediatr Crit Care Med 2000; 1: 51-54 25. Allen CE, Yu X, Kozinetz CA, Mc Clain KL. Highly elevated ferritin levels and the diagnosis of hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2008; 50 (6): 1227-1235

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Case Report

10.5505/tjh.2012.78553

Treatment of Priapism with Automated Red Cell Exchange and Hyperbaric Oxygen in an 11-year-old Patient with Sickle Cell Disease Orak Hücre Hastalığı olan 11 Yaşında Bir Çocukta Gelişen Priapizmin Otomatik Eritrosit Değişimi ve Hiperbarik Oksijen ile Tedavisi Fatih Mehmet Azık1, Avni Atay1, Ahmet Emin Kürekçi1, Hakan Ay2, Yusuf Kibar3, Okan Özcan1 Gülhane Military Medical Academy, Department of Pediatric Hematology, Ankara, Turkey Gülhane Military Medical Academy, Department of Underwater and Hyperbaric Medicine, Ankara, Turkey 3 Gülhane Military Medical Academy, Department of Urology, Ankara, Turkey 1 2

Abstract Priapism affects up to 50% of all males with sickle cell disease, and there is no standard treatment. Delayed and unsuccessful treatment leads to corporal fibrosis and impotence. It is therefore necessary to determine the best treatment methods for this complication in order to offer effective interventions to all affected patients. Herein we report an 11-year-old patient with sickle cell disease that presented with priapism 72 h after onset, and was successfully treated with automated red cell exchange and hyperbaric oxygen following unsuccessful surgical and conventional interventions.

Key Words: Treatment of priapism, Automated red cell exchange, Hyperbaric oxygen therapy, Sickle cell disease

Özet Priapizm orak hücre hastalığı olan tüm erkeklerin yaklaşık yarısında ortaya çıkabilir ve tedavisinde henüz bir fikir birliği oluşmamıştır. Tedavinin başarısız olması ve gecikme korporal fibrozise ve impotansa neden olur. Priapizm gelişen hastalarda en etkili girişimsel yaklaşımı önerebilmek için bu komplikasyonun tedavisinin en iyi şekilde bilinmesine gereklilik vardır. Burada, orak hücre hastalığı olan,72 saattir süregelen priapizm komplikasyonu olan 11 yaşında bir çocuk hastanın cerrahi ve konvansiyonel girişimler yetersiz kaldıktan sonra, otomatik eritrosit değişimi ve hiperbarik oksijen ile başarılı tedavisini bildiriyoruz.

Anahtar Sözcükler: Priapizm tedavisi, Otomatik eritrosit değişimi, Hiperbarik oksijen tedavisi, Orak hücre hastalığı Introduction Priapism is the persistence of an erection in the absence of sexual desire that fails to subside despite orgasm. There are 2 types of priapism: low-flow ischemic (veno-occlusive priapism), which is the form seen in sickle cell disease (SCD), and high-flow priapism (non-ischemic), which is

associated with external trauma that damages the cavernosal artery. Sickle cell disease is one of the most common underlying diseases responsible from venous occlusion in low-flow ischemic priapism [1,2]. Priapism occurs in 30%-89% of males with SCD [3-5]. The prevalence of priapism in children with SCD was estimated to be 2%-6%

Address for Correspondence: Fatih Mehmet Azık, M.D., Gülhane Askeri Tıp Akademisi, Pediatrik Hematoloji Bilim Dalı, Ankara, Turkey Phone: +90 312 596 99 72 E-mail: mfatihazik@yahoo.com Received/Geliş tarihi : June 1, 2010 Accepted/Kabul tarihi : March 5, 2011

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[6,7]; however, the prevalence of priapism in children and adolescents with SCD is much higher than previously described [5]. Delayed and unsuccessful treatment leads to corporal fibrosis and impotence. Herein, we report an 11-year-old patient with SCD that presented with priapism 72 h after onset, and was successfully treated with automated red cell exchange and hyperbaric oxygen following unsuccessful surgical and conventional interventions.

Case Report An 11-year-old boy with SCD presented to the emergency department with a painful and persistent erection 72 h after onset. He was known to have SS hemoglobin and had been previously admitted to other hospitals for painful sickle cell crises on 3 occasions: once at the age of 4 years and twice when he was 5 years old. These 3 crises involved upper and lower extremity pain that resolved spontaneously. The diagnosis of homozygous SS hemoglobinopathy was established at the second admission when he presented extremity pain at the age of 5 years.. The patient’s history of recurrent stuttering episodes of priapism was negative. He first presented to another regional hospital with priapism and underwent corporal aspiration in the urology department, which was not successful, and was subsequently referred to our hospital.

Patient history of trauma, difficulty voiding, dysuria, and hematuria was negative. Causative factors (dehydration, infections, or the use of drugs) of priapism were not noted. Physical examination was unremarkable, except for a swollen, erect penis that was erythematous and very tender. Other symptoms of SCD, including veno-occlusive crisis, acute chest syndrome, acute splenic sequestration, sepsis, and aplastic crisis, were not associated with the priapism. Initial investigations confirmed sickle cell abnormality (92% SS homozygous), based on hemoglobin electrophoresis, and showed severe anemia (Hb: 5.7 g dL–1). The patient was treated with hydration, sedation, oxygenation, and transfusion of packed red blood cells.

As the patient’s priapism had persisted for 72 h, we immediately performed a corpora cavernosa-glans penis shunt. Despite the surgical intervention, detumescence did not occur. The patient was taken into a large, walk-in hyperbaric chamber at 2.5 ATA and 45 ft H2O for 90 min session–1. Hyperbaric oxygen treatment was initiated. (5 sessions/weekly; totally 11 sessions). On the fourth session of hyperbaric oxygen therapy, erythrocyte apheresis was performed automatically and erythrocyte apheresis was repeated on the fifth session of hyperbaric oxygen therapy.

Peripheral access was achieved using a 19-gauge needle.

Azık FM, et al: Treatment of Priapism in Sickle Cell Disease

The machine was primed with cross-matched compatible red blood cells. This achieved an Hb level of 10.6 g dL–1 with an Hb S fraction of 7.7%. There were no procedural complications. Upon examination after the first erythrocyte aphaeresis, improvement in the degree of erection and pain was evident, and priapism almost completely resolved after the second aphaeresis treatment. Hyperbaric oxygen treatment were applied 11 sessions. The patient’s priapism continued for 6 d before detumescence was complete. We did not observe any complications related to hyperbaric oxygen treatment in our patient. During a 4-year follow up period priapism did not reoccur.

Discussion It is thought that priapism is caused by sickle cells occluding the venous outflow of blood from the corpora of the penis. Episodes most commonly start during sleep or upon awakening, last about 2 h, and first occur during adolescence and pre-puberty. Some episodes resolve spontaneously within 2-3 h. Prolonged episodes can cause irreversible ischemic injury and lead to impotence. Symptoms of priapism are classified as acute (lasting >3 h), recurrent or stuttering (lasting ≤3 h and resolving spontaneously), and chronic, which is a rare condition in which the penis is persistently semi-erect, but not painful [8]. Priapism is usually accompanied by pain and tenderness.

The presented patient was an 11-year-old boy with SCD. Priapism can occur in all age groups, with reported peak incidences in SCD patients between the ages of 5 and 10 years, and 20 and 50 years [2]. Treatment for acute attacks includes hydration, sedation, oxygenation, aspiration, and irrigation with an α-adrenergic agonist and exchange transfusion [9-12]; these interventions may be helpful within the initial 10-12 h of onset [13]. Surgical treatments for priapism are designed to remove blood from the corpora cavernosa. Early intervention with corporeal aspiration and irrigation with adrenergic agonists was recently reported to be successful in an outpatient clinic [14]. Prior to presentation to our hospital, the presented patient underwent corporal aspiration that was unsuccessful, and the initial therapies; hydration, sedation, oxygenation was not applied.

At presentation to our hospital (72 h after the onset of symptoms) it was too late for conventional treatment approaches in the presented case. A corpora cavernosaglans penis shunt was performed immediately in addition to conventional management with hydration, sedation, and oxygenation, but the symptoms were not relieved. As reported, the success of surgical treatment is associated

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with its timing (<48 h of the onset of symptoms). Patients suffer physically and most progress to impotency, in part because of delayed treatment [15,16]. The precise mechanism of priapism in patients with SCD remains unclear. It is assumed that a normal erection decreases oxygen tension in the corpora cavernosa, predisposing to erythrocyte sickling [17]. With regard to pathophysiology, hyperbaric oxygen treatment has been successfully used for SCD complications, especially retinopathy, hyphema, and painful crisis [18-21]. To the best to our knowledge the literature does not contain any data on hyperbaric oxygen treatment for priapism in SCD patients.

In consideration of the presented patient’s serious condition, we initiated hyperbaric oxygen treatment. During this period automated red cell exchange was performed for 2 d and Hb S decreased to 7.7% from 92%, which is a desired level. The benefit of red cell exchange transfusion in SCD patients with priapism remains unclear [22].

McCarthy et al. reported that automated red cell exchange is not useful when initiated ≥1 d after the onset of priapism and when conservative management fails [8]. In contrast, we successfully performed automated red cell exchange 4 d after the onset of priapism, even though previous surgical and conventional treatments failed. Although hyperbaric oxygen appears to have no effect on the morphology of sickle cells in vitro [23], we think it was beneficial in the presented patient.

Hyperbaric oxygen therapy can result in arterial oxygen tension in the range of 1000-1600 mmHg and tissue oxygen tension close to 400 mmHg. Several indications for the use of hyperbaric oxygen therapy have been described [24], including carbon monoxide poisoning, necrotizing soft tissue infection, decompression sickness, and arterial gas embolism. On the other hand, there are some complications and side effects related to hyperbaric oxygen therapy [25]; pressure equalization problems in the middle ear, sinus squeeze, and tooth squeeze are common complications associated with hyperbaric oxygen therapy. No randomized or controlled studies on the complications of hyperbaric oxygen therapy in pediatric patients have been reported, but a few case reports have been reported. The complications associated with hyperbaric oxygen therapy are strongly correlated with the number of treatments.

Ambirou et al. reported a complication rate of 8.1% in patients that received 11-29 treatments, and that when the number of hyperbaric oxygen treatments was ≥30 the complication rate was 17.1% [26]. In the presented patient hyperbaric oxygen treatment was completed after 11 sessions, without any complications. Despite the use

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of combination treatments, low-flow priapism may result in impotence or erectile dysfunction secondary to corporeal fibrosis [15]. In conclusion, the results obtained in the present case suggest that automated red cell exchange and hyperbaric oxygen may be a good combination for the treatment of resistant priapism in SCD patients.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Muthall JP, Honig SC. Priapism: Etiology and Management.

Acad Emerg Med 1996: 3: 810-816 2. Lue TF. Physiology of penile erection and pathophysiology of erectile dysfunction and priapism. In: Walsh PC, Retik AB, Darracott Vaughan E Jr, Wein AJ editor(s). Campbell’s Urology. 7th Edition. Vol. 2, Philadelphia: W.B. Saunders, 1998: 1155–1180 3. Emond AM, Holman R, Hayes RJ, Serjeant GR. Priapism and impotence in homozygous sickle cell disease. Arch Intern Med 1980; 140: 1434-1437 4. Fowler JE Jr, Koshy M, Strub M, Chinn SK. Priapism associated with the sickle cell hemoglobinopathies: Prevalence, natural history and sequelae. J Urol 1991; 145: 65-68 5. Mandatakis E, Cavender JD, Rogers ZR, Ewalt DH, Buchanan GR. Prevalence of priapism in children and adolescents with sickle cell anemia. J Ped Hematol Oncol 1999; 21: 518-522 6. Hasen HB, Raines SL. Priapism associated with sickle cell disease. J Urol 1962; 88: 71-76 7. Tarry WF, Duckett JW, Synder HM 3rd. Urological complications of sickle cell disease in pediatric population.

J Urol 1987; 138: 592-594 8. McCarthy LJ, Vattuone J, Weidner J, Skipworth E, Fernandez C, Jackson L, Rothenberger S, Waxman D, Miraglia C, Porcu P, Danielson CF. Do Automated Red Cell Exchanges Relieve Priapism In Patients with Sickle Cell Anemia? Ther Apher 2000; 4: 256-258 9. Powars DR, Johnson CS. Priapism. Hematol Oncol Clin North Am 1996; 10: 1363-1372 10. Serjeant GR, Serjeant BE. Sickle cell disease. 3rd edition.

New York: Oxford University Press; 2001 11. Hamre MR, Harmon EP, Kirkpatrick DV, Stern MJ, Humbert JR. Priapism as a complication of sickle cell disease. J Urol 1991; 145: 1-5

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12. Serjeant GR, De Ceulaer K, Maude GH. Stilboestrol and stuttering priapism in homozygous sickle cell disease.

Lancet 1985; 2: 1274-1276 13. Dewan PA, Tan HL, Auldist AW, Moss DI. Priapism in childhood. Br J Urol 1989; 64: 541-545 14. Adeyoju AB, Olujohungbe AB, Morris J, Yardumian A, Bareford D, Akenova A, Akinyanju O, Cinkotai K, O’Reilly PH. Priapism in sickle-cell disease; incidence, risk factors and complications-an international multicentre study. BJU Int 2002; 90: 898–902 15. Chakrabarty A, Upadhyay J, Dhabuwala CB, Sarnaik S, Perlmutter AD, Connor JP. Priapism associated with sickle cell hemoglobinopathy in children: Long-term effects on potency. J Urol 1996; 155: 1419–1423 16. Maples BL, Hagemann TM. Treatment of priapism in pediatric patients with sickle cell disease. Am J Health Syst Pharm 2004; 61: 355–363 17. Francis RB, Johnson CS. Vascular occlusion in sickle cell disease. Current concepts and unanswered questions. Blood 1991; 77: 1405–1414 18. Wallyn CR, Jampol LM, Goldberg MF, Zanetti CL. The use of hyperbaric oxygen therapy in the treatment of sickle cell hyphema. Invest Ophthalmol Vis Sci 1985; 26: 1155-1158 19. Freilich DB, Seelenfreund MH. Further studies in the use of hyperbaric oxygen in retinal detachment with sickle cell anemia. Mod Probl Ophthalmol 1975; 15: 313-317

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20. Freilich DB, Seelenfreund MH. Long-term follow-up of scleral buckling procedures with sickle cell disease and retinal detachment treated with the use of hyperbaric oxygen. Mod Probl Ophthalmol 1977; 18: 368-372 21. Reynolds JD. Painful sickle cell crisis. Successful treatment with hyperbaric oxygen therapy. JAMA 1971; 216 (12): 1977-1978 22. Danielson CF. The Role of Red Blood Cell Exchange Transfusion in the Treatment and Prevention of Complications of Sickle Cell Disease. Ther Apher 2002; 6: 24-31 23. Mychaskiw G 2nd, Woodyard SA, Brunson CD, May WS, Eichhorn JH. In vitro effects of hyperbaric oxygen on sickle cell morphology. J Clin Anesth 2001; 13: 255-258 24. Moon RE, Feldmeier JJ. Hyperbaric oxygen: An evidence based approach to its application. Undersea Hyperb Med 2002; 29: 1-3 25. Jain KK. Indications, contraindications, and complications of HBO therapy. In: Jain KK, editor. Textbook of hyperbaric medicine. 4th revised and expanded edition. Cambridge: Hogrefe & Huber Publishers 2004: 73-78 26. Ambiru S, Furuyama N, Aono M, Otsuka H, Suzuki T, Miyazaki M. Analysis of risk factors associated with complications of hyperbaric oxygen therapy. J Crit Care 2008; 23: 295-300

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10.5152/tjh.2011.80

Primary Breast Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma Transformation to Diffuse Large B-cell Lymphoma: A Case Report Diffüz Büyük B Hücreli Lenfomaya Dönüşen Primer Meme Mukoza İlişkili Lenfoid Doku (MALT) Lenfoması: Bir Olgu Sunumu Şerife Hülya Arslan1, Ümmügül Üyetürk2, Emre Tekgündüz1, Sultan Çiğdem Irkkan3, Meltem Yüksel Kurt1, Itır Şirinoğlu Demiriz1, Fevzi Altuntaş1 Dr. Abdurrahman Yurtarslan Oncology Education and Research Hospital, Department of Hematology, Ankara, Turkey Dr. Abdurrahman Yurtarslan Oncology Education and Research Hospital, Department of Medical Oncology, Ankara, Turkey 3 Dr. Abdurrahman Yurtarslan Oncology Education and Research Hospital, Department of Pathology, Ankara, Turkey 1 2

Abstract Primary non-Hodgkin’s lymphoma (NHL) of the breast constitutes 0.04%-0.53% of all malignancies and 2.2% of extra nodal lymphomas. In total, 7%-8% of all B-cell lymphomas are the mucosa-associated lymphoid tissue (MALT) type, of which up to 50% of primary gastric MALT lymphoma. Herein we present a patient with breast MALT lymphoma that transformed to diffuse large B-cell lymphoma (DLBCL). A 69-year-old female presented with a mass on her left breast. Physical examination showed a 3 × 3-cm mass located 1 cm from the areola on the upper lateral quadrant of the breast at the 1 o’clock position, which was fixed and firm. Excisional biopsy was performed and pathologic examination of the specimen showed MALT lymphoma transformation to DLBCL. The patient was staged as II-EA. The rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) protocol was scheduled as treatment. Following 6 courses of R-CHOP, 2 additional courses of rituximab were administered. Positron emission tomography (PET)-CT was done at the end of the treatment. PET showed that the patient was in complete remission. At the time this report was written, the patient was being followed-up at the outpatient clinic on a regular basis. Lymphoma of the breast is a rarity among malignant tumors of the breast. The most common type of lymphoma is DLBCL. Breast MALT lymphoma is extremely rare. Primary MALT lymphoma of the breast can transform from low grade to high grade and recurrence is possible; therefore, such patients should be monitored carefully for transformation.

Key Words: Primary breast mucosa-associated lymphoid tissue (MALT) lymphoma, Diffuse large B-cell lymphoma

Özet Memenin primer non-Hodgkin lenfomaları (NHL), meme dokusu malignitelerinin %0.04-0.53’ünü; ekstranodal lenfomaların ise %2.2’sini oluşturmaktadır. B hücreli lenfomaların %7-8’ini mukoza ilişkili lenfoid doku (mucosaassociated lymphoid tissue-MALT) tipi lenfomalar, bunların da %50’den fazlasını primer gastrik MALT lenfomalar oluşturmaktadır. Burada memede nadir görülen MALT lenfoma zemininde diffüz büyük B hücreli lenfomaya (DBBHL) dönüşen bir olgunun sunulması amaçlanmıştır. Sol memede şişlik şikayeti ile başvuran 69 yaşındaki kadın hastanın

Address for Correspondence: Ümmügül Üyetürk, M.D., Dr. Abdurrahman Yurtarslan Onkoloji Eğitim ve Araştırma Hastanesi, 2. Tıbbi Onkoloji Kliniği, Demetevler Ankara, Turkey Phone: +90 312 336 09 09 E-mail: ummuguluyeturk@yahoo.com.tr Received/Geliş tarihi : August 17, 2010 Accepted/Kabul tarihi : November 26, 2010

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Arslan ŞH, et al: Primary Breast Lymphoma

yapılan muayenesinde, sol meme üst dış kadranda saat 1 hizasında, areoladan 1 cm uzaklıkta 3x3 cm boyutlarında sert kitle tespit edildi. Yapılan eksizyonel biyopsisinin patolojik incelemesinde DBBHL’ya progresyon gösteren MALT lenfoma tanısı konuldu. Hasta evre II EA olarak evrelendi. Hastaya rituksimab, siklofosfamid, doksorubisin, vinkristin, prednizolon tedavisi planlandı (R-CHOP). Hastaya 6 kür R-CHOP ve sonrasında 2 kür R verildi. Yanıt değerlendirmesi için çekilen pozitron emisyon tomografisi (PET)-BT ile tam remisyon sağlandığı görüldü. Halen hastanın takibine devam edilmektedir. Memenin malign tümörleri içerisinde meme lenfomaları nadir görülmektedir. Meme lenfoma olgularının en sık görülen alt tipi DBBHL’dır. Memede MALT lenfoma ise çok daha nadir görülmektedir. Memenin primer MALT lenfomasının düşük dereceli lenfomadan, yüksek dereceliye dönüşüm ve nüks olasılığı nedeniyle dikkatli takibi gerekmektedir.

Anahtar Sözcükler: Primer meme mukoza ilişkili lenfoid doku (MALT) lenfoma, Diffüz büyük B hücreli lenfoma Introduction Primary, non-epithelial breast tumors constitute 5% of all malignancies originating from breast, and must be included in the differential diagnosis of breast masses. In patients with lymphoma of the breast secondary breast involvement occurs often, although primary breast lymphoma is extremely rare [1]. Primary non-Hodgkin’s lymphoma (NHL) of the breast constitutes 0.04%-0.53% of all malignancies and 2.2% of extranodal lymphomas [2].

Lymphocytes in breast tissue are located close to the axillary region, upper lateral quadrant, in lymph nodes, and in the lymphatic ductus. These lymphoid aggregations are the major cause of lymphoid neoplasia [3]. Informed consent was obtained.

In all, 7%-8% of B-cell lymphomas are the mucosaassociated lymphoid tissue (MALT) type, of which up to 50% of primary gastric MALT lymphoma. Furthermore, during the course of autoimmune diseases MALT lymphomas often involve such tissues as skin, ocular adnexal, lung, salivary gland, thyroid, and breast [4]. Herein we present a case with primary breast MALT lymphoma that transformed to diffuse large B-cell lymphoma (DLBCL).

Figure 1: Small lymphoid cells infiltrating along the LCA and breast ducts, forming aggregates around the ducts (400x).

Case A 69-year-old female presented with a mass on her left breast, which was first noticed approximately 3 months earlier. Physical examination showed a 3 x 3-cm mass located 1 cm from the areola on the upper lateral quadrant of the breast at the 1 o’clock position, which was fixed and firm. The patient did not have sweats, weight loss, or fever. Her medical history was unremarkable. Complete blood count was normal, biochemistry was normal, except for LDH of 274 U L-1 (normal range: 0-200 U L-1), and the erythrocyte sedimentation rate (ESR) was 56 mm h-1.

Excisional biopsy was performed and pathologic examination of the specimen showed a lymphoepithelial lesion rich in plasma cells, centroblasts, and immunoblast-like

Figure 2: Diffuse infiltration pattern of the tumor removed from the breast tissue. Frequent mitosis and apoptosis can be seen inside the diffuse large B-cell lymphoma region, in which large centroblastic lymphoid cells are observed (HE, 400x).

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cells. These regions stained diffusely with LCA (Figure 1), CD20, and CD79a, and focally with bcl2. An extensive invasion pattern had destroyed the breast tissue; frequent mitosis and apoptosis were observed. Large centroblastictype and polymorphic lymphoid cells were interpreted as MALT lymphoma transforming to DLBCL (Figure 2).

Analysis of a bone marrow biopsy specimen was negative for lymphoma infiltration. Cervical and abdominal computed tomography (CT) showed no involvement.

Thoracic CT showed a 15-mm lymphadenopathy in the left axillary zone. The patient was staged as II-EA. The rituximab (375 mg m-²) cyclophosphamide (750 mg m-²), doxorubicin (50 mg m-²), vincristine (1.4 mg m-² [maximum: 2 mg]), methylprednisolone (80 mg d-1) (R-CHOP) protocol was scheduled as the treatment. After 6 courses of R-CHOP, 2 additional courses of rituximab were administered. Positron emission tomography (PET-CT) was done at the end of the treatment. PET showed that the patient was in complete remission.

Discussion Primary breast lymphomas present in elderly woman as painless, unilateral masses. Most cases are consistent with B-cell NHL. The most common subtypes are DLBCL and marginal zone lymphoma [5]. Prognosis depends on histological subtype and stage. Stage I disease is treated with involved-field radiation only. Marginal zone lymphomas rarely transform to the aggressive DLBCL [6,7].

Median age at the time primary breast lymphoma is diagnosed is 60 years, although high-grade lymphomas are seen in patients younger than 60 years [8]. Diagnostic criteria include involvement of breast and lymphoid tissue in the tumor, negative history of extra-mammary lymphoma, absence of extensive lymphoma involvement, and the breast as the primary region of involvement [9].

The presented case had simultaneous pathological breast and lymphoid tissue involvement. Therefore the primary focus of lymphoma is a matter of speculation. Left axillary lymph node involvement was interpreted as adjacent lymph node extension, as the mass was located within the left breast.

Fine needle aspiration is often unsuccessful in diagnosing such cases; therefore, as in the presented case, excisional biopsy should be performed [10]. Histologically, MALT lymphomas invade epithelial tissues and exhibit proliferation of neoplastic marginal zone cells that form the characteristic lymphoepithelial lesions [11]. MALT lymphomas are low-grade lymphomas and rarely progress

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to high-grade, which occurs in only 10% of cases and is related to such genetic abnormalities as p16INK and p53 activation [11].

Primary breast MALT lymphomas generally tend to be limited; therefore, mastectomy is not recommended, except in cases with bulky disease and aggressive histopathology; breast-conserving surgery is the preferred treatment method. Although the present case had stage II disease, transformation to aggressive lymphoma indicated that systemic treatment was the best option, which resulted in complete remission and complication-free follow-up.

Differential diagnosis of breast masses includes lymphomas, although they are extremely rare. Primary MALT lymphoma of the breast can transform from low grade to high grade, and careful patient monitoring is necessary because of the risk of recurrence.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Berg JW, Hutter RV. Breast cancer. Cancer 1995; 75 (1 Suppl): 257-269 2. Rajendran RR, Palazzo JP, Schwartz GF, Glick JH, Solin LJ. Primary mucosa-associated lymphoid tissue lymphoma of the breast. J Breast Cancer 2008; 8 (2): 187-188 3. Dao AH, Adkins RB Jr, Glick AD. Malignant lymphoma of the breast: A review of 13 cases. Am Surg 1992; 58: 792796 4. Bea S, Zettl A, Wright G, Salaverria I, Jehn P, Moreno V, Burek C, Ott G, Puig X, Yang L, Lopez-Guillermo A, Chan WC, Greiner TC, Weisenburger DD, Armitage JO, Gascoyne RD, Connors JM, Grogan TM, Braziel R, Fisher RI, Smeland EB, Kvaloy S, Holte H, Delabie J, Simon R, Powell J, Wilson WH, Jaffe ES, Montserrat E, Muller-Hermelink HK, Staudt LM, Campo E, Rosenwald A. Diffuse large B-cell lymphoma subgroups have distinct genetic profiles that influence tumor biology and improve gene-expression-based survival prediction. Blood 2005; 106: 3183-3190 5. Brogi E, Haris NL. Lymphomas of the breast: Pathology and clinical behavior. Semin Oncol 1999; 26 (3): 357-364 6. Pinheiro RF, Colleoni GW, Baiocchi OC, Kerbauy FR, Duarte LC, Bordin JO. Primary Breast Lymphoma: An uncommon but curable disease. Leuk Lymphoma 2003; 44 (1): 149-151

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7. Ezzat H, Filipenko D, Vickars L, Galbraith P, Li C, Murphy K, Montaner JS, Harris M, Hogg RS, Vercauteren S, Leger CS, Zypchen L, Leitch HA. Improved survival in HIVassociated diffuse large B-cell lymphoma with the addition of rituximab to chemotherapy in patients receiving highly active antiretroviral therapy. HIV Clin Trials 2007; 8: 132144 8. Briggs JH, Algan, Stea B. Primary T-Cell lymphoma of the breast: A case report. Cancer Invest 2003; 21: 68-72

Arslan Ĺ&#x17E;H, et al: Primary Breast Lymphoma

9. Wiseman C, Liao KT. Primary lymphoma of the breast. Cancer 1972; 29: 1705-1712 10. Burke JS. Are there site-specific differences among the MALT lymphomas morphologic, clinical. Am J Clin Pathol 1999; 111: 133-143 11. Thieblemont C, Berger F, Dumontet C, Moullet I, Bouafia F, Felman P, Salles G, Coiffier B. Mucosa-associated lymphoid tissue lymphoma is disseminated disease in one third of 158 patients analyzed. Blood 2000; 95 (3): 802-806

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10.5505/tjh.2012.94809

Myeloid Sarcoma: An Unusual Presentation of Acute Promyelocytic Leukemia Causing Spinal Cord Compression Miyeloid Sarkom: Akut Promyelositik Löseminin Spinal Kord Kompresyonuna Neden Olan Olağandışı Bir Klinik Tablosu Tay Za Kyaw1, Jayaranee A.S. Maniam1, Ping Chong Bee2, Edmund Fui Min Chin2, Veera Sekaran Nadarajan1, Hemalatha Shanmugam1, Khairul Azmi Abd Kadir3 University of Malaya, Faculty of Medicine, Department of Pathology, Kuala Lumpur, Malaysia University of Malaya, Faculty of Medicine, Department of Medicine, Kuala Lumpur, Malaysia 3 University of Malaya, Faculty of Medicine, Department of Radiology, Kuala Lumpur, Malaysia 1 2

Abstract Acute promyelocytic leukemia with concurrent myeloid sarcoma is a rare clinical event. Herein we describe a patient that presented with back pain and bilateral leg weakness caused by spinal cord compression due to extramedullary deposition of leukemic cells. Acute promyelocytic leukemia was suspected based on immunophenotypic findings of malignant cells in bone marrow aspirate. The diagnosis was confirmed by the presence of PML-RARα fusion copies.

MRI showed multiple hyperintense changes on the vertebral bodies, together with intraspinal masses causing spinal cord compression. The patient immediately underwent radiotherapy, and was treated with all-trans retinoic acid and idarubicin. Reassessment MRI showed complete resolution of all intraspinal masses and the disappearance of most of the bony lesions. Post-treatment bone marrow aspirate showed complete hematological and molecular remission. The motor power of his legs fully recovered from 0/5 to 5/5; however, sensory loss below the T4 level persisted.

Key Words: Myeloid sarcoma, Promyelocytic leukemia, Cord compression, Chloroma

Özet Eş zamanlı miyeloid sarkomlu akut promiyelositik lösemi nadir bir klinik olaydır. Burada ekstramedüller lösemik hücre birikimi nedeniyle spinal kord kompresyonu sonucunda bel ağrısı ve bilateral bacak zayıflığı ile gelen bir hastayı tanımlıyoruz Kemik iliği aspiratında malign hücreler immünofenotipik bulguları temelinde akut promiyelositik lösemiden şüphelenildi. Tanı PML-RARα füzyon kopyalarının varlığıyla doğrulandı. MRG, vertebral cisimlerde çok sayıda hiperintens değişiklik ve beraberinde spinal kord kompresyonuna neden olan intraspinal kitleler gösterdi.

Hastaya hemen radyoterapi başlandı ve all-trans retinoik asit ve idarubisin ile tedavi edildi. Tekrar değerlendirmede MRG tüm intraspinal kitlelerin tam olarak geçtiğini ve kemik lezyonlarının çoğunun kaybolduğunu gösterdi. Tedavi sonrası kemik iliği aspiratı tam hematolojik ve moleküler remisyon gösterdi. Bacaklarda motor güç 0/5’ten 5/5’e tam olarak iyileşti ancak T4 seviyesi altındaki duyu kaybı devam etti.

Anahtar Sözcükler: Miyeloid sarkom, Promiyelositik lösemi, Kord kompresyonu, Kloroma Address for Correspondence: Tay Za Kyaw, MPath, Department of Pathology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia Phone: +60 3 79492375 E-mail: kyawtayza79@gmail.com Received/Geliş tarihi : April 12, 2011 Accepted/Kabul tarihi : August 22, 2011

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Introduction Myeloid sarcoma (MS) is a rare extramedullary tumor consisting of immature myeloid cells [1]. MS may develop de novo or concurrently with acute myeloid leukemia (AML), myeloproliferative disorders, or myelodysplastic syndrome. It may proceed or coincide with the occurrence of AML or may present as the initial manifestation of relapse in a previously treated AML patient in remission [2]. MS may involve any organ system, from the more common involvement of the skin, bone, soft tissue of the head and neck (frequently the orbits) and lymph nodes, to rare cases involving the heart or small intestine [3].

The occurrence of MS in an acute promyelocytic leukemia (APL) patient is a rare clinical event. Herein we report a patient with APL that presented with spinal cord compression due to underlying MS. Case Presentation Written informed consent was obtained from the patient. A 26-year-old male was referred to our hospital due to suspected AML in August 2010. He presented with progressive back pain and bilateral leg weakness, and was unable to walk. Initial neurological examination showed

Kyaw TZ, et al: Promyelocytic Sarcoma With Cord Compression

paraparesis with a power of 0/5 in both lower limbs, presence of the Babinski sign, and loss of pain and sensory perception below the T4 level. There were no other abnormal findings on physical examination. Urgent MRI of the spine showed multiple hyperintense bony lesions on the T12, L1, L2, L4, and L5 vertebral bodies, and sacrum.

Intraspinal masses located extradurally were also observed between the T2 and T4, and between the T12 and L2 vertebral levels. Theses masses caused anterior displacement and compression of the spinal cord (Figure 1A and B).

The patient was immediately referred to a spinal surgeon due to cord compression caused by the intraspinal masses.

Radiotherapy was administered immediately at a dose of 20 Gy to 2 regions (T1-T7 and T12-L2) for 5 d.

Complete blood count upon admission were as follows: hemoglobin: 10.7 g dL-1; white blood cell (WBC) count: 2.8 x 109 L-1; platelet count: 102 x 109 L-1. Occasional blasts were noted in the peripheral blood smear.

There wasnâ&#x20AC;&#x2122;t significant abnormality in the coagulation profile. Cerebrospinal fluid cytology was negative. No conclusive finding was obtained on morphological examination of the bone marrow aspirate, because almost all of the nucleated cells were ruptured. Repeat bone mar-

Figure 1: T2 weighted MRI of the patientâ&#x20AC;&#x2122;s spine. (A+B): Images taken at presentation showing an intraspinal mass (white arrows) located extradurally between T2 and T4 vertebral bodies with multiple hyperintense bony lesions (black arrows) on T12, L1, L2, L4 and L5 vertebral bodies. (C+D): Images taken 1 month after treatment showing no evidence of intraspinal mass but the bony lesions on thoracic and lumbar vertebral bodies were persistent (black arrows). (E): Image taken after the third consolidation therapy showing a few residual bony lesions on L1 and L2 vertebral bodies (black arrows).

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row aspirate analysis showed similar findings. Viability of cell suspension in the flow cytometric examination of bone marrow aspirate was 96.6%; this was assessed using 7-amino-actinomycin-D DNA binding dye. Flow cytometric immunophenotyping of bone marrow leukemic cells was suggestive of APL (CD45+, CD13+, CD33+, cytoplasmic MPO+, CD117+, CD64+, CD34-, HLADR-, CD14-, CD11b-, CD19-, CD22-, CD10-, cytoplasmic CD79a-, and cytoplasmic CD3-) (Figure 2). Reverse transcriptasepolymerase chain reaction of the bone marrow sample showed BCR1-type PML-RARα fusion copies, confirming the diagnosis of APL. Bone marrow biopsy also showed

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diffuse infiltration of the marrow tissue by neoplastic cells (Figure 3). Immunohistochemical staining showed that these infiltrating cells were reactive for CD45, CD117, and MPO. T-cell and B-cell markers (CD3 and CD20), as well as CD34 and HLA-DR were negative (Figure 3).

Induction therapy consisting of all-trans retinoic acid (ATRA) 45 mg·m-2·d-1 for 8 weeks, and intravenous idarubicin 12 mg·m-2·d-1 on d 1, d 3, and d 5 was initiated following molecular confirmation of APL. The treatment course was uneventful. Clinically, motor power of the patient’s lower limbs gradually improved and was restored to 5/5; however, sensory deficits persisted. Repeat MRI of

Figure 2: Flow cytometric immunophenotyping of the bone marrow aspirate showing leukemic cells which are CD45 (+), CD13 (+), CD117 (+), cytoplasmic MPO (+), CD34 (-), HLA-DR (-), CD22 (-) and cytoplasmic CD3 (-).

Figure 3: Diffuse infiltration of neoplastic cells in the bone marrow biopsy (H&E, x40). Immunohistochemical analysis of the infiltrating neoplastic cells showed positive reaction with CD117 (CD117 x40) and negative reaction with CD34 (CD34 x40) and HLA-DR (HLA-DR x40).

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the spine 1 month after completion of radiotherapy showed normal signal intensity of the spinal cord and no evidence of the intraspinal masses, but the bony lesions remained (Figure 1C and D). MRI 17 weeks after the completion of the third cycle of consolidation therapy showed that the bony lesions on the T12, L4, and L5 vertebral bodies, and part of the lesion on the L2 vertebral body disappeared (Figure 1E). No intraspinal masses or new lesions were detected. A biopsy of the residual bony lesions was not performed, as the patient did not consent to the procedure. Bone marrow examination and molecular analysis were performed 3 times—8 weeks after the commencement of ATRA therapy, and 6 weeks and 22 weeks after the third consolidation chemotherapy—and showed complete hematological and molecular remission with undetectable PML-RARα fusion copies. The patient completed 3 cycles of consolidation chemotherapy, and then intrathecal chemotherapy was given for central nervous system (CNS) prophylaxis. At the time this manuscript was prepared the patient was undergoing maintenance therapy.

Discussion MS was previously referred to as chloroma due to its characteristic greenish color caused by high myeloperoxidase content [4]. MS at diagnosis or relapse occurs in 3%-8% of AML patients—more frequently in those with myelomonocytic and monocytic morphology (M4 and M5 French-American-British subtypes) [5]. Cytogenetically, MS occurs in association with a variety of chromosomal abnormalities, including MLL gene rearrangement and t(8;21) [6]; the latter more often occurs in childhood and/ or is seen in lesions occurring in the orbit [7].

MS in most cases of APL occurs at the time of relapse [6]. In rare cases MS can precede or, as in the presented case, coincide with APL [5,6]. In patients with APL extramedullary disease most commonly occurs in the CNS and ≥10% of hematologic relapses are accompanied by CNS involvement [8]. The occurrence of extramedullary disease in patients with APL has increased since the introduction of ATRA, and 2 possible explanations for this have been considered. The first suggests that there is a direct effect of ATRA on adhesion molecules, resulting in increased infiltrative capability of leukemic cells [9]. The second theory is that the occurrence of MS in relapsed APL patients is a consequence of prolonged survival [10]. A large-scale study that included patients that did and did not receive ATRA therapy failed to demonstrate a correlation between the occurrence of extramedullary disease and ATRA therapy [10]. It has also been suggested that extramedullary tumors may be associated with a high WBC count (>10 x 109 L-1) at

Kyaw TZ, et al: Promyelocytic Sarcoma With Cord Compression

presentation, the presence of BCR3-type PML/RARα fusion transcripts, and microgranular morphology. de Bottom et al. reported that 8 of 10 APL patients with extramedullary relapse had elevated WBC counts at initial presentation, and BCR3-type PML/RARα fusion transcripts were present in 6 of the 8 patients [11]. Similar risk factors were not present in the presented patient, who had pancytopenia and BCR1-type PML/RARα fusion copies at presentation.

Patients with MS can present with various clinical manifestations, depending on the site of involvement. The presented patient presented with the features of spinal cord compression due to lesions in the spine, but tissue biopsy was not performed. Although the bony lesions were persistent in the first repeat MRI, there was complete resolution of the intraspinal masses. Significant improvement was noted in the second MRI, with the disappearance of most of the bony lesions. Post-treatment neurological examination showed complete recovery of the patient’s motor function. The initial MRI findings (concurrent leukemia together with evidence of significant clinical and radiological improvement following treatment), established the diagnosis of MS. The persistence of a few bony lesions in the patient might have been due to residual disease; however, the possibility of radiation-induced changes cannot be completely excluded in the absence of tissue biopsy.

Post-treatment bone marrow examination and molecular reassessment, which were performed at 3 time points, showed complete morphological and molecular remission.

In patients with MS the presence of concurrent AML or APL is easily diagnosed in typical cases in which blasts or abnormal promyelocytes are present in the peripheral blood smear or bone marrow aspirate. In the present patient the morphological findings were not conclusive for the diagnosis of leukemia, as almost all nucleated cells in the bone marrow smears were ruptured. It is likely that the fragile leukemic cells were damaged in the process of making the blood smears. The diagnosis of APL was suspected based on the findings of flow cytometric analysis of viable leukemic cells in the same bone marrow sample. Molecular assessment of the bone marrow aspirate confirmed the presence of PML-RARα transcripts.

MS and concurrent APL causing spinal cord compression is a rare clinical event, but must be considered in the differential diagnosis in patients that present with neurological symptoms and multiple spinal lesions. Treatment options for MS include surgical decompression, radiotherapy, chemotherapy, and any combination of these treatment methods [12]. The presented patient had significant improvement following local radiotherapy and systemic

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chemotherapy; however, the presence of residual disease could not be completely excluded, as tissue biopsy was not performed.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Neiman RS, Barcos M, Berard C, Bonner H, Mann R, Rydell RE, Bennett JM. Granulocytic sarcoma: Clinicopathologic study of 61 biopsied cases. Cancer 1981; 48: 1426-1437 2. Pileri SA, Ascani S, Cox MC, Campidelli C, Bacci F, Paccioli M, Piccaluga PP, Agostinelli C, Asioli S, Novero D, Bisceglia M, Ponzoni M, Gentile A, Rinaldi P, Franco V, Vincelli D, Pileri Jr A, Gasbarra R, Falini B, Zinzani PL, Baccarani M. Myeloid sarcoma. Clinico-pathologic, phenotypic and cytogenetic analysis of 92 adult patients. Leukemia 2007; 21: 340-350 3. Antic D, Verstovsek S, Elezovic I, Grujicic D, Gotic M, Bila J, Perunicic M, Jakovic L. Spinal epidural granulocytic sarcoma in non-leukemic patient. Int J Hematol 2009; 89: 95-97 4. Dâ&#x20AC;&#x2122;Alteroche L, Mor C, Durand V, De Muret A, Benbouker L, Colombat P, Danquechin Dorval E. Gastric granulocytic sarcoma revealed by a massive digestive hemorrhage.

Gastroenterol Clin Biol 1999; 23: 779-782 5. Pacilli L, Lo Coco F, Ramadan SM, GiannĂŹ L, Pingi A, Remotti D, Majolino I. Promyelocytic sarcoma of the spine: a case report and review of the literature. Adv Hematol 2010; 2010:137608

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6. Campidelli C, Agostinelli C, Stitson R, Pileri SA. Extramedullary Manifestation of Myeloid Disorders. Am J Clin Pathol 2009; 132: 426-437 7. Schwyzer R, Sherman GG, Cohn RJ, Poole JE, Willem P. Granulocytic Sarcoma in Children with Acute Myeloblastic Leukamia and t(8:21). Med Pediatr Oncol 1998; 31: 144-149 8. Evans GD, Grimwade DJ. Extramedullary disease in acute promyelocytic leukemia. Leuk Lymphoma 1999; 33 (3-4): 219-229 9. Ko BS, Tang JL, Chen YC, Yao M, Wang CH, Shen MC, Tien HF. Extramedullary relapse after all-trans retinoic acid treatment in acute promyelocytic leukemia-the occurrence of retinoic acid syndrome is a risk factor. Leukemia 1999; 13: 1406-1408 10. Specchia G, Lo Coco F, Vignetti M, Avvisati G, Fazi P, Albano F, Di Raimondo F, Martino B, Ferrara F, Selleri C, Liso V, Mandelli F. Extramedullary involvement at relapse in acute promyelocytic leukemia patients treated or not with ATRA: A report by the GIMEMA Group. J Clin Oncol 2001; 19: 4023-4028 11. de Botton S, Sanz MA, Chevret S, Dombret H, Martin G, Thomas X, Mediavilla JD, Recher C, Ades L, Quesnel B, Brault P, Fey M, Wandt H, Machover D, Guerci A, Maloisel F, Stoppa AM, Rayon C, Ribera JM, Chomienne C, Degos L, Fenaux P. European APL Group; PETHEMA Group: Extramedullary relapse in acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy.

Leukemia 2006; 20: 35-41 12. Kalayci M, Sumer M, Yenidunya S, Ozdolap S, Acikgoz B. Spinal granulocytic sarcoma (chloroma) presenting as acute cord compression in a nonleukaemic patient. Neurol India 2005; 53: 221-223

Case Report

10.5152/tjh.2011.17

Hodgkin’s Lymphoma with Leptomeningeal Involvement Leptomeningeal Tutulumlu Hodgkin Lenfoma Rahşan Yıldırım1, Mehmet Gündoğdu1, Fuat Erdem1, İlhami Kiki1, Betül Gündoğdu2 Atatürk University, School of Medicine, Department of Internal Medicine, Division of Hematology, Erzurum, Turkey Atatürk University, School of Medicine, Department of Pathology, Erzurum, Turkey

1 2

Abstract A 31-year-old male patient with Hodgkin’s lymphoma (HL) and leptomeningeal involvement presented to at Hematology Departments of Atatürk University . The patient developed dizziness, limb weakness, involuntary contractions of the hands and feet, incontinence, headache, fever, nausea, and vomiting following the first cycle of treatment with the ABVD protocol (adriamycin 25 mg m⎯2, bleomycin 10 mg m⎯2, vinblastin 6 mg m⎯2, and dacarbazine 375 mg m⎯2 on d 1 and 15 of a 28-d cycle). Magnetic resonance imaging (MRI) showed that there were regions with infiltration in particularly the thoracic vertebrae (T3, T4, T9, and T10-T12). In the lumbar region leptomeningeal involvement beginning at the end of the medulla spinalis that gave rise to a separation of nerves in the cauda equine was observed. Contrast agent uptake was observed, especially at the dural sac, with edema in the nerve roots.

Key Words: Leptomeningeal involvement, Hodgkin’s lymphoma

Özet Otuz bir yaşında leptomeningeal tutulumu olan Hodgkin’s Lenfoma (HL)’lı erkek hasta sunuldu. Hastada, ABVD (Adriamycin 25 mg/m2, bleomycin 10 mg/m2, vinblastine 6 mg/m2, dacarbazine 375 mg/m2 on days 1 and 15 of a 28-day cycle) tedavisinin ilk siklusundan sonra baş dönmesi, ekstremitelerde güçsüzlük, el ve ayaklarda istemsiz kasılma, inkontinans, baş ağrısı, ateş, bulantı ve kusma gelişti. Magnetic resonance imaging (MRI)’da özellikle torakal vertebralarda (T3, T4, T9, T10-T12), infiltrasyon ile uyumlu alanlar görüldü. Lomber bölgede medulla spinalisin son kısmından başlayan, kauda equinadaki sinirlerde ayrılmaya sebep olan leptomeningeal tutulum görüldü. Sinir köklerinde ödem ile birlikte özellikle dural kesede kontrast madde tutulumu görüldü.

Anahtar Sözcükler: Leptomeningeal tutulum, Hodgkin lenfoma

Address for Correspondence: Rahşan Yıldırım, M.D., Atatürk Üniversitesi, Tıp Fakültesi, Yakutiye Araştırma Hastanesi İç Hastalıkları Anabilim Dalı, 25100 Erzurum, Turkey Phone: +90 442 231 71 95 E-mail: rahsan.yildiri@hotmail.com Received/Geliş tarihi : February 9, 2010 Accepted/Kabul tarihi : May 25, 2010

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Introduction Although primary and secondary involvement of the central nervous system (CNS) can be seen in patients followed-up for various hematological malignancies, they are not very common in patients with malignant lymphomas. CNS involvement leads to increases in morbidity and mortality [1]. The frequency of CNS involvement is 0.2%-0.5% in patients with advanced stage Hodgkin’s lymphoma (HL) [2]. Leptomeningeal involvement in various malignancies is not common [3,4] and in Hodgkin’s disease it is rare [5]. As such, herein we present an HL patient with involvement of the leptomeninges.

Case Report A 31-year-old, male presented with complaints of night sweats, fever, weight loss (8 kg), and cervical and axillary lymphadenopathy, which began 2 months earlier at Hematology Departments of Atatürk University. Written informed consent to publish the details of his clinical course and treatment was obtained from the patient at the time of presentation. The patient’s personal and family medical histories were non-specific. Physical examination showed that his vital functions were normal; body temperature was 38.7 °C, and blood pressure and pulse were 110/70 mmHg and 108 min-1, respectively. The patient had generalized, mobile elastic lymphadenopathies (maximum diameter of 4 cm) in the cervical, axillary, and inguinal regions. The liver and spleen were palpable 3 and 5 cm below the costal margin, respectively.

Admission of hospital, the patient had the following findings: white blood cell count: 3 x 109 L-1; hemoglobin: 11 g dL-1; platelet count: 158 x 109 L-1. The erythrocyte sedimentation rate was 55 mm h-1. Serum biochemistry findings were within normal limits, except for serum lactate dehydrogenase, which was 560 IU L-1 (normal range: 150-450 IU L-1). C-reactive protein was 18 mg L-1 (normal range: 0-0.5 mg L-1). In posterior-anterior radiography hilar enlargement was the only pathology observed.. on 30 March 2009, nodular sclerosing HL was diagnosed via histopathological evaluation of a cervical lymph node biopsy specimen, and treatment with the ABVD protocol (adriamycin 25 mg m—2, bleomycin 10 mg m—2, vinblastin 6 mg m—2, and dacarbazine 375 mg m—2 on d 1 and 15 of a 28-d cycle) started on 1 April 2009.

The patient’s diameter of lymph nodes regressed to 1.5-2 cm, and night sweats and fever became less severe 15 d after starting the ABVD chemotherapy protocol. The patient presented on 1 May 2009 for the second cycle of chemotherapy and reported having experienced dizziness,

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limb weakness, involuntary contractions of the hands and feet, which lasted a few minutes, and occurred 1-2 times d–1 and gradually increased in frequency, and incontinence, headache, fever, nausea, and vomiting began the previous week. Physical examination showed bilateral cervical and axillary lymphadenopathies with a maximum diameter 2 cm. Neurological examination showed bilateral weakness (3/6) in the lower extremities associated with loss of deep tendon reflexes, hypoesthesia in the lower extremities (mostly in distal regions), proprioceptive sensory disturbance, and epileptic convulsions. Blood count and serum biochemistry findings were unremarkable. Fundus examination showed that the optic disc margins in both eyes were blurred. Craniospinal involvement was considered based on clinical symptoms and lumbar puncture, and cerebrospinal fluid cytology and cytocentrifugation were planned; however, due to abnormal fundoscopic findings they could not be performed at that time.

Cranial brain magnetic resonance imaging (MRI) showed that the patient had hydrocephalus. Vertebral MRI showed changes in intensity indicative of infiltration; thoracic vertebral corpuses appeared hypointense in T1-weighted images and hyperintense in T2 weighted images (Figure 1), particularly vertebrae T3, T4, T9, and T10-12. Similar findings were noted in vertebrae L1-L2-L3 and the sacrum. Additionally, the appearance of the lumbar region starting from the end of the medulla spinalis that caused edema in nerve roots and contrast agent uptake—particularly in the dural sac—giving rise to a separation of nerves in cauda equina were interpreted as leptomeningeal involvement (Figure 2). Electro encephalo myelography findings were normal.

As the patient was considered to have leptomeningeal involvement, on 10 May 2009 radiotherapy was commenced; 3600 cGy was administered primarily to the regions of vertebral involvement, and then to the entire cranium for 20 days. Following radiotherapy, the frequency of epileptic convulsions decreased gradually and eventually ceased. As post-radiotherapy fundoscopic findings were normal, cerebrospinal fluid testing could be performed. Glucose was 22 mg dL–1 (normal range: 40-70 mg L–1) and microprotein was 58 mg dL–1 (normal range: 15-45 mg L–1). No tumor cells were observed via cytocentrifugation or cytology. Event hough the patient’s epileptic convulsions gradually decreased in frequency and ceased following radiotherapy, other neurological symptoms persisted and were progressive; therefore the patient underwent physiotherapy for paraplegia. The patient was treated with 1 cycle of ifosfamide, carboplatin, and etopo-

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Yıldırım R, et al: Hodgkin’s Lymphoma with Central Nervous System Involvement

via cranial and meningeal involvement [6-8], or via the hematogenous route [9-11]. Neurological hydrocephaly occurred together with leptomeningeal involvement in the presented case. Intramedullary cord metastasis does not occur frequently in HL and when it does it indicates a poor prognosis [12].

Figure 1: Bone lesions in lumbosacral vertebrae.

Figure 2: Leptomeningeal involvement (white arrow).

side (ICE) on 20 June 2009; however, the patient’s clinical condition deteriorated gradually and he died due to respiratory failure on 15. day of the chemotherapy cycle.

Discussion Neurological complications of HL have been reported frequently and are generally observed after the disease is diagnosed [6]. In the presented case neurological involvement was detected 1 month after the diagnosis of HL. Although neurological involvement in HL manifests as spinal cord lesions rather than intracranial lesions, intracranial involvement usually appears via tumoral invasion

The frequency of CNS involvement is 0.2%-0.5% in advanced stage HL patients [2]. CNS involvement in HL can be seen in patients that relapse frequently (as in the presented case) and in immunodeficient patients at the time of diagnosis [13]. CNS involvement leads to increases in morbidity and mortality [1]. Leptomeningeal involvement, as observed in the presented case, occurs more frequently in patients with such malignancies as non-Hodgkin’s lymphoma and carcinomas than in those with HL, and is diagnosed based on evaluation of clinical presentation and detection of malignant cells during cytological analysis of cerebrospinal fluid. It could be necessary to repeat the analysis because of the diagnostic yield of the cytologic analysis of cerebrospinal fluid [13,14].

Although the clinical findings in the presented case strongly suggested neurological involvement, cerebrospinal fluid analysis and intrathecal therapy could not be performed due to abnormal fundoscopic findings; however, leptomeningeal involvement was confirmed via cranial or vertebral MRI findings. Following radiotherapy, cytological analysis and cytocentrifugation of the patient’s cerebrospinal fluid showed that there were no cells, whereas biochemical analysis showed an increase in microprotein levels and a decrease in the glucose level, suggesting leptomeningeal involvement. According to previous reports, remission can be achieved with intrathecal chemotherapy and cranial radiotherapy in patients with leptomeningeal involvement [15], although it was not achieved in the presented patient.

Figueroa et al. reported a 23-year-old female patient with cerebral mass imaging findings consistent with meningioma and nodular sclerosis-type HL that underwent excisional biopsy. As in the presented case, corpus vertebrae involvement was noted, and subsequent analysis of her cerebrospinal spinal showed no sign of involvement.

She achieved complete remission following systemic chemotherapy, radiotherapy, and intrathecal treatment [16].

Akyüz et al. reported 3 patients with intracranial involvement that had intracranial recurrence about 36 months after initial diagnosis despite receiving systemic chemotherapy and low-dose radiotherapy to the involved area.

In this case, intracranial involvement was detected using computerized tomography and MRI in these 3 patients,

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and cerebrospinal fluid was analyzed cytologically. Again, as in the presented case, these 3 patients died despite systemic chemotherapy and external radiotherapy to the cranial lesions. While involvement was only in the form of metastatic lesions in these 3 patients, leptomeningeal and metastatic lesions coexisted in the presented case. In addition, CNS involvement was noted 1 month after the diagnosis, in other words, only at the start of the second cycle of treatment [17]. Morawa et al. reported a male patient with Down syndrome and mixed cellular-type HL that had tonic clonic seizures; CNS involvement was noted after 5 cycles of treatment, even though complete remission was achieved following 4 cycles of the ABVD protocol. In the presented case CNS involvement was noted after 1 cycle of the ABVD chemotherapy protocol and clinical presentation were similar to that in the their patient. The researchers reported that the patient’s mass in the right ventricle observed with the MRI was consistent with the HL and that radiotherapy resulted in remission [18]. Leptomeningeal involvement was not present in any of the last four cases described. The presented case is different from these four cases in this regard.

The presented case had nodular sclerosing HL with a rapidly progressive course due to leptomeningeal involvement during chemotherapy. Although it is difficult to diagnose such neurological complications as leptomeningeal involvement or cranial nerve paralysis, it is important that they be diagnosed and treated early in order to improve patient quality of life and lower the mortality rate.

CNS and leptomeningeal involvement occurred in the presented nodular sclerosing HL patient. While nodular sclerosing HL has relatively better prognosis than those of other types of HL, the progression of the case while getting chemotherapy lead us to report this case.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Lister A, Abrey LE, Sandlund JT. Central nervous system lymphoma. Hematology Am Soc Hematol Educ Program 2002; 1: 283-296 2. Blake PR, Carr DH, Goolden AW. Intracranial Hodgkin’s disease. Br J Radiol 1986; 59: 414-416

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3. Olson ME, Chernik NL, Posner JB. Infiltration of the leptomeninges by systemic cancer. Arch Neurol 1974; 30: 122-137 4. Shapiro WR, Posner JB, Ushio Y, Chernik NL, Young DF. Treatment of meningeal neoplasms. Cancer Treat Rep 1977; 61: 733-743 5. Ultmann JE, Moran EM. Hodgkin’s disease: Clinical course and complications. Arch Intern Med 1973; 131: 322-353 6. Eyre DP. Neurological complications of Hodgkin’s disease.

Postgrad Med J 1966; 42: 723-724 7. Marshall G, Roesmann U, Van den Noort S. Invasive Hodgkin’s disease of the brain: Report of two new cases and review of the American and Eurepean literature with clinical pathological correlations. Cancer 1968; 22 (3): 621-630 8. Kaufman G. Hodgkin’s disease involving the central nervous system. Arch Neurol 1965; 13: 555-558 9. Sapozink MD, Kaplan HS. Intracranial Hodgkin’s disease: A report of 12 cases and review of the literature. Cancer 1983; 52: 1301-1307 10. Scheithauer BW. Cerebral metastases in Hodgkin’s disease.

Arch Pathol Lab Med 1979; 103: 284-287 11. Ashby MA, Barber PC, Holmes AE, Freer CE, Collins RD. Primary intracranial Hodgkin’s disease. A case report and discussion. Am J Surg Pathol 1988; 12: 294-299 12. Guermazi A, Brice P, de Kerviler E E, Fermé C, Hennequin C, Meignin V, Frija J. Extranodal Hodgkin’s disease: spectrum of disease. Radiographics 2001; 21: 161-179 13. Anselmo AP, Proia A, Cartoni C, Baroni CD, Maurizi Enrici R, Delfini R, Avvisati G. Meningeal localization in a patient with Hodgkin’s disease: Description of a case and review of the literature. Ann Oncol 1996; 7: 1071-1075 14. Young RC, Howser DM, Anderson T, Fisher RI, Joffe E, De Vita VT Jr. Central nervous system complications of nonHodgkin‘s lymphoma. Am J Med 1979; 66: 435-443 15. Orlowski EP, Hansen RM, Anderson T, Hanson GA, Kun LE, Pisciotta AV. Hodgkin’s disease with leptomeningeal involvement. Report of a case with long survival. Cancer 1984; 53: 1833-1835 16. Figueroa BE, Brown JR, Nascimento A, Fisher DC, Tuli S. Unusual Sites of Hodgkin’s Lymphoma CASE 2. Hodgkin’s Lymphoma of the CNS Masquerading As Meningioma. J Clin Oncol 2004; 22: 4228-4230 17. Akyüz C, Yalcin B, Atahan IL, Varan A, Kutluk MT, Büyükpamukçu M. Intracranial involvement in Hodgkin’s disease. Pediatr Hematol Oncol 2005; 22: 589-596 18. Morawa E, Ragam A, Sirota R, Nabhan C. Hodgkin’s lymphoma involving the CNS. J Clin Oncol 2007; 25: 14371438

Letter to the Editor

10.5505/tjh.2012.50470

HbA2-Yokoshima (delta 25(B7)Gly >Asp) and Hb A2-Yialousa (delta 27(B9)Ala>Ser) in Turkey Türkiye’de Gözlenen HbA2- Yokoshima (delta25(B7)Gly >Asp) ve HbA2–Yialousa (delta27(B9)Ala>Ser) Olguları Aylin Köseler, Ayfer Atalay, Erol Ömer Atalay Pamukkale University, School of Medicine, Department of Biophysics, Denizli, Turkey

1

To the Editor, Heterozygous beta-thalassemia (β-thal) carriers are characterized by microcytosis, hypochromia, and elevated HbA2 levels (≥3.5%) [1]. Although an elevated HbA2 level is a diagnostic parameter for b-thal, the interaction between d-globin gene mutation and b-thal can result in a normal HbA2 level, leading to misdiagnosis [2]. As δ-thalassemia (δ-thal) has no clinical significance, a reduced HbA2 level in β-thal carriers is an important parameter in the presence of d-thalassemia [3]. δ-globin gene mutations (http:// globin.cse.psu.edu/hbvar/menu.html) have been reported [4]. HbA2-Yialousa (delta 27(B9)Ala>Ser) is the most common δ-thal mutation in the Mediterranean Region and was first identified by Trifillis et al. in a Sardinian family in 1991 [1,5]. HbA2-Yokoshima (delta 25(B7)Gly >Asp) was first identified in a Japanese family in which 1 member was homozygous [6].

Altay et al. reported the presence of abnormal hemoglobin variants in their review, including a-globin, b-globin, and d-globin variants [7]. In 2007 Akar et al. reported that there were 88 hemoglobin variants [8]. As most hemoglobin variants are asymptomatic, they are often detected during family and population studies, and premarital screening programs. As an example, during premarital screening in Denizli, Turkey, several variants of hemoglobin were observed and identified; Hb-Yaizu, Hb-Ouled Rabah, and Hb-Tunis were the first reported cases in Turkey [9,10].

In total, the DNA of 12 b-thalassemia carriers with a low HbA2 level was studied. Written informed consent was obtained from the patients during donation of their DNA for use as anonymous samples. DNA amplification and sequencing were performed using a BECKMAN Coulter CEQ8000 non-radioactive fluorescence dye-based genetic analysis system, according to Pavlou et al. [11]. We identified IVS-1/nt-6 (T>C), IVS-1/nt-110 (G>A), IVS-2/nt-1 (G>A), and Cd44 (-C) mutations in all the DNA samples.

Only 3 of the 12 b-thal carriers had a low HbA2 level and d-globin gene mutation. DNA sequencing showed that the mutation at d-globin gene codon 25 (GGT/GAT) caused Hb-Yokoshima (Figure 1a) and mutation at d-globin gene codon 27 (GCC/TCC) caused Hb-Yialousa (Figure 1b). All

a

b

Figure 1: DNA sequencing of the HbA2-Yokoshima (a) and HbA2-Yialousa (b) cases.

Address for Correspondence: Aylin Köseler, M.D., Pamukkale Üniversitesi, Tıp Fakültesi, Biyofizik Anabilim Dalı, Kınıklı 20020, Denizli, Turkey Phone: +90 258 211 90 27 E-mail: akoseler@pau.edu.tr Received/Geliş tarihi : December 18, 2011 Accepted/Kabul tarihi : December 19, 2011

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of the patients that carried d-globin mutations also carried (b-thal) IVS-1-110 (G>A) mutation at the b-globin gene.

In conclusion, we would like to emphasize the importance of HbA2 variants in premarital diagnosis, and that the presence of variants of this δ-globin resulting in decreased HbA2 expression could lead to misdiagnosis of β-thal carrier status.

Acknowledgments We are grateful to Professor Dr. Ali Keskin from Pamukkale University, School of Medicine, Department of Hematology, and Dr. Hasan Koyuncu from the Turkish Ministry of Health, Denizli Hemoglobinopathy Center, Denizli, Turkey, for their valuable contributions.

Conflict of Interest Statement None of the authors has any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.

References 1. Morgado A, Picanço I, Gomes S, Miranda A, Coucelo M, Seuanes F, Seixas MT, Romão L, Faustino P. Mutational spectrum of delta-globin gene in the Portuguese population.

Eur J Haematol 2007: 79 (5): 422-428 2. De Angioletti M, Lacerra G, Gaudiano C, Mastrolonardo G, Pagano L, Mastrullo L, Masciandaro S, Carestia C. Epidemiology of the delta globin alleles in southern Italy shows complex molecular, genetic and phenotypic features.

Hum Mutat 2002: 20 (5): 358-367

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3. Bouva MJ, Harteveld CL, van Delft P, Giordano PC. Known and new delta globin gene mutations and their diagnostic significance. Haematologica 2006: 91 (1): 129-132 4. Huisman, THJ, Carver MFH, Efremov GD. A Syllabus of Human Hemoglobin Variants, 2nd edition; The Sickle Cell Anemia Foundation: Augusta, GA, USA, 1998; (http:// globin.cse.psu.edu) 5. Trifillis P, Ioannou P, Schwartz E, Surrey S. Identification of four novel delta-globin gene mutations in Greek Cypriots using polymerase chain reaction and automated fluorescence-based DNA sequence analysis. Blood 1991: 15; 78 (12): 3298-3305 6. Ohba Y, Igarashi M, Tsukahara M, Nakashima M, Sanada C, Ami M, Arai Y, Miyaji T. Hb A2 Yokoshima, alpha(2) delta(2)25(B7)Gly----Asp, a new delta chain variant found in a Japanese family. Hemoglobin 1985; 9 (6): 613-615 7. Altay C. Abnormal hemoglobins in Turkey. Turk J Haematol 2002:19: 63-74.

8. Akar E, Akar N. A review of abnormal hemoglobins in Turkey, Turk J Hematol 2007: 24:143-145.

9. Atalay EO, Atalay A, Koyuncu H, Ozturk O, Köseler A, Yıldız S, Bahadır A. Molecular, genetic and hematological analysis of a rare hemoglobin variant Hb Yaizu observed in Turkey. Med Princ Pract 2008; 17 (4): 321-324 10. Köseler A, Koyuncu H, Öztürk O, Bahadır A, Demirtepe S, Atalay A, Atalay E.O. Hb Tunis First Observation of Hb Tunis [beta124(H2)Pro>Ser] in Turkey, Turk J Hematol 2010; 27: 120-122.

11. Pavlou E, Phylactides M, Kyrri A, Kalogerou E, Makariou C, Georgiou I, Kleanthous M. Delta-thalassemia in Cyprus.

Hemoglobin 2006; 30 (4): 455-462

Letter to the Editor

10.5505/tjh.2012.78736

Spontaneous Subdural Hematoma Associated with Kasabach-Merritt Syndrome: A Case Report Kasabach-Merritt Sendromu ile İlişkili Spontan Subdural Hematom: Bir Olgu Sunumu Ufuk Emre1, Ayla Gökmen2, Banu Özen1, Enes Demiryürek1, Şanser Gül3, Dilvin Gökçe4 Bülent Ecevit University, School of Medicine, Department of Neurology, Zonguldak, Turkey Bülent Ecevit University, School of Medicine, Department of Hematology, Zonguldak, Turkey 3 Bülent Ecevit University, School of Medicine, Department of Neurosurgery, Zonguldak, Turkey 4 Zonguldak Ataturk State Hospital, Zonguldak, Turkey 1 2

Kasabach-Merritt syndrome (KMS) is characterized by consumption coagulopathy (thrombocytopenia, anemia, hypofibrinogenemia, and high D-dimer level), angioma, and kaposiform hemangioendothelioma [1-3]. Herein we present the first reported case of spontaneous subdural hematoma associated with KMS. Written informed consent was obtained from the patient.

rhage in the right tentorial region (Figure A-C). Fresh frozen plasma (3 units) was administered to treat hypofibrinogenemia within 3 days after admission, and the patient was given bed rest with head elevated for 15 days.Amitriptyline and analgesic drugs were initiated from the first day of admission. Two months later after discharge from hospital, she did not state any complaints and cranial CT showed a reduction in the size of the subdural hematoma.

A 24-year-old female with KMS was referred to our hospital with a subdural hematoma. Persistent, severe headache and neck pain began 10 days earlier, and she had a negative history of trauma. She had migraine without aura, psoriasis, and KMS. KMS was diagnosed at another medical center. Her general physical examination showed large cutaneous hemangiomas in her extremities, with left side predominance. Two fingers on her left hand had been amputated. Her neurological examination was normal.

Blood analysis findings were thrombocytopenia (platelet count: 87 x 109/L), hypofibrinogenemia, and a high D-dimer level (5000 ng/ mL [normal: 147 ng /mL]). The fibrinogen level was 40 mg /dL (normal range: 175-400 mg/ dL). Other hemogram parameters (hemoglobin level: 12.6 g/dl, white blood cell count: 6.1 x 109/L, hematocrit level: 36.1 %) and laboratory findings were normal. Cranial MRI showed a bilateral subacute subdural hematoma in the frontotemporoparietal region and subacute hemor-

Central nervous system involvement in KMS is rare. A search of the literature showed that there are no reports of intracranial bleeding as a complication of KMS. In the presented case cranial MRI did not show a hemangioma or tumoral lesion in the parenchymal region, but did show a large bilateral subdural hematoma. In patients with a subdural hematoma imaging findings (hematoma volume, degree of midline shift, and compression of the brainstem) and neurological examination (low Glasgow Coma Scale score) may help indicate prognosis and appropriate treatment options (surgery or medical) [4]. In the presented case surgical treatment wasn’t considered because hematoma size didn’t increase and patient’s clinic did not deteriorate during follow-up. Corticosteroid is considered the first-line therapy for KMS—either high-dose methylprednisolone (30 mg/kg for 3 d, followed by dose tapering) or oral corticosteroid (2–5 mg/kg per day), and intravenous corticosteroid may be more efficacious than oral cortico-

To the Editor,

Address for Correspondence: Ufuk Emre, M.D., Bülent Ecevit Üniversitesi, Tıp Fakültesi, Nöroloji Anabilim Dalı, Zonguldak, Turkey Phone: +90 372 261 01 69 E-mail: ufuemr@gmail.com Received/Geliş tarihi : October 22, 2011 Accepted/Kabul tarihi : March 6, 2012

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a

b

Turk J Hematol 2012; 29: 291-292

c

Figure : The cranial magnetic resonance imaging revealed bilateral subacute subdural haematomas in the frontotemporoparietal region on axial T2-weighted image (a), T1-weighted (b), FLAIR image (c) respectively.

steroid [5]. Alfa-IFN-2a and chemotherapeutic agents are used to prevent tumor expansion [1,5]. Packed red cells, fresh frozen plasma, cryoprecipitate, and pentoxifyline may be considered for treatment [6]. Fresh frozen plasma (15 mL/kg) is recommended in patients with prolonged clotting time and generalized bleeding, and prior to invasive procedures [2]. Surgical excision, embolization, and radiotherapy can be used in appropriate patients [1,2].

The presented patient received medical therapy based on the clinical findings. In conclusion, the present case report is the first to describe a subdural hematoma in a KMS patient; thus, KMS should be considered in the differential diagnosis of patients with intracranial hemorrhage.

Conflict of Interest Statement None of the authors has any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.

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References 1. Maguiness S, Guenther L. Kasabach-Merritt syndrome. J Cutan Med Surg 2002; 6 (4): 335-339 2. Hall GW. Kasabach-MerrÄątt Syndrome: Pathogenesis And Management. Br J Haematol 2001; 112: 851-862 3. Mizuno J, Nakagawa H, Watabe T, Sugimoto I. Spontaneous spinal epidural haematoma in association with KasabachMerritt syndrome. J Clin Neurosci 1999; 6: 518-520 4. Dent DL, Croce MA, Menke PG, Young BH, Hinson MS, Kudsk KA, Minard G, Pritchard FE, Robertson JT, Fabian TC. Prognostic factors after acute subdural hematoma.

J Trauma 1995; 39: 36-42 5. Ryan C, Price V, John P, Mahant S, Baruchel S, BrandĂŁo L, Blanchette V, Pope E, Weinstein M. Kasabachâ&#x20AC;&#x201C;Merritt phenomenon: A single centre experience Miriam Weinstein.

Eur J Haematol 2010; 84: 97-104 6. Sarkar M, Mulliken JB, Kozakewich HP, Robertson RL, Burrows PE. Thrombocytopenic coagulopathy (KasabachMerritt Phenomenon) is associated with Kaposiform hemangioendothelioma and not with common infantile hemangioma. Plas Reconstr Surg 1997; 100: 1377-1386

Letter to the Editor

10.5505/tjh.2012.66563

Erythema Annulare Centrifugum and Relapsing Polychondritis Associated with Acute Myeloid Leukemia: A Case Report Erythema Annulare Centrifugum ve Tekrarlayan Polikondritle Birliktelik Gösteren Akut Miyeloid Lösemi: Bir Olgu Sunumu Selami K. Toprak1, Sema Karakuş1, Aysun Halaçoğlu1, Efe Hasdemir1, Halit Üner2 Baskent University, School of Medicine, Department of Hematology, Ankara, Turkey Baskent University, School of Medicine, Department of Pathology, Ankara, Turkey

1 2

To the Editor, Erythema annulare centrifugum (EAC) is a slow progressing and recurring skin disease with unknown etiology, characterized by ring shaped erythematous skin rashes. EAC was first described by Darier in 1916, and classified in 1978 by Ackerman into a superficial and a deep type [1]. EAC has been associated with many different entities, including infections, food allergy and drug reactions, polycythemia vera, cryoglobulinaemia, myelodysplastic syndrome, hypereosinophilic syndrome, hyperthyroidism, autoimmune thyroidities, hepatic diseases, pregnancy and malignant neoplasms [2]. Relapsing polychondritis (RP) is a disease which progresses with inflammatory attacks on articular and non-articular cartilaginous tissue and is most commonly encountered as auricular chondritis. In auricular chondritis, that the earlobe is not affected and existence of one or two sided fulminant rubescence, sensitivity and oedema on external ear cartilaginous tissue are salient [3]. We report a case, who was diagnosed with acute myeloblastic leukemia (AML) two months after EAC was detected. Bilateral auricular chondritis developed in the third day of her chemotherapy and EAC lesions relapsed during the course of chemotherapy.

A 53 year old female patient with complaints of a sore throat was assessed in otorhinolaryngology polyclinic.

While diagnosed with tonsillitis and her antibiotherapy arranged, she was redirected to us upon detection of anemia, moderate thrombocytopenia and leukocytosis (hemoglobin 11.2 g/dL, hematocrit 33%, mean corpuscular volume 91 fL, white blood cell 19.2 x 109/L, neutrophile 0.9 x 109/L, thrombocyte 120 x 109/L). In her physical examination no symptom was found except cryptic tonsillitis.

She did not have a known systemic disease nor she did regularly use a medication. Her liver, kidney and thyroid functions tests were normal. In her bone marrow biopsy, conducted upon monitoring of blastic cell with a rate of 90% during her peripheral smear, diffuse blastic cell infiltration was found. In bone marrow aspiration, usually large, potential undifferentiated blasts with heterogenous structure and agranular cytoplasm, but not involving Auer rods, were detected. After immunohistochemical examination the patient was diagnosed with AML M0, and classic 7+3 induction chemotherapy regimen involving cytosine arabinoside and idarubicin combination protocol was initiated. On the third day of treatment palpable rash on her abdominal skin (Figure 1), and swelling, rubescence and sensitivity on both ears that do not prominently effect the earlobe (Figure 2a -2b) occurred. A biopsy from the lesions on the abdominal skin was performed.

Lesions on the ear auricle of the patient were assessed as

Address for Correspondence: Selami K. Toprak, M.D., Başkent Üniversitesi, Tıp Fakültesi, Hematoloji Bilim Dalı, Ankara, Turkey Phone: +90 312 212 29 12 E-mail: sktoprak@yahoo.com Received/Geliş tarihi : December 17, 2011 Accepted/Kabul tarihi : February 21, 2012

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Figure 1: EAC lesions on the abdomen that occurred during the course of chemotherapy.

A

B

Figure 3: Benign, self-limited lymphocytic infiltration around the veins in superficial and deep dermis.

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chondritis by dermatology department and corticosteroid (CS) treatment (methylprednisolone 40 mg/day) was initiated. However, while chondritis regressed on the third day of CS treatment, no change was detected on lesions of unknown cause (probably disease related or adverse effect of drugs?), which developed on the abdominal skin.

Results of biopsy taken from abdominal skin a couple of days later came as EAC (Figure 3). When auricular chondritis started to evidently regress under treatment, CS treatment was decreasingly ceased within seven days. During the time when remission was achieved by means of bone marrow assessment carried out on the 21st day of the course of chemotherapy, it was found that the rashes on the abdominal skin, which previously did not respond to CS treatment, decreasingly disappeared. It was also found that lesions similar to those on abdominal skin also developed on her both legs two months before the patient was diagnosed with AML and the results of biopsy that had

Figure 2A and 2B: Bilateral auricle and external auditory canal erythema, induration, and edema.

Figure 4: Prominent lymphoid infiltration around the veins in the dermis and among collagen fibers.

Turk J Hematol 2012; 29: 293-296

Toprak SK, et al: Erythema Annulare Centrifugum and Relapsing Polychondritis Associated with Leukemia

been performed were likewise available as EAC (Figure 4).

The patient is currently in remission and is being monitored in the course of allogeneic hematopoietic stem cell transplantation to follow consolidation chemotherapy.

Malignant diseases associated with EAC include myelomatosis, Hodgkinâ&#x20AC;&#x2122;s lymphoma, acute leukemia, prostatic adenocarcinoma, nasopharyngeal carcinoma and histiocytosis [4].

In a study where 66 cases with EAC were assessed it was found that 13% of these were related to malignancies and at the same time there was no enough response to topical and systemic CS treatment [5]. In literature there is a case with anaplastic large cell lymphoma where a close relationship between the activity of disease and EAC was found [4]. Likewise, while our case did not respond to CS treatment, with the disease regression, skin lesions prominently regressed similarly to limited literature information.

Diagnostic algorithms for RP were established in 1976

by McAdam et al. These are; a) bilateral auricular chondritis, b) nonerosive, seronegative inflammatory polyarthritis, c) nasal chondritis, d) ocular inflammation, e) chondritis of respiratory system, and f) audiovestibular damage. Therefore, at least three criterion validities or an histopathological diagnosis and a criterion validity, or , along with response to CS and/or dapsone, existence of chondritis on two or more anatomic regions are sufficient for establishing a diagnosis of recurring polychondritis [6]. Our patient had bilateral auricular chondritis and she responded to CS. Our patient did not show any symptoms such as cough or dyspnoea that would suggest respiratory system cartilaginous tissue involvement. Additionally, no symptom regarding cartilaginous tissue involvement was found in her thorax computerized tomography taken when she had fever. No ocular, nasal or inner ear involvement was detected in our patient. While in RP skin involvement is most commonly encountered as vasculitis, erythema nodosum or superficial thrombophlebitis, when the literature was examined it was found that there is only one case where recurring polychondritis and EAC were detected [7, 8]. Therefore, since our patient had the risk of cartilage involvement on a different anatomic region, we planned her follow-up also in this respect.

EAC is currently considered to be a distinctive hypersensitivity reaction that may be triggered by many different antigens and disappears after 1â&#x20AC;&#x201C;2 weeks. Lesions tend to appear on the trunk, proximal parts of the extremities and, more frequently, in the superficial type appear especially on the buttocks [2].This is a chronic and recurrent

disease despite treatment. EAC is thought to be highly associated with internal disease as well as with superficial fungal infection. However, it was difficult to prove a causal association. The recognition and exact diagnosis of EAC is important.

Another issue was the relationship between AML and EAC in our case. It is not clear whether these represent two separate diseases (AML and EAC) or a continuous range. The skin eruption may be the presenting sign of an underlying disease. In most cases, the etiology of EAC is unknown. However, numerous skin lesions may occur during AML. They more frequently have a nonleukemic infiltrate, mainly represented by eruption secondary to chemotherapeutic agents and antibiotics. Cutaneous AML due to skin infiltration by malignant blasts (leukemia cutis) is less frequent. For the present case; clinicopathological and molecular evidence was not suggestive of cutaneous AML. Many pathological associations have been reported in EAC, mainly in isolated clinical case reports [9]. Similarly, in a publication of Weyers et al., pathological associations were less frequent and patients with neoplasm-associated EAC were on average 20 years older than those with isolated EAC [1]. This might suggest a fortuitous association between cancer and EAC or at least an overestimation of this association mainly due to the fact that pathological associations with EAC tend to be more reported [9]. Nevertheless, in our patient, despite her young age, the low frequency of EAC in the general population and the concomitant discovery of EAC and AML argue against a fortuitous association.

The presented case is important because she was diagnosed with AML M0 only two months after EAC diagnosis and that auricular chondritis was detected alongside EAC on the third day of remission-induction chemotherapy.

The probability of an underlying hematologic malignancy, especially in cases where EAC is detected, should always be kept in mind.

Written informed consent was obtained from the patient.

Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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References 1. Weyers W, Diaz-Cascajo C, Weyers I. Erythema annulare centrifugum: results of a clinicopathologic study of 73 patients. Am J Dermatopathol 2003; 25 (6): 451-462 2. Carlesimo M, Fidanza L, Mari E, Pranteda G, Cacchi C, Veggia B, Cox MC, Camplone G. Erythema annulare centrifugum associated with mantle b-cell non-Hodgkin’s lymphoma. Acta Derm Venereol 2009; 89 (3): 319-320 3. Rapini RP, Warner NB. Relapsing polychondritis. Clin Dermatol 2006; 24 (6): 482-485 4. Ural AU, Ozcan A, Avcu F, Kaptan K, Taştan B, Beyan C, Yalçin A. Erythema annulare centrifugum as the presenting sign of CD 30 positive anaplastic large cell lymphoma-association with disease activity. Haematologia (Budap) 2001; 31 (1): 81-84 5. Kim KJ, Chang SE, Choi JH, Sung KJ, Moon KC, Koh JK. Clinicopathologic analysis of 66 cases of erythema annulare centrifugum. J Dermatol 2002; 29 (2): 61-67

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6. McAdam LP, O’Hanlan MA, Bluestone R, Pearson CM. Relapsing polychondritis: Prospective study of 23 patients and a review of the literature. Medicine (Baltimore) 1976; 55 (3): 193-215 7. Ananthakrishna R, Goel R, Padhan P, Mathew J, Danda D. Relapsing polychondritis--case series from South India. Clin Rheumatol 2009; 28 Suppl 1: 7-10 8. Ingen-Housz S, Venutolo E, Pinquier L, Cavelier-Balloy B, Dubertret L, Flageul B. Erythema annulare centrifugum and relapsing polychondritis Ann Dermatol Venereol 2000; 127 (8-9): 735-739 9. Stokkermans-Dubois J, Beylot-Barry M, Vergier B, Bouabdallah K, Doutre MS. Erythema annulare centrifugum revealing chronic lymphocytic leukaemia. Br J Dermatol 2007; 157 (5): 1045-1047

Letter to the Editor

10.5505/tjh.2012.72325

Mirtazapine-Induced Thrombocytopenia and Neutropenia Mirtazapin İlişkili Trombositopeni ve Nötropeni Selami K. Toprak 1, Elçin Erdogan1, Özlem Kurt Azap2 Baskent University, School of Medicine, Department of Hematology, Ankara, Turkey Baskent University, School of Medicine, Department of Clinical Microbiology and Infectious Disease, Ankara, Turkey

1 2

To the Editor, Neutropenia is a rare and commonly reversible side effect of antidepressant treatment [1]. Mirtazapine is a noradrenergic and specific serotonergic antidepressant that is approved for use in the treatment of major depression in many countries [2]. A major hematological side effect of mirtazapine is agranulocytosis. Herein we report a patient with depression that developed severe neutropenia-associated thrombocytopenia during treatment with mirtazapine; the patient was safely treated by switching to another drug.

A 72-year-old Caucasian male presented to the emergency room with fever and epistaxis. On presentation his white blood cell, neutrophil, and platelet counts were 3.27 x 109 L–1, 0.17 x 109 L–1, and 28.10 x 109 L–1, respectively.

The patient was hospitalized with neutropenic fever and thrombocytopenia. Complete blood cell count from 20 d earlier was essentially normal.

The patient had a medical history of diabetes mellitus type 2 (regulated by diet), hypertension, and cervical spondylosis. His medication use included isosorbide5-monohydrate and metoprolol succinate for 5 years. He had no obvious past history of hematological disorders.

He did have a 1-month history of depressive symptoms, including low mood and alysosis. Because of his symptom profile 10 d earlier he was started on mirtazapine 15 mg at night by the neurology department.

On physical examination he did not have organomegaly or lymphadenopathy; nor did he have ecchymoses or

petechia/purpura. Laboratory findings, including immunoglobulin, vitamin B12, folate, and immunofixation levels were within normal limits. Other tests performed to determine a possible infectious etiology were negative.

Peripheral blood smear showed thrombocytopenia and leucopenia/neutropenia. Bone marrow aspiration biopsy was performed with the permission of the patient. Bone marrow examination showed hypocellularity with differentiation of the myeloid lineage and mildly decreased megakaryocytes. There was no evidence of malign infiltration.

The absence of any other etiological mechanism, the patient’s unremarkable laboratory work-up, and the close temporal association between administration of mirtazapine and the decrease in his neutrophil and thrombocyte counts strongly suggested that the bicytopenia was related to mirtazapine. As such, mirtazapine treatment was withdrawn due to the suspicion of its link to his symptoms.

One week after discontinuation of mirtazapine escitalopram and alprazolam were initiated following psychiatric consultation due to his labile and bad affect. The patient’s platelet count increased to 89.00 x 109 L–1, and then to 100.00 x 109 L–1 on the 7th and 14th d of mirtazapine withdrawal, respectively. The absolute neutrophil and platelet counts increased to 2.5 x 109 L–1 and 219.00 x 109 L–1, respectively, 21 d after discontinuation of mirtazapine.

To the best of our knowledge like other tetracyclic antidepressants, mirtazapine is associated with severe neutropenia, but the mechanism of mirtazapine-associ-

Address for Correspondence: Selami K. Toprak, M.D., Başkent Üniversitesi, Tıp Fakültesi, Hematoloji Bilim Dalı, Ankara, Turkey Phone: +90 312 212 29 12 E-mail: sktoprak@yahoo.com Received/Geliş tarihi : November 23, 2011 Accepted/Kabul tarihi : January 2, 2012

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Turk J Hematol 2012; 29: 297-298

ated leucopenia (neutropenia) is not well understood [3].

Hypersensitivity and immune-mediated mechanisms have been suggested, including complement-mediated toxicity and drug-induced antibodies against committed stem cells, proliferating precursors, and mature blood cells [4].

Mirtazapine-induced thrombocytopenia is a rare hematological disorder; this serotonin reuptake inhibitor is not currently accepted as a definitive or probable cause of thrombocytopenia [5]. In a single case report that was published in 2003 Liu and Sahud researched the immunological nature of mirtazapine-induced thrombocytopenia [6]. The researchers identified mirtazapine-induced GPIIb/IIIa reactive antibodies in the patient’s serum.

other than mirtazapine in the presented patient, such as different medications and disease, supported the hypothesis of mirtazapine-induced thrombocytopenia, suggesting an immune mechanism.

Tests to demonstrate drug-dependent antibodies were not performed in the presented patient and therefore the drug causality assessment was based on the relationship between drug intake and the onset of thrombocytopenia, as previously reported. Furthermore, the rapid increase in the patient’s platelet count after discontinuation of mirtazapine therapy further indicated that neutropenia was due to mirtazapine treatment. Common causes of thrombocytopenia and leucopenia (neutropenia), including infections, were investigated using bone marrow aspiration biopsy and other laboratory investigations. Malignancy was excluded based on the clinical and histological pattern of thrombocytopenia/neutropenia.

To the best of our knowledge in vitro data show that platelet aggregation decreases when exposed to isosorbide, but thrombocytopenia and leucopenia (neutropenia) were not documented in clinical practice. Hematological side effects, including agranulocytosis, non-thrombocytopenic purpura and thrombocytopenic purpura have been associated with beta-blocking agents, in general. These side effects occur short-medium term after initiation of metoprolol therapy. In the presented case, metoprolol was used for 5 years and it was not stopped during the patient’s hospitalization. These findings showed us that metoprolol and isosorbide were not the cause of thrombocytopenia/ neutropenia.

Neutropenia with cross intolerance between 2 tricyclics has been described, and there is evidence that patients can be successfully treated with another class of drug after such an incidence [1]. The absence of etiologic factors

298

While collaboration with psychiatrists is important, clinicians (hematologist) should keep in mind that a primary care physician (psychiatrist) can fail to warn patients about thrombocytopenia/neutropenia or monitor thrombocyte and total-differential white blood cell counts in patients receiving mirtazapine at minimum several times during the first year of the therapy. As such, in patients treated with mirtazapine —especially older adults —clinicians should be aware that the drug can cause neutropenia/thrombocytopenia.

Written informed consent was obtained from the patient.

Conflicts of Interest Statement None of the authors of this letter have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.

References 1. Ozcanli T, Unsalver B, Ozdemir S, Ozmen M. Sertraline and mirtazapine-induced severe neutropenia. Am J Psychiatry 2005; 162 (7): 1386 2. Croom KF, Perry CM, Plosker GL. Mirtazapine: A review of its use in major depression and other psychiatric disorders.

CNS Drugs 2009; 23 (5): 427-452 3. Davis J, Barkin RL. Clinical pharmacology of mirtazapine: Revisited. Am Fam Physician 1999; 60 (4): 1101 4. Dholakia R, Schleifer SJ, Ahmad YJ, Narang IS. Delayedonset mirtazapine-related leucopenia and rechallenge. J Clin Psychopharmacol 2010; 30 (6): 758 5. Reese JA, Li X, Hauben M, Aster RH, Bougie DW, Curtis BR, George JN, Vesely SK. Identifying drugs that cause acute thrombocytopenia: An analysis using 3 distinct methods.

Blood 2010; 116 (12): 2127-2133 6. Liu X, Sahud MA. Glycoprotein IIb/IIIa complex is the target in mirtazapine-induced immune thrombocytopenia.

Blood Cell Mol Dis 2003; 30 (3): 241-245

Letter to the Editor

10.5505/tjh.2012.02438

Non-Secretory Multiple Myeloma in a Hemodialysis Patient with Hypercalcemia Hiperkalsemi Saptanan Hemodiyaliz Hastasında Nonsekretuar Multipl Miyelom Tanısı Fatih Mehmet Erdur1, Yasemin Usul Soyoral1, Habib Emre1, Hüseyin Beğenik1, Mehmet Taşdemir2, Reha Erkoç3 Department of Nephrology, School of Medicine, Yuzuncu Yil University, Van, Turkey Department of Internal Medicine, School of Medicine, Yuzuncu Yil University, Van, Turkey 3 Department of Nephrology, School of Medicine, Bezmialem University, Istanbul, Turkey 1 2

To the Editor, Multiple myeloma (MM) develops due to clonal expansion of malign plasma cells. Abnormal plasma cells secrete abnormal immunoglobulins that can be detected monoclonally (M peak) in serum and/or urine via electrophoresis.

MM manifests clinically and radiologically with anemia, hypercalcemia, renal failure, and lytic lesions in bones [1]. When the characteristic MM clinical and radiological findings are observed, but there is no monoclonal M peak based on electrophoresis, it is known as non-secretory multiple myeloma (NSMM)—a rare variant of MM [2,3]. NSMM is observed 1%-5% of all MM patients [4].

Hypercalcemia, which can present as mild to severe and life threatening, is the most common metabolic abnormality associated with MM, occurring in approximately 33% of patients [5]. Herein we report a patient diagnosed with NSMM after detecting hypercalcemia during follow-up 7 months after the start of hemodialysis treatment.

A 62-year-old male presented to our hospital with a 1-month history of left knee-joint pain. This patient began peritoneal dialysis following the diagnosis of chronic renal failure (CRF) 7 months earlier at another hospital, and then 1 month later began hemodialysis due to resistant peritonitis. Written informed consent was obtained from the patient. Physical examination findings were as follows:

blood pressure: 100/60 mmHg; pulse: 90 bpm; temperature: 36.7 °C; respiration rate: 18 breaths min–1. Except for left knee-joint tenderness and movement restriction, examination of all other systems was normal. X-ray imaging of the left knee-joint was normal. Laboratory findings were as follows: hemoglobin: 8.3 g dL–1; MCV: 91.2fL; urea: 105 mg dL–1; creatinine: 8.39 mg dL–1; corrected calcium: 14.92 mg dL–1 (normal range: 8.4-10.7 mg dL–1); phosphorus: 5.1 mg dL–1; total protein: 5.71 g dL–1; albumin: 3.0 g dL–1; globulin: 2.7 g dL–1; sedimentation: 40 mm h–1; parathormone: 6.6 pg mL–1 (normal range: 15-68 pg mL–1).

The patient was hemodialyzed with calcium dialysate 1.25 mmol L–1 and was hydrated. Steroid and zoledronic acid were initiated because the corrected calcium level increased to 17.12 mg dL–1. On d 5 of treatment the calcium level returned to normal (9.3 mg dL -1). The patient’s history of using drugs containing vitamin D and calcium was negative. Malignancy screening showed no pathological findings. Abdominal ultrasonography showed that kidney size was within the normal range. We performed protein electrophoresis due to the suspicion of MM in this patient with hypercalcemia, a positive CRF history, and normal kidney size. Monoclonal gammopathy was not observed in serum and urine protein electrophoresis, or immunofixation electrophoresis. Osteolytic bone lesions

Address for Correspondence: Fatih Mehmet Erdur, M.D., Yüzüncü Yıl Üniversitesi, Tıp Fakültesi, Eğitim ve Araştırma Hastanesi, Nefroloji Kliniği 65300 Van, Turkey Phone: +90 432 215 04 70 E-mail: drfme@yahoo.com Received/Geliş tarihi : April 21, 2011 Accepted/Kabul tarihi : February 21, 2012

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were observed via pelvic X-ray. Examination of specimens obtained via bone marrow aspiration and biopsy showed diffuse interstitial plasma cell infiltration (35% plasma cells). Immunohistochemical examination showed CD38 and kappa (Îş) staining in infiltrative plasma cells, but no lambda (Îť) staining. The patient was diagnosed as NSMM and referred to the hematology clinic.

In conclusion, in end-stage CRF patients with undefined hypercalcemia and bone pain, radiographically observed lytic bone lesions, and normal kidney size, but no M peak based on electrophoresis NSMM should be considered, and bone marrow aspiration and biopsy should be performed.

Conflicts of Interest Statement None of the authors of this letter have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.

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References 1. Laubach J, Richardson P, Anderson K. Multiple Myeloma.

Annu Rev Med 2011; 62: 249-264 2. Bourantas K. Nonsecretory multiple myeloma. Eur J Haematol 1996; 56: 109-111 3. Doster DR, Folds J, Gabriel DA. Nonsecretory multiple myeloma. Arch Pathol Lab Med 1988; 112: 147-150 4. Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy MQ, Dispenzieri A, Fonseca R, Rajkumar SV, Offord JR, Larson DR, Plevak ME, Therneau TM, Greipp PR. Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 2003; 78: 21-33 5. Oyajobi BO. Multiple myeloma/hypercalcemia. Arthritis Res Ther 2007; 9 (Suppl 1): S4

Letter to the Editor

10.5505/tjh.2012.90377

Bortezomib-Associated Severe Orthostatic Hypotension and Hyponatremia Bortezomib İlişkili Ortostatik Hipotansiyon ve Hiponatremi Elif Suyanı1, Zeynep Akı1, Zeynep Arzu Yeğin1, Gülsan Türköz Sucak1 Gazi University, School of Medicine, Department of Hematology, Ankara, Turkey

1

To the Editor, The most frequent side effects of bortezomib are fatigue, hematologic toxicity with cytopenias, and peripheral neuropathy, whereas postural hypotension and hyponatremia are less common late side effects [1,2]. Hyponatremia is a potentially fatal complication that has been reported in 3%-22% of patients treated with bortezomib, and has been attributed to direct bortezomib toxicity and syndrome of inappropriate anti-diuretic hormone secretion (SIADH) [3]. The direct toxicity of bortezomib might be dose dependent [1]; however; there is insufficient evidence to support this mode of action. We think hyponatremia in the presented patient was due to direct bortezomib toxicity, as it resolved following cessation of bortezomib and saline infusion.

A 68-year-old male with IgG kappa multiple myeloma had undergone autologous hematopoetic stem cell transplantation (ASCT). The disease progressed despite posttransplantation thalidomide maintenance. Cyclophosphamide and dexamethasone (CD) was commenced 6 months post-transplantation; however, disease progression continued and 2 years post ASCT bortezomib monotherapy was started at the dose of 1.3 mg m–2 on d 1, 4, 8, and 11, every 3 weeks. The first 2 courses of bortezomib were uneventful; however, on d 7 of the third cycle the patient was admitted to hospital due to dizziness and a syncope episode. Physical examination showed severe orthostatic hypotension in the absence of compensatory tachycardia, with blood pressure of 120/80 mmHg in the supine posi-

tion and 60/50 mmHg while standing. He was not clinically dehydrated and there were no signs of infection or edema. Laboratory investigations showed isolated hyponatremia without any other electrolyte abnormality (Na: 126 mmol L–1; K: 3.5 mmol L–1; Cl: 98 mmol L–1). Serum biochemistry, including the renal profile, was normal. A written consent was obtained from the patient about the treatments and use of data in his file.

Bortezomib was withdrawn and isotonic saline infusion was commenced. Sodium returned to normal without requiring fluid restriction; however, there was no improvement in orthostatic hypotension, despite normal cardiac findings. Holter electrocardiography, echocardiography, and carotid Doppler ultrasound were also normal, which excluded the possibility of cardiac etiology. Neurologic examination was suspicious for peripheral sensorial neuropathy, which was confirmed via electromyography. Pregabalin 75 mg b.i.d. was started and the dose was increased to 150 mg b.i.d. 7 days later The patient’s clinical condition improved 2 weeks after starting pregabalin treatment, without a recurrent syncope and with decreased dizziness.

Postural hypotension occurs in 10% of patients treated with bortezomib and is associated with dehydration, concomitant anti-hypertensive treatment, and/or autonomic neuropathy [4]. The presented patient had no signs of dehydration and postural hypotension did not improve with saline infusion. Moreover, the presence of widespread peripheral neuropathy and the absence of compensatory

Address for Correspondence: Elif Suyanı, M.D., Gazi Üniversitesi Hastanesi, Erişkin Hematoloji, Beşevler 06500 Ankara, Turkey Phone: +90 312 202 63 17 E-mail: elifsuyani@hotmail.com Received/Geliş tarihi : May 24, 2011 Accepted/Kabul tarihi : February 21, 2012

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tachycardia suggested that autonomic neuropathy was the cause of orthostatic hypotension.

As the number of myeloma patients treated with bortezomib increases, so is awareness of bortezomib-related side effects. Orthostatic hypotension associated with autonomic neuropathy and hyponatremia seems to be reversible with cessation of bortezomib and use of pregabalin.

In conclusion, orthostatic hypotension and hyponatremia should be included in the differential diagnosis of treatment-related toxicity in myeloma patients treated with bortezomib.

Conflicts of Interest Statement None of the authors of this letter have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.

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References 1. Orlowski RZ, Stinchcombe TE, Mitchell BS, Shea TC, Baldwin AS, Stahl S, Adams J, Esseltine DL, Elliott PJ, Pien CS, Guerciolini R, Anderson JK, Depcik-Smith ND, Bhagat R, Lehman MJ, Novick SC, O’Connor OA, Soignet SL. Phase I trial of the proteasome inhibitor PS–341 in patients with refractory hematologic malignancies. J Clin Oncol 2002; 20 (22): 4420-4427 2. O’Connor OA, Wright J, Moskowitz C, Muzzy J, MacGregorCortelli B, Stubblefield M, Straus D, Portlock C, Hamlin P, Choi E, Dumetrescu O, Esseltine D, Trehu E, Adams J, Schenkein D, Zelenetz AD. Phase II clinical experience with the novel proteasome inhibitor bortezomib in patients with indolent non-Hodgkin’s lymphoma and mantle cell lymphoma. J Clin Oncol 2005; 23 (4): 676-684 3. Brodmann S, Gyr Klaas E, Cathomas R, Girardi V, von Moos R. Severe hyponatremia in a patient with mantle cell lymphoma treated with bortezomib: A case report and review of the literature. Onkologie 2007; 30 (12): 651-654 4. Rajkumar SV, Richardson PG, Hideshima T, Anderson KC. Proteasome inhibition as a novel therapeutic target in human cancer. J Clin Oncol 2005; 23 (3): 630-639

Letter to the Editor

10.5505/tjh.2012.48568

Autoimmune Hemolytic Anemia Otoimmün Hemolitik Anemi Şinasi Özsoylu Retired Prof of Pediatrics, Hematology, Hepatology, Ankara, Turkey Honorary fellow of American Academy of Pediatrics Honorary member of American Pediatric Society

To the Editor, I would like to make few comments on the Sarper and her colleagues’ article entitled “Management of autoimmune hemolytic anemia in children and adolescents: a single center experience” published in the recent issue of the Journal (2011;28:198-205) [1].

First of all, Coombs test was negative two out of 13 patients with hemolytic anemia (patients 3 and 10).

Although Coombs positivity may not correspond to hemolysis, because of low titer, reversals may not be expected.

As recently brought to the attention once more, the disease may differ despite of similar pathogenesis [2]. Therefore, Evans syndrome (ES) should be presented separetly then autoimmune hemolytic anemia (AIHA) as of WiskottAldrich syndrome (WAS) in which Coombs positivity may develop as of alloimmune reaction.

In addition “Patients with ES have sequentiel or spontaneous” thrombocytopenia, but it is not called ITP [3].

Although corticosteroids are the main drugs for the treatment of AIHA, it should not be used in divided doses as subtitution treatment as of surrenal insufficiency; which has been applied previously; Rather it should be given at once around 6 a.m preferentially as Megadose methylprednisolone (MDMP) [4] intravenously or orally as desribed by us which was used in parttially one of the authors’ patient (30 mg/kg/day, for 3 days).

May I also remind that blood group changes may occur in autoimmune disaeses, which should be correlated with the parents blood groups.

Since the patients retrospectively reviewed, was ethic comimette approval required?

References 1. Nazan S, Suar ÇK, Emine Z, Sema Aylan G. Management of autoimmune hemolytic anemia in children and adolescents: a single center experience.Turk J Hemato 2011;28:198-205 2. Fere W, Gregory, Guttmacher AC. Genomics and the multifactorial nature of human autoimmune disaese. NEJM 2011;365:1612-23 3. Özsoylu Ş. Is every immune hrombocytopenia. Idıopathic thrombocytopenic purpura. Acta Paediatrica 2004;93: 1129-1130 4. Özsoylu Ş, İrken G, Karabent A. High-dose intravenous methylprednisolone for acute childhood idiopathic thrombocytopenic purpura. Eur J Hematol 1989;42: 431-435

Reply, In discussion we reported that false negative direct Coombs test may be found in 2-4% of patients with autoimmune hemolytic anemia (AIHA) due to low titers of antibodies or low sensitivity of the test. We have already refered, reference 7 and 8 on the subject in our study [1,2].

Our patients with negative Coombs test were responsive to steroids. Özsoylu et al says that false positive Coombs test may be observed but false negative can not be observed in OIHA. He might not have such an experience.

Address for Correspondence: Şinasi ÖZSOYLU, M.D., Beysukent Altınşehir Sitesi No:30, Ankara, Turkey Phone: +90 312 235 41 88 E-mail: sinasiozsoylu@hotmail.com Received/Geliş tarihi : November 21, 2011 Accepted/Kabul tarihi : November 21, 2011

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The aim of presenting cases with Evans syndrome and W.Aldrich syndrome in the same article was to attract attention to these cases presenting with hemolytic anemia and underlying immunodeficiency. In the last paragraphs of the abstract and discussion we said that hemolytic reactions of these cases may require other immunosupressive agents and each case must be considered indiviually before splenectomy. In the recent literature Evans syndome is presented under the title of autoimmune hemolytic anemia [3]. In Wiscott-Aldrich syndrome autoimmune hemolytic anemia develops as many other autoimmune and inflammatory complications [4], but Özsoylu says that it is an alloimmune reaction. Özsoylu says that ‘’Patients with ES have sequentiel or spontaneous thrombocytopenia, but it is not called ITP2 ‘’.

I think he wants to say ‘’simultaneous’’ instead of ‘’spontaneous’’. In the study we already reported that thrombocytopenic and hemolytic anemia attack may be sequential even second cytopenia may emerge over the course of ten years. When the first attack is thrombocytopenia you have to diagnose it as immune thrombocytopenic purpura because at that moment you do not know that this patient will also develop an autoimmune hemolytic anemia attack.

In the recent literature Evans syndrome is described as follows [5]: ‘’Evans syndrome (ES) is characterized by the coexistence of an autoimmune hemolytic anemia (AIHA) and immune thrombocytopenic purpura (ITP)’’. Özsoylu says that corticosteroids should not be used in divided doses as subtitution treatment as of surrenal in-sufficiency; which has been applied previously. Rather it should be given at once around 6 a.m preferentially as megadose methylprednisolone (MDMP)3. In text book ‘’Williams Hematology’’ ‘’ it is written that, in adults oral methylprednisolone should be used at an initial daily dose of 60-100 mg. Critically ill patients with rapid hemolysis may receive 100-200 mg in divided dose over the first 24 hr.’’[6]. If hemoglobin level is already stabilized at a reasonable level, I do not think megadose methylprednisolone is required. Özsoylu may prefer such high doses. In Manual of Pediatric Hematology and Oncology by Philip Lanzkowsky also, 2-6 mg/kg/day prednisolone or methyl

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prednisolone is recommended [7]. I do not think divided doses of corticosteroids are used always as subtitution treatment of surrenal in-sufficiency. Divided doses are also used in leukemia. Özsoylu says that, blood group changes rarely occurs in autoimmune disaeses. So we want to attract attention to rare cases. Patient 2 was refered to us from another center after some transfusions and corticosteroid replacement and still ongoing hemolysis. We observed that blood group typing was false and transfusion with incompatible packed red cells also contributed to hemolysis.

As a conclusion, I could not find any scientific error in our article regarding Özsoylu’s comments.

Nazan SARPER Kocaeli University, School of Medicine, Department of Pediatric Hematology, Kocaeli, Turkey

References 1. Agarwal B. Autoimmune hemolytic anemia. 1998; 65: 663668 2. Collins PW, Newland AC. Treatment modalities of autoimmune blood disorders. 1992; 29: 64-74 3. Baek SW, Lee MW, Ryu HW, Lee KS, Song IC, Lee HJ, Yun HJ, Kim S, Jo DY. Clinical features and outcomes of autoimmune hemolytic anemia: A retrospective analysis of 32 cases. Korean J Hematol 2011; 46: 111-117 4. Dupuis-Girod S, Medioni J, Haddad E, Quartier P, Cavazzana-Calvo M, Le Deist F, de Saint Basile G, Delaunay J, Schwarz K, Casanova JL, Blanche S, Fischer A. Autoimmunity in Wiskott-Aldrich syndrome: Risk factors, clinical features and outcome in a single-center cohort of 55 patients. Pediatrics 2003; 111: 622-627 5. Eddou H, Helissey C, Konopacki J, Souleau B, de Revel T, Malfuson JV. Evans syndrome: Be careful of over-diagnosis.

Rev Med Interne 2012; 33 (3): 155-158 6. Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn U. Williams Hematology. Sixth edition 2001, McGraw-Hill Companies, New York 7. Philip Lanzkowsky. Manual of Pediatric Hematology and Oncology, Fifth edition, 2011 Academic Press, London

Letter to the Editor

10.5505/tjh.2012.23922

Oral Findings and Inherited Disorders of Platelet Function Oral Bulgular ve Kalıtsal Trombosit İşlevi Bozuklukları Beuy Joob1, Viroj Wiwanitkit2 Sanitation 1, Medical Academic Center, Bangkhae, Bangkok, Thailand Wiwanitkit House, Bangkhae, Bangkok, Thailand

1 2

Reply,

To the Editor, The recent report on oral findings and inherited disorders of platelet function (IDPF) was very interesting [1]. Hatipoglu et al. concluded that, “IDPF have a negative effect on periodontal tissues [1].” I would like to raise some relevant issues. First, there are many factors that can cause periodontal disorders, such as behavior, nutrition, and other underlying diseases [2], which were not completely investigated in their study. Hence, the conclusion of a direct relationship might not be warranted. Second, the measured platelet parameters and their relationship to periodontal disease would be an interesting focus of further study. A recent publication showed that there wasn’t a clinical correlation between the platelet count and periodontal disorder, but between mean platelet volume and periodontal disorder in the study group [3].

References 1. Hatipoglu MG, Kansu O, Büyükaşık Y. Clinical investigation of oral findings in inherited disorders of platelet function.

Turk J Hematol. 2011; 28(4): 294-298.

2. Kornman KS.Mapping the pathogenesis of periodontitis: a new look. J P eriodontol. 2008 Aug;79(8 Suppl):1560-8.

3. López R, Loos BG, Baelum V. Hematological features in adolescents with periodontitis. Clin Oral Investig. 2011 Oct 19. [Epub ahead of print]

I would like to thank authors of the letter for their suggestions. This study used a case-controlled design and, as we noted in the report, the aim of the study was to identify clinical oral findings in IDPF patients. Care was taken for similar hematological aspects. Patients with other systemic diseases or conditions were excluded from the study and control groups, and we attempted to differentiate behavioral, nutritional, and underlying disease etiologies from those that were hematological.

Inherited platelet function disorders were the primary focus of the study, which showed that was indicated molecular and biochemical studies in this topic which has been started in recent times [1-3]. We think that more comprehensive studies are needed to further delineate the effect of IDPF on periodontium.

Assist. Prof. Müjgan Güngör Hatipoğlu, Department of Oral, Tooth and Jaw Radiology, University of Dumlupinar, Faculty of Dentistry, Kütahya, Turkey

References 1. Spolidorio LC, Herrera BS, Coimbra LS, Figueiredo MN, Spolidorio DM, Muscará MN. Short-term induction of thrombocytopenia delays periodontal healing in rats with periodontal disease: Participation of endostatin and vascular endothelial growth factor. J Periodontal Res 2010; 45 (2): 184-192

Address for Correspondence: Beuy Joob, M.D., Sanitation1, Medical Academic Center, Bangkok, Thailand Phone: 662 413 24 36 E-mail: beuyjoob@hotmail.com Received/Geliş tarihi : December 18, 2011 Accepted/Kabul tarihi : December 19, 2011

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2. Javed F, Al-Askar M, Al-Rasheed A, Al-Hezaimi K. Significance of the platelet-derived growth factor in periodontal tissue regeneration. Arch Oral Biol 2011; 56 (12): 1476-1484

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3. Coimbra LS, Rossa C Jr, Guimarรฃes MR, Gerlach RF, Muscarรก MN, Spolidorio DM, Herrera BS, Spolidorio LC. Influence of antiplatelet drugs in the pathogenesis of experimental periodontitis and periodontal repair in rats. J Periodontol 2011; 82 (5): 767-777

Letter to the Editor

10.5505/tjh.2012.24382

Extramedullary Plasmacytoma Presenting as a Mediastinal Mass: A Comment Mediastende Kitle ile Beliren İlik Dışı (Ekstramedüller) Plazmositom: Bir Yorum Selami K. Toprak1, Sema Karakuş1, Pervin Topçuoğlu2 Baskent University, School of Medicine, Department of Hematology, Ankara, Turkey Ankara University, School of Medicine, Department of Hematology, Ankara, Turkey

1 2

To the Editor, We read with great interest the recent publication about extramedullary plasmacytoma (EMP) by Shukla et al., in which they concluded that plasmacytoma involving the mediastinum is an early indication of the diagnosis of occult multiple myeloma (MM), which should be included in the differential diagnosis of mediastinal masses [1].

About 3%-5% of plasma cell neoplasms may present with isolated extramedullary involvement [2]. Because this disease can be confused with several malignancies—including lymphomas—microscopic and immunohistochemical evaluation are essential for accurate diagnosis [3,4]. We would like to share our experience concerning the refractory course of EMP. A 62-year-old male presented in 2009 with a swollen left-anterior cervical lesion. MRI showed lytic lesions on several bony elements (suggestive of MM) and a plasmacytoma-like appearance of the swollen region. The patient’s medical history was positive for similar swelling of the same region in 2000. The lesion was totally excised and diagnosed as EMP. Subsequently, the patient received radiation therapy (XRT) and the lesion remained silent for the following years. In 2009 he was diagnosed as stage II MM, subsequently given systemic chemotherapy, and had a very good partial response. The lesion persisted, decreasing in size by only 10%. Although the patient’s medical history and MRI findings were suggestive of the diagnosis,

biopsy samples were histopathologically assessed to better establish the final status of the lesion, and showed fibrotic tissue remnants.

The patient underwent autologous hematopoietic stem cell transplantation (AHSCT) using high-dose melphalan.

Urine immunofixation electrophoresis was positive during the front-line chemotherapy, but was negative post AHSCT. The treatment was considered successful in terms of MM response, but the lesion (thought to be a plasmacytoma) persisted, at 30% of its original size. Our opinion—and that of several surgeons—was that any surgical intervention could lead to major life-threatening complications; therefore, routine outpatient follow-up provided.

MRI is a useful tool for evaluating the extent and size of EMP lesions; however, the role of MRI in staging EMP is not clear. Diagnosis of EMP is based on immunohistochemistry and the exclusion of a systemic plasma-cell proliferative disorder [5]. In the presented case, although not proven histopathologically, relapse and subsequent progression to MM, as is expected in 30% of cases, occurred 9 years after the initial diagnosis. Interestingly, front-line high-dose chemotherapy resulted in an excellent MM response, similar success rates could not be obtained in terms of lesion size, which persisted with a lower than 50% decrease. As EMPs are highly radiosensitive tumors, XRT alone is considered the treatment of choice, with local control rates of 80%-100% [5]. Yet, this treatment modal-

Address for Correspondence: Selami K. Toprak, M.D., Başkent Üniversitesi, Tıp Fakültesi, Hematoloji Bilim Dalı, Ankara, Turkey Phone: +90 312 212 29 12 E-mail: sktoprak@yahoo.com Received/Geliş tarihi : February 2, 2012 Accepted/Kabul tarihi : February 6, 2012

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ity requires a definitive diagnosis, which in the presented case was lacking due to biopsy sampling concerns. Relapse and progression to MM are the main concerns during the course of EMP. In 2000 the presented patient received only XRT following surgical excision of the lesion and did not receive adjuvant chemotherapy; however, given the lack of published evidence supporting its use in the management of EMP, this is acceptable [1]. Ahmed et al. reported a patient with an adrenal EMP that had a partial response to initial AHSCT and complete remission after repeat AHSCT [6]. Their case may also be an explanation for the merely 30% decrease in the lesion size in our case.

Although EMP has a relatively good prognosis and responds well to local treatment, adjuvant chemotherapy should be a consideration for the management of the disease. Additionally, EMP patients should remain under close lifelong observation, with yearly evaluation to rule out MM. Written informed consent was obtained from the patient.

Conflict of Interest Statement None of the authors has any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.

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References 1. Shukla A, Bansal V, Bhutani R, Kumar G, Sharma J, Solanki S, Mehta A. Extramedullary plasmacytoma presenting as a mediastinal mass. Turk J Hematol 2011; 28: 228-231 2. Alexiou C, Kau RJ, Dietzfelbinger H, Kremer M, Spiess JC, Schratzenstaller B, Arnold W. Extramedullary plasmacytoma: Tumor occurrence and therapeutic concepts. Cancer 1999; 85; 2305-2314 3. Boll M, Parkins E, O’Connor SJ, Rawstron AC, Owen RG. Extramedullary plasmacytoma are characterized by a ‘myeloma-like’ immunophenotype and genotype and occult bone marrow involvement. Br J Haematol 2010; 151 (5): 525-527 4. Dimopoulos MA, Kiamouris C, Moulopoulos LA. Solitary plasmacytoma of bone and extramedullary plasmacytoma.

Hematol Oncol Clin North Am 1999; 13 (6): 1249-1257 5. Vanan I, Redner A, Atlas M, Marin L, Kadkade P, Bandovic J, Jaffe ES. Solitary extramedullary plasmacytoma of the vocal cord in an adolescent. J Clin Oncol 2009; 27 (35): e244-247 6. Ahmed M, Al-Ghamdi A, Al-Omari M, Aljurf M, Al-Kadhi Y. Autologous bone marrow transplanation for extramedullary plasmacytoma presenting as adrenal incidentaloma. Ann Saudi Med 2009; 29 (3): 219-222

Letter to the Editor

10.5505/tjh.2012.24865

Megadose Methylprednisolone (MDMP) for the Treatment of Steroid Refractory Patient with Diamond-Blackfan (D-B) Anemia Diamond-Blackfan Anemisinin Megadoz Metilprednizolon ile Tedavisi Şinasi Özsoylu Retired Prof of Pediatrics, Hematology, Hepatology, Ankara, Turkey Honorary fellow of American Academy of Pediatrics Honorary member of American Pediatric Society

To the Editor, Dr. Malbora [1] and his colleagues reported metoclopramide in addition to corticosteroid in the treatment of a patient with steroid refractory Diamond-Blackfan (D-B) anemia in the recent issue of the Journal [1].

Although corticosteroid treatment had been used in the treatment of patients with D-B anemia, I reported first time that MDMP is effective (in appropriate doses) and safe in those patients including refractory and resistant cases [1-14], which was supported by Bernini et al’[15] from USA. Therefore, I was underimpression that MDMP would be preferred treatment for those patients including steroid refractory and resistant ones.

I would like to reemphasize that MDMP is different than pulse methylprednisolone and conventional corticosteroid administration as was used for D-B anemia [5,6] long period as advised. In conventional corticosteroid treatment 1-2 mg/kg/day dose in gven 6-8 or 12 hours intervals. In pulse methyprednisolone treatment 1 g methlyprednisolone (MP) is giving i.v within 4 hours, at any time of the day.

In MDMP treatment, methylprednisolone MP initial dose 30-100 mg/kg day is given within 10 to 15 minutes IV or at once orally arouned 6 am, 3 days then 20-50 mg/ kg, day for 4 days subsquently 10,5,2,1 mg/kg dose given for 1 week each according to response).

Although the authors also used 30 mg/kg day for 3 days followed by 20 mg/kg day for 4days, which were suggested by us for only acule ITP cases [15].

I would like to bring to the attention that clorpromide is potentially carcinogenic drug but methylprednisolone when administered as suggested by us, does not have any serious side effcs as supported by Bernini et al [16].

Importantly, I would like take to add that the very first patient treated by MDMP has been in remission about 30 years.

References 1. Malbora B, Avcı Z, Özbek N. Successful treatment of refractory Diamond-Blackfon anemia using metoclopromid prednisolon. Turk J Hematol 2012;29:191-192.

2. Özsoylu Ş. High-dose intranevous corticosteroid for a patient with Diamond-Blackfan syndrome refractory to classical prednisone treatment. Acta Haematol 1984;71:207-210.

Address for Correspondence: Şinasi ÖZSOYLU, M.D., Beysukent Altınşehir Sitesi No:30, Ankara, Turkey Phone: +90 312 235 41 88 E-mail: sinasiozsoylu@hotmail.com Received/Geliş tarihi : November 21, 2011 Accepted/Kabul tarihi : November 21, 2011

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3. Özsoylu Ş. Bolus methylprednisonole for refractory Diamond-Blackfan syndrome. Lancet 1984;2:1033.

4. Özsoylu Ş. High-dose intravenous corticosteroid treatmnt in refractory Diamond-Blackfan syndrome. Türkiye Klinikleri Tıp Bilimleri Araştırma Dergisi 1985;3:178-181.(in Turkish) 5. Özsoylu Ş. Hihg-dose intravenous methylprednisolone for the refractory or resistent Diamond Blackfan anemia. Eur J Hematol 1987;39:190-191.

6. Özsoylu Ş. High-dose intravenous corticosteroid treatment for patient with Diamond-Blackfan syndrome resistant or refractory to conventional treatment. Am J pediatr Hematol/ Oncol 1988;10:210-217.

7. Özsoylu Ş. High-dose methylprednisolone for constitional pure red cell apasia. Blood 1988;72.1839.

8. Özsoylu Ş. High-dose intravenous methylprednisolone (HIVMP) in hematologic disorders. Hematology Reviews 1990;4(4):197-207.

9. Özsoylu Ş. Oral megadose methylprednisolone for treatment of Diamond-Blackfan anemia. Brit J Hematol 1992;81:135136.

10. Özsoylu Ş, Albayrak D, Güler E, Duru F. Oral megadose methylprednisolone for Diamond-Blackfan anemia. Eur J Pediatr 1992;151:629.

11. Özsoylu Ş. Megadose methylprednisolone for DiamondBlackfan anemia. Am J Hematol 1993;42:335.

12. Özsoylu Ş. Oral megadose methylprednisolone the treatment of Diamond-Blackfan anemia. Pediatr Hematol/Oncol 1994;11:561-562.

13. Özsoylu Ş. Oral megadose methylprednisolone DiamondBlackfan anemia. Blood 1994;84:3245-6.

14. Özsoylu Ş. Megadose methylprednisolone for hematologic and non-hematologic disorders. Turk J Hematol 2010;27:130-131.

15. Özsoylu Ş. Megadose methylprednisolone for childhood idiopathic thrombocytopenic purpura (ITP). Turk J Med Sci 2005;35:347-356.

16. Bernini JC, Carillo M, Buchanon GR. High-dose intravenous methylprednisolone therapy for patients with DiamondBlackfan anemia refractory to conventional doses of prednisone. J Pediatr 1995;127:1654-1659.

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Dear Editor, We are grateful for correspondence by Prof. Özsoylu.

In our patient, we administered pulse steroid with a dose of 30 mg/kg/day for three days, followed by 20 mg/kg/ day which is tapered weekly (Figure 1 of our letter). Our patient used steroid treatment for 20 weeks before metoclopramide treatment. We administered all steroid doses at once in the morning between 6-8 a.m. We believe this protocol is in line with the references mentioned by Prof. Özsoylu. It has also been mentioned that clorpromide is a potentially carcinogenic drug. However, we administered metoclopramide to our patient instead of clopromide. Although in some studies metoclopramide is accused to have an indirect relation with breast cancer, this effect has not been confirmed yet (ref. 9).

Sincerely, Barış Malbora, M.D.

Dr. Sami Ulus Research and Training Hospital of Women’s and Children’s Health and Diseases, Babür Caddesi No:44 (06080) Altındağ, Ankara, Turkey Tel: +90 305 60 00 Fax: +90 317 03 53 GSM: +90 533 6413841 E mail: barismalbora@gmail.com

bmalbora@yahoo.com

Images in Hematology

10.5505/tjh.2012.60973

EDTA-Dependent Pseudothrombocytopenia EDTA-İlişkili Yalancı Trombositopeni Ayşenur Bahadır, Erol Erduran, Beril Dilber, Nalan Üçüncü Karadeniz Technical University, School of Medicine, Department of Pediatric Hematology, Trabzon, Turkey

Ethylenediaminetetraacetic acid-dependent pseudothrombocytopenia (EDTA-DP) occurs rarely, with an incidence rate of 0.09%-0.21%. EDTA-DP may lead to misdiagnosis since low platelet count may determine by outomated blood counter in which EDTA as anticoagulant is used [1,2]. It is diagnosed via microscopic detection of platelet aggregates in a peripheral blood smear. Additionally, in vitro clumping of the platelets occurs due to autoantibodies that develop against platelet surface antigens [1]. Although automatic blood analyzers are widely used, peripheral blood smear is the hematological gold standard for definitive diagnosis. When the thrombocyte number is low the morphology and appearance of platelets must be confirmed via a peripheral blood smear. As such, laboratory errors and excessive expenditure of time can be avoided.

A

A 7-year old boy was referred to our hospital due to thrombocytopenia The patient’s platelet count was determined by a different laboratory to be between 10x109/L and 15 x109/L. History of familial or acquired hemorrhagic disorders, lymphadenopathy, drug intake, blood transfusion, systemic disease, and recent viral infection was negative. Physical examination was normal. Laboratory findings were as follows: platelet count: 13 x109/L; hemoglobin: 12.2 g/dL; white blood cell count: 14,8 x109/L; mean platelet volume: 8.8 fL. Aggregation of platelets was observed via microscopic examination of the peripheral blood smear (Figure 1A and 1B). Direct Coombs’ test, and cold agglutinin, serum immunoglobulins, antinuclear antibody, total protein, and lipid levels were normal.

Using sodium citrate, the patient’s platelet count was 249 x109/L (Figure 2).

B

Figure 1A and 1B: Aggregation of platelets in the peripheral blood smear with EDTA (Wright-Giemsa, 100x).

Address for Correspondence: Ayşenur BahadIr, M.D., Karadeniz Teknik Üniversitesi, Tıp Fakültesi, Pediatrik Hematoloji Bilim Dalı, Trabzon, Turkey Phone: +90 462 377 57 76 E-mail: aysenurkbr@yahoo.com Received/Geliş tarihi : July 4, 2011 Accepted/Kabul tarihi : August 15, 2011

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Bahadır A, et al: Pseudothrombocytopenia

Turk J Hematol 2012; 29: 311-312

tion of platelets in EDTA-DP is prevented by other anticoagulants, such as sodium citrate or heparin [3]. The presented patient was referred to our clinic due to thrombocytopenia. The patient’s peripheral blood smear had not been evaluated prior to presentation to our facility EDTADP was diagnosed when clustered platelets were separately seen on the peripheric blood smear in our hematology clinic. Nowadays, automatic blood count device has been used. But still, peripheric blood semear is the gold standart for hematologists for definitive diagnosis. When thrombocytopenia is detected, peripheric blood smear should be performed from the finger tip and examined to see whether or not it is a real or pseudothrombocytopenia.

Conflict of Interest Statement Figure 2: Normal peripheral blood smear with sodium citrate (Wright-Giemsa, 100x).

We considered that EDTA-DP induced platelet agglutination in vitro, resulting in the observed decrease in the platelet count, and therefore diagnosed the patients as EDTA-DP. The patient was not given any treatment. During 4 months of outpatient follow-up the patient’s platelet count varied between 200 and 409 x109/L. Written informed consent was obtained.

EDTA-DP may be erroneously diagnosed due to a false low platelet count measured in an automated blood analyzer in which EDTA was used as anticoagulant. Aggrega-

None of the authors has any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.

References 1. Yoneyama A, Nakahara K. EDTA-dependent pseudothrombocytopenia-differentiation from true thrombocytopenia.

Nippon Rinsho 2003; 61 (4): 569-574 2. Carrillo-Esper R, Contreras-Dominguez V. Pseudothrombocytopenia induced by ethylenediaminetetraacetic acid in burned patients. Cir Cir 2004; 72 (4): 335-338 3. Celkan T, Toptan H, Dogru O. Spurious thrombocytopenia.

Turk Arch Ped 2009; 44 (2): 73-74

Erratum Please kindly be informed that the units of laboratory measurements should be corrected in a recent article by Özkaya et al (TJH 2012;29:195-196), entitled “Carbamazepine -induced red blood cell aplasia: A case report”, as follows: Red blood cell counts x1012 L-1, white blood cell counts x109 L-1, platelet counts x109 L-1, serum iron level and total iron binding capasity µg dL-1.

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