tjh-2010-3 by LookUs Scientific

Issue 3

September 2010

40 TL

ISSN 1300-7777

Volume 27

Review Article Iron transport to mitochondria NamÕk Özbek; Ankara, Turkey

Research Articles Clinical course in children with ITP IúÕn Yaprak et al.; øzmir, Turkey

Active and passive immunization in acute leukemia Tuphan Kanti Dolai et al.; India

Thrombophilia and cerebral venous thrombosis Osman Yokuú et al.; østanbul, Ankara, Turkey

Posterior reversible leukoencephalopathy syndrome in children BarÕú Malbora et al.; Ankara, Turkey

Hematological effect of calabash chalk Amabe Otoabasi Akpantah et al.; Nigeria

Flow cytometry and CD87 in myeloma Murat Albayrak et al.; Ankara, østanbul, Turkey

Cover Picture: Evren Özdemir Mostar Bridge

NATIONAL HEMATOLOGY CONGRESS

November 3-6, 2010 Gloria Congress Center

Belek - Antalya, Turkey

Scientific Secretariat

Congress Organisation

Turkish Society of Hematology Address: Turan GĂźneĹ&#x; Bulv. Sancak Mah.613 Sok. No: 8 Ă&#x2021;ankaya - Ankara / TĂźrkiye 1IPOF t 'BY & NBJM UIEPĂśT!UIE PSH US t 8FC XXX UIE PSH US

Serenas Tourism Congress & Organisation Address: Turan GĂźneĹ&#x; BulvarÄą 5. Cadde No:13 06550 YÄąldÄąz, Ă&#x2021;ankaya-Ankara / TĂźrkiye 1IPOF t 'BY E-mail: ulusalhematoloji2010@serenas.com.tr / gokce.onay@serenas.com.tr Web: www.serenas.com.tr

www.ulusalhematoloji2010.org

Editor-in-Chief

International Review Board

Aytemiz Gürgey

Nejat Akar (Turkey) Görgün Akpek (USA) Serhan Alkan (USA) Çi÷dem Altay (Turkey) Ayhan Çavdar (Turkey) M.SÕraç Dilber (Sweden) Ahmet Do÷an (USA) Jawed Fareed (USA) Gösta Gahrton (Sweden) Dieter Hoelzer (Germany) Marilyn Manco-Johnson (USA) Andreas Josting (Germany) Emin Kansu (Turkey)

Winfried Kern (Germany) Nigel Key (USA) Korgün Koral (USA) Abdullah Kutlar (USA) Ghulam Mufti (UK) Gerassimos A. Pangalis (Greece) Santiago Pavlovsky (Argentina) Antonio Piga (Italy) Ananda Prasad (USA) Orhan Sezer (Germany) Nüket Tüzüner (Turkey) Catherine Verfaillie (USA) Claudio Viscoli (Italy)

Contact Information

All other inquiries should be adressed to TURKISH JOURNAL OF HEMATOLOGY Address: ølkbahar Mahallesi, Turan Güneú BulvarÕ 613. Sk. No:8 06550 Çankaya, Ankara/Turkey Phone: +90 312 490 98 97 Fax: +90 312 490 98 68 E-mail: info@tjh.com.tr ISSN: 1300-7777

Corinne Can

Editorial Correspondence should be addressed to Dr. Aytemiz Gürgey Editor-in-Chief Address: Hacettepe University Faculty of Medicine Pediatric Hematology Department 06100 SÕhhiye, Ankara/Turkey Phone: +90 312 305 41 17 Fax: +90 312 305 41 17 E-mail: agurgey@hacettepe.edu.tr

Statistic Editor

Turkish Society of Hematology

Associate Editors

Mutlu Arat Muzaffer Demir Reyhan Diz Küçükkaya Mehmet Ertem Hale Ören Mehmet Ali Özcan Teoman Soysal Ayúegül Ünüvar Celalettin Üstün Neúe YaralÕ Past Editors

Erich Frank Orhan Ulutin Hamdi Akan Senior Advisory Board

Orhan Ulutin Yücel Tangün Osman ølhan Language Editor

Web page www.tjh.com.tr

Mutlu Hayran

Cover Picture:

Muhit Özcan, President Mutlu Arat, General Secretary Hale Ören, Vice President Muzaffer Demir, Research Secretary Teoman Soysal, Treasurer Fahir Özkalemkaú, Member Mehmet Sönmez, Member

Evren Özdemir was born in 1969, Bahçe, Turkey. He is currently working in Hacettepe University Oncology Institute, Department of Clinical Oncology, Ankara, Turkey.

Türk Hematoloji Derne÷i, 07.10.2008 tarihli ve 6 no’lu kararÕ ile Turkish Journal of Hematology’nin Türk Hematoloji Derne÷i økdisadi øúletmesi tarafÕndan yayÕnlanmasÕna karar vermiútir.

Editorial Secretary

øpek Durusu Bengü Timoçin

Sahibi Türk Hematoloji Derne÷i adÕna Muhit Özcan

Sorumlu YazÕ øúleri Müdürü Aytemiz Gürgey

YayÕn ve Yönetim Yeri Türk Hematoloji Derne÷i Türk Oca÷Õ Cad. 17/6 Ca÷alo÷lu-Eminönü-østanbul Üç ayda bir yayÕnlanan yerel bilimsel dergidir.

Publisher: AVES YayÕncÕlÕk Address: KÕzÕlelma cad. 5/3 34096 FÕndÕkzade-østanbul-Turkey Phone: +90 212 589 00 53 Fax: +90 212 589 00 94 E-mail: info@avesyayincilik.com Place of printing: Görsel Dizayn Ofset MatbaacÕlÕk Tic. Ltd. ùti. - +90 212 671 91 00 Date of printing: August 2010 Broadcast as: Local periodical

A-I

AIMS AND SCOPE The Turkish Journal of Hematology is the regular publishing organ of the Turkish Society of Hematology. This periodical journal covers subjects on hematology. The journal is an independent, peer-reviewed international periodical, published quarterly (March, June, September and December) in English language. The Turkish Journal of Hematology is a nonprofit scientific peer reviewed journal. The journal's editorial and publishing process is adherent to standards of ICMJE, WAME, and COPE. The aim of the Turkish Journal Hematology is to publish original research papers of highest scientific and clinical value on hematology. Additionally, educational material, reviews on basic developments, editorial short notes, case reports, original views and letters from specialists on hematology, and hematology medicine covering their experience and comments as well as social subjects are published. General Practitioners interested in hematology, and internal medicine specialists, are also our target audience, and we will arrange the Turkish Journal of Hematology according to their needs. The Turkish Journal of Hematology is indexed in - Science Citation Index Expanded - Embase - Scopus - Cinahl - Index Copernicus - Gale Cengage Learning - EBSCO - DOAJ - ProQuest - Tübitak Ulakbim Türk TÕp Dizini Subscription Information The Turkish Journal of Hematology is sent free of charge to hematologists and academicians in our country as well as to other specialists interested in hematology. All published volumes in full text can be reached free of charge through the web site www.tjh.com.tr Adress: Ilkbahar mah. Turan Güneú BulvarÕ 613. sok. No: 8 Çankaya-Ankara, Turkey Telephone: +90 312 490 98 97 Fax: +90 312 490 98 68 Web page: www.tjh.com.tr E-mail: info@tjh.com.tr Permissions Requests for permission to reproduce published material should be sent to the editorial office. Editor: Prof.Dr. Aytemiz Gürgey Adress: Ilkbahar mah. Turan Güneú BulvarÕ 613. sok. No: 8 Çankaya-Ankara, Turkey Telephone: +90 312 490 98 97 Fax: +90 312 490 98 68 Web page: www.tjh.com.tr E-mail: info@tjh.com.tr Instructions for Authors Instructions for authors are published in the journal and on the web page www.tjh.com.tr Material Disclaimer The author(s) is (are) responsible from the articles published in the Turkish Journal of Hematology. The editor, editorial board and publisher do not accept any responsibility for the articles. The journal is printed on acid-free paper.

A-II

INSTRUCTION TO AUTHORS The Turkish Journal of Hematology accepts invited review articles, research articles, brief reports, case reports, letters to the editor, and images in Hematology on subjects within the scope of hematology, on the condition that they have not been previously published elsewhere. All papers are subject to editorial revision for purpose of conformity to the style adopted by the Journal. Evaluation is a double blind kind of evaluation.

Images in Haematology

Original research articles

Preparation of Manuscript

Regular Articles Maximum length for a Regular Article is 4,000 words of text. Abstracts must not exceed 300 words with subheadings; objective, material and methods, results, conclusion. Submissions are limited to a total of 7 figures/ tables. References should be limited to 50. The sections of a Regular Article should include Abstract, Introduction, Material and Methods, Results, Discussion, References, Figure Legends. The journal's editorial and publishing process is adherent to standards of ICMJE, WAME, and COPE.

Each of the following sections of the manuscript should be typed on separate pages. Title Page should include (in Turkish when possible): (a) title of the article in a concise but informative style, (b) first name, middle initial, last name of each author, (c) name of department(s) and institution(s) to which the work should be attributed, (d) name and address of author responsible for correspondence for the manuscript, (e) name and address of author to whom requests for reprints should be addressed, (f) source(s) of support in the form of grants, equipments, drugs, etc., and (h) short running title of no more than 40 characters.

Brief Reports Short manuscripts definitively documenting either experimental results or informative clinical observations will be considered as brief report. Brief Reports should not exceed 1,000 words of text not counting the abstract, figure legends, and references; abstracts must not exceed 300 words. Review Articles Review articles should not exceed 4,000 words in length, must include an abstract of 300 words or fewer, and may not have more than 100 references. Letters to the Editor Letters can include no more than 400 words of text, 5-10 references, and 1 figure or table. No abstract is required, but please include a brief title.

A-III

Authors can submit for consideration an illustration (or, where appropriate, two or more related images) which is interesting, instructive and visually attractive, with a few lines of explanatory text and references. The images (e.g. a clinical photograph, radiology, cytology, histology, a laboratory test) should be submitted in a digital format.

Authorship Each author should have participated sufficiently in the work to take public responsibility for the content. Any part of an article critical to its main conclusions must be the responsibility of at least one author. All authors’ signatures should be included in the title page. The signed statement on absence of conflict of interests between authors is required. Acknowledgments Acknowledge support received from individuals, organizations, grants, corporations, or any other sources. For work involving a biomedical product or potential product partially or wholly supported by

corporate funding, a note must be included stating: This study was supported (in part) by research funding from (company name) to (authors’ initials). Grant support, if received, needs to be stated and the specific granting institution(s) name(s) and grant numbers provided when applicable. Authors are expected to disclose, on the title page of their manuscripts, any commercial or other associations that might pose a conflict of interest in connection with the submitted article. All funding sources supporting the work, and institutional or corporate affiliations of the authors, should be acknowledged on the title page. Ethics When reporting experiments on human subjects indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation. An approval of research protocols by ethic committee in accordance with international agreements (Helsinki Declaration of 1975, revised 2002 available at http://www.wma.net/e/policy/b3.htm, “Guide for the care and use of laboratory animals www.nap.edu/catalog/5140.html/) is required for experimental, clinical and drug studies. Do not use patient names, initials, or hospital numbers, especially in any illustrative material. Manuscripts reporting the results of experimental investigations on human subjects must include a statement to the effect that procedures had received official institutional approval. The statement on the informed consent of patients is required. We frown upon unethical practices such as plagiarism, duplicate publication, ‘salami’ publication, and efforts to influence the review process with practices such as gifting authorship, inappropriate acknowledgements and references. Also, authors must respect patients’ right to privacy.

Abstract and key words: The second page should include an Abstract which does not exceed 300 words. For manuscripts sent from Turkey, a title and abstract in Turkish are required. The abstract should state the purpose of the study or investigation, basic procedures, methods, main findings, specific data, statistical significance and the principal conclusions. Provide 3 to 10 key words below the abstract to assist indexers. Use terms from the Medical Subject Headings List of Index Medicus. The text should be divided into sections with headings as follows: Objective, Materials and Methods, Results and Conclusion. Other types of articles such as case reports, reviews, perspectives and editorials will be published according to uniform requirements. Introduction: State the purpose of the article and summarize the rationale for the study. Materials and Methods: Describe your selection of the observational or experimental subjects clearly. Identify the methods and procedures in sufficient detail to allow other workers to reproduce the results. Give references to established methods (including statistical methods), provide references and brief modified methods, give reasons for using them and evaluate their limitations. Identify all drugs and chemicals used, including generic name(s), dose(s) and route(s) of administration. Statistics: Describe statistical methods in enough detail to enable a knowledgeable reader with access to the original data to verify the reported results. Give details about randomization, describe treatment complications, give number of observations, and specify any computer program used. Results: Present your results in logical sequence in the text, tables and illustrations. Do not repeat in the text all the data in the tables or illustrations; emphasize or summarize only important observations.

A-IV

Discussion: Emphasize the new and important aspects of the study and the conclusions that follow them. Link the conclusions with the goals of the study but avoid unqualified statements and conclusions not completely supported by your data. References: Identify references in text, tables and legends by Arabic numerals in parentheses. Number references consecutively in the order in which they are first mentioned in the text. The titles of the journals should be abbreviated according to the style used in Index Medicus; consult List of Journals Indexed in Index Medicus. Include among the references any papers accepted but not yet published, designating the journal and followed by “in press”. Articles in Journals 1. List all authors Williams RL, Hilton DJ, Pease S, Wilson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukemia inhibitory factor (LIF) maintains the developmental potential of embryonic stem cells. Nature 1988;336:684-687. 2. Organization as author Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of syngeneic bone marrow graft without preconditioning in post-hepatitis marrow aplasia. Lancet 1977;2:742-744. 3. Complete book Adams DO, Edelson PJ, Koren HS. Methods for studying mononuclear phagocytes. San Diego: Academic Press, 1981. 4. Chapter of book Smolen JE, Boxer LA. Functions of Neutrophils. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA, eds. Hematology. 4th ed. New York: McGraw-Hill, 1991: 780-794.

5. Abstract Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract). 6. Letter to the Editor Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further definition of 20q deletion in myeloid leukemia using fluorescence in situ hybridization. Blood 1994;84:2821-2823. Tables Type each table on a separate sheet. Number tables consecutively in the order of appearance in the text and supply a brief title for each. Give each column a short or abbreviated heading. Place explanatory statistical measures of variations such as standard deviation or standard error of mean. Be sure that each table is cited in the text. Illustrations Figures should be professionally drawn and photographed. Please send sharp, glossy, black and white photographic prints, usually 9 x 13 cm. Affix a label to the back of each figure indicating the number of the figure, first author’s name and top of the figure. Type legends for illustrations double-spaced, starting on a separate page with Arabic numerals corresponding to the illustrations. Explain the internal scale and identify method of staining. Units of Measurement Measurements should be reported in the metric system in terms of the International System of Units (SI). Consult SI Unit Conversion Guide, New England Journal of Medicine Books 1992, when necessary. Abbreviations and Symbols Use only standard abbreviations. Avoid abbreviations in the title and abstract. The full term for which an abbreviation stands should precede its first use in the text unless it is a standard abbreviation.

ONLINE MANUSCRIPT SUBMISSION PROCESS Manuscripts can be submitted online at www.journalagent.com/tjh/ The online system consists of four main parts: manuscript submission module (MSM), editorial module, admin module and referee module. The editorial module, admin module and referee module work on the background and will not be open to the end user. The term module used in this document refers only to the MSM. As part of the peer-review system, authors will also receive the referee reports and can observe he current status of their manuscript(s) online. An online help is also available during the submission process. The module accepts the body of the manuscript as a whole document; thus, documents should be completed as a .doc or .rtf file before submission. The supported file extensions, Table 1.

Supported formats .doc (MS Office for Windows or Macintosh) .rtf (rich text format)

Supported fonts Arial Times Helvetica Times New Roman Courier

fonts and other formats are given in Table 1. Tables, figures and pictures Tables should be created in your original wordprocessing software or inserted in the original file from Excel or another compatible software. Please ensure the table or figure created complies with the limitations mentioned in Table 1. Tables created as a picture file are problematic and are not advised. Figures should be embedded in the original file, but the system also requires that they be sent separately. The supported image files are given in Table 1. Symbols Special characters not available on the keyboard can be accessed either from the insert menu (select symbol) or by selecting

A-V

symbol as a font from the font window of the Formatting toolbar. Please check these characters in your original file and proofs as the softwares tend to replace these characters with others if they are unreadable. Submission online To submit a manuscript, you must first establish a login name and password, which can then be used indefinitely. After you login to the module, the first page accessed allows you to track the status of your current or previous manuscripts. To submit a new manuscript, login with your user name and password, then click on “Author” on your main menu. From this page, select “new article” pull-down menus. The next page is for the details of the institutions of the authors. On the next page where the authors are listed, the instructions and menus allow you to select the appropriate institution for each author Supported images .bmp .jpg .jpeg .gif .tif

from the list. Please remember to check the box next to the corresponding author. The title page requires only the title; special characters listed on the bottom of the title input window can be used when needed. On the summary page which follows, please write the abstract of your manuscript in the appropriate window. This is followed by a keyword input page, which allows up to 10 keywords. Any special notes to the editor can be recorded on the next page. The body and figures of the manuscript are submitted on the next page. Locate the manuscript in your PC, then write the type of the file you are sending and give a description. Use this page to also send your images. Send your manuscript using the designated button on the next page. All pages are supported with help menus; if you require additional help or experience a problem, please send an e-mail to info@tjh.com.tr

Manuscripts that have passed an initial screening by the. Editors are reviewed by members of the Editorial Board and/or other experts in the field. The Editors select the reviewers and make the final decision on the manuscript. Referees who review a manuscript remain unknown to the authors. Every manuscript is treated by the Editors and reviewers as privileged information, and they are instructed to exclude themselves from review of any manuscript that may involve a conflict of interest or the appearance of such. Following initial peer-review, articles judged worthy of further consideration often require revisions. Revised manuscript generally must be received within 3 months of the date on the initial decision. Extensions must be requested from the Associate Editor at least 2 weeks before the 3-month revision deadline expires. Otherwise Turkish Journal of Hematology will reject manuscripts which do not received within 3 months of the date on the initial revesion decision. A Copyright transfer and conflict of interest form signed by all authors, must also be submitted by fax to +90 3124909868. Both forms can be found at the web site www.tjh. com.tr Authors of accepted manuscripts will receive electronic page proofs directly from the printer and are responsible for proofreading and checking the entire article, including tables, figures, and references. Page proofs must be returned within 48 hours to avoid delays in publication. English-language editing All manuscripts are professionally edited by English language editor before publication. Online Early Turkish Journal of Hematology published abstracts of accepted articles online in advance of their publication in a printed issue.

CONTENTS Review Article 137

Iron transport: From enterocyte to mitochondria NamÕk Özbek, Ankara, Turkey

Research Articles 147

Variant clinical courses in children with immune thrombocytopenic purpura: Sixteen year experience of a single medical center

156

Ef¿cacy of immunization against hepatitis B virus infection in acute leukemia

IúÕn Yaprak, Berna Atabay, økbal Durak, Meral Türker, Haldun Öniz, Esra Arun Özer, øzmir, Turkey

Tuphan Kanti Dolai, Manoranjan Mahapatra, Hara Prasad Pati, Pravas Mishra, Tulika Seth, Rahul Bhargava, Shyam Rathi, Niranjan Rathod, Renu Saxena, New Delhi, India

162

Evaluation of risk factors for thrombophilia in patients with cerebral venous thrombosis

168

Posterior reversible leukoencephalopathy syndrome in children with hematologic disorders

Osman Yokuú, Özlem ùahin BalçÕk, Murat Albayrak, Funda Ceran, Simten Da÷daú, Mesude YÕlmaz, Gülsüm Özet, østanbul, Ankara, Turkey

BarÕú Malbora, Zekai AvcÕ, Fulden Dönmez, Bülent Alio÷lu, Esra BaskÕn, Füsun Alehan, NamÕk Özbek, Ankara, Turkey

177

The effect of calabash chalk on some hematological parameters in female adult Wistar rats Amabe Otoabasi Akpantah, Ofon Samuel Ibok, Moses Bassey Ekong, Mokutima Amarachi Eluwa, Theresa Bassey Ekanem, Calabar, Nigeria

182

Role of Àow cytometry in multiple myeloma and the prognostic signi¿cance of CD87 (uPAR) expression Murat Albayrak, Özlem ùahin BalçÕk, Simten Da÷daú, Mesude YÕlmaz, Funda Ceran, Osman Yokuú, Gülsüm Özet, Ankara, østanbul, Turkey

Case Reports 190

Enzyme replacement therapy in type 1 Gaucher disease and a review of the literature Gökhan Kabacam, Gülúah Kabacam, Pervin Topçuo÷lu, IúÕnsu Kuzu, Mutlu Arat, Ankara, Turkey

196

Cyclic thrombocytopenia: A case report

200

HbQ-India associated with microcytosis: an uncommon hemoglobin variant associated with a common hematologic condition

Abdullah ùumnu, Reyhan Diz-Küçükkaya, østanbul, Turkey Amit Kumar Yadav, Usha Rusia, New Delhi, India

204

Severe iron overload and hyporegenerative anemia in a case with rhesus hemolytic disease: Therapeutic approach to rare complications Fatih Demircio÷lu, ùule Ça÷layan Sözmen, ùebnem YÕlmaz, Hale Ören, Nur Arslan, Abdullah Kumral, Erdener Özer, Gülersu ørken, øzmir, Turkey

Letters to the Editor 209

Idiopathic thrombocytopenic purpura with venous thrombosis: A case report

211

The prevalence of factor V 1691G-A mutation in Van region of Turkey

Gül ølhan, Selami K. Toprak, Neslihan AndÕç, Sema Karakuú, Ankara, Turkey Sinan Akbayram, Cihangir Akgün, Murat Do÷an, Mehmet Selçuk Bektaú, Ahmet Faik Öner, Van, Turkey

213

Portal vein thrombosis secondary to Klebsiella oxytoca bacteriemia

216

Late onset of isovaleric acidemia presenting with bicytopenia

Ebru Uz, AlÕcÕ Özlem, Özlem ùahin BalçÕk, Mehmet Kanbay, Ayúe IúÕk, Burak Uz, Arif Kaya, Ali Koúar, Ankara, Turkey BarÕú Malbora, Zekai AvcÕ, Alev Hasano÷lu, Füsun Alehan, NamÕk Özbek, Ankara, Turkey

219

Primary uterine lymphoma of the uterine cervix in advanced age M. Murat Naki, Hasniye Çelik, Oluú Api, Nagehan Özdemir, Sadullah Bulut, Orhan Ünal, østanbul, Turkey

Images in Hematology 221

Leishman-Donovan (LD) bodies in bone marrow biopsy of an adult male with AIDS Abbas Hashim Abdulsalam, Fatin Al-Yassin, Baghdad, Iraq

A-VI

Review

137

Iron transport: From enterocyte to mitochondria Demir taü×n×m×: Enterositten mitokondriye Nam×k Özbek

Department of Pediatric Hematology, Baükent University Faculty of Medicine, Ankara, Turkey

Abstract Transport of iron to tissues is of vital importance. Remarkable advances have been made concerning the mechanisms involving iron metabolism after its absorption. Studies assessing cellular and mitochondrial iron metabolism have resulted in interesting findings. This review highlights the recent advances in the mechanisms involving transport and delivery of iron to tissues, cellular and mitochondrial iron metabolism, iron-related molecules, and mitochondrial disorders. (Turk J Hematol 2010; 27: 137-46) Key words: Iron, iron transport, mitochondria, mitochondrial iron Received: February 8, 2010

Accepted: April 6, 2010

Özet Demirin dokulara taü×nmas× yaüamsal önem taü×r. Demirin emiliminden sonraki metabolizmas×yla ilgili büyük ilerlemeler kaydedilmiütir. Selüler ve mitokondriyal demir metabolizmas×yla ilgili araüt×rmalar ilginç sonuçlar vermektedir. Bu derleme demirin dokulara taü×nmas×, verilmesi, hücresel ve mitokondriyal demir metabolizmas×, demirle iliükili moleküller ve mitokondriyal hastal×klarla ilgili yeni geliümeleri vurgulamaktad×r. (Turk J Hematol 2010; 27: 137-46) Anahtar kelimeler: Demir, demir taü×n×m×, mitokondri, mitokondriyal demir Geliü tarihi: 8 ûubat 2010

Kabul tarihi: 6 Nisan 2010

A current theme in the field of hematology as well as in other disciplines is the molecular mechanisms concerning iron metabolism. Mechanisms involving absorption of iron by enterocytes has been reviewed elsewhere [1-3]. In this review, delivery of iron to tissues, mechanisms concerning cellular and mitochondrial iron metabolism, iron-related molecules, and mitochondrial disorders are presented (Table 1).

Transport of iron in plasma Binding to transferrin (Tf) is the first step in iron transport. Tf has high affinity for ferric iron; therefore, the oxidation of ferrous (Fe+2) to the ferric (Fe+3) form by hephaestin is an essential step for transport. Hephaestin is a ceruloplasmin homolog, and colocalizes with ferroportin at the basolateral membrane of enterocytes. The role of hephaestin in iron absorption was first shown in the sla (sex-

Address for Correspondence: Prof. Nam×k Özbek, 6. Cadde, 70/4, Bahçeli, 06490, Ankara, Turkey Phone: +90 312 212 68 68/1806 E-mail: nozbek@tr.net doi:10.5152/tjh.2010.20

138

Ă&#x2013;zbek N. Iron transport to mitochondria

Turk J Hematol 2010; 27: 137-46

Table 1. Genetic and functional identification of some iron-related proteins OMIM#

Gene

Locus

Function

Reference

Oxidizes Fe+2 to Fe+3

4, 5 6, 7

Hephaestin

300167

HEPH

Xq11-q12

Hepcidin

606464

HAMP

19q13

Induces internalization, ubiquitination and degradation of ferroportin via Janus kinase 2

Transferrin

190000

3q21

Transports iron from enterocyte to cells

Transferrin receptor 1 (CD71)

190010

TFR1

3q29

Transports Tf through the cell membrane

Transferrin receptor 2

604720

TFR2

7q22

Transports Tf through the cell membrane especially in hepatocytes 14

STEAP3

609671

STEAP3 (TSAP6)

2q14.2

IRP1

100880

IRP1 (ACO1)

IRP2

147582

MRCK_

Converts Fe+3 to Fe+2 in endosomes

9p22-p13

Bifunctional protein, acts as aconitase or iron response protein due to cellular iron levels

IRP2 (IREB2)

15q25.1

Binds to IRE in low-iron conditions

603412

MRCKA

1q41-q42

Regulates Tf-vesicle movement through actin/myosin filament assembly in response to decreased intracellular iron level

CDC14

603504

CDC14A

1p21

Operates in cell cycle, acts as a tumor suppressor

Poly r(C)-Binding Protein 1

601209

PCBP1

2p13-p12

Delivers iron from endosomes to ferritin

Mitoferrin

610387

MFRN

8p21

An iron importer on the inner membrane of the mitochondria

Frataxin

606829

FXN

9q13

Bifunctional mitochondrial protein that acts as a chaperone or stores iron due to cellular iron levels

Mitochondrial ferritin

Stores iron in mitochondrial matrix

608847

FTMT

5q21.3

ATP-Binding Cassette, Subfamily 300135 B, Member 7 (ABCB7)

ABCB7

Xq13.1-q13.3

Transports ISCs to cytoplasm, a putative role in heme synthesis 43, 44 is also suggested

ATP-Binding Cassette, Subfamily 605452 B, Member 6 (ABCB6)

ABCB6

2q36

Transports porphyrin to mitochondria and functionally linked to 45, 46 heme biosynthesis

Glutaredoxin

609588

GLRX5

14q32

Required for ISC assembly

BCS1L

603647

BCS1L

2q33

A chaperone that facilitates insertion of Rieske Fe/S protein into mitochondrial respiratory chain complex III

linked anemia) mouse with moderate to severe microcytic hypochromic anemia [4,5]. Enterocytes of the sla mouse are able to absorb iron from lumen; however, there is a block in transport to blood due to defective hephaestin. Another molecule that has a very important influence on iron absorption at this step is hepcidin. If total body iron is high, hepatic synthesis of hepcidin increases. Binding of hepcidin to ferroportin in its exterior segment causes upregulation of Janus kinase 2 [6,7]. Activation of Janus kinase 2 results in internalization, ubiquitination and degradation of ferroportin, which results in decreased iron transfer to blood. Transferrin is the major protein that binds and delivers iron to tissues. It is member of a family of homologous transport proteins that includes lactoferrin, ovotransferrin, and melanotransferrin. All of these proteins share the same structure. Due to a suggested ancestral gene duplication, N- and C- terminals (lobes) of these proteins show approximately 40% sequence identity. These terminals are sepa-

rated by a short spacer sequence. Each of these two lobes (N- and C-) is divided into two equally sized domains: N1, N2 and C1, C2. Clefts between these domains are designed for transport of Fe+3; therefore, each Tf molecule can transport 2 iron molecules [8,9]. Side chains of 1 asparagine, 2 tyrosines and 1 histidine provide 3 negative charges within the cleft matching 3 positive charges of Fe+3. Also, binding of CO23- ion is critical for the binding and release of Fe+3. Ferric iron and CO23- ion could not bind to Tf in the absence of the other. Conformation of the binding site is suitable with ferric iron through a delicate balance. Binding constant for Fe+3 is approximately 1020 whereas it is only 103 for Fe+2 [10]. Binding and release of iron result in conformational changes in the Tf molecule enabling rapid exchange [11]. Transferrin receptors Transferrin receptor (TfR), the binding site of Tf on the cell, has two types; namely TfR1 and TfR2.

Ă&#x2013;zbek N. Iron transport to mitochondria

Turk J Hematol 2010; 27: 137-46

TfR1 has an extremely important role in embryogenesis; knockout embryos do not survive due to defective erythropoiesis and neurological development [12,13]. However, other tissues are not affected in the absence of TfR1, which indicates additional mechanism(s) for iron transport in these cells. TfR1 is expressed in all tissues except mature erythrocytes. On the other hand, TfR2 is mainly expressed in the liver, normal erythroid precursors and some leukemic cells [14,15]. Although protein structures of TfR1 and TfR2 have a high degree of homology, their functions and regulation are not the same [16]. Expression of TfR1 is tightly regulated by cellular iron levels through HFE (hereditary hemochromatosis) protein. In normal conditions, HFE protein decreases the affinity of TfR1 for Tf by a magnitude of 10 to 50 [17]. Mutations in the HFE gene may alter the iron absorption and cause hemochromatosis phenotype. Nonetheless, cellular iron levels have no effect on the regulation of TfR2. Furthermore, TfR2 has no interaction with the HFE protein in contrast to TfR1. High levels of TfR2-mRNA expression in erythroid precursors and erythroleukemia cells point out that TfR2 expression is both cell-cycle and differentiation dependent [15]. Similar to TfR1, TfR2 has high affinity for diferric Tf. TfR2-mediated endocytosis of the Tf/iron complex is also the same as with TfR1 [18]. On the other hand, affinity of TfR2 for Tf is approximately 25-30-fold less than that of TfR1. Mutations in TfR2 cause a rare type of hereditary hemochromatosis (HH), namely HFE 3. A recent study in zebrafish embryos showed that TfR2 was required for hepcidin expression [19]. In summary, TfR2 senses the body iron status by sensing the Tf saturation. Consequently, it modulates hepcidin expression adjusted to the actual body iron level.

139

Therefore, loss of TfR2 or its function results in failure to sense body iron status by hepatocytes, which causes increased hepcidin and iron deposition, especially in the liver [20]. Transfer of iron into cells After binding of diferric Tf to TfR, the Tf/TfR complex on the clathrin-coated cell membrane is internalized through a receptor-mediated endocytosis. Clathrin coat is needed for invagination of the cell membrane. Newly formed vesicles lose the clathrin coat and fuse with endosomes [21]. After formation of endosomes, the acidification process by means of an ATPase proton pump (pH 5.5-6) takes place in order to dissociate iron from Tf. A protein called STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3), has been shown to convert Fe+3 to Fe+2 in erythroid precursor cells [22]. An animal model revealed that STEAP3 is highly expressed in hematopoietic tissues, colocalizes with the Tf cycle endosome, and facilitates Tf-bound iron uptake. Moreover, mutations in the STEAP3 gene were found to be responsible for the iron deficiency anemia in the mouse mutant nm1054. Homologs of this protein, STEAP1, 2 and 4, are expressed in hepatocytes as well as other tissues [23]. Free ferrous iron released from Tf in the endosome enters into cytoplasm via DMT1 on the endosomal membrane. Then, the endosome containing Tf and TfR fuses back to the plasma membrane. This recycling process enables Tf and TfR to be used in new cycles. Hepatic iron uptake Hepatic iron transport has different aspects compared to the other cells [24]. Hepatocytes can acquire both transferrin-bound iron (TBI) and nontransferrin-bound iron (NTBI) from plasma (Table 2).

Table 2. Hepatocyte uptake of iron-related proteins Form of iron

Hepatic uptake mechanism

Entry into hepatocyte

TBI

TfR1- or TfR2-mediated endocytosis Direct uptake of iron after releasing from Tf on hepatocyte surface

Through DMT1 on endosomal membrane Through DMT1, ZIP14 or calcium channels on hepatocyte membrane

NTBI

Direct uptake of iron after releasing from carrier molecule (mostly citrate) on hepatocyte surface

Through DMT1, ZIP14 or calcium channels on hepatocyte membrane

Specific receptor-mediated uptake

Through endocytosis

Ferritin Lactoferrin Heme-hemopexin complex Hemoglobin-haptoglobin complex

140

Özbek N. Iron transport to mitochondria

Turk J Hematol 2010; 27: 137-46

Table 3. Some iron-related molecules containing IREs on their mRNA Molecule

IRE localization on mRNA

Function

3’UTR

Iron uptake

DMT1

3’UTR

Iron uptake

Ferroportin

5’UTR

Iron export

H- and L-ferritin

5’UTR

Iron storage

ALAS1 and ALAS2

5’UTR

Iron utilization

MRCK_

3’UTR

Tf vesicle movement

CDC14A

3’UTR

Tumor suppressor

TfR1

TBI uptake may occur through TfR-dependent or -independent mechanisms. As discussed above, both TfR1 and TfR2 operate in the uptake of TBI. In the process of TfR-independent delivery of Tf-bound iron, Tf releases iron at the hepatocyte surface. Following this step, a ferrireductase activity converts Fe+3 to Fe+2, which also takes place at the surface [25]. Afterwards, Fe+2 enters the cell via membrane transporters. The most common form of NTBI is iron citrate, usually increased in iron overload disorders [26]. Iron citrate releases iron at the hepatocyte surface like TBI. Iron delivered by NTBI also enters hepatocytes via membrane transporters, especially DMT1 [27]. Other molecules transporting iron through the hepatocyte membrane are ZIP14 and calcium channels. There are some other iron-related proteins or complexes transported to hepatocytes by specific membrane receptors. These include ferritin, lactoferrin, heme-hemopexin complex, and hemoglobinhaptoglobin complex. The fate of these molecules in hepatocytes following endocytosis may be different: iron and some proteins (i.e. ferritin) may reside in the hepatocyte, some proteins may go into degradation (i.e. ferritin, heme, hemoglobin, haptoglobin, lactoferrin), whereas others may recycle back to the plasma (i.e. hemopexin, receptors of these proteins) or to bile (i.e. ferritin, hemoglobin) [24]. Molecular control of iron content in cells The need for iron is different in different body cells. For example, cardiomyocytes, muscle cells and neurons require higher amounts of iron compared to the other cells. Therefore, uptake of iron is individually regulated in each cell regardless of total body iron need. Important molecules concerning cellular iron uptake are iron-responsive proteins (IRP1 and IRP2) and iron-responsive elements (IRE). Two IRPs have been defined to date: IRP1 and

IRP2 [28,29]. In iron-depleted conditions, IRP1 and 2 bind to IREs of a number of molecules related to iron metabolism (Table 3). These molecules are assigned to iron uptake (TfR1, DMT1), utilization [5-aminolevulinate synthase (ALAS1) and erythroid ALAS (ALAS2)], storage (H- and L-ferritin) or export (ferroportin). Recent studies showed that at least two cell cycle-related molecules, MRCK_ (myotonic dystrophy kinase-related Cdc42-binding kinase) and CDC14A (cell division cycle 14A), also bear IREs in their mRNA at the 3’ untranslated region (UTR). It has been suggested that MRCK_, a serine/threonine kinase, regulates Tf-vesicle movement through actin/ myosin filament assembly in response to decreased intracellular iron level. Supporting this suggestion, MRCK_-mRNA levels in various tissues strongly positively correlated with the level of TfR-mRNA levels [30]. The second cell cycle-related molecule, CDC14A, is a phosphatase involved in the dephosphorylation of several critical cell cycle proteins and is suggested to act as a tumor suppressor [31]. Synthesis of IRPs is regulated by IRPs to maintain optimum iron levels in each cell. Depending on the location of IRE, the IRE/IRP interaction results in stabilization or translational repression of mRNA [32]. During low-iron conditions, synthesis of both IRP1 and IRP2 increases. Interaction of IRPs with IREs in the 3’ UTR of mRNA results in stabilization; therefore, increased expression of molecules such as TfR1 and DMT1. Meanwhile, interaction of IRPs with IREs in the 5’ UTR of mRNA results in translational repression; the expression of molecules such as ferritin, ferroportin, and ALAS2 will be suppressed. During high-iron levels, both IRP1 and IRP2 lose their affinity for IRE. As a result, mRNAs bearing IRE in the 5’ UTR stabilize and expression of molecules such as ferritin, ferroportin, and ALAS2 increases whereas synthesis of TfR1 and DMT1 decreases. IRP1 is a bifunctional protein that binds iron-sulfur clusters (ISC) in case of high intracellular iron. After binding ISC [4Fe-4S], IRP1 functions as aconitase, and loses its IRE-related functions. However, the high intracellular iron levels do not cause a conformational change but rather cause degradation of IRP2. Erythroid cells require more iron than any other cell in the body. Therefore, these cells need specific adaptation mechanism(s) for high iron influx. A recent study revealed that terminal erythropoiesis caused a switch of regulation to a different mode in the IRP/IRE system [33]. Since committed erythro-

Özbek N. Iron transport to mitochondria

Turk J Hematol 2010; 27: 137-46

blasts need much more iron than any other cell, they should maintain high TfR1 expression despite high intracellular iron levels. In this context, the erythroid IRP/IRE system should sense a low-iron state despite increased cytosolic iron. Furthermore, although both ferritin- and ALAS2-mRNAs bear IRE on their 5’ UTR, strong inhibition of ferritin-mRNA translation and efficient ALAS2-mRNA translation have been observed in differentiating, iron-rich erythroblasts. Another conflicting data regarding IRE/IRP regulation is of ferroportin. Ferroportin-mRNA also bears IRE on its 5’ UTR. Therefore, during low-iron conditions, mRNA stability of ferroportin should decrease in all cells. However, this is not the case for duodenal absorptive cells and erythroid precursors. During low-iron conditions, increased absorption of iron by enterocytes and release to blood via ferroportin is a physiologically relevant way. A very recent study documented that duodenal epithelial and erythroid precursor cells utilize an alternative upstream promoter to express a ferroportin transcript, ferroportin1B, which lacks the IRE and is not repressed in irondeficient conditions [34]. The identification of ferroportin1B may explain how high ferroportin expression is possible in duodenal epithelial and erythroid precursor cells during high intracellular iron levels. This study strongly suggests that different regulatory systems operate influx of iron into different cells. Mitochondrial iron metabolism After entering from the endosome to cytoplasm, iron can be stored within ferritin or it can be used

141

for cellular reactions. A newly discovered protein, PCBP1 [Poly r(C)-Binding Protein 1], has been reported to transfer iron from the endosome to ferritin [35]. Although the exact mechanism of iron transport to mitochondria is not well defined, iron can also enter the mitochondria. A recent report defined a new way, namely “kiss and run” [36]. It was suggested that delivery of iron to mitochondria happens directly from iron-containing endosomes bypassing the cytoplasm. Molecules, including iron, cross the mitochondrial outer membrane via a large diameter voltage-dependent anion channel called porin [37]. Transport of iron through the inner mitochondrial membrane occurs with the aid of mitoferrin, a special carrier. Mutation in the mitoferrin gene has been described in a zebrafish mutant that caused profound hypochromic anemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake [38]. After its transport into mitochondria, iron can be stored or used for vital reactions. Two important iron-related reactions that happen within mitochondria are heme synthesis and ISC biogenesis [39]. Iron in the mitochondrial matrix is in potentially redox-active ferrous (Fe+2) form. Therefore, tight control of iron influx and maintenance in bound-form is mandatory to achieve low toxic iron levels in the mitochondrial matrix. Two newly described proteins store iron within the matrix: frataxin (FXN) and mitochondrial ferritin. FXN is a bifunctional protein like IRP1 [40]. It works as a Fe+2 chaperone for metabolic actions when mitochondrial iron is limited. When iron is in

Table 4. Some disorders related to mitochondrial iron metabolism Disease Friedreich ataxia

OMIM# 229300

Inheritance Autosomal recessive

X-linked sideroblastic anemia

300751

Pyridoxine-unresponsive sideroblastic anemia due to GLRX5 mutation

205950

X- linked recessive Autosomal recessive

X-linked sideroblastic anemia with ataxia

301310

Myopathy associated with ISCU1 defect GRACILE syndrome

Related gene locus Related molecular defect 9q13, 9p23-p11 Expansion of GAA triplet repeats in intron 1 (97%) or point mutations in FXN gene

Clinical features Progressive ataxia, cardiomyopathy, skeletal deformities, and impaired glucose tolerance or diabetes mellitus

Xp11.21

Mutation in ALAS2 gene

14q32, 3p22.1

Mutation in GLRX5 gene

Hypochromic microcytic anemia, variable response to pyridoxine treatment Severe microcytic anemia, jaundice, hepatosplenomegaly, iron overload, ringed sideroblasts, and cirrhosis

X- linked recessive

Xq13.1-q13.3

Mutation in mitochondrial ABCB7 gene

Hypochromic microcytic anemia, early onset nonprogressive ataxia

255125

Autosomal recessive

12q24.1

Mutation in ISCU1 gene

Early fatigue, dyspnea, and palpitations followed by hard and tender muscles and muscle cramps

603358

Autosomal recessive

2q33

Mutation in BCS1L gene

Growth retardation, amino aciduria, cholestasis, iron overload, lactic acidosis, and early death

142

Özbek N. Iron transport to mitochondria

excess, it works as a storage compartment forming ferrihydrite. The mitochondrial ferritin precursor protein is coded by an unusual intronless gene and targeted to mitochondria. Within mitochondria, this precursor is processed to a smaller protein, mitochondrial ferritin. It is remarkably similar to H-ferritin that assembles into typical ferritin shells within mitochondria and has ferroxidase activity [41]. Immunohistochemical analysis showed that erythroblasts of subjects with impaired heme synthesis had high amounts of mitochondrial ferritin accumulation. In pathological conditions, it can be easily detected as “ringed sideroblast” [42]. One of the main functions of mitochondria is ISC biosynthesis. These clusters are cofactors of several proteins including electron transport, Krebs cycle, regulation of gene expression, and redox reactions [39]. Iron sulfur clusters have also been shown in cytoplasm within IRP1 and in the nucleus within an enzyme that is involved in base excision repair. After synthesis within mitochondria, ISCs are transported to cytoplasm through mitochondrial inner membrane channels by a specific carrier, ABCB7 (ATP-Binding Cassette, Subfamily B, Member 7) [43]. ABCB7 also interacts with ferrochelatase, suggesting a putative role in heme metabolism [44]. The mitochondrion is a major site for heme synthesis. Out of 8 steps in heme biosynthesis, 4 steps, including the first ratelimiting step of the pathway, take place within mitochondria. This step involves condensation of succinyl CoA and glycine to delta-aminolevulinate catalyzed by delta-ALAS. There are 2 types of ALAS: ALAS1 and ALAS2 (erythroid ALAS). Although the rate-limiting step in non-erythroid cells is the deltaaminolevulinate formation, in erythroid cells, the rate-limiting step is acquisition of iron to mitochondria. ABCB6 (ABCB Member 6), a protein very similar to ABCB7, has been shown to localize at the outer mitochondrial membrane [45]. A recent study suggested that ABCB6 is upregulated by elevation of cellular porphyrins and has a function in porphyrin transport to mitochondria. The authors also predicted that ABCB6 is functionally linked to heme biosynthesis [46]. Mitochondrial disorders related to iron metabolism The current understanding of mitochondrial iron metabolism has led to elucidation of pathophysiologic mechanisms in some disorders (Table 4).

Turk J Hematol 2010; 27: 137-46

These disorders usually affect erythroid cells as well as neural tissues. Friedreich ataxia Friedreich ataxia (FRDA) is the most common form of autosomal recessive ataxia. The disease is characterized by progressive ataxia of all limbs, skeletal and cardiac muscle myopathy, skeletal deformities, impaired glucose tolerance or diabetes mellitus, sensorineural deafness, and optic neuropathy [47]. The molecular abnormality in more than 97% of patients is GAA trinucleotide repeat expansion in intron 1 of the FXN gene [48]. Normal alleles of the FXN gene have 5 to 30 GAA repeat expansions; however, disease alleles have from 70 to more than 1,000 GAA triplets. Increased GAA repeats causes triplex DNA structures, called “sticky DNA”, which interfere with FXN-mRNA transcription [49]. As in other triplet repeat disorders, the size of triplet expansion correlates inversely with the age of onset and directly with the rate of disease progression [50]. Since known mitochondrial diseases and FRDA share some clinical manifestations, mitochondrial disturbance in the etiology of FRDA has long been suspected. For example, isolated vitamin A deficiency, a disease with mitochondrial involvement, has strikingly similar signs and symptoms with FRDA [51]. After demonstration of a yeast that has mitochondrial iron accumulation due to a defective protein homolog with FTX, subsequent studies revealed defective FTX in the etiology of FRDA [52]. FTX plays a vital role in the regulation of mitochondrial iron metabolism. However, the pathophysiology of FRDA has not been completely understood to date. Oxidative stress due to FTX deficiency has usually been cited in the FRDA pathophysiology. The decrease in the ISC-containing proteins (aconitase and mitochondrial respiratory chain complexes) observed in heart biopsies of patients with FRDA supports that this finding was related to a mitochondrial damage caused by iron overload [53]. Another study on cultured fibroblasts carrying homozygous FRDA mutation showed that these fibroblasts were more sensitive to oxidative stress than were the controls [54]. Treatment with deferoxamine and apoptosis inhibitors rescued fibroblasts with FRDA mutation. A recent study that demonstrated upregulation of stress pathways in FTXdeficient cells further supports the role of oxidative stress in FRDA as well [55].

Turk J Hematol 2010; 27: 137-46

X-linked sideroblastic anemia X-linked sideroblastic anemia (XLSA) is a rare disorder caused by mutations in the ALAS2 gene [56]. The peripheral smear of patients typically includes two distinct erythrocyte populations: normochromic normocytic erythrocytes together with hypochromic microcytic cells. Bone marrow examination reveals ringed sideroblasts due to increased iron within mitochondrial ferritin [57]. Since neuronal cells rely on ALAS1 for iron metabolism, no neurological finding is present in these patients in contrast with XLSA with ataxia. Although the inheritance is X-linked recessive, some female carriers may present with severe anemia. This finding is attributed to an additional event following inheritance of the ALAS2 mutation, i.e. congenital skewing led to dominance of hematopoietic cells expressing the X chromosome with the mutant gene [58]. Interestingly, a case study revealed a significantly higher frequency of coinheritance of HFE mutant allele C282Y in patients with XLSA compared to the normal population [57]. Interaction of two mutant alleles may point out the severity of iron overload. Response to pyridoxine treatment is variable among XLSA patients. This study [57] also revealed that iron overload suppressed response to pyridoxine. Another factor suggested for variability in pyridoxine response is the location of the mutation on the ALAS2 gene. Analysis of the ALAS2 crystal structure in a recent study revealed that patients with mutations close to the 5â&#x20AC;&#x2122; pyridoxal phosphate binding site respond to pyridoxine [59]. Pyridoxine-unresponsive sideroblastic anemia due to GLRX5 mutation In 2007, a 60-year-old southern Italian man who presented with severe microcytic anemia, jaundice, hepatosplenomegaly, iron overload, ringed sideroblasts, and cirrhosis was reported [60]. His unaffected parents were consanguineous, consistent with autosomal recessive inheritance. Interestingly, anemia of the patient worsened with transfusion but improved with iron chelation. Striking similarities of his phenotype with mutant shiraz zebrafish directed the authors to the Glutaredoxin 5 (GLRX5) gene, which was largely deleted in shiraz zebrafish. They found a homozygous mutation in this gene that interferes with intron 1 splicing and causes a drastic reduction in GLRX5-RNA. GLRX5 deficiency

Ă&#x2013;zbek N. Iron transport to mitochondria

143

causes impaired ISC synthesis that activates cytosolic IRP1 and finally leads to increased cellular iron import. As discussed above, increased IRP1 results in increased TfR and decreased ferritin and ALAS2 synthesis. As a result, decreased ALAS2 level causes anemia in spite of increased intracellular iron level. In this low-heme environment, IRP2 does not undergo proteosomal degradation. Prolonged activity of IRP2 may contribute to increased mitochondrial iron. The authors concluded that redistribution of iron into cytoplasm by iron chelation might relieve iron excess, improving heme synthesis and anemia. X-linked sideroblastic anemia with ataxia X-linked sideroblastic anemia with ataxia is a mitochondrial disease caused by a mutation in the nuclear genome. The disease is characterized by hypochromic microcytic anemia with ring sideroblasts and early onset, nonprogressive spinocerebellar ataxia. Missense mutations on the ABCB7 gene cause partial loss of function in patients with this disease [61]. Nonsense mutations have not been identified, possibly due to the lethal effect of complete loss of function that was shown in a knock out mouse model [62]. The defect in the ABCB7 gene causes deficiency in ISCs in cytoplasm but not in mitochondria. However, iron is accumulated within mitochondria by an unknown mechanism [43]. Myopathy associated with ISCU1 defect The disease was first described in patients from Sweden and characterized by low physical performance. Physical activity results in early fatigue, dyspnea, and palpitations followed by hard and tender muscles and muscle cramps. Recently, a splice mutation in the ISC scaffold protein ISCU has been reported in these patients [63]. The loss of FTX in FRDA and GLRX5 in pyridoxine-unresponsive sideroblastic anemia causes deficiency in ISCs as in the myopathy associated with ISCU1 defect. Typically, iron accumulation in mitochondria is a common event in these three disorders. The authors suggested that this observation supported the possibility that one or more ISC proteins act as sensor for regulation of mitochondrial iron homeostasis. GRACILE syndrome GRACILE syndrome (Growth Retardation, Amino aciduria, Cholestasis, Iron overload, Lactic acidosis,

144

Ă&#x2013;zbek N. Iron transport to mitochondria

and Early death) has been frequently reported in neonates from Finland [64]. Necropsy findings have revealed increased liver iron content, paucity of intrahepatic bile ducts and periportal fibrosis in the oldest patient, who died at the age of 4 months. Electron microscopic studies revealed an abundance of hemosiderin granules; however, the number and structure of mitochondria were normal. Subsequently, case reports including Turkish patients together with patients from other countries have been followed [65]. Distinctive clinical findings were reported among patients from different countries: Turkish and British patients had variable neurological symptoms and findings. Recently, mutations in the BCS1L gene as a cause of this disorder have been identified [66]. This gene encodes a mitochondrial inner-membrane protein. It is a chaperone that is presumed to facilitate insertion of Rieske Fe/S protein into precursors of mitochondrial respiratory chain complex III. The Rieske iron-sulfur protein is a nuclear-encoded subunit of the mammalian cytochrome complex III of the mitochondrial respiratory chain [67]. Interestingly, the British and Turkish patients with GRACILE syndrome had mitochondrial complex III deficiency, whereas in the Finnish patients, complex III activity was within the normal range. Also, a distinctive mutation in the BCS1L gene has been found in Turkish patients. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1. 2. 3. 4.

Mackenzie B, Garrick MD. Iron Imports. II. Iron uptake at the apical membrane in the intestine. Am J Physiol Gastrointest Liver Physiol 2005;289:G981-6. Ma Y, Yeh M, Yeh KY, Glass J. Iron Imports. V. Transport of iron through the intestinal epithelium. Am J Physiol Gastrointest Liver Physiol 2006;290:G417-22. West AR, Oates PS. Mechanisms of heme iron absorption: current questions and controversies. World J Gastroenterol 2008;14:4101-10. Vulpe CD, Kuo YM, Murphy TL, Cowley L, Askwith C, Libina N, Gitschier J, Anderson GJ. Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse. Nature Genet 1999;21:195-9. Anderson GJ, Murphy TL, Cowley L, Evans BA, Halliday JW, McLaren GD. Mapping the gene for sex-linked

Turk J Hematol 2010; 27: 137-46

9. 10. 11. 12. 13.

14.

15.

16. 17.

18.

19.

20.

anemia: an inherited defect of intestinal iron absorption in the mouse. Genomics 1998;48:34-9. De Domenico I, Nemeth E, Nelson JM, Phillips JD, Ajioka RS, Kay MS, Kushner JP, Ganz T, Ward DM, Kaplan J. The hepcidin-binding site on ferroportin is evolutionarily conserved. Cell Metab 2008;8:146-56. De Domenico I, Lo E, Ward DM, Kaplan J. Hepcidininduced internalization of ferroportin requires binding and cooperative interaction with Jak2. Proc Natl Acad Sci U S A 2009;106:3800-5. Yang F, Lum JB, McGill JR, Moore CM, Naylor SL, van Bragt PH, Baldwin WD, Bowman BH. Human transferrin: cDNA characterization and chromosomal localization. Proc Natl Acad Sci U S A 1984;81:2752-6. Hirose M. The structural mechanism for iron uptake and release by transferrins. Biosci Biotechnol Biochem 2000;64:1328-1336. Harris WR. Estimation of the ferrous-transferrin binding constants based on thermodynamic studies of nickel(II)transferrin. J Inorg Biochem 1986;27:41-52. Baker HM, Anderson BF, Baker EN. Dealing with iron: common structural principles in proteins that transport iron and heme. Proc Natl Acad Sci U S A 2003;100:3579-83. Levy JE, Jin O, Fujiwara Y, Kuo F, Andrews NC. Transferrin receptor is necessary for development of erythrocytes and the nervous system. Nat Genet 1999;21:396-9. Casey JL, Di Jeso B, Rao K, Klausner RD, Harford JB. Two genetic loci participate in the regulation by iron of the gene for the human transferrin receptor. Proc Natl Acad Sci U S A 1988;85:1787-91. Kawabata H, Yang R, Hirama T, Vuong PT, Kawano S, Gombart AF, Koeffler HP. Molecular cloning of transferrin receptor 2. A new member of the transferin receptorlike family. J Biol Chem 1999;274:20826-32. Kawabata H, Nakamaki T, Ikonomi P, Smith RD, Germain RS, Koeffler HP. Expression of transferrin receptor 2 in normal and neoplastic hematopoietic cells. Blood 2001;98:2714-9. Trinder D, Baker E. Transferrin receptor 2: a new molecule in iron metabolism. Int J Biochem Cell Biol 2003;35:292-6. Feder JN, Penny DM, Irrinki A, Lee VK, Lebron JA, Watson N, Tsuchihashi Z, Sigal E, Bjorkman PJ, Schatzman RC. The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. Proc Natl Acad Sci USA 1998;95:1472-7. Kawabata H, Germain RS, Vuong PT, Nakamaki T, Said JW, Koeffler HP. Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo. J Biol Chem 2000;275:16618-25. Fraenkel PG, Gibert Y, Holzheimer JL, Lattanzi VJ, Burnett SF, Dooley KA, Wingert RA, Zon LI. Transferrin-a modulates hepcidin expression in zebrafish embryos. Blood 2009;113:2843-50. Fleming RE, Ahmann JR, Migas MC, Waheed A, Koeffler HP, Kawabata H, Britton RS, Bacon BR, Sly WS. Targeted mutagenesis of the murine transferrin receptor-2 gene

Özbek N. Iron transport to mitochondria

Turk J Hematol 2010; 27: 137-46

21. 22.

23. 24. 25. 26.

27.

28.

29.

30. 31.

32. 33.

34.

produces hemochromatosis. Proc Natl Acad Sci U S A 2002;99:10653-8. Dautry-Varsat A. Receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor. Biochimie 1986;68:375-81. Ohgami RS, Campagna DR, Greer EL, Antiochos B, McDonald A, Chen J, Sharp JJ, Fujiwara Y, Barker JE, Fleming MD. Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells. Nat Genet 2005;37:1264-9. Ohgami RS, Campagna DR, McDonald A, Fleming MD. The Steap proteins are metalloreductases. Blood 2006;108:1388-94. Graham RM, Chua AC, Herbison CE, Olynyk JK, Trinder D. Liver iron transport. World J Gastroenterol 2007;13:4725-36. Thorstensen K, Romslo I. Uptake of iron from transferrin by isolated rat hepatocytes. A redox mediated plasma membrane process? J Biol Chem 1988;263:8844-50. Grootveld M, Bell JD, Halliwell B, Aruoma OI, Bomford A, Sadler PJ. Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy. J Biol Chem 1989;264:4417-22. Shindo M, Torimoto Y, Saito H, Motomura W, Ikuta K, Sato K, Fujimoto Y, Kohgo Y. Functional role of DMT1 in transferrin-independent iron uptake by human hepatocyte and hepatocellular carcinoma cell, HLF. Hepatol Res 2006;35:152-62. Povey S, Slaughter CA, Wilson DE, Gormley IP, Buckton KE, Perry P, Bobrow M. Evidence for the assignment of the loci AK1, AK3 and ACONs to chromosome 9 in man. Ann Hum Genet 1976;39:413-22. Rouault TA, Tang CK, Kaptain S, Burgess WH, Haile DJ, Samaniego F, McBride OW, Harford JB, Klausner RD. Cloning of the cDNA encoding an RNA regulatory protein-the human iron-responsive element-binding protein. Proc Natl Acad Sci USA 1990;87:7958-62. Cmejla R, Petrak J, Cmejlova J. A novel iron responsive element in the 3'UTR of human MRCKalpha. Biochem Biophys Res Commun 2006;341:158-66. Sanchez M, Galy B, Dandekar T, Bengert P, Vainshtein Y, Stolte J, Muckenthaler MU, Hentze MW. Iron regulation and the cell cycle: identification of an iron-responsive element in the 3'-untranslated region of human cell division cycle 14A mRNA by a refined microarray-based screening strategy. J Biol Chem 2006;281:22865-74. Wallander ML, Leibold EA, Eisenstein RS. Molecular control of vertebrate iron homeostasis by iron regulatory proteins. Biochim Biophys Acta 2006;1763:668-89. Schranzhofer M, Schifrer M, Cabrera JA, Kopp S, Chiba P, Beug H, Müllner EW. Remodeling the regulation of iron metabolism during erythroid differentiation to ensure efficient heme biosynthesis. Blood 2006;107:4159-67. Zhang DL, Hughes RM, Ollivierre-Wilson H, Ghosh MC, Rouault TA. A ferroportin transcript that lacks an ironresponsive element enables duodenal and erythroid

35. 36. 37.

38.

39. 40

41.

42.

43. 44. 45.

46.

47. 48.

145

precursor cells to evade translational repression. Cell Metab 2009;9:461-73. Shi H, Bencze KZ, Stemmler TL, Philpott CC. A cytosolic iron chaperone that delivers iron to ferritin. Science 2008;320:1207-10. Sheftel AD, Zhang AS, Brown C, Shirihai OS, Ponka P. Direct interorganellar transfer of iron from endosome to mitochondrion. Blood 2007;110:125-32. Dahout-Gonzalez C, Nury H, Trézéguet V, Lauquin GJ, Pebay-Peyroula E, Brandolin G. Molecular, functional, and pathological aspects of the mitochondrial ADP/ATP carrier. Physiology (Bethesda) 2006;21:242-9. Shaw GC, Cope JJ, Li L, Corson K, Hersey C, Ackermann GE, Gwynn B, Lambert AJ,Wingert RA, Traver D, Trede NS, Barut BA, Zhou Y, Minet E, Donovan A, Brownlie A, Balzan R, Weiss MJ, Peters LL, Kaplan J, Zon LI, Paw BH. Mitoferrin is essential for erythroid iron assimilation. Nature 2006;440:96-100. Levi S, Rovida E. The role of iron in mitochondrial function. Biochim Biophys Acta 2009;1790:629-36. Park S, Gakh O, O'Neill HA, Mangravita A, Nichol H, Ferreira GC, Isaya G. Yeast frataxin sequentially chaperones and stores iron by coupling protein assembly with iron oxidation. J Biol Chem 2003;278:31340-51. Levi S, Corsi B, Bosisio M, Invernizzi R, Volz A, Sanford D, Arosio P, Drysdale J. A human mitochondrial ferritin encoded by an intronless gene. J Biol Chem 2001;276:24437-40. Cazzola M, Invernizzi R, Bergamaschi G, Levi S, Corsi B, Travaglino E, Rolandi V, Biasiotto G, Drysdale J, Arosio P. Mitochondrial ferritin expression in erythroid cells from patients with sideroblastic anemia. Blood 2003;101:1996-2000. Napier I, Ponka P, Richardson DR. Iron trafficking in the mitochondrion: novel pathways revealed by disease. Blood 2005;105:1867-74. Zutz A, Gompf S, Schägger H, Tampé R. Mitochondrial ABC proteins in health and disease. Biochim Biophys Acta 2009;1787:681-90. Mitsuhashi N, Miki T, Senbongi H, Yokoi N, Yano H, Miyazaki M, Nakajima N, Iwanaga T, Yokoyama Y, Shibata T, Seino S. MTABC3, a novel mitochondrial ATPbinding cassette protein involved in iron homeostasis. J Biol Chem 2000;275:17536-40. Krishnamurthy PC, Du G, Fukuda Y, Sun D, Sampath J, Mercer KE, Wang J, Sosa-Pineda B, Murti KG, Schuetz JD. Identification of a mammalian mitochondrial porphyrin transporter. Nature 2006;443:586-9. Delatycki MB, Williamson R, Forrest SM. Friedreich ataxia: an overview. J Med Genet 2000;37:1-8. Al-Mahdawi S, Pinto RM, Varshney D, Lawrence L, Lowrie MB, Hughes S, Webster Z, Blake J, Cooper JM, King R, Pook MA. GAA repeat expansion mutation Mouse models of Friedreich ataxia exhibit oxidative stress leading to progressive neuronal and cardiac pathology. Genomics 2006;88:580-90.

146 49.

50.

51. 52.

53.

54.

55.

56.

57.

58.

Özbek N. Iron transport to mitochondria

Sakamoto N, Ohshima K, Montermini L, Pandolfo M, Wells RD. Sticky DNA, a self-associated complex formed at long GAA*TTC repeats in intron 1 of the frataxin gene, inhibits transcription. J Biol Chem 2001;276:27171-7. Dürr A, Cossee M, Agid Y, Campuzano V, Mignard C, Penet C, Mandel JL, Brice A, Koenig M. Clinical and genetic abnormalities in patients with Friedreich's ataxia. N Engl J Med 1996;335:1169-75. Wilson RB. Iron dysregulation in Friedreich ataxia. Semin Pediatr Neurol 2006;13:166-75. Babcock M, de Silva D, Oaks R, Davis-Kaplan S, Jiralerspong S, Montermini L, Pandolfo M, Kaplan J. Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin. Science 1997;276:1709-12. Rötig A, de Lonlay P, Chretien D, Foury F, Koenig M, Sidi D, Munnich A, Rustin P. Aconitase and mitochondrial iron-sulphur protein deficiency in Friedreich ataxia. Nat Genet 1997;17:215-7. Wong A, Yang J, Cavadini P, Gellera C, Lonnerdal B, Taroni F, Cortopassi G. The Friedreich's ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis. Hum Mol Genet 1999;8:425-30. Pianese L, Busino L, De Biase I, De Cristofaro T, Lo Casale MS, Giuliano P, Monticelli A, Turano M, Criscuolo C, Filla A, Varrone S, Cocozza S. Up-regulation of c-Jun N-terminal kinase pathway in Friedreich's ataxia cells. Hum Mol Genet 2002;11:2989-96. Cotter PD, Willard HF, Gorski JL, Bishop DF. Assignment of human erythroid delta-aminolevulinate synthase (ALAS2) to a distal subregion of band Xp11.21 by PCR analysis of somatic cell hybrids containing X; autosome translocations. Genomics 1992;13:211-2. Cotter PD, May A, Li L, Al-Sabah AI, Fitzsimons EJ, Cazzola M, Bishop DF. Four new mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causing X-linked sideroblastic anemia: increased pyridoxine responsiveness after removal of iron overload by phlebotomy and coinheritance of hereditary hemochromatosis. Blood 1999;93:1757-69. Cazzola M, May A, Bergamaschi G, Cerani P, Rosti V, Bishop DF. Familial-skewed X-chromosome inactivation as a predisposing factor for late-onset X-linked sideroblastic anemia in carrier females. Blood 2000;96:4363-5.

Turk J Hematol 2010; 27: 137-46

59.

60.

61.

62.

63.

64.

65.

66.

67.

Astner I, Schulze JO, van den Heuvel J, Jahn D, Schubert WD, Heinz DW. Crystal structure of 5-aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans. EMBO J 2005;24:3166-77. Camaschella C, Campanella A, De Falco L, Boschetto L, Merlini R, Silvestri L, Levi S, Iolascon A. The human counterpart of zebrafish shiraz shows sideroblastic-like microcytic anemia and iron overload. Blood 2007;110:1353-8. Allikmets R, Raskind WH, Hutchinson A, Schueck ND, Dean M, Koeller DM. Mutation of a putative mitochondrial iron transporter gene (ABC7) in X-linked sideroblastic anemia and ataxia (XLSA/A). Hum Mol Genet 1999;8:743-9. Pondarre C, Campagna DR, Antiochos B, Sikorski L, Mulhern H, Fleming MD. Abcb7, the gene responsible for X-linked sideroblastic anemia with ataxia, is essential for hematopoiesis. Blood 2007;109:3567-9. Mochel F, Knight MA, Tong WH, Hernandez D, Ayyad K, Taivassalo T, Andersen PM, Singleton A, Rouault TA, Fischbeck KH, Haller RG. Splice mutation in the ironsulfur cluster scaffold protein ISCU causes myopathy with exercise intolerance. Am J Hum Genet 2008;82:652-60. Fellman V, Rapola J, Pihko H, Varilo T, Raivio KO. Ironoverload disease in infants involving fetal growth retardation, lactic acidosis, liver haemosiderosis, and aminoaciduria. Lancet 1998;351:490-3. de Lonlay P, Valnot I, Barrientos A, Gorbatyuk M, Tzagoloff A, Taanman JW, Benayoun E, Chrétien D, Kadhom N, Lombès A, de Baulny HO, Niaudet P, Munnich A, Rustin P, Rötig A. A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure. Nat Genet 2001;29:57-60. Visapää I, Fellman V, Vesa J, Dasvarma A, Hutton JL, Kumar V, Payne GS, Makarow M, Van Coster R, Taylor RW, Turnbull DM, Suomalainen A, Peltonen L. GRACILE syndrome, a lethal metabolic disorder with iron overload, is caused by a point mutation in BCS1L. Am J Hum Genet 2002;71:863-6. Pennacchio LA, Bergmann A, Fukushima A, Okubo K, Salemi A, Lennon GG. Structure, sequence and location of the UQCRFS1 gene for the human Rieske Fe-S protein. Gene 1995;155:207-11.

Research Article

147

Variant clinical courses in children with immune thrombocytopenic purpura: Sixteen year experience of a single medical center Çocukluk çaù×nda immun trombositopenik purpura ve farkl× klinik seyir: Tek merkezin 16 y×ll×k deneyimi Iü×n Yaprak, Berna Atabay, úkbal Durak, Meral Türker, Haldun Öniz, Esra Arun Özer úzmir Tepecik Training and Research Hospital, úzmir, Turkey

Abstract Objective: Immune thrombocytopenic purpura (ITP) is the most common cause of acquired thrombocytopenia in children. The objective of this study was to evaluate the presenting features, variation in the clinical courses, initial response rate to therapy, and long-term outcome in patients with ITP. Materials and Methods: Three hundred and fifty out of 491 newly diagnosed patients with ITP between the initial diagnosis ages of 6 months to 16 years were included in this retrospective, descriptive study. Patients with acute vs chronic ITP, acute vs recurrent ITP and chronic vs recurrent ITP were compared in terms of age at diagnosis, gender, initial platelet count, response rate to initial therapy, long-term outcome, and total duration of follow-up. Results: The clinical courses of the patients were determined as acute, chronic and recurrent in 63.8%, 29.1%, and 7.1%, respectively. Platelet count >20x109/L and initial diagnosis age >10 years were found to increase the probability of chronic outcome by at least two-fold. Conclusion: It is concluded that ITP in childhood is a common disease with low morbidity and mortality. In addition to the acute and chronic form, a rare recurrent form, which accounts for about 4-7% of all ITP patients, should be considered. (Turk J Hematol 2010; 27: 147-55) Key words: Immune thrombocytopenic purpura, children, clinical course, chronic, recurrent, outcome Received: June 2, 2009

Accepted: March 22, 2010

Özet Amaç: úmmun trombositopenik purpura (ITP) çocukluk çaù×nda en s×k görülen edinsel trombositopeni nedenlerindendir. Bu çal×ümada, ITP’li çocuklarda baüvuru bulgular×, klinik seyirlerindeki farkl×l×klar, tedaviye ilk yan×t ve uzun dönem prognozlar×n×n deùerlendirilmesi amaçland×. Yöntem ve Gereçler: Bu retrospektif, tan×mlay×c× araüt×rmaya tan× yaü× 6 ay-16 y×l olan ITP’li 491 hastadan 350’si dahil edildi. Akut, kronik ve rekürren ITP’li olgular tan× yaü×, cinsiyet, baüvurudaki trombosit say×s×, ilk tedavi yan×t×, prognoz ve izlem süreleri yönünden ikili gruplar halinde karü×laüt×r×ld×. Bulgular: Hastalar×n %63.8’i akut, %29.1’i kronik ve %7.1’i rekürren ITP olarak tan×mland×. Trombosit Address for Correspondence: M.D. Berna Atabay, Saùl×k Bakanl×ù× Tepecik Eùitim ve Araüt×rma Hastanesi, Yeniüehir, úzmir, Turkey Phone: +90 505 479 26 62 E-mail: batabay@ttmail.com doi:10.5152/tjh.2010.21

148

Yaprak et al. Clinical course in children with ITP

Turk J Hematol 2010; 27: 147-55

say×s× >20x109/L ve baüvuru yaü×n×n >10 yaü olman×n kronik ITP olas×l×ù×n× 2 kat artt×rd×ù× görüldü. Sonuç: Sonuç olarak ITP, çocukluk yaü grubunda mortalite ve morbiditesi düüük bir hastal×kt×r. Akut ve kronik seyirli ITP d×ü×nda, tüm ITP olgular×n×n %4-7’sini oluüturan rekürren formlar×n da olabileceùi ak×lda tutulmal×d×r. (Turk J Hematol 2010; 27: 147-55) Anahtar kelimeler: úmmun trombositopenik purpura, çocuk, klinik seyir, kronik, rekürren, prognoz Geliü tarihi: 2 Haziran 2009

Kabul tarihi: 22 Mart 2010

Introduction Immune thrombocytopenic purpura (ITP) is a common acquired bleeding disorder characterized by increased destruction of antibody-sensitized platelets with normal to increased megakaryocytes in the bone marrow, presence of thrombocytopenia with otherwise normal red cells and leukocytes, absence of splenomegaly, and absence of other causes of thrombocytopenia [1,2]. Variability in the natural history and response to therapy suggest that ITP comprises a heterogeneous pathophysiology [3]. ITP affects infants, children, and adults; in the majority of the children, it is an acute, self-limiting disorder with complete resolution of the illness occurring within six months. However, 15%-30% of the children with ITP develop the chronic form of the disease defined as persistence of thrombocytopenia beyond six months. Children with chronic ITP (cITP) manifest variable clinical features and management is controversial [1-5]. Response rate to treatment is usually low in cITP; however, in some studies, high rates of spontaneous remission have been reported [6-8]. In addition to the acute and chronic forms of the disease, a recurrent course is also encountered in children. Recurrent ITP (rITP) is characterized by intermittent episodes of thrombocytopenia followed by periods of recovery, unrelated to therapeutic intervention [9,10]. Although first described in 1961 [11], literature on this clinical course of the disease is limited. At present, the natural course of ITP is not fully understood. There are no established criteria regarding the clinical characteristics of the variant clinical courses, in order to predict the time and risk factors for a chronic outcome, response to treatment and/or ultimate outcome. Furthermore, there is controversy regarding the classification of patients with recurrences. The objective of our study was to evaluate the presenting features, diversity in clinical courses (including patients with recurrences), response to initial therapy, and long-term outcome in patients

with ITP treated in our Pediatric Hematology Division and to share our single-institution, long-term, hospital-based experience together with related information in the literature.

Materials and Methods Data Collection and Diagnosis This retrospective, descriptive, cohort study was performed on patients with ITP diagnosed and followed from January 1990 to December 2006 in the Pediatric Hematology Division, Tepecik Training and Research Hospital, úzmir, Turkey. Data regarding 491 children with ITP were evaluated. There is ethical committee approval for the study. Patients less than 6 months and over 16 years at initial diagnosis, noncompliant patients and patients with proven secondary ITP were excluded. A total of 350 patients (186 females, 164 males) between the ages of 6 months to 16 years were included in the study. Diagnosis of ITP was made by detailed physical examination and history, presence of thrombocytopenia (<150x109/L) with otherwise normal red cells and leukocytes, evaluation of Giemsa-stained peripheral blood smear and bone marrow aspiration, and serological tests for infectious causes and autoimmune diseases [1]. Data collected from patients’ records included presenting hemorrhagic symptoms, age at diagnosis, gender, platelet count (PC) at diagnosis, the onset of each recurrence, number of recurrences, time elapsed between recurrences and from the initial diagnosis, treatment modality, response to treatment, follow-up duration, type of clinical course, preceding viral or bacterial infection, and medication or vaccination history. Definitions Patients having low PC (<150x109/L) for less than 6 months were defined as acute ITP (aITP) and patients with thrombocytopenia persisting for more than 6 months after initial diagnosis were defined as cITP. Complete remission was defined as PC >150x109/L whereas partial remission was PC

Turk J Hematol 2010; 27: 147-55

between 50-150x109/L. Patients achieving complete remission after initial diagnosis, but showing recurrences after at least 3 months of remission (with normal PCs) sustained without treatment and then relapsed were defined as rITP [10] (they achieved complete response to therapy after each recurrence and then relapsed). cITP patients were also evaluated in late-remission and non-remission subgroups. Chronic patients achieving normal PC after at least 6 months (usually 1-10 years) and maintaining the normal PC values thereafter were defined as late-remission cITP. Patients who did not achieve normal PC values within the entire follow-up period were defined as non-remission cITP. Treatment Patients with PC <20x109/L and/or bleeding symptoms at initial diagnosis were treated. Treatment consisted of high-dose methyl prednisolone (HDMP) or intravenous immunoglobulin (IVIG) and combination therapy of HDMP and IVIG, if the third-day PCs were still <20x109/L. In patients Â&#x201D;2 years, the first choice of therapy was IVIG, whereas HDMP was the first choice in patients >2 years. IVIG was given at a dose of 1 g/kg/day for 2 days; HDMP was given at a dose of 30 mg/kg/day for 3 days and 20 mg/kg/ day for 4 days, consecutively and intravenously. For children >6 years, HDMP was given either orally or intravenously at the same dosage [5]. Regarding criteria for response to treatment, complete response (CR) was defined as PC >150x109/L and partial response (PR) as PC 50-149x109/L, including clinical recovery. PC <50x109/L was defined as nonresponsive (NR). Evaluation and Statistical Analysis For statistical analysis, SPSS for Windows version 13.0 was used. Independent samples t-test, Pearson r2 test, Mann-Whitney U test and linear regression analysis were used. p<0.05 was accepted as statistically significant.

Results Among 350 patients with ITP, 186 were females; 96.7% had presented with minor bleeding symptoms, either skin and/or mucosal hemorrhage. Three patients presented with epistaxis requiring nasal packing, 5 with gross hematuria and 5 with gastrointestinal bleeding in addition to minor bleed-

Yaprak et al. Clinical course in children with ITP

149

ing symptoms. 71% of the patients gave a history of viral illness 3-6 weeks prior to admission. Median age at diagnosis was 60 months (6 months-16 years) and median initial PC was 9x109/L. Overall, patients were followed for a median 42 months (range: 7 months-16 years). Clinical forms were determined as aITP in 63.8%, cITP in 29.1% and rITP in 7.1%. Demographic, clinical and laboratory data of the aITP, cITP and rITP patients are shown in Table 1. Patients with aITP presented at a younger age (median 54 months) than both recurrent (median 75 months) and chronic (median 78 months) patients (p<0.05, p<0.05). 22.5% of chronic patients, 20% of recurrent patients and 11.7% of the aITP patients were >10 years of age. Initial PCs in aITP and rITP patients were significantly lower than in cITP (p<0.05, p<0.05). PC >20x109/L and initial diagnosis age >10 years were found to increase the probability of chronic outcome (odds ratio [OR]=2) by at least two-fold. Concerning the initial response rate to treatment, CR plus PR rates were highest in acute cases (87.4%), whereas 37.2% (n=38) of the chronic patients were found to be nonresponders. Three patients (12%) with rITP did not respond initially to HDMP but achieved CR by combination therapy (IVIG and HDMP) within 45-90 days. Comparison of the general characteristics of the aITP and cITP are shown in Table 2. Statistical differences were found in age at diagnosis, PC at diagnosis and response rate to initial treatment between acute and overall cITP (p<0.05). Age at diagnosis was not different in cITP vs rITP patients, whereas a significant difference was shown in aITP vs rITP patients (p>0.05). PC at diagnosis and response rate to initial treatment were not found statistically different between acute and recurrent patients (p>0.05). However, PC at diagnosis showed significant difference in cITP vs rITP (p<0.05). Initial response rate to treatment showed marginal significance between cITP and rITP (p=0.05). Regarding the 102 cITP patients, 77.5% were found to be in the non-remission group, whereas 22.5% (n=23) achieved remission in a median 18 months (8-66 months). Median age at diagnosis of the patients in non-remission and late-remission groups was 96 months and 42 months, respectively (p<0.05). 27.8% of the patients with non-remission cITP were >10 years of age, whereas 4.3% of the late remission group was >10 years at initial diagno-

150

Yaprak et al. Clinical course in children with ITP

Turk J Hematol 2010; 27: 147-55

sis (p=0.00). PC at initial diagnosis was similar but PC distribution within the follow-up period approached statistical significance between the two groups (p=0.000). Current status revealed no complete remission and 27.8% partial remission in patients with non-remission cITP, whereas all of the patients in late-remission cITP achieved complete remission during the follow-up period (Table 3). Detailed data on the patients with rITP are presented in Table 4 and general characteristics and outcome of patients with rITP are given in Table 5. Median age at initial diagnosis of these patients was 75 months (range: 22-146 months) with female pre-

dominance. PC at initial diagnosis was a median 6x109/L. After achieving complete remission in a median 20 days (at initial diagnosis) and sustaining normal PC for a median 20 months, these patients experienced 1-4 recurrences within the follow-up period of 18-141 months. Fifteen patients (60%) had only one recurrence, whereas 7 (28%) patients had 2, 2 (8%) patients had 3, and 1 (4%) patient had 4 recurrences. The first recurrence was seen between 8-109 months (median 20 months) after initial diagnosis. Time elapsed to 2nd, 3rd and 4th recurrence after diagnosis was 29, 67 and 58 months, respectively (range: 12-117 months). Duration of each

Table 1. Demographic, clinical and laboratory data of the patients with aITP, cITP and rITP No. of patients Gender, F/M Age at initial diagnosis (months) *

Acute

Chronic

Recurrent

223

102

119/104

53/49

14/11

54 (6-164)

78 (6-192)

75 (22-146)

Age distribution at diagnosis, n (%) 6-24 months

48 (21.5)

12 (11.8)

1 (4)

25-60 months

76 (34.1)

30 (29.4)

9 (36)

61-120 months

73 (32.7)

37 (36.3)

10 (40)

>120 months

26 (11.7)

23 (22.5)

5 (20)

Platelet count at diagnosis (x109/L)*

8 (1-77)

14 (1-83)

6 (1-30)

Platelet count at 1 month (x109/L)*

21 (7-72)

30 (1-43)

131 (27-324)

Complete response

130 (58.2)

27 (26.5)

12 (48)

Partial response

65 (29.2)

37 (36.3)

9 (36)

No response

28 (12.6)

38 (37.2)

3 (12)

36 (7-132)

54 (8-174)

72 (18-141)

Acute

Chronic

119/104

53/49

0.81

54 (6-164)

78 (6-192)

0.00

PC at diagnosis (x109/L)*

8 (1-77)

14 (1-83)

0.00

<20x109/L, n(%)

184 (82.5)

65 (63.7)

0.00

>20x109/L, n(%)

39 (17.5)

37 (36.3)

Complete response

130 (58.2)

27 (26.5)

Partial response

65 (29.2)

37 (36.3)

No response

28 (12.6)

38 (37.2)

36 (7-132)

54 (8-174)

Response to initial treatment, n (%)

Follow-up duration (months) * * Data are presented as median values (range)

Table 2. Comparison of the general characteristics of the patients with aITP and cITP Gender, F / M Age at diagnosis (months) *

Response to initial treatment n(%)

Follow-up duration (months) * * Data are presented as median values (range)

0.00

Yaprak et al. Clinical course in children with ITP

Turk J Hematol 2010; 27: 147-55

151

Table 3. Comparison of the 102 patients with cITP with different clinical courses Non-remission

Late-remission

44/35

9/14

0.16

96 (12-193)

42 (6-149)

0.00

No. of patients Gender, F /M Age at diagnosis (months) *

Age distribution at diagnosis, n (%) 6-24 months

6 (7.6)

6 (26.1)

25-120 months

51 (64.6)

16 (69.6)

>120 months

22 (27.8)

1 (4.3)

14 (1-83)

13 (1-76)

0.81

18 (8-66)

42 (8-118)

54 ( 8-180)

72 (18-156)

0.12

23 (100)

Partial remission

22 (27.8)

No remission

57 (72.2)

PC at diagnosis (x109/L)* Months to achieve remission * Post-remission follow-up (months) * Total follow-up (months) *

0.00

Current status n (%) Complete remission

* Data are presented as median values (range)

Table 4. Clinical and laboratory characteristics of thrombocytopenic episodes in rITP Initial Diagnosis

1st

2nd

3rd

4th

No. of patients

x109/L*

6 (1-30)

Bleeding manifestations**

9 (4-45) 11 (8-22) 4 (3-8) 37

IVIG or HDMP

24/3

22/4

8/0

2/0

IVIG+HDMP

3/0

4/0

No treatment

1/-

3/-

2/-

1/-

Treatment/no response

*Data are presented as median (range) **Bleeding manifestations were all minor manifestations. IVIG: Intravenous immunoglobulin; HDMP: High-dose methylprednisolone

recurrence was 5-11 days. Time interval between the recurrences was a median 12 months (range: 4-50 months). Time elapsed after last remission was a median 24 months (6-99 months). Currently, these patients are all in complete remission. Out of 102 cITP patients, 30 (29.4%) underwent splenectomy. Median ages at diagnosis and at splenectomy of these patients were 8.5 years and 10.7 years, respectively. Patients were followed 12-170 months after splenectomy, and within this period, 73.3% achieved complete remission. Of overall patients, 2 non-remission and 1 lateremission patients with cITP revealed antinuclear antibody (ANA) positivity (1/400). Median age at

diagnosis of these patients was 6.5 years and median follow-up duration was 5 years, during which neither clinical nor pathological laboratory data were detected. One male patient with non-remission cITP developed intracranial hemorrhage at the eighth month of diagnosis with a PC of 7x109/L; he did not respond to medical treatment and was splenectomized. None of the patients in the study group died.

Discussion Immune thrombocytopenic purpura (ITP) is a common hemorrhagic disease with a heterogeneous background during childhood. It is likely that all pediatricians will encounter children with ITP at some time in their practice. There are still many questions to be answered regarding the natural progress of the clinical forms and the prediction of chronic or recurrent outcome, response to treatment and ultimate outcome [1-10]. Our results concerning the acute and chronic patients were comparable with other series in terms of the incidence of clinical courses, mean/ median age at diagnosis, male/female ratio, initial PCs, and initial response rates to therapy [12-15]. At present, it is difficult to foresee at the time of diagnosis the patients who will develop a chronic course; although age older than 10 years, adoles-

152

Yaprak et al. Clinical course in children with ITP

Turk J Hematol 2010; 27: 147-55

Table 5. General characteristics and outcome of 25 patients with rITP No/sex

1/F

Age at Dx Months

PC at Dx (x109/L)

Days to achieve initial CR**

Time of 1st recurrence after Dx*

124

13,000

Time of 2nd recurrence after Dx*

Time of 3rd Months elapsed Total recurrence after last remission follow-up* after Dx* 41

2/M

2000

3/F

19,000

4/F

6000

5/M

20,000

6/F

7,000

7/F

13,000

8/M

5,000

136

9/F

5,000

109

117

141

10/M

8,000

120

11/F

125

4,000

12/F

30,000

141

13/M

120

4,000

132

14/M

20,000

15/M

151

5,000

16/F

6,000

17F

7,000

18/F

4,000

19/M

10,000

20/F

138

4,000

21/F

3,000

22/M

23/M 24/F 25/M

3,000

146

19,000

1,000

4,000

*months Dx: Diagnosis; **CR: Complete response

cence in females, and an initial PC >20x109/L have been associated with a chronic course [12-16]. In our study, initial PC >20x109/L and initial diagnosis age >10 years were found to increase the probability of chronic outcome by at least two-fold (OR=2). Glanz et al. [16] reported an approximate five-fold risk for progressing to chronic disease in children whose illness was diagnosed at 10 years of age and who had PC 20x109/L. Kühne et al. [12] reported a high incidence of cITP (47.3%) in older children and adolescents. The majority of the children with cITP have mild bleeding episodes not requiring treatment despite persistent thrombocytopenia. In addition, rates of spontaneous recovery of 30-60% have been report-

ed many years after initial diagnosis [6-8]. In our cohort, 102 patients with cITP were evaluated in non-remission and late-remission subgroups. Twenty-three patients (22.5%) achieved complete remission in a median 18 months (8 months-66 months). In 16 of the 23 patients, PC normalized at 8-24 months. Currently, all of the patients in the lateremission group achieved complete remission, whereas 27.8% and 72.2% of the patients in the nonremission cITP subgroup revealed partial remission and no remission, respectively. Ruggiero et al. [6] reported 5 (41.6%) out of 12 cITP patients approaching complete remission within 15-90 months. Donato et al. [8] reported a 32.9% rate of spontaneous remission in 325 nonsplenectomized children

Turk J Hematol 2010; 27: 147-55

with cITP between 6 months to 11 years; 44.9% approached remission 6-12 months from diagnosis. In Jayabose et al.’s [7] series of 62 cITP patients, a 56% spontaneous remission rate was reported. All of these authors recommended delay or omission of surgical treatment and reservation of splenectomy for patients with severe symptoms and non-responders. Imbach et al. [14] and Donato et al. [8] also reported that the cut-off value to define cITP should be changed from 6 months to 12 months. Our observation, too, showed that the cut-off point of cITP should be changed to 12 months since immune thrombocytopenia could persist beyond 6 months and could recover spontaneously in months to years. Recurrent ITP (rITP) is a rare clinical course of ITP. It was first described by Walker and Walker [11] in 1961, who defined it as recurrence of ITP after a prolonged remission. Later, Imbach [9] defined it as the recurrence of ITP after at least 3 months of remission (with normal PCs) sustained without any treatment, including splenectomy. Vranou et al. [19] defined rITP as isolated episodes of thrombocytopenic purpura following complete remission, nonrelated to therapeutic intervention, regardless of the time interval elapsed between the episodes, and reported that the time interval between two episodes less than 1-3 months was identified in 31.1% of the recurrences. Therefore, there is no consensus in the literature about the definition of rITP as to the interval between the episodes. Since 1961, only series with small numbers of patients have been published on rITP [10,17-20]. Jayabose et al. [10] reported 14 (4.1%) children with rITP among 340 pediatric patients. Median time to recurrence of these patients was 33 months (4-120 months). Twelve (86%) patients had favorable outcome, with 71% achieving complete plus partial remission and 14.2% achieving complete remission after splenectomy. Vranou et al. [19] gave the incidence of rITP as 6% in their childhood ITP series. The majority of their rITP patients had only one recurrence (1-4 recurrences). The time interval between the two recurrences was longer than 3 months (up to 96 months) in two-thirds of the episodes. They observed that the initial episode and the first recurrence generally shared the features of aITP. The incidence of rITP in our series was 7.1% (25 of 350 patients), which is the highest of those reported by Khalifa et al. [18], Jayabose et al. [10] and Vranou et al. [19]. Within the follow-up period of 18-141 months, these patients experienced 1 to 4 recurrences. Sixty percent (n=15) had only one,

Yaprak et al. Clinical course in children with ITP

153

28% (n=7) had two, 8% (n=2) had three, and 4% (n=1) had four recurrences. Median time elapsed to first recurrence after diagnosis was 20 months (8-109 months). The time interval before any recurrence was longer than four months (up to 50 months). It was also noted that these patients had low PC at initial diagnosis and at 1st, 2nd and 3rd recurrences and had achieved complete remission at 5-11 days during the recurrence. In addition, rITP and aITP were found comparable in terms of PC at initial diagnosis and response rate to initial treatment, whereas rITP was found comparable to cITP regarding age at diagnosis. PCs at initial diagnosis and during the thrombocytopenic episodes in rITP patients were as low as seen in patients with aITP. We found that the bleeding manifestations in our rITP patients were more prominent in the initial presentation and in the first recurrence, in accordance with the study by Vranou et al. [19], and the bleeding manifestations were less than expected for the low PC in the 2nd, 3rd and 4th recurrences. There are contradictory reports on the classification of the patients with recurrences. rITP is believed by some to be an exacerbation during the course of a compensated cITP, probably triggered by viral infection [21,22]. In some reviews, it is mentioned as a separate form [10,18,19]. However, Roganovic et al. [20] reported that it could be characterized as “recurrent acute ITP” or what has been called by Dameshek et al. [17] “intermittent acute ITP”. Dameshek et al. [17] showed that recurrent acute ITP was distinct from cITP since platelet survival between episodes was found normal. Our experience with rITP showed us that the majority of these patients generally shared the clinical and hematological features of aITP (low initial PC, high response rate to initial treatment, mild and self-limited course and excellent prognosis). None of these patients presented with major bleeding at any recurrence and none of the patients needed splenectomy. Even though most of these patients presented with low PCs at recurrences, responses to treatment were excellent. Preceding viral infection was noted in 16 patients at initial diagnosis. No previous history of vaccination or medication of any kind was detected at any recurrence. Unlike our favorable results in patients with rITP, Vranou et al. [19] and Jayabose et al. [10] reported 2 cases with rITP who developed intracranial hemorrhage, 1 being fatal. Also, 2 of 14 patients of Jayabose et al. [10] achieved complete remission after splenectomy. The discrepancy between the results of the study groups shows that

154

Yaprak et al. Clinical course in children with ITP

definition, pathogenesis and outcome of rITP requires more data to be reported. Splenectomy remains an effective therapeutic option for patients with cITP with severe symptoms and/or nonresponders to pharmacological treatment [23]. The rate of splenectomy performed for children with cITP ranges between 9% and 39% in the literature [23-27]. However, there are no currently available reliable factors predictive of whom and when to splenectomize and/or foresee the response rate to splenectomy. In our population of 102 cITP patients, 29.4% (n=30) underwent splenectomy with a CR rate of 73.3% and a PR rate of 6.7%. Median ages at initial diagnosis and at splenectomy were 8.5 years and 10.7 years, respectively. Fourteen patients underwent splenectomy between 8-16 months, 6 patients between 2-3 years and 8 patients more than 3 years after the initial diagnosis. None of the children with ITP had serious bleeding manifestations within the total follow-up period except a three-year-old male patient who experienced an intracranial hemorrhage at the seventh month of diagnosis with a PC=7x109/L, who had shown no response to pharmacological therapy; he was splenectomized. He is now six years old and is being followed in our hematology division without neurological sequelae and with PC at 30-50x109/L. In conclusion, ITP in childhood is a hemorrhagic disease with diverse natural history. Its heterogeneous background and lack of long-term clinical data cause controversies in classifying and managing patients with ITP. Our experience shows that a late-remission course in patients with cITP should be considered and the cut-off value to define cITP should be changed to 12 months. Children with PC >20x109/L at initial diagnosis and age at diagnosis >10 years have a two-fold risk for progression to chronic disease. rITP, a rare clinical course that seems to be mild and self-limiting and generally shares the clinical and hematological features of aITP, should also be considered. Splenectomy should be delayed and reserved for children with severe symptoms and/or non-responders. Prospective international long-term studies are needed to enlighten the natural history of the clinical courses and the final outcome in children with ITP. Acknowledgement The authors thank Prof. Hale Ören (Dokuz Eylül University, School of Medicine, úzmir, Turkey) for her contribution in revising the manuscript.

Turk J Hematol 2010; 27: 147-55

Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1. 2. 3. 4.

10.

11. 12.

Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med 2002;346:995-1008. Nugent DJ. Childhood immunothrombocytopenic purpura. Blood Rev 2002;16:27-9. Kühne T. Immune thrombocytopenic purpura in childhood: controversies and solutions. Pediatr Blood Cancer 2006;47(5 Suppl):650-2. Belletrutti M, Ali K, Barnard D, Blanchette V, Chan A, David M, Luke B, Price V, Ritchie B, Wu J; Canadian Pediatric Chronic ITP Working Group; Canadian Pediatric Thrombosis and Hemostasis Network. Chronic immune thrombocytopenic purpura in children: a survey of the Canadian experience. J Pediatr Hematol Oncol 2007;29:95-100. Ozer EA, Yaprak I, Atabay B, Türker M, Aküit S, Sar×oùlu B. Oral cyclic megadose methylprednisolone therapy for chronic immune thrombocytopenic purpura in childhood. Eur J Hematol 2000;64:411-5. Ruggiero A, Annunziata ML, Lazzareschi I, Ridola V, Attinà G, Riccardi R. Chronic immune thrombocytopenic purpura in children: case reports of spontaneous recovery without splenectomy. Pediatr Hematol Oncol 2006;23:471-5. Jayabose S, Levendoglu-Tugal O, Ozkaynkak MF, Visintainer P, Sandoval C.. Long-term outcome of chronic immune thrombocytopenic purpura in children. J Pediatr Hematol Oncol 2004;26:724-6. Donato H, Picón A, Martinez M, Rapetti MC, Rosso A, Gomez S, Rossi N, Bacciedoni V, Schvartzman G, Riccheri C, Costa A, Di Santo J. Demographic data, natural history, and prognostic factors of immune thrombocytopenic purpura in children: a multicentered study from Argentina. Pediatr Blood Cancer 2009;52:491-6. Imbach P, Lilleyman JS, Hann IM and Blanchetter, V. S. (eds) (1999) Immune thrombocytopenic purpura. Pediatric Hematology, ed 2 Churchill Livingstone, London: Churchill Livingstone, 1999: 437-53. Jayabose S, Levendoglu-Tugal O, Ozkaynak MF, Sandoval C. Recurrent immune thrombocytopenic purpura in children. Pediatr Hematol Oncol 2006;23:677-82. Walker JH, Walker W. Immune thrombocytopenic purpura in childhood. Arch Dis Child 1961;36:649-57. Kuhne T, Buchanan GR, Z×mmerman S. Michaels LA, Kohan R, Berchtold W, Imbach P. A prospective comparative study of 2540 infants and children with newly diagnosed immune thrombocytopenic purpura (ITP) from the intercontinental childhood ITP study group. J Pediatr 2003;143:605-8.

Yaprak et al. Clinical course in children with ITP

Turk J Hematol 2010; 27: 147-55

13.

14.

15.

16.

17.

18.

19.

20.

Watts RG. Immune thrombocytopenic purpura: a 10-year natural history study at the childrens hospital of Alabama. Clin Pediatr 2004;43:691-702. Imbach P, Kühne T, Müller D, Berchtold W, Zimmeman S, Elalfy M, Buchanan GR. Childhood ITP: 12 months follow-up data from the Prospective Registry I of the Intercontinental Childhood ITP Study Group (ICIS). Pediatr Blood Cancer 2006;46:351-6. Paling A, Stefan DC. Immune thrombocytopenic purpura in childhood: a 10-year audit. Hematology 2008;13:175-80. Glanz C, France E, Xu S, Hayes T, Hambidge S. A population-based multisite, cohort study of the predictors of chronic idiopathic thrombocytopenic purpura in children. Pediatrics 2008;121:e506-12. Dameshek W, Ebbe S, Greenberg L, Baldini M. Recurrent acute idiopathic thrombocytopenic purpura. N Engl J Med 1963;269:649-53. Khalifa AS, Tolba KA, el-Alfy MS, Gadallah M, Ibrahim FH. Idiopathic thrombocytopenic purpura in Egyptian children. Acta Haematol 1993;90:125-9. Vranou M, Platokouki H, Pergantou H, Aronis S. Recurrent immune thrombocytopenic purpura in childhood. Pediatr Blood Cancer 2008;51:261-4. Roganovic J, Letica-Crepulja M, Dordevic L. Recurrent immune thrombocytopenic purpura in children. Pediatr Hematol Oncol 2008;25:243-4.

21.

22.

23.

24. 25.

26.

27.

155

Wilson DB. Acquired platelet disorders. In: Nathan DG, Orkin SH, Ginsburg D, Look AT, editors. Nathan and Oski’s Hematology of Infancy and Childhood. 6th ed. Philadelphia: WB Saunders, 2003: 1597-630. Conradi A, Diaz de Heredia C, Tussel Puigbert J, Tobena Boada L, Ortega Aramburu JJ. Chronic and recurrent immune thrombocytopenic purpura. Ann Pediatr (Barc) 2003;59:6-12. Donato H, Picón A, Rapetti MC, Rosso A, Schvartzman G, Drozdowski C, Di Santo JJ. Splenectomy and spontaneous remission in children with chronic immune thrombocytopenic purpura. Pediatr Blood Cancer 2006;47:737-9. Mantadakis E, Buchanan GR. Elective splenectomy in children with immune thrombocytopenic purpura. J Pediatr Hematol Oncol 2000;22:148-53. El-Alfy MS, El-Tawil MM, Shahein N. 5- to 16-year follow-up following splenectomy in chronic immune thrombocytopenic purpura in children. Acta Haematol 2003;110:20-4. Wang T, Xu M, Ji L, Yang R. Splenectomy for chronic immune thrombocytopenic purpura in children: a single center study in China. Acta Haematol 2006;115:39-45. Yetgin S, Olcay L, Ozsoylu S, Hiçsönmez G, Gurgey A, Tuncer AM. Retrospective analysis of 78 children with chronic ITP: follow-up from 1976-1996. Pediatr Hematol Oncol 1997;14:399-412.

156

Research Article

Efficacy of immunization against hepatitis B virus infection in acute leukemia Akut lösemide hepatit B virüsü enfeksiyonuna karü× aü×lama etkililiùi Tuphan Kanti Dolai, Manoranjan Mahapatra, Hara Prasad Pati, Pravas Mishra, Tulika Seth, Rahul Bhargava, Shyam Rathi, Niranjan Rathod, Renu Saxena All India Institute of Medical Sciences (AIIMS), New Delhi, India

Abstract Objective: The aim of this study was to assess the antibody response to combined passive-active immunization versus active immunization against hepatitis B in 71 patients with acute leukemia with negative hepatitis B virus serology at presentation. Materials and Methods: The first group (n=28) received a double dose of hepatitis B vaccine at 0, 1, 2 and 6 months and immunoglobin (HBIG) at 0 and 1 month concurrently with vaccine but at a different intramuscular site. The second group (n=43) received double dose of hepatitis B vaccine at 0, 1, 2, and 6 months. HBsAg and anti-HBs titers were determined one month after the 1st, 2nd, 3rd and 4th doses of vaccine. Results: In the vaccine-only group, 2.56%, 8.33%, 14.28% and 34.29% of patients developed anti-HBs titer 10 IU/L after the 1st, 2nd, 3rd and 4th doses of vaccine, respectively. In the HBIG group, 91.30%, 91.30%, 69.56% and 73.91% of patients developed anti-HBs titer 10 IU/L after the 1st, 2nd, 3rd and 4th doses of vaccine, respectively. Those in the vaccine-HBIG group maintained their anti-HBs titer 10 IU/L from the 1st to the 4th doses. In the vaccine-only group, 34.29% of patients gained protective antibody titer after receiving the 4th dose of vaccine. Subgroup analysis of age (pediatric vs adult) and disease (acute lymphoblastic leukemia vs acute myeloid leukemia) groups showed no effect of either on the development of protective antibody titer. The incidence of HBsAg positivity one month after the 4th dose of vaccine was 8.62%. No patient became positive for anti-HCV or HIV antibody before or after chemo therapy. Conclusion: Combined HBIG and vaccine may protect acute leukemia patients during the intensive chemotherapy period. (Turk J Hematol 2010; 27: 156-61) Key words: Acute leukemia, hepatitis B vaccine, hepatitis B immunoglobin Received: May 27, 2009

Accepted: April 30, 2010

Özet Amaç: Bu çal×ümada, prezantasyonda negatif Hepatit B virüsü serolojisine sahip akut lösemili 71 hastada Hepatit B virüsüne karü× aktif aü×lama karü×s×nda birleüik pasif aktif aü×lamaya yönelik antikor yan×t×n×n deùerlendirilmesi amaçlanm×üt×r. Address for Correspondence: M.D. Tuphan Kanti Dolai, Department of Hematology, 1st Floor, All India Institute of Medical Sciences (AIIMS) Ansari Nagar, 11002 New Delhi, India Phone: 09968182523 E-mail: tkdolai@hotmail.com doi:10.5152/tjh.2010.22

Dolai et al. Active and passive immunization in acute leukemia

Turk J Hematol 2010; 27: 156-61

157

Yöntem ve Gereçler: Birinci grup (n=28), 0. ay, 1. ay, 2. ay ve 6. ay zaman noktalar×nda ikili hepatit B aü×s× dozu ve 0. ay, 1. ay zaman noktalar×nda aü× ile eüzamanl× olarak (ancak kas içi olarak) immunoglobin (HBIG) alm×üt×r. úkinci grup (n=43), 0. ay, 1. ay, 2. ay ve 6. ay zaman noktalar×nda ikili hepatit B aü×s× dozu alm×üt×r. HBsAg ve anti HBs titreleri, aü×n×n 1. dozu, 2. dozu, 3. dozu ve 4. dozundan sonra yap×lm×üt×r. Bulgular: Yaln×zca aü× yap×lan grupta hastalar×n %2.56, %8.33, %14.28 ve %34.29’unda, s×ras×yla 1., 2., 3. ve 4. aü× dozu verildikten sonra anti HBs titresi 10 IU/l oluümuütur. HBIG grubunda, hastalar×n %91.30, %91.30, %69.56 ve %73.91’inde, s×ras×yla 1., 2., 3. ve 4. aü× dozu verildikten sonra anti HBs titresi 10 IU/l oluümuütur. HBIG alan hastalar, 1. dozdan 4. doza kadar 10 IU/l HBs titre deùerini muhafaza etmiütir. Yaln×zca aü× verilen grupta, hastalar×n %34.29’u 4. aü× dozu verildikten sonra koruyucu antikor titresi edinmiütir. Yaü (yetiükin karü×s×nda pediatrik) ve hastal×k (akut myeloid lösemi karü×s×nda akut lenfoblastik lösemi) gruplar×nda, koruyucu antikor titresi geliüimi bak×m×nda herhangi bir etki gözlenmemiütir. Aü×n×n 4. dozundan bir ay sonar HBsAg pozitivite insidans× %8.62’dir. Kemoterapiden önce sonra, hiçbir hastada anti HCV ve HIV antikoru için pozitivite geliümemiütir. Sonuç: Kombine HBIG ve aü×, yoùun kemoterapi periyodu s×ras×nda akut lösemi hastalar×n× koruyabilir.

(Turk J Hematol 2010; 27: 156-61)

Anahtar kelimeler: Akut lösemi, Hepatit B aü×s×, Hepatit B immunoglobin Geliü tarihi: 27 May×s 2009

Kabul tarihi: 30 Nisan 2010

Introduction Hepatitis B is one of the most important causes of acute and chronic hepatitis. Children with malignant disease are at an especially high risk for developing hepatitis B virus (HBV) infection from immunosuppression secondary to chemotherapy, radiotherapy and multiple blood transfusions. Most of the children infected with HBV develop chronic hepatitis. This plays an adverse prognostic role in terms of their disease-free survival because of delays in chemotherapy. The increasing potential for the cure of childhood malignant diseases emphasizes the need for a method of reducing hepatitis and its sequelae in these children. Hepatitis B virus infection is prevalent in India. Among blood donors, pregnant women and the general population whose carrier frequency is 2%-4% and hepatitis B surfact antibody (anti-HBs) positivity is around 18%-20% [1]. A high proportions of children treated for malignant disorders demonstrate seroconversion for HBV infection markers [2]. The high prevalence of HBV infection and relative failure of active immunization in patients with leukemia on therapy have prompted a search for alternative forms of prophylaxis [3]. Various studies have observed a decreased rate of transmission of the infection with passive immunization [4]. Thus, for protection against HBV infection, both active and passive immunization have been tried [5,6]. Since there are very few studies in this context, this study was undertaken to determine the efficacy of immunization (both active and passive) against HBV infection in acute leukemia patients.

Materials and Methods The aims and objectives of this study were to determine the incidence of hepatitis B surface antigen (HBsAg) positivity before treatment, the efficacy of hepatitis B vaccine along with HBIG (hepatitis B immunoglobulin) in patients with acute leukemia, the anti-HBs titer level in the course of treatment and up to six months, and the HBsAg positivity rate in the course of treatment up to six months. A total of 114 patients with acute leukemia were tested for HBsAg and anti-HBs titer before starting treatment. Of the 114 patients, 3 were positive for HBsAg and 111 patients were negative for HBsAg. Out of 111 patients, 40 had anti-HBs titer 10 IU/L and 71 patients had anti-HBs titer <10 IU/L. Thus, 71 patients were included in this study. This project was approved by the All India Institute of Medical Sciences (AIIMS) Ethics Committee. It is a prospective comparative trial including patients with acute leukemia with HBsAg, anti-HBs negativity before treatment and with anti-HBs titer <10 IU/L. Those patients already immunized and with protective anti-HBs titer 10 IU/L were excluded from the study. Those who already completed immunization against hepatitis B and with anti-HBs titer <10 IU/L were also included in this study. Immunization was started from induction. The first group (n=28) received hepatitis B vaccine (Engerix B GlaxoSmithKline Biologicals, double dose, i.e. 11 years 20 mcg and >11 years 40 mcg) at 0, 1, 2, and 6 months and HBIG (Hepabig- VHB Life Sciences Limited, Mumbai, India at a dose of 40 IU/kg maximum of 800 IU) at 0 and 1 month concurrently with

158

Dolai et al. Active and passive immunization in acute leukemia

Engerix B but at a different intramuscular site. The second group (n=43) received only hepatitis B vaccine (Engerix B - double dose, i.e. 11 years 20 mcg and >11 years 40 mcg) at 0, 1, 2, and 6 months. HBsAg and anti-HBs titer were determined one month after the 1st, 2nd, 3rd and 4th doses of vaccine. In those patients who were given Engerix B and HBIG and became HBsAg positive after starting treatment, subsequent doses of Engerix B and HBIG were not given. Anti-hepatitis C virus (HCV) total and human immunodeficiency virus (HIV) antibody were also tested before starting treatment and one month after the 4th dose of vaccine. HbsAg was tested using Hepanostika® HBsAg Ultra (bioMerieux bv Boseind 15, 5281RM Boxtel, The Netherlands). Anti-HBs was tested using VIDAS® (bioMérieux® sa, France). Fisher’s exact test and chi-square test were used to compare proportions between the groups. McNemar test was used to compare proportions within the group. Wilcoxon rank-sum (MannWhitney U) test was used to compare the medians. Two-sample t test was used to compare the median between the groups in equal variances. The statistical tests were performed by using STATA 9.0 and SPSS software version 11.5 for Windows®.

Results Of these 114 patients, 3 (3%) were positive for HBsAg and 111 patients (97%) were negative for HBsAg. Out of 111 patients, 40 (36%) had anti-HBs titer 10 IU/L and 71 (64%) had anti-HBs titer <10 IU/L. Of those patients with anti-HBs titer 10 IU/L, 7 (18%) patients had past history of jaundice, 13 (33%) had history of previous vaccination and 20 (49%) had no history of jaundice or previous vaccination. A significant statistical difference (p=0.04) was found between pediatric and adult patients in anti-HBs titer 10 IU/L and anti- HBs titer <10 IU/L groups. The majority of pediatric patients had antiHBs titer 10 IU/L compared to adults before starting chemotherapy. The median age was 22 years (range, 1-52); 49 (69.01%) were male and 22 (30.99%) were female (Table 1). The total number of pediatric ( 18 yrs) patients was 31 (43.66%) and of adult (>18 yrs) patients was 40 (56.34%). The total number of acute lymphoblastic leukemia (ALL) patients was 38 (53.52%) and of acute myeloid leukemia (AML)

Turk J Hematol 2010; 27: 156-61

Table 1. Baseline patient characteristics according to vaccine group Parameters HBIG-Vaccine Vaccine-only P value n=28 n=43 (Mann-Whitney Median Median test) (range) (range) Age in years

18 (3-44)

25 (1-52)

0.20

DDTT days

10 (2-142)

14 (1-147)

0.42

2 (0-18)

3 (0-40)

0.87

0.8 (0.6-5.8)

0.8 (0.5-4.6)

1.00

AST U/dl

27 (12-451)

29 (10-127)

0.96

ALT U/dl

31.5 (10-169)

36 (11-295)

0.87

ALP U/dl

243 (129-552)

232 (96-1270)

0.71

6.35 (4-7.6)

6.8 (4-9.5)

0.02*

S. Albumin g/dl

3.55 (2.1-4.6)

3.9 (2.3-4.8)

0.02*

S. Globulin g/dl

2.75 (1.9-4.3)

2.9 (1.7-5.1)

0.21*

22 (44.9)/

27 (55.10)/

0.12 #

6 (39.44)

16 (72.73)

20 (52.63)/

18 (47.37)/

8 (24.24)

25 (75.76)

20 (71.42)/

11 (25.58)/

8 (28.58)

32 (74.42)

BT units S. Bilirubin mg/dl

S. Protein g/dl

Sex M/F ALL/AML Age 18 yrs/ >18 yrs

0.01# 0.0002#

HBIG: Hepatitis B immunoglobin; DDTT: Duration of diagnosis to received treatment; BT: Blood transfusion; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase *done by two sample t test; #done by Fisher’s exact test

patients was 33 (46.48%). Nine patients (12.67%) died due to their disease and chemotherapy complications before the 2nd dose of vaccine; thus, 62 patients received a 2nd vaccine dose. Three patients died before receiving their 3rd dose of vaccine; thus, 59 patients received a 3rd vaccine dose. One patient died before receiving their 4th dose of vaccine; thus, 58 patients received a 4th vaccine dose. In the vaccine-only group, 2.56%, 8.33%, 14.28% and 34.29% patients developed anti- HBs titer 10 IU/L after the 1st, 2nd, 3rd and 4th doses of vaccine, respectively. In the HBIG-vaccine group, 91.30%, 91.30%, 69.56% and 73.91% patients developed antiHBs titer 10 IU/L after the 1st, 2nd, 3rd and 4th doses of vaccine, respectively, and 8.69%, 8.69%, 30.43% and 26.09% patients had anti-HBs titer <10 IU/L after the 1st, 2nd, 3rd and 4th doses of vaccine, respectively. Significantly statistical differences (Table 2) in anti-HBs titer ( 10 U/L) were seen between the two vaccine groups after the 1st, 2nd, 3rd and 4th doses of vaccine. Those who received HBIG maintained their anti-HBs titer 10 IU/L even one month after the 4th dose of vaccine. In the vac-

Dolai et al. Active and passive immunization in acute leukemia

Turk J Hematol 2010; 27: 156-61

Table 2. Anti-HBs titer according to vaccine group at the different time points Parameters <10 IU/L, 10 IU/L P value (Fisher’s exact test) One month after 1st dose HBIG-Vaccine

2 (8.69)

21 (91.30)

n=23, no (%) Vaccine-only

0.0001 38 (97.43)

1 (2.56)

2 (8.69)

21 (91.30)

33 (91.66)

3 (8.33)

7(30.43)

16 (69.56)

30 (85.71)

5 (14.28)

6 (26.09)

17 (73.91)

23 (65.71)

12 (34.29)

n=39, no (%) One month after 2nd dose HBIG-Vaccine n=23, no (%) Vaccine-only

0.0001

n=36, no (%) One month after 3rd dose HBIG-Vaccine n=23, no (%) Vaccine-only

0.0001

n=35, no (%) One month after 4th dose HBIG-Vaccine n=23, no (%) Vaccine-only

0.003

n=35, no (%) HBIG: Hepatitis B immunoglobin

cine-only group, after the 4th dose, anti-HBs titer 10 IU/L was achieved in 34.29% of patients, which was a very small proportion compared to the HBIG group. In both groups, a subgroup analysis was done between the ALL vs AML groups and pediatric vs adult groups. No significant differences were found between the ALL vs AML groups or between the pediatric vs adult groups. In the pediatric age group, 72.72%, 58.33%, 47.61% and 41.37% patients developed anti-HBs titer 10 IU/L after the 1st, 2nd, 3rd and 4th doses of vaccine, respectively, whereas in the adult age group, 27.28%, 41.67%, 52.38% and 58.63% patients developed anti-HBs titer 10 IU/L after the 1st, 2nd, 3rd and 4th doses of vaccine, respectively. This showed that pediatric patients lost their anti-HBs titer gradually from the 1st to the 4th dose of vaccine. On the other hand, an increasing number of adult patients gained anti-HBs titer 10 IU/L gradually from the 1st to the 4th dose of vaccine. Statistical differences in antiHBs titer 10 IU/L were seen between the two age

159

groups after the 1st (p=0.002) and 2nd (p=0.01) dose of vaccine, but not after the 3rd (p=0.40) and 4th (p=1.00) dose of vaccine. Thus, more pediatric patients developed anti-HBs titer 10 IU/L after the 1st and 2nd doses of vaccine compared to adult patients. Though more adult patients gained protective anti-HBs titer gradually from the 3rd to 4th doses of vaccine, when compared to the pediatric age group, the difference was statistically insignificant. In the ALL group, 81.81%, 70.84%, 47.62% and 55.17% patients developed anti-HBs titer 10 IU/L after the 1st, 2nd, 3rd and 4th doses of vaccine, respectively, whereas in the AML group, 18.19%, 29.16%, 52.38% and 44.83% patients developed antiHBs titer 10 IU/L after the 1st dose, 2nd, 3rd and 4th doses of vaccine, respectively. After the 1st dose of vaccine, a statistical difference in anti-HBs titer 10 IU/L was seen between the ALL and AML groups. No statistically significant differences were found after administration of the other doses (p values: 0.06 after 2nd dose, 0.28 after 3rd dose and 1.00 after 4th dose). A significant number of ALL patients developed protective antibody titer after the 1st vaccine dose compared to AML patients; thereafter, both groups behaved similarly. One month after the 4th dose of vaccine, out of 58 patients, 5 patients (8.6%) were found HBsAgpositive. Among the HBsAg-positive patients, 1 (4.34%) was in the HBIG group and the remaining 4 (11.42%) were in the vaccine-only group. Of the HBsAg-positive patients, 3 had ALL and 2 had AML. No significant difference (p=0.63) in HBsAg positivity was found between the vaccine groups. After 210 days of follow up, none of the patients was found to be positive for anti-HCV or HIV antibody.

Discussion A study from India showed that 47.8% of ALL patients were positive for HBsAg after therapy. In order to reduce the impact of HBV infection, schedules for active immunization, double doses of active immunization and both active and passive immunization against this infection have been investigated. The HBsAg positivity rate (3%) in our study is quite similar to that of other studies performed in the general population in India. But the anti-HBs positivity rate in our study is quite high (40%) compared to other studies (18-20%) performed from India. This is probably due to the increased awareness of hepatitis B vaccination in the general popu-

160

Dolai et al. Active and passive immunization in acute leukemia

lation. The HBsAg positivity rate of acute leukemia in this study was slightly high (3%) compared to that of an older study (1.85%) in ALL done by Marwaha et al. [2] from Chandigarh, India. It is probably due to the less sensitive method used for detection of HBsAg in 2001. Anti-HBs titer 10 IU/L was seen in more pediatric patients compared to adults, due to the increased awareness of hepatitis B vaccination in the pediatric population. Goyal et al. [3] showed that after administration of double-dose vaccine during induction, consolidation and maintenance (0-1-2-12 months) chemotherapy, 19.7% patients developed anti-HBs titers and anti-HBs titer 10 IU/L was achieved in only 10.5% of patients. They detected HBsAg in 48.79% of their patients during the course of treatment. Another study from India by Somjee et al. [7] showed that at the end of six doses of vaccine (0-1-2-3-4-12), 29.75% of patients developed anti-HBs titers, and of them, only 18.9% had anti-HBs titer 10 IU/L. In the course of treatment, 43% of patients developed HBsAg positivity. A similar dose schedule as in our study (0-1-2-6) was tried in adult patients with acute leukemia (ALL & AML) by Gurina et al. [8]. In 30 patients, the rate of antibody positivity was 50% after the 4th dose of vaccine and 6.7% became infected with HBV over the three-year follow-up. A more intensive vaccination schedule (0-1-2-6-12) was used by Yetgin et al. [9] in 82 patients with childhood ALL. They found that the rate of antibody positivity was 35.4% after the 5th dose of vaccine, and 4.8% became infected with HBV after the end of vaccination. Our results are quite similar with the Yetgin et al. trial. An exact comparison with other studies is not possible because different vaccination schedules were used. In our study, more patients (34.29%) developed protective antibody titer after receiving their 4th dose of vaccine compared with other trials done by Goyal et al. [3] and Somjee et al. [7]. This is possibly due to the more severe immunosuppressive chemotherapy schedule used in their study. In our study, after a seven-month follow-up period, a small number (11.42%) of patients developed HBsAg positivity compared with other trials done by Goyal et al. and Somjee et al. This is due to improved infection control measures, safer blood supply in our hospital and the short duration of follow-up in our study. In spite of active immunization with recombinant DNA vaccine, some patients became infected with HBV infection. Thus, active immunization with recombinant DNA vaccine has a minimal role in these

Turk J Hematol 2010; 27: 156-61

immunosuppressed patients while they are on aggressive therapy. A total of 28 patients received both vaccine and HBIG. Passively transferred immunity generated high titers of antibodies in these patients in the first 3 months. However, once these were eliminated over a period of another 3 months, active immunization was unable to confer sustained protection at 7 months. Therefore, it should be stressed that passive prophylaxis alone needs to be administered during the entire course of aggressive chemotherapy. Kavakli et al. [4] studied 22 patients with leukemia who received HBIG (800 IU once per month for 3 doses), together with vaccine at a different intramuscular site. HBV infection was not observed in any of the patients at the 4th and 12th months of serological follow-up. At the end of the first year, the antibody response reached 85%. In another study by Meral et al. [6], passive immunization with immunoglobulin (monthly for four doses) was given at the time of aggressive chemotherapy, and subsequently these patients were actively immunized with vaccine (40 mcg, 1-2-12 months) from the third month of maintenance therapy. Their rate of antiHBs positivity was 90.3% at the end of the four doses, and at the end of the three-year follow-up, only 5 patients (16%) lost the protective antibody titers. In another Indian study by Somjee et al. [5], five doses of hepatitis B vaccine (ENGERIXSmithKline Beecham) along with HBIG (HEPABIGVHB Pharmaceuticals) were given in 31 ALL patients. Their rates of anti-HBs positivity were 89.6% at the end of 6 months, and at the end of 9 months, only 8 (42%) patients had lost the protective antibody titer. The rate of HBV infection was 27% at the end of 9 months. In our study, 73.91% of patients had developed anti-HBs titer 10 IU/L 1 month after the 4th dose of vaccine. This is a slightly inferior response compared to results of the Kavakli et al. [4], Meral et al. [6] and Somjee et al. [7] trials. The inferior response is possibly due to lesser doses of HBIG used in our trial (i.e. 2 doses of HBIG in our trial, 3 doses in Kavakli et al. trial, 4 doses in Meral et al. trial and 5 doses in Somjee et al. trial). Thus, a comparison of our study and other studies clearly shows that more doses of HBIG are required to achieve the maximum protective anti-HBs titers. Apart from that, HBIG may protect patients during the intensive chemotherapy period, but there must be sufficient antibody level during the entire period to decrease the rate of HBV infection in these patients. HBIG

Dolai et al. Active and passive immunization in acute leukemia

Turk J Hematol 2010; 27: 156-61

could be given during aggressive treatment where maximum immunosuppression exists in addition to increased chances of exposure. This could be used in combination with more doses of active immunization, which may result in higher rates of antibody response. In normal individuals, the rate of seroconversion following hepatitis B vaccine is almost 96%, and protective antibody levels can be achieved in 93% [3]. In the present study, despite the use of a double dose of vaccine and a more intensified vaccination program, seroconversion could be achieved in only 34.29% and 73.91% of the patients in the vaccineonly and vaccine-HBIG groups, respectively. This is because immunosuppression in leukemic patients resulting from the disease as well as the use of steroids lead to diminished response to initial or booster vaccination. Therefore, passive immunization with hyperimmunoglobulin followed by active immunization starting during maintenance therapy or after cessation of intensive chemotherapy may be a better alternative to achieve permanent protective antibody titers. In the HBIG group, subgroup analysis was done between the ALL vs AML and pediatric vs adult groups. No statistically significant differences were found between the ALL vs AML groups or between the pediatric vs adult groups. Thus, differences in age and disease were not responsible for development of protective antibody titer. HBsAg positivity rate was similar between the two vaccine groups in the short follow-up period (7 months). The outcome of long-term follow-up might be different in terms of HBsAg positivity. Thus, long- term followup will be required to determine the efficacy of vaccination and the HBsAg positivity rate. In conclusion, HBIG may protect patients during the intensive chemotherapy period, but there must be sufficient antibody level during the entire period to decrease the rate of HBV infection in these patients. To increase the level of protective antibody

161

titers (Â&#x2022;10 IU/L), both active and passive immunization are required. A large prospective multicenter randomized controlled trial is needed to address this issue. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1.

Acharya SK, Panda SK. Hepatitis E virus: epidemiology, diagnosis, pathology and prevention. Trop Gastroenterol 2006;27:63-8. Marwaha R, Rawat D, Chawla Y. Seroprevalence of hepatitis B and C viral infections at diagnosis and during the course of treatment in childhood malignancies. Med Pediatr Oncol 2001;37:166. Goyal S, Pai SK, Kelkar R, Advani SH. Hepatitis B vaccination in acute lymphoblastic leukemia. Leuk Res 1998;22:193-5. Kavakli K, Cetingul N, Oztop S. Combined administration of hepatitis B vaccine and hepatitis B immunoglobin in children with cancer. Pediatr Hematol Oncol 1996;13:295-8. Somjee S, Pai S, Parikh P, Banavali S, Kelkar R, Advani S. Passive active prophylaxis against hepatitis B in children with acute lymphoblastic leukemia. Leuk Res 2002;26:989-92. Meral A, Sevinir B, Gunay U. Efficacy of immunization against hepatitis B virus infection in children with cancer. Med Pediatr Oncol 2000;35:47-51. Somjee S, Pai S, Kelkar R, Advani S. Hepatitis B vaccination in children with acute lymphoblastic leukemia: results of an intensified immunization schedule. Leuk Res 1999;23:365-7. Gurina NM, Svedentsov EP, Shardakov VI, Cherepanova VV. Vaccine prophylaxis of viral hepatitis B in patients with acute leukemia. Ter Arkh 2008;80:27-9. Yetgin S, Tavil B, Aytac S, Kuskonmaz B, Kanra G. Unexpected protection from infection by two booster hepatitis B virus vaccination in children with acute lymphoblastic leukemia. Leuk Res 2007;31:493-6.

162

Research Article

Evaluation of risk factors for thrombophilia in patients with cerebral venous thrombosis Serebral venöz tromboz geçiren hastalar×n trombofilik risk faktörleri aç×s×ndan deùerlendirilmesi Osman Yokuü1, Özlem ûahin Balç×k2, Murat Albayrak3, Funda Ceran4, Simten Daùdaü4, Mesude Y×lmaz4, Gülsüm Özet4 1Department

of Hematology, Sema Hospital, ústanbul, Turkey of Hematology, Fatih University Medical School, Ankara, Turkey 3Department of Hematology, D×ükap× Y×ld×r×m Beyaz×t Education and Research Hospital, Ankara, Turkey 4Department of Hematology, Numune Education and Research Hospital, Ankara, Turkey 2Department

Abstract Objective: The increased risk for thrombosis is known as hypercoagulability or thrombophilia. In our study, we aimed to determine the frequency of the identified defects for thrombophilia in patients with central venous thrombosis aged under 50 years and to compare results with the findings in the current literature. Materials and Methods: Forty-three patients (16-50 years old) were retrospectively evaluated. Thrombophilia investigation included determinations of protein C, protein S, antithrombin, and activated protein C resistance, factor V Leiden (FVL), prothrombin 20210A (PT 20210) and methylene tetrahydrofolate reductase (MTHFR) C677T mutations, antiphospholipid antibodies (APA), factor VIII levels, and homocysteine levels. Results: We detected a single thrombophilic defect in 67.4%, two defects in 27.9% and three defects in 4.7% of our patients. The most common thrombophilic defect was mutation in the MTHFR gene (41.8%), and this was followed by the FVL mutation (34.9%). Conclusion: Since the prevalence of individual thrombophilic defects varies in each population, ethnic group and geographical location, screening for thrombophilic defects in patients presenting with cerebral venous thrombosis should primarily investigate the most frequent thrombophilia risk factors.

(Turk J Hematol 2010; 27: 162-7)

Key words: Cerebral venous thrombosis, thrombophilia, thrombophilic defects Received: October 4, 2009

Accepted: March 22, 2010

Özet Amaç: Tromboz riskinin artmas× hiperkoagulabilite ya da trombofili olarak bilinmektedir. Çal×ümam×zda, serebral venöz tromboz geliüen ve trombofilik defekt saptanan 50 yaü ve alt× hastalar×m×zda tespit edilen trombofilik defektleri, s×kl×ù×n× deùerlendirmek ve literatür bilgileriyle karü×laüt×rmak amaçlanm×üt×r. Address for Correspondence: M.D. Özlem ûahin Balç×k, Dizgi Sok. 9/6 Bas×nevleri, 06120 Ankara, Turkey Phone: +90 312 321 66 97 E-mail: drozlembalcik@yahoo.com doi:10.5152/tjh.2010.23

Yokuü et al. Thrombophilia and cerebral venous thrombosis

Turk J Hematol 2010; 27: 162-7

163

Yöntem ve Gereçler: 16-50 yaü aras×nda 43 hasta retrospektif olarak deùerlendirildi. Trombofili tetkiki olarak protein C, protein S, antitrombin eksikliùi, aktive protein C rezistans×, faktör V Leiden (FVL), protrombin 20210A (PT 20210), metilentetrahidrofolat redüktaz (MTHFR) gen mutasyonlar×, antifosfolipid antikorlar× (AFA), faktör VIII ve homosistein yüksekliùi araüt×r×ld×. Bulgular: Hastalar×m×z×n %67.4’ünde tek, %27.9’unda iki, %4.7’sinde üç trombofilik defekt saptad×k. En s×k saptanan defekt MTHFR gen mutasyonu (%41.8) idi, bunu FVL mutasyonu (%34.9) takip etti. Sonuç: Trombofili etkenlerinin s×kl×ù×, etnik farkl×l×k ve coùrafi bölgelere göre deùiüebildiùinden, her toplum serebral venöz tromboz kliniùi ile baüvuran hastalarda öncelikle s×k görülen trombofili etkenlerini araüt×rmal×d×r. (Turk J Hematol 2010; 27: 162-7) Anahtar kelimeler: Serebral venöz tromboz, trombofili, trombofilik defektler Geliü tarihi: 4 Ekim 2009

Kabul tarihi: 22 Mart 2010

Introduction Thrombosis is abnormal clot formation in the vascular system through the interaction of various vascular, cellular and humoral factors. It has been long known that alterations in the vascular wall, blood flow and blood composition (widely known as ‘Virchow’s triad’) cause thrombosis. The impairment of the delicate balance in the hemostatic process due to the deficiency of natural anticoagulants or defects in the fibrinolytic pathway may result in hypercoagulability [1]. An increased risk for thrombosis is known as hypercoagulability or thrombophilia. Generally, thrombophilia is the general term covering a range of hereditary or acquired conditions [2]. Currently, arterial and venous thrombosis are among the most common causes of morbidity and mortality [3]. In random patient populations, thrombophilic factors leading to a predisposition to thrombosis can be detected in approximately one-third of patients with venous thromboembolism. Hereditary thrombophilia is detected in more than half of the patients in whom thrombophilic defects are present. The risk for thrombosis increases in the presence of multiple thrombophilic defects [3]. Hereditary thrombophilic factors and their frequencies exhibit some variations in different populations or patient groups [4-8]. Ischemic stroke (IS) is usually associated with arterial diseases (e.g. carotid stenosis) or heart diseases (e.g. atrial fibrillation). In studies assessing the role of hereditary thrombophilia in the etiology of cerebral arterial and cerebral venous thrombosis (CAT, CVT), the risk for stroke was found to increase in the presence of different thrombophilic factors [9-16]. Hypercoagulable states are responsible for 5-10% of all IS cases, and hereditary thrombophilia is especially more common in the etiology of IS in the younger subgroup of patients [17].

In the present study, we aimed to assess the frequency of the identified defects for thrombophilia in patients with CVT and thrombophilic defects aged 50 years and to compare our data with the reports in the literature.

Materials and Methods Patients Forty-three patients (30 females, 13 males; median age: 36 years; range: 16-50) with CVT and thrombophilic defects aged 50 years were included in the study. All subjects were diagnosed to have CVT by the neurology clinics, and each patient was consulted with the hematology clinic to explore the etiology of the underlying thrombophilic factor. CVT was diagnosed by means of magnetic resonance imaging (MRI). Chronic liver diseases, diabetes mellitus, dyslipidemia, presence of central venous catheter, history of recent surgical procedures, myeloproliferative diseases, paroxysmal nocturnal hemoglobinuria, malignancies, autoimmune diseases, hormone replacement therapy, and oral contraceptive usage were the exclusion criteria. Laboratory tests were performed under the following conditions: 15 days after discontinuation of heparin or its derivatives, four weeks after discontinuation of coumadin derivatives, and three months later in patients with a history of thrombosis. The tests were repeated in the event of positive results. Oral informed consent were obtained from all patients. Assessments Antithrombin (AT) activity, protein C (PC) activity, total and free protein S (PS) antigen and activity, activated protein C resistance (APC-R), factor V Leiden (FVL) G1691A, prothrombin 20210A (PT 20210), and methylene tetrahydrofolate reductase (MTHFR) C677T mutations, factor VIII (FVIII) levels, fasting homocysteine levels, and antiphospholipid

164

Yokuü et al. Thrombophilia and cerebral venous thrombosis

antibodies (APA) (anticardiolipin antibodies [ACA] IgG and ACA IgM, and lupus anticoagulant [LA]) were assessed in our study. MTHFR C677T mutations were assessed by commercial polymerase chain reaction (PCR) kits (MTHFR LC PCR, QIAGEN). Factor II (prothrombin) G20210A and FVL mutations were determined by commercial kits (Roche Diagnostics). “LightCycler 1.5 Roche” real-time PCR device was used in this study. AT, PC, PS, APC-R, LA, and FVIII levels were assessed in citrated peripheric blood samples drawn after a 12-hour fast, which were centrifuged at 1500 x g for 15 minutes. Citrated samples were stored at -20°C until testing. PC, PS, AT and FVIII were studied using commercial kits (Dade Behring), and APC-R was determined by a Dade Behring proC global kit. These analyses were performed on the BCS instrument (Dade Behring). Homocysteine levels were evaluated using Recipe commercial kits (Recipe Chemical and Instruments GmbH, Munich, Germany) by Shimadzu HPLC analyzer. A laboratory reference range study performed with 40 healthy individuals was established to determine the cut-off value to define PC (70-140%), PS (58-127%), AT (75-125%) and FVIII (70-150%) activity. LA-sensitive activated partial thromboplastin time (aPTT) reagent PTT-LA (Diagnostica Stago) test was performed for the patients with high levels of aPTT. Presence of heparin, intrinsic pathway factor deficiencies, factor inhibitors, and dysfibrinogenemia was ruled out. In the presence of LA, the plasma clotting time increases. A difference of 8 seconds between the two clotting times is thought to be positive for LA [18,19]. These analyses were performed on the Diagnostica Stago STart® 4 Hemostasis Analyzer (Diagnostica Stago, France). ACA IgM and ACA IgG levels were evaluated by Intec cardiolipin IgM and IgG commercial ELISA kits on a Biomaster instrument. ACA IgM and ACA IgG of less than 2.0 PL-U/ml were accepted as normal range. The determination of >12 PL-U/ml value in at least two separate measurements was considered positive. The normal range of homocysteine in plasma/serum was accepted to be between 5.5-14 mol/L. A minimum of two determinations of homocysteine level >14 mol/L was considered as “high”. All blood samples were drawn after formal written consent was obtained from the patients. The study was carried out in accordance with the Helsinki Declaration.

Turk J Hematol 2010; 27: 162-7

Statistical analysis SPSS 11.5 for Windows Statistical Software Package was used to analyze the data. The descriptive statistics used for continuous variables were percentage (%) and mean±standard deviation for categorical variables. Fisher’s exact or chi-square test was used for categorical comparisons. Statistical significance was assigned as p values lower than 0.05.

Results Demographical properties of the patients are shown in Table 1. A previous thrombotic episode was present in 2 patients (4.7%) and a family history of a thrombotic episode was observed in 3 patients (6.9%). Among all cases, 38 patients (88%) were on antithrombotic medication (aspirin, coumadin, and low molecular weight heparin, either as single agents or in combinations). We detected a single thrombophilic defect in 29 (67.4%), two defects in 12 (27.9%) and three defects in 2 (4.7%) of our patients. There was no significant difference between male and female patients with regard to the presence of a single or combined thrombophilic defects (p=0.491). The frequencies of the thrombophilic defects that we could detect were as follows: The most frequent thrombophilic defect was a defect in the MTHFR gene, present in 41.8% (n=18) of the patients; 13 of the patients were heterozygote and 5 were homozygote. This defect was present as a single defect in 50% and as a joint defect with others in 50% of our patients. The second most common thrombophilic defect was FVL mutation, which was observed in 34.9% (n=15) of the patients, with 13 patients being heterozygote and 2 cases homozygote. Other frequent thrombophilic defects were the presence of APA and PS deficiency, which were Table 1. Demographic characteristics of the patients Patients (n)

Males (n)

13 (30.2%)

Females (n)

30 (69.8%)

Median age (overall) (years)

36 (16-50)

Median age (females) (years)

35 (16-49)

Median age (males) (years)

37 (20-50)

Previous thrombotic events

2 (4.7%)

Family history of thrombotic events

3 (6.9%)

Antithrombotic medication

38 (88%)

YokuĂź et al. Thrombophilia and cerebral venous thrombosis

Turk J Hematol 2010; 27: 162-7

observed in 18% (n=8) of the patients. This was followed by elevated FVIII and homocysteine levels, detected in only 2 patients. Similarly, PC deficiency was also detected in 2 patients. AT deficiency was in the screening program but was not detected in any of our patients. Nine of the patients with a mutation in the MTHFR gene, 6 patients with a FVL mutation and 5 patients with APA had combined thrombophilic defects. The most frequent pair of thrombophilic defects was the combination of a defect in the MTHFR gene + FVL mutation (n=5), followed by a defect in the MTHFR gene + APA (9%, n=4). The difference in frequencies between male and female patients was not statistically significant in any of the above-mentioned thrombophilic defects. The frequencies of the various thrombophilic defects are shown in Tables 2 and 3. Recurrent abortus coexisted with CVT in 3 of our patients and in 1 CVT patient, retinal artery thrombosis was also present.

Discussion Thromboembolism manifests itself mostly in adults and is a multicausal clinical entity [2]. Increasingly more reports in the literature demonstrate that hereditary defects are as important as acquired factors in the etiology of thromboembolic disease in adults [5,6,8,20]. However, several issues are still under investigation, such as in which populations, age groups and in the presence of which acquired factors will hereditary defects cause a tendency towards clinical thrombosis and which combination(s) constitute a strong synergy [21,22]. In a study conducted on patients with venous thromboembolism [23], thrombophilia was found in 41% of the patients, and in 32%, it involved a genetic cause. The FVL was found in 15%, followed by natural anticoagulant dysfunction (11%). Similar to our results, the authors observed that thrombophilia frequency did not change significantly with age or gender. Hankey et al. [24] performed a casecontrol study of 219 hospital cases with a first-ever IS and 205 randomly selected community control subjects in order to identify the role of hereditary thrombophilia in IS. They did not observe significant differences in the prevalence of thrombophilia between cases and control subjects or between pathogenic subtypes of IS. In another case-control study reported by Cantu et al. [25], the potential association among homocysteine, folate and vita-

165

Table 2. Prevalence of hereditary thrombophilic factors and gender distribution Males Females Total (n=13) (n=30) (n=43) n % n % n % PC

6.7

4.7

7.7

23.3

18.6

MTHFR

46.2

41.8

PT 20210

15.4

6.7

9.3

FVL

38.5

33.3

34.9

FVIII

7.7

3.3

4.7

APA

7.7

23.3

18.6

Homocysteine Elevation

7.7

3.3

4.7

PC: Protein C deficiency; PS: Protein S deficiency; FVL: Factor V Leiden G1691A mutation; MTHFR: 5,10-Methylenetetrahydrofolate reductase C677T mutation; PT 20210: Prothrombin G20210A mutation; FVIII: Elevated factor VIII levels; APA: Antiphospholipid antibody (anticardiolipin antibody IgG, IgM and lupus anticoagulant)

Table 3. The distribution of single and combined mutations in cases of cerebral venous thrombosis Single thrombophilic defect: 29 (67.4%) n=43

Two defects: 12 (27.9%) Three defects: 2 (4.7%) MTHFR: 41.8 (n=18; 13 heterozygotes, 5 homozygotes)

Most frequent thrombophilic defects (%)

FVL: 34.9 (n=15; 13 heterozygotes, 2 homozygotes) APA: 18.6 (n=8) Protein S: 18.6 (n=8) PT 20210: 9.3 (n=4; 4 heterozygotes) Most frequent: 20.9 MTHFR (n=9)

Frequency of the combined mutations (%)

2nd most frequent: 13.9 FVL (n=6) and 11.6 APA (n=5) MTHFR+FVL: 6.9 (n=3) MTHFR+APA: 6.9 (n=3) MTHFR+PS, MTHFR+FVL+PS, MTHFR+FVL+PT 20210, FVL+PS, PS+PT 20210, FVIII+APA, APA+Homocysteine Elevation: 2.3 (n=1)

min B12 levels and the common C677T mutation in the MTHFR gene was examined in patients with CVT. The authors also performed thrombophilic tests and reported a higher prevalence of APC-R

166

Yokuü et al. Thrombophilia and cerebral venous thrombosis

and presence of FVL and mutation in the PT gene (G20210A) in CVT patients [25]. In another study conducted in patients with CVT [26], infections and postpartum state were found to be the most common predisposing factors for CVT, and the prevalence of genetic thrombophilia was 5%. In our study, CVT did not develop during the gestation or puerperal period in female cases. In populations with low folate levels and low socioeconomic status, mutation in the MTHFR gene and hyperhomocysteinemia increase the risk for IS [9,10,15,25]. In a study performed on 125 patients by Nadir et al. [27], there was no correlation between high plasma homocysteine levels and C677T mutation in the MTHFR gene. In our study, only two cases among 43 were determined to have homocysteine levels above the normal (14 mol/L). No MTHFR gene mutation was detected in these cases. The fasting homocysteine levels of the remaining 41 cases were within the normal range. In the present study, we detected two or more thrombophilic defects in 32% of our patients. This finding shows that approximately one-third of all patients presented with combined thrombophilic defects. Thus, the search for these defects should not be stopped when one defect is detected; rather it would be an appropriate approach to continue the scan for at least 3-5 of the most common thrombophilic defects. The presence of MTHFR and FVL mutations and APA in our patients with CVT was in accordance with a study performed on pregnant women with venous thromboembolism [28]. Similar to our results, in a study performed in Australia [29], the MTHFR C677T polymorphism was the most common inherited thrombophilic defect. In that study, the birth prevalences of heterozygosity and homozygosity for the MTHFR C677T polymorphism were 37.3% and 12.4%, respectively. The authors concluded that inherited thrombophilic polymorphisms are common in the Caucasian Australian population, but the reason why thromboembolic disorders are not observed in most of these subjects may be the absence of additional risk factors, which facilitate the clinical manifestation of thromboembolism [29]. The association between the MTHFR C677T polymorphism and vascular disease is still a matter of debate [9,10,15,16,30]. The high number of polymorphic alleles and the differential distribution of these polymorphisms among various ethnic groups (more prevalent among subjects of Caucasian decent than blacks) are some of

Turk J Hematol 2010; 27: 162-7

the factors that make it difficult to comment on the results of various studies and to derive general conclusions [16]. In conclusion, since the prevalence of individual thrombophilic defects varies in different ethnic groups and geographical locations, in each population, screening for thrombophilic defects in patients presenting with CVT should primarily involve the most frequent thrombophilia risk factors. Such an approach will enable the initiation of prophylactic and therapeutic measures in the appropriate cases and thus decrease the economic burden for healthcare. Further studies involving larger patient populations are mandatory for the evaluation of the relevance of our results with current clinical practice. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1. 2. 3.

Esmon CT. Protein C. The regulation of natural anticoagulant pathways. Science 1987;235:1348-52. Rosendaal FR. Venous thrombosis, a multicausal disease. Lancet 1999;353:1167-73. Friedman KD, Rodgers GM. Thrombosis and antithrombotic therapy. In: Greer JP, Foerster J, Lukens J, Rodgers G, Paraskevas F, Glader B, editors. Wintrobe's Clinical Hematology. 11th ed. USA: Lippincott Williams & Wilkins, 2004: 1713-28. Majluf-Cruz A, Moreno-Hernandez M, Ruiz-de-ChavezOchoa A, Monroy-Garcia R, Majluf-Cruz K, GuardadoMendoza R, Molina-Avila I, Isordia-Salas I, Corona-de la Peña N, Vargas-Vorackova F, Vela-Ojeda J, GarcíaChávez J. Activated protein C resistance and factor V Leiden in Mexico. Clin Appl Thromb Hemost 2008;14:428-37. Okumus G, Kiyan E, Arseven O, Tabak L, Diz-Kucukkaya R, Unlucerci Y, Abaci N, Unaltuna NE, Issever H. Hereditary thrombophilic risk factors and venous thromboembolism in Istanbul, Turkey: the role in different clinical manifestations of venous thromboembolism. Clin Appl Thromb Hemost 2008;14:168-73. Kovac M, Mikovic D, Antonijevic N, Rakicevic L, Djordjevic V, Radojkovic D, Elezovic I. FV Leiden mutation and risk of recurrent venous thromboembolism in Serbian population. J Thromb Thrombolysis 2008;25:284-7. Kabukcu S, Keskin N, Keskin A, Atalay E. The frequency of factor V Leiden and concomitance of factor V Leiden with prothrombin G20210A mutation and methylene

Yokuü et al. Thrombophilia and cerebral venous thrombosis

Turk J Hematol 2010; 27: 162-7

10.

11.

12.

13.

14.

15.

16.

17. 18.

tetrahydrofolate reductase C677T gene mutation in healthy population of Denizli, Aegean region of Turkey. Clin Appl Thromb Hemost 2007;13:166-71. Shen MC, Lin JS, Tsay W. High prevalence of antithrombin III, protein C and protein S deficiency, but no factor V Leiden mutation in venous thrombophilic Chinese patients in Taiwan. Thromb Res 1997;87:377-85. Kawamoto R, Kohara K, Oka Y, Tomita H, Tabara Y, Miki T. An association of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene polymorphism and ischemic stroke. J Stroke Cerebrovasc Dis 2005;14:67-74. Sazci A, Ergul E, Tuncer N, Akpinar G, Kara I. Methylenetetrahydrofolate reductase gene polymorphisms are associated with ischemic and hemorrhagic stroke: dual effect of MTHFR polymorphisms C677T and A1298C. Brain Res Bull 2006;71:45-50. Glueck CJ, Munjal J, Aregawi D, Agloria M, Winiarska M, Khalil Q, Wang P. Thrombophilia-hypofibrinolysis and atherothrombotic cardiovascular disease < or = age 45 years. Transl Res 2007;150:93-100. Tänäsescu R, Ticmeanu M, Nicolau A, Uscätescu V, Cojocaru M, Grecu V, Serbänescu A. Anticardiolipin antibodies, recurrent stroke and vascular events: a prospective study on 210 patients. Rom J Intern Med 2005;43:79-88. Carod-Artal FJ, Nunes SV, Portugal D, Silva TV, Vargas AP. Ischemic stroke subtypes and thrombophilia in young and elderly Brazilian stroke patients admitted to a rehabilitation hospital. Stroke 2005;36:2012-4. Aznar J, Mira Y, Vayä A, Corella D, Ferrando F, Villa P, Estelles A. Factor V Leiden and prothrombin G20210A mutations in young adults with cryptogenic ischemic stroke. Thromb Haemost 2004;91:1031-4. Madonna P, de Stefano V, Coppola A, Cirillo F, Cerbone AM, Orefice G, Di Minno G. Hyperhomocysteinemia and other inherited prothrombotic conditions in young adults with a history of ischemic stroke. Stroke 2002;33:51-6. Voetsch B, Damasceno BP, Camargo EC, Massaro A, Bacheschi LA, Scaff M, Annichino-Bizzacchi JM, Arruda VR. Inherited thrombophilia as a risk factor for the development of ischemic stroke in young adults. Thromb Haemost 2000;83:229-33. Levine SR. Hypercoagulable states and stroke: a selective review. CNS Spectr 2005;10:567-78. Triplett DA. Assays for detection of antiphospholipid antibodies. Thromb Haemost 1994;3:281-7.

19. 20. 21.

22. 23. 24.

25.

26.

27.

28.

29.

30.

167

Triplett DA, Barna LK, Unger GA. A hexagonal(II) phase phospholipid neutralization assay for lupus anticoagulant identification. Lupus 1993;70:787-93. Bombeli T, Basic A, Fehr J. Prevalence of hereditary thrombophilia in patients with thrombosis in different venous systems. Am J Hematol 2002;70:126-32. Lane DA, Mannucci PM, Bauer KA, Bertina RM, Bochkov NP, Boulyjenkov V, Chandy M, Dahlbäck B, Ginter EK, Miletich JP, Rosendaal FR, Seligsohn U. Inherited thrombophilia: part 1. Thromb Haemost 1996;76:651-62. Lawson SE, Butler D, Enayat MS, Williams MD. Congenital thrombophilia and thrombosis: a study in a single centre: Arch Dis Child 1999;81:176-8. Paschóa AF, Guillaumon AT. Impact of screening on thrombophilia for patients with venous thrombosis. Int Angiol 2006;25:52-9. Hankey GJ, Eikelboom JW, van Bockxmeer FM, Lofthouse E, Staples N, Baker RI. Inherited thrombophilia in ischemic stroke and its pathogenic subtypes. Stroke 2001;32:1793-9. Cantu C, Alonso E, Jara A, Martínez L, Ríos C, Fernández Mde L, Garcia I, Barinagarrementeria F. Hyperhomocysteinemia, low folate and vitamin B12 concentrations, and methylene tetrahydrofolate reductase mutation in cerebral venous thrombosis. Stroke 2004;35:1790-4. Khealani BA, Wasay M, Saadah M, Sultana E, Mustafa S, Shohab Khan F, Kamal AK. Cerebral venous thrombosis. A descriptive multicenter study of patients in Pakistan and Middle East. Stroke 2008;39:2707-11. Nadir Y, Hoffman R, Brenner B. Association of homocysteine, vitamin B12, folic acid, and MTHFR C677T in patients with a thrombotic event or recurrent fetal loss. Ann Hematol 2007;86:35-40. Ogunyemi D, Cuellar F, Ku W, Arkel Y. Association between inherited thrombophilias, antiphospholipid antibodies, and lipoprotein A levels and venous thromboembolism in pregnancy. Am J Perinatol 2003;20:17-24. Gibson CS, MacLennan AH, Rudzki Z, Hague WM, Haan EA, Sharpe P, Priest K, Chan A, Dekker GA; South Australian Cerebral Palsy Research Group. The prevalence of inherited thrombophilias in a Caucasian Australian population. Pathology 2005;37:160-3. Yeh PS, Lin HJ, Li YH, Lin KC, Cheng TJ, Chang CY, Ke DS. Prognosis of young ischemic stroke in Taiwan: impact of prothrombotic genetic polymorphisms. Thromb Haemost 2004;92:583-9.

168

Research Article

Posterior reversible leukoencephalopathy syndrome in children with hematologic disorders Hematolojik hastal×ù× olan çocuklarda posterior geri dönüüümlü lökoensefalopati sendromu Bar×ü Malbora1, Zekai Avc×1, Fulden Dönmez2, Bülent Alioùlu1, Esra Bask×n3, Füsun Alehan4, Nam×k Özbek1 1Department

of Pediatric Hematology, Baükent University Faculty of Medicine, Ankara, Turkey of Radiology, Baükent University Faculty of Medicine, Ankara, Turkey 3Department of Pediatric Nephrology, Baükent University Faculty of Medicine, Ankara, Turkey 4Department of Pediatric Neurology, Baükent University Faculty of Medicine, Ankara, Turkey 2Department

Abstract Objective: Posterior reversible leukoencephalopathy syndrome (PRES) is characterized by headache, altered mental status, cortical blindness, and seizures associated with neuroradiological findings. It involves predominantly white matter of the parieto-occipital lobes. Several medications and disorders play a role in the etiology of PRES. In this study, we aimed to show how the prognosis of PRES in hematological diseases of childhood might be according to the etiological factors. Materials and Methods: Here, we report PRES in six patients, aged 4 to 14 years, with diagnoses of leukemia and aplastic anemia. Results: Suggested causes in our patients were chemotherapeutics, hypertension, infection and antimicrobial drug administration, tumor lysis syndrome, acute renal failure and hemodialysis, immunosuppressive drug administration, and hypomagnesemia. One of the patients died of sepsis, renal failure and pulmonary hemorrhage and another died of relapse after total recovery from PRES. The other four patients are under follow-up without problems. Conclusion: We suggest that PRES can recover fully with early diagnosis and treatment whereas it can show poor prognosis depending on the etiology. (Turk J Hematol 2010; 27: 168-76) Key words: Acute leukemia, aplastic anemia, posterior reversible leukoencephalopathy syndrome, tumor lysis syndrome Received: November 6, 2009

Accepted: April 30, 2010

Özet Amaç: Posterior geri dönüüümlü lökoensefalopati sendromu (PGDLS) nöroradyolojik bulgularla birlikte baü aùr×s×, bilinç deùiüiklikleri, kortikal körlük ve nöbet ile seyreden bir sendromdur. Hastal×k aù×rl×kl× olarak parieto-oksipital loblar×n beyaz cevherini tutar. Çeüitli ilaçlar ve hastal×klar PGDLS etiyolojisinde Address for Correspondence: M.D. Bar×ü Malbora, Baükent University Faculty of Medicine, Department of Pediatric Hematology 6. Cadde, No: 72/3, 06490, Bahçelievler 06490 Ankara, Turkey Phone: +90 312 212 68 68 E-mail: barismalbora@gmail.com doi:10.5152/tjh.2010.24

Turk J Hematol 2010; 27: 168-76

Malbora et al. Posterior reversible leukoencephalopathy syndrome in children

169

rol oynar. Bu çal×ümada, çocukluk çaù× hematolojik hastal×klar×nda altta yatan nedenlere göre PGDLS’nun nas×l seyrettiùini göstermeyi amaçlad×k. Yöntemve Gereçler: Burada, yaülar× 4 ile 14 aras×nda olan, lösemi ve aplastik anemi tan×lar× ile izlenen PGDLS’lu 6 hastay× sunduk. Bulgular: Hastalar×m×zda kemoterapotikler, hipertansiyon, infeksiyon, antimikrobiyal ilaç kullan×m×, tümör lizis sendromu, akut böbrek yetmezliùi, hemodiyaliz, immünsüpresif ilaç kullan×m× ve hipomagnezemiyi PGDLS nedenleri olarak tespit ettik. Hastalar×m×zdan birini sepsis, böbrek yetmezliùi ve pulmoner hemaroji nedenleriyle, bir diùerini ise PGDLS tamamen düzeldikten sonra relaps nedeniyle kaybettik. Diùer dört hastam×z herhangi bir problemi olmaks×z×n izlenmektedir. Sonuç: PGDLS erken tan× ve tedavi ile tamamen düzeltilebilir bir hastal×k olmas×na raùmen altta yatan nedene baùl× olarak kötü seyirli de olabilen bir sendromdur. (Turk J Hematol 2010; 27: 168-76) Anahtar kelimeler: Akut lösemi, aplastik anemi, posterior geri dönüüümlü lökoensefalopati sendromu, tümör lizis sendromu Geliü tarihi: 6 Kas×m 2009

Kabul tarihi: 30 Nisan 2010

Introduction Posterior reversible leukoencephalopathy syndrome (PRES) is a disorder that typically presents with headache, nausea and vomiting, visual disturbances, a disturbed level of consciousness, seizures, and occasionally focal neurologic deficits often in the setting of accelerated hypertension [1]. The entity has become increasingly recognized over recent years, with the term PRES first being used in 1996 [1]. Some of the suspected factors in the etiology of this syndrome are sickle cell disease, severe hypertension, solid organ and bone marrow transplantation, acute renal failure, tumor lysis syndrome (TLS), immunosuppressive/cytotoxic drugs, and chemotherapeutic agents [2-5]. The pathophysiology of this complex syndrome is still unknown. In uncomplicated patients, magnetic resonance imaging (MRI) of the brain illustrates edema primarily in the cortex and subcortical white matter of posterior brain regions, such as the occipital lobes and posterior parietal lobes. These findings generally resolve with normalization of blood pressure or discontinuation of the offending drugs [6]. Although recognized to occur in pediatric patients, PRES has not been well evaluated in this age group [7]. The aim of this study was to analyze a group of pediatric patients with hematologic disorders who developed PRES. We also aimed to identify possible predisposing factors and describe the common clinical and radiographic features.

Materials and Methods Patient 1 A five-year-old male had been diagnosed with T-cell acute lymphoblastic leukemia (ALL) (initial white blood cell (WBC) count: 450×109/L), and

ALL-BFM 90 chemotherapy protocol had been started at a local center. He had been given oral lowdose steroid and intrathecal methotrexate. Although alkali hydration and oral allopurinol for TLS prophylaxis were administered, he had developed acute renal failure after administration of intravenous vincristine. Biochemical analysis at that time revealed the following values: blood urea nitrogen (BUN): 83 mg/dl (normal range, 6-21 mg/dl), creatinine: 1.27 mg/dl (normal range, 0.5-1.0 mg/dl), sodium (Na): 130 mmol/L (normal range, 135-146 mmol/L), potassium (K): 7.1 mmol/L (normal range, 3.5-5.2 mmol/L), uric acid: 5.2 mg/dl (normal range, 2.5-6 mg/dl), calcium (Ca): 6.2 mg/dl (normal range, 8.510.5 mg/dl), and phosphorus: 16 mg/dl (normal range, 3-6 mg/dl). He had developed TLS and was referred to our hospital for hemodialysis on the third day of chemotherapy. The physical examination at admission revealed normal blood pressure (90/60 mmHg), confusion, lethargy, and abdominal distention due to hepatomegaly. Complete blood count showed hemoglobin (Hb): 7.4 g/dl, WBC count 40.4×109/L, and platelet count: 54×109/L, and biochemical analysis revealed BUN 85 mg/dl, creatinine 1.3 mg/dl, Ca 5.6 mg/dl, phosphorus 11.9 mg/dl, uric acid 9.7 mg/dl, Na 135 mmol/L, K 4 mmol/L, total protein 5.7 g/dl (normal range, 6-8 g/dl), albumin 3.3 g/dl (normal range, 3.5-5.5 g/dl), and total cholesterol 160 mg/dl (normal range 140-200 mg/dl). For the treatment of TLS, intravenous alkali hydration, allopurinol and calcium-acetate were administered. He also had hemodialysis treatment six times until his biochemical values returned to normal. Imipenem and amikacin were started because of neutropenic fever. On the fourth day of hospitalization, vomiting, headache, focal seizures, and paresis on the right arm were observed. The physical examina-

170

Malbora et al. Posterior reversible leukoencephalopathy syndrome in children

tion revealed blood pressure 120/80 mmHg and right hemiparesis. Brain MRI disclosed bilateral hyperintense signal changes at the bilateral frontoparietal, occipital and temporal areas on fluid-attenuated inversion recovery (FLAIR) and T2-weighted images, consistent with vasogenic edema. Diffusion-weighted imaging (DWI) confirmed the presence of vasogenic edema (Figure 1a). Electroencephalogram (EEG) showed epileptiform activity in the right temporal lobe. Phenytoin was started; however, due to insistent seizures, phenobarbital was added to the treatment. On the eighth day of the treatment, the neurological findings began to regress. The follow-up MRI performed 15 days after the first seizure showed almost complete regression of the initial findings (Figure 1b). His seizures did not recur, so phenobarbital and phenytoin were stopped five and six weeks after the initiation of the antiepileptic therapy, respectively. He completed the chemotherapy protocol three years ago, and has had no problems over the last four years. Patient 2 A 12-year-old male had been admitted to a local center with the diagnosis of abdominal Burkitt lym-

Turk J Hematol 2010; 27: 168-76

phoma. The physical examination had revealed a mass of 10x10 cm in diameter at the upper right quadrant of the abdomen. His blood pressure was normal. Complete blood count at the time showed Hb: 9.4 g/dl, WBC count: 14.7x109/L, and platelet count: 613x109/L. Biochemical analysis was normal except for lactate dehydrogenase (LDH) (3235 U/L [normal range: 120-300 U/L]) and aspartate aminotransferase (AST) (65 U/L [normal range: 0-40 U/L]). Following the intravenous alkali hydration and oral allopurinol, he had been given intravenous cyclophosphamide. Immediately after cyclophosphamide treatment, he developed anuria and hypertension (150/100 mmHg) and tumor lysis syndrome (TLS) within 24 hours. The biochemical analysis at the time revealed BUN 53 mg/dl, creatinine 2.25 mg/ dl, Ca 7.6 mg/dl, phosphorus 13 mg/dl, uric acid 8.4 mg/dl, Na 131 mmol/L, K 3.5 mmol/L, total protein 5.9 g/dl, and albumin 3.2 g/dl. After administration of captopril and furosemide, the patient was referred to our hospital for hemodialysis treatment. At admission, his blood pressure was 145/95 mmHg. The complete blood count revealed Hb 9.1 g/dl, WBC count 13.3x109/L and platelet count 683x109/L.

Figure 1. There are hyperintensities on both frontal and parietal white matter, consistent with vasogenic edema on initial axial FLAIR image (A). There are also similar signal changes in temporal lobes (not shown). On follow-up MRI, the FLAIR image shows regression of the edema at the frontal and parietal lobes (B)

Turk J Hematol 2010; 27: 168-76

Malbora et al. Posterior reversible leukoencephalopathy syndrome in children

Biochemical analysis showed BUN 23 mg/dl, creatinine 3.73 mg/dl, Na 135 mmol/L, K 3.5 mmol/L, uric acid 1.3 mg/dl, Ca 8.7 mg/dl, phosphorus: 4.1 mg/dl, magnesium (Mg) 1.5 mg/dl, total protein: 5.7 g/dl, albumin: 2.9 g/dl, AST 25 U/L, alanine aminotransferase (ALT) 10 U/L, and LDH: 693 U/L. Amlodipine was added to the anti-hypertensive therapy due to persisting high blood pressure. Renal function had partially improved. He received hemodialysis treatment 12 times over 20 days, and his NHL BFM 90 course AA protocol (doses adjusted for creatinine clearance) was started due to the enlargement of the abdominal mass. At the time, he developed febrile neutropenia and intravenous broad-spectrum antibiotics (cefepime and amikacin) were started. On the second day of the antibiotic treatment, generalized tonic-clonic seizure and lateral deviation on the left eye were observed. His general status was poor on his physical examination with continuing high blood pressure. He had hyperreactive deep tendon reflexes (DTR) with additional clonus on his right lower extremity. His biochemical analysis disclosed the following values: BUN: 41 mg/dl, creatinine: 1.97 mg/dl, Mg: 0.9 mg/dl (normal range, 1.6-3 mg/dl), albumin: 2.9 mg/dl, Na: 132 mmol/L, K: 3.9 mmol/L, Ca: 9.4 mg/dl, phosphorus: 3 mg/dl, AST: 27 U/L, ALT: 16 U/L, and total cholesterol: 162 mg/dl. Brain MRI showed bilateral patchy increase in signal intensity of the cortical and subcortical areas on the parietal and frontal lobes on FLAIR images (Figure 2a). Dexamethasone and phenytoin were started for his convulsions. Due to persistent seizures, valproic acid was added to the

171

treatment; however, the seizures were refractory to both agents so additional midazolam and thiopental infusions were started. The brain MRI performed one week later showed progressive findings on FLAIR and T2-weighted images. Although the lesions were increased in distribution, no evidence of cytotoxic edema was found on DWI (Figure 2b, 2c). Twenty days after beginning anti-epileptic therapy, the patient died due to sepsis, renal failure and pulmonary hemorrhage. Patient 3 A 10-year-old male had been admitted to a local center with polyuria and swelling of the eyelids. Blood pressure had been measured as 130/90 mmHg on physical examination. Complete blood count had shown Hb: 10.6 g/dl, WBC count: 43.2x109/L, and platelet count: 89x109/L with 65% blasts on peripheral blood smear. Diffuse L-3 type blastic infiltration was present on bone marrow smear and biopsy. Biochemical analysis had shown BUN: 49.7 mg/dl, creatinine: 1.4 mg/dl, uric acid: 15.6 mg/dl, total protein: 6.1 g/dl, albumin: 3.8 g/dl, ALT: 99 U/L, AST: 129 U/L, Na: 133 mmol/L, K: 4.1 mmol/L, Ca: 9.3 mg/dl, phosphorus: 3.1 mg/dl, and Mg: 2.1 mg/dl. With these findings, the patient had been diagnosed with ALL and acute renal failure due to TLS. Alkali hydration, allopurinol and lowdose dexamethasone (0.5 mg/d) were administered. Despite this therapy, the patientâ&#x20AC;&#x2122;s WBC count had increased, and blood pressure had risen, and he developed dyspnea secondary to hypervolemia and impaired renal functions. The patient was then

Figure 2. Axial initial FLAIR image shows mild signal increase on the frontal and parietal lobes (A). On follow-up MRI, FLAIR images show severe progression of the lesions on bilateral frontal, parietal and temporal lobes and new lesions are seen on bilateral cerebellar hemispheres (B,C)

172

Malbora et al. Posterior reversible leukoencephalopathy syndrome in children

referred to our hospital for hemodialysis. The biochemical analysis at that time revealed BUN: 31 mg/ dl, creatinine: 2.75 mg/dl, uric acid: 9.5 mg/dl, total protein: 6.1 g/dl, albumin: 3.3 g/dl, ALT: 30 U/L, AST: 61 U/L, Na: 128 mmol/L, K: 3.2 mmol/L, Ca: 8.1 mg/ dl, phosphorus: 8.5 mg/dl, Mg: 1.5 mg/dl, and LDH 3080 U/L. Low-dose dexamethasone (1 mg/d) and allopurinol were started. He received hemodialysis treatment 4 times over 4 days. After hemodialysis, the patient’s blood pressure levels normalized. He was given NHL BFM 95 prephase block and immediately after the prephase, Course AA block was started. On the third day of his chemotherapy, he developed visual disturbance, headache and vomiting before etoposide and cytarabine treatment. At the time, his blood pressure was 150/95 mmHg. Six hours later he had generalized tonic-clonic seizures. On MRI, FLAIR images showed diffuse signal increase in the cortex and subcortical white matter of the bilateral frontal, parietal and temporal lobes, and also in bilateral cerebellar hemispheres, which was demonstrated as vasogenic edema on DWI. Serum methotrexate level (42nd hour of methotrexate infusion) was 2.4 mol/L. EEG was normal. Chemotherapy was stopped and phenytoin was given. Fifteen days after PRES, the patient had no neurological problems and his chemotherapy was re-started. MRI performed two months after the diagnosis showed disappearance of all of the lesions; no evidence of signal changes was found. Three months after PRES, phenytoin dose was decreased gradually and then stopped. Patient 4 A 14-year-old girl had been diagnosed with AMLM2 at a local center. AIE block of AML BFM 93 treatment protocol including cytosine arabinoside, idarubicin and etoposide had been started. Due to febrile neutropenia and invasive pulmonary aspergillosis, voriconazole, meropenem and metronidazole had been administered. She had developed visual disturbance and weakness in both lower extremities, in addition to cerebellar findings (hypometria, incompetency in knee-toe test). She developed generalized convulsions three times after cerebellar findings. The patient had been normotensive during this period. At the time, Hb was 9.2 g/ dl, WBC count was 0.82x109/L and platelet count was 21.0x109/L; the biochemical analysis showed total cholesterol of 88 mg/dl, Ca 6.9 mg/dl and albu-

Turk J Hematol 2010; 27: 168-76

min 2.2 g/dl. The patient was referred to our hospital with these findings for intensive care. Brain MRI revealed symmetrical hyperintensities in the bilateral thalami and frontal lobes on FLAIR and T2-weighted sequence. As this kind of localization is rare, she was diagnosed as atypical PRES. Her EEG was normal. Phenytoin was started initially; however, due to persistent convulsions, phenobarbital was added. From the 10th day of the anti-epileptic treatment, neurologic symptoms started to improve and at the end of the second week, the symptoms had totally regressed. One month later, the followup MRI showed marked regression of the lesions. The neurologic symptoms and the invasive fungal infection had regressed, so the chemotherapy protocol was continued. Anti-epileptic treatment was stopped at the 4th month. Her chemotherapy was completed and she has been in remission for the last six months. Patient 5 A four-year-old male was given St. Jude Total XIII I/H risk chemotherapy protocol with the diagnosis of Pre-B ALL. On the 15th day of induction therapy, vincristine, prednisolone, daunorubicin, and L-asparaginase were administered. Seven days after this treatment, generalized tonic-clonic seizure had occurred. The patient was monitored with mechanical ventilation because of status epilepticus. The physical examination revealed blood pressure of 125/95 mmHg. His Hb was 8.7 g/dl, WBC count 3.2x109/L and platelet count 112x109/L; the biochemical analysis was normal. On cranial MRI, there were asymmetric cortico-subcortical hyperintense signal changes at bilateral occipital and parietal areas on FLAIR images. DWI also showed vasogenic edema. EEG revealed bilateral temporal irregular background activity. The chemotherapy protocol was stopped and carbamazepine was administered. On the 7th day of the anti-epileptic treatment, the neurological symptoms recovered. Two months after the anti-epileptic treatment, follow-up cranial MRI revealed that the lesions had totally regressed. His seizures did not re¨cur, so the anti-epileptic treatment was stopped and the chemotherapy was continued. At the 93rd week of the maintenance chemotherapy, he developed a bone marrow relapse. During the administration of relapse protocol, he died of sepsis.

Turk J Hematol 2010; 27: 168-76

Malbora et al. Posterior reversible leukoencephalopathy syndrome in children

Patient 6 A 13-year-old girl had received a treatment protocol consisting of anti-thymocyte globulin, cyclosporine, danazol, prednisolone, and filgrastim for severe acquired aplastic anemia. During the 2nd month of the treatment, headache, contractions of the right arm and staring at a point had developed. Physical examination revealed blood pressure of 140/85 mmHg and DTR were hyperactive. Complete blood count showed Hb: 7.38 g/dl, WBC count: 7.2x109/L and platelet count: 149x109/L. Biochemical analysis revealed BUN 21 mg/dl, creatinine 0.49 mg/dl, Ca 8.5 mg/dl, Na 132 mmol/L, K 4.4 mmol/L, AST 30 U/L, ALT 188 U/L, gamma-glutamyl transpeptidase (GGT) 34 U/L, alkaline phosphatase (ALP) 217 U/L, total cholesterol 186 mg/dl, and Mg 1.5 mg/dl. Brain MRI showed hyperintense signal changes on the posterior-inferior part of the right cerebellar hemisphere and right parietal subcortical white matter at the level of the vertex on FLAIR and T2-weighted sequences, which were confirmed to be vasogenic edema on DWI. During this period, the serum level of cyclosporine was found to be high (530 ng/ml; normal range: 100-400 ng/ml) and it was then stopped. Phenytoin was started for the convulsions and following the treatment, there was no convulsion recurrence. The brain MRI performed five months after anti-epileptic treatment showed that edema had regressed; however, hemorrhage was observed in the subcortical white matter of the bilateral frontal lobes and right posterior parietal and left occipital lobes on gradient-echo images. The control EEG was normal. During the follow up, phenytoin treatment was stopped after one year. The patient has been in partial remission for the last three years.

Results and Discussion Various factors have been defined in the etiology of PRES. The most common factors are immunosuppressive drugs (cyclosporine, anti-thymocyte globulin, tacrolimus, rituximab, interferon), and chemotherapeutic agents (methotrexate, L-asparaginase, adriamycin, cyclophosphamide, cytosine arabinoside, vincristine) [2,4,7,8-11]. Sickle cell disease, hypertension, acute blood pressure changes, renal failure, TLS, infection, sepsis, shock, and organ transplantation are some factors that can also cause PRES [3,5,12-15]. The exact etiopathogenesis of posterior leukoencephalopathy syn-

173

drome is still unknown. Two opposing hypotheses are commonly cited, but the issue is controversial. The more popular theory suggests that severe hypertension exceeds the limits of autoregulation, with injury to the capillary bed and hyperperfusion leading to breakthrough brain edema [16]. Another theory suggests that hypertension leads to cerebral autoregulatory vasoconstriction, ischemia and subsequent cytotoxic edema and then extracellular edema [1,17]. On computerized tomography (CT)/MRI, the brain typically demonstrates focal regions of symmetric hemispheric edema. The parietal and occipital lobes are most commonly affected, followed by the frontal lobes, the inferior temporal-occipital junction and the cerebellum [10]. Lesion confluence may develop as the extent of edema increases. DWI was instrumental in establishing and consistently demonstrating that the areas of abnormality represent vasogenic edema [18]. Focal/patchy areas of PRES vasogenic edema may also be seen in the basal ganglia, brain stem, and deep white matter [18-20]. In our patients, frontal lobe (n=4), parietal lobe (n=5), occipital lobe (n=4), temporal lobe (n=2), cerebellum (n=2), and thalamus (n=1) were involved. (Table 1) The above-mentioned abnormalities were bilaterally symmetric in five patients (not in Case 6). Research indicates that imaging abnormalities in posterior leukoencephalopathy syndromes are often symmetrical; however, asymmetrical involvement is not unusual [1,10]. In our patients, chemotherapeutics (n=5), hypertension (n=5), infection and antimicrobial drug administration (n=3), acute renal failure and hemodialysis with TLS (n=3), and immunosuppressive drug administration and hypomagnesemia (n=1) were suggested to be the causes of PRES (Table 2). The blood pressures according to age, sex and height of all our patients were high in our patient group with the exception of Case 4. PRES may also develop and reverse in the face of systemic toxicity but in the absence of hypertension. In 20-30% of patients who develop PRES, blood pressure is essentially normal at toxicity [8]. The blood pressure of Case 4 was in normal ranges. In this patient, intravenous and intrathecal cytosine arabinoside, idarubicin, etoposide, and antibiotics for the treatment of febrile neutropenia may have been the causative factors for PRES. Infection and/or inflammation are some other factors accused in the pathogenesis of PRES. The

174

Malbora et al. Posterior reversible leukoencephalopathy syndrome in children

Turk J Hematol 2010; 27: 168-76

Table 1. Distribution of lesions with regard to the brain lobes on initial and follow-up magnetic resonance imagings Case

Parietal

Occipital

Frontal

Temporal

Cerebellar

Talamus

Control MRI

Bilateral

Regression

Bilateral

No regression

Bilateral

Bilateral asymmetric

Right

Regression (New hemorrhagic areas)

MRI: magnetic resonance imaging; N: normal

Table 2. Demographic and clinical data for the 6 patients of posterior leukoencephalopathy syndrome Case Age Sex Disease (Year)

Medication

Mean arterial BP TLS Infection EEG Treatment syst/diast (mmHg)

Signs and symptoms

Outcome

T-ALL

Prednisolone Vincristin, MTX (I.T.) Allopurinol Ca-asetat, Imipenem Amicasin

125/85*

Phenytoin

Vomiting Headache Focal seizure Paresis on the right arm

Remission without neurologic sequel

Burkitt Prednisolone Cytarabine, 150/100* lymphoma Ifosfamid, Etoposid, Cefepime, Amikacin, Amlodipine, Captopril, Allopurinol

Phenytoin Dexhamethasone Valproic acid Midazolam Thiopental

GTC Lateral deviation on the left eye Hyperactive DTR Clonus on right lower extremity

Died due to sepsis, renal failure and pulmonary hemorrhage

ALL L-3

Dexametasone, Ifosfamid, Vincristin MTX, MTX (IT), Cytarabine (IT), Prednisolone (IT)

150/95*

Phenytoin

Vomiting Headache GTC

Chemotherapy continues without neurologic sequel

AML

Cytarabine Idarubicin, Etaposid, Meropenem, Metronidazole, Voriconazole

100/60

Phenytoin Phenobarbital

Visual disturbance Hypometry, Incompetency in knee-toe test GTC

Remission without neurologic sequel

Pre-B ALL Prednisolone, Vincristin, Daunorubicin, L-asparaginase

125/95*

Carbamazepine

GTC

Died due to sepsis

Aplastic anemia

140/85*

Phenytoin

Headache, Contractions at the right arm, Staring to a point, Hyperactive, DTR

Partially remission without neurologic sequel

Cyclosporin, ATG, G-CSF, Danazol, Prednizolone

M: male; F: female; ALL: Acute lymphoblastic leukemia; AML: Acute myeloblastic leucemia; ATG: antitimocyte globulin; BP: blood pressure; Ca: calcium; DTR: deep tendon reflex; EEG: electroencephalogram; G-CSF: granulocyte- colony stimulating factor; GTC: generalized tonic-clonic convulsions; IT: intrathecal; NA: non available; AN: abnormal; N: normal; MTX: methotrexate; TLS: tumor lysis syndrome * Increased blood pressure according to age, sex and height

Turk J Hematol 2010; 27: 168-76

Malbora et al. Posterior reversible leukoencephalopathy syndrome in children

septic shock response likely reflects systemic toxicity similar to systemic inflammatory response syndrome or multiorgan dysfunction syndrome and bacteremia, or endotoxins/exotoxins are considered as potential triggers [21,22]. Cytokine response (tumor necrosis factor [TNF]-_, interleukin [IL]-1) plays a critical role in development of this effect [23]. Grampositive organisms are commonly obtained in infection/sepsis/shock-associated PRES [14]. In our study, three of our patients had infection. Case 4 was in febrile neutropenia, receiving antibiotics and antifungals after the treatment, when she developed neurologic findings. However, during this period, all the cultures were negative. An alternative mechanism of PRES also implicates endothelial dysfunction, such a notion underpinning the treatment of affected individuals with immunosuppressive therapy, mainly cyclosporine [1]. Immunosuppressive agents could damage the blood-brain barrier by various means: direct toxic effects on the vascular endothelium, vasoconstriction caused by elaboration of endothelin and microthrombosis [1]. The situation for bone marrow transplant recipients who feature cyclosporine neurotoxicity may be related to abnormalities of the blood-brain barrier, increased blood pressure and renal failure prior to the onset of various neurological symptoms [13]. Under most circumstances, the neurological symptoms and signs associated with cyclosporine use are reversible when the drugâ&#x20AC;&#x2122;s administered dosage is decreased or stopped, but symptoms may recur when the drug is reintroduced. Although neurotoxicity appears to be more frequent when blood cyclosporine levels are substantially elevated, neurotoxicity may also occur when the drug is administered within the normal therapeutic range [1]. Hypocholesterolemia and hypomagnesemia are found in more than 50% of patients with cyclosporine neurotoxicity [2]. The steroids administered simultaneously with cyclosporine can also aggravate the neurologic complications of cyclosporine [9]. In Case 6, blood cyclosporine level (530 ng/ml) was only marginally above the recommended normal range (200-400 ng/ml). This patient also had a low magnesium level (1.5 mg/dl); however, the cholesterol level was normal. Additionally, this patient was receiving both prednisolone and cyclosporine, and both may have contributed to PRES. There are reports on the development of PRES in patients with TLS. TLS causes hypertension and

175

various electrolyte imbalances, which result in PRES. Recent literature reports PRES patients secondary to TLS, seen after chemotherapy administration for hematologic malignancies, similar to our patients (Cases 1, 2, and 3) [3,10,11]. In summary, PRES is a multi-factorial syndrome. As in our patients, one or more factors such as TLS, chemotherapeutics, immunosuppressive drugs, and sepsis may cause PRES. This syndrome usually improves without any complications. However, it may progress to irreversible neurologic deficits, even death, depending on the severity of the primary disease and delays in diagnosis and treatment. Acknowledgement Oral and written informed consent was taken from the parents. This study conforms to the principles outlined in the Declaration of Helsinki (1975) and later revisions, and was approved by Ethical Committee of Baskent University Faculty of Medicine, Ankara, Turkey. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1.

5. 6.

Hinchey J, Chaves C, Appignanin B, Breen J, Pao L, Wang A, Pessin MS, Lamy C, Mas JL, Caplan LR. A reversible posterior leukoencephalopathy. N Engl J Med 1996; 334: 494-500. Gupta A, Swaroop C, Rastogi R, Garg R, Bakhshi S. Simultaneous occurrence of posterior reversible leukoencephalopathy syndrome in two cases of childhood acute lymphoblastic leukemia induction chemotherapy. Pediatr Hematol Oncol 2008;25:351-8. Ozkan A, Hakyemez B, Ozkalemkas F, Ali R, Ozkocaman V, Ozcelik T, Taskapilioglu O, Altundal Y, Tunali A. Tumor lysis syndrome as a contributory factor to the development of reversible posterior leukoencephalopathy. Neuroradiology 2006;48:887-92. Saito B, Nakamaki T, Nakashima H, Usui T, Hattori N, Kawakami K, Tomoyasu S. Reversible posterior leukoencephalopathy syndrome after repeat intermediatedose cytarabine chemotherapy in a patient with acute myeloid leukemia. Am J Hematol 2007;82:304-6. Frye RE. Reversible posterior leukoencephalopathy syndrome in sickle-cell anemia. Pediatr Neurol. 2009;40:298-301. Pavlakis SG, Frank Y, Chusid R. Hypertensive encephalopathy, reversible occipitoparietal encephalopathy, or

176

9. 10. 11.

12.

13. 14.

15.

Malbora et al. Posterior reversible leukoencephalopathy syndrome in children

reversible posterior leukoencephalopathy: three names for an old syndrome. J Child Neurol 1999;14:277-81. Suminoe A, Matsuzaki A, Kira R, Fukunaga N, Nishio T, Hoshina T, Hara T. Reversible posterior leukoencephalopathy syndrome in children with cancers. J Pediatr Hematol Oncol 2003;25:236-9. Bartynski WS, Zeigler Z, Spearman MP, Lin L, Shadduck RK, Lister J. Etiology of cortical and white matter lesions in cyclosporin-A and FK-506 neurotoxicity. AJNR Am J Neuroradiol 2001;22:1901-14. Durrant S, Chipping PM, Palmer S, Gordon-Smith EC. Cyclosporin A, methylprednisolone, and convulsions. Lancet 1982;2:829-30. Lee VH, Wijdicks EM, Manno EM, Rabinstein AA. Clinical spectrum of reversible posterior leukoencephalopathy syndrome. Arch Neurol 2008;65:205-10. Honkaniemi J, Kahara V, Dastidar P, Latvala M, Hietaharju A, Salonen T, Keskinen L, Ollikainen J, Vähämäki L, Kellokumpu-Lehtinen P, Frey H. Reversible posterior leukoencephalopathy after combination chemotherapy. Neuroradiology 2000;42:895-9. Servillo G, Bifulco F, De Robertis E, Piazza O, Striano P, Tortora F, Striano S, Tufano R. Posterior reversible encephalopathy syndrome in intensive care medicine. Intensive Care Med 2007;33:230-6. Sloane JP, Lwin KY, Gore ME, Powles RL, Smith JF. Disturbance of blood-brain barrier after bone marrow transplantation. Lancet 1985;2:280-1. Bartynski WS, Boardman JF, Zeigler ZR, Shadduck RK, Lister J. Posterior reversible encephalopathy syndrome in infection, sepsis, and shock. AJNR Am J Neuroradiol 2006;27:2179-90. Alehan F, Erol I, Agildere AM, Ozcay F, Baskin E, Cengiz N, Alioglu B, Haberal M. Posterior leukoencephalopathy

16.

17.

18.

19.

20. 21.

22.

23.

Turk J Hematol 2010; 27: 168-76

syndrome in children and adolescents. J Child Neurol 2007;22:406-13. Schwartz RB, Jones KM, Kalina P, Bajakian RL, Mantello MT, Garada B, Holman BL. Hypertensive encephalopathy: findings on CT, MR imaging, and SPECT imaging in 14 cases. AJR Am J Roentgenol 1992;159:379-83. Bartynski WS. Posterior reversible encephalopathy syndrome, part 2: controversies surrounding pathophysiology of vasogenic edema. AJNR Am J Neuroradiol 2008;29:1043-9. Covarrubias DJ, Luetmer PH, Campeau NG. Posterior reversible encephalopathy syndrome: prognostic utility of quantitative diffusion-weighted MR images. AJNR Am J Neuroradiol 2002;23:1038-48. Oliverio PJ, Restrepo L, Mitchell SA, Tornatore CS, Frankel SR. Reversible tacrolimus-induced neurotoxicity isolated to the brain stem. AJNR Am J Neuroradiol 2000;21:1251-4. Casey SO, Truwit CL. Pontine reversible edema: a newly recognized imaging variant of hypertensive encephalopathy? AJNR Am J Neuroradiol 2000;21:243-5. Munford RS. Sepsis, severe sepsis and septic shock. In: Mandell GL, Bennett JE, Dolin R, eds. Principles and Practice of Infectious Disease. 6th ed. Philadelphia: Elsevier Churchill Livingstone, 2005:906-26. Varon J, Marik PE. Multiple organ dysfunction syndrome. In: Irwin RS, Rippe JM, eds. Irwin and Rippe’s Intensive Care Medicine. 5th ed. Philadelphia: Lippincott-Williams & Wilkins, 2003:1834-8. McGuire TR, Bociek GR, Pavletic SZ, Hock L, Lynch J, Schneider J, Hoie EB, Tarantolo S, Haire WD. Organ dysfunction following stem cell transplantation: relationship to plasma cytokine concentrations. Bone Marrow Transplant 2001;28:889-93.

Research Article

177

The effect of calabash chalk on some hematological parameters in female adult Wistar rats Kalabaü tebeüiri (Calabash chalk) diüi yetiükin Wistar s×çanlar×n baz× hematolojik parametreleri üzerindeki etkisi Amabe Otoabasi Akpantah, Ofon Samuel Ibok, Moses Bassey Ekong, Mokutima Amarachi Eluwa, Theresa Bassey Ekanem Department of Anatomy, University of Calabar, Calabar, Nigeria

Abstract Objective: Calabash chalk is a naturally occurring mineral consumed among the Nigerian community for pleasure and commonly by pregnant women as a remedy for morning sickness. Reports have shown that it contains different toxic substances, with lead being the most abundant. This study was therefore undertaken to ascertain the effect of two commonly available preparations of this chalk on some hematological parameters. Materials and Methods: Twenty-four adult female Wistar rats with average weight of 100 g were assigned into three groups (1, 2, 3). Group 1 served as the control and the animals received distilled water, while Groups 2 and 3 were treated by oral gavage with 40 mg/kg of non-salted (NSCC) and salted calabash chalk (SCC), respectively, for 14 days. Results: The hemoglobin (Hb) concentration and red blood cell (RBC) count were significantly (p<0.05, 0.001 respectively) lower in the NSCC group, while erythrocyte sedimentation rate (ESR) was significantly (p<0.05) higher in the NSCC group compared to the control. There were no significant differences in packed cell volume (PCV), white blood cell (WBC) and platelet (Pl) counts compared to the control. The SCC group presented no significant difference in all blood count parameters compared to the control. Conclusion: This infers that calabash chalk, particularly the non-salted form, alters the normal concentration of Hb, RBC and Pl counts, and ESR, as observed in the female Wistar rats studied. (Turk J Hematol 2010; 27: 177-81)

Key words: Calabash chalk, blood parameters, wistar rat Received: December 19, 2008

Accepted: January 28, 2010

Özet Amaç: Kalabaü tebeüiri, genellikle gebe ve diùer kad×nlar taraf×ndan keyfi olarak ve sabah bulant×s×n× iyileütirmek için tüketilen, doùan oluüumlu bir mineraldir. Baüta kurüun olmak üzere farkl× toksik maddeler içerdiùi raporlarda gösterilmiütir. Dolay×s×yla bu çal×ümada, bu taü×n en yayg×n iki preparat×n×n baz× hematolojik parametreler üzerindeki etkisinin belirlenmesi amaçlanm×üt×r. Address for Correspondence: Mr. Moses Bassey Ekong, ETA-AGBOR ROAD 234 Calabar, Nigeria Phone: +2348030868505 E-mail: mbe_flashpoint@yahoo.com doi:10.5152/tjh.2010.25

178

Akpantah et al. Hematological effect of calabash chalk

Turk J Hematol 2010; 27: 177-81

Yöntem ve Gereçler: Ortalama 100 g aù×rl×ù×nda 24 yetiükin diüi Wistar s×çan× üç gruba (1, 2 ve 3) ayr×lm×üt×r. Grup 1 kontrol grubu olup, hayvanlara distile su verilmiü ve grup 2 ve 3, 14 gün süreyle her biri 40 mg/kg tuzsuz (NSCC) ve tuzlu Kalabaü tebeüiri (SCC) ile birlikte oral besleme yoluyla tedavi edilmiütir. Bulgular: Hemoglobin (Hb) konsantrasyonu ve k×rm×z× kan hücresi (RBC) say×m× anlaml× derecede NSCC grubunda düüüktü (S×ras×yla P<0.05, 0.001), eritrosit sedimentasy×on h×z× da kontrol grubuna oranla anlaml× olarak yüksekti (p<0.05). Hematokrit, beyaz kan hücresi ve trombosit say×mlar×nda kontrol grubuna gore anlaml× deùiüiklik olmam×üt×r. SCC grubunun tüm kan parametrelerinde kontrol grubuna göre anlaml× deùiüiklik gözlemlenmemiütir. Sonuç: Özellikle tuzsuz olan Kalabaü tebeüiri, incelenen diüi Wistar s×çanlarda normal Hb konsantrasyonu, RBC ve PI say×mlar×n× ve ESR deùerini deùiütirdiùi gösterilmiütir. (Turk J Hematol 2010; 27: 177-81)

Anahtar kelimeler: Kalabaü tebeüiri, kan parametreleri, wistar s×çan Geliü tarihi: 19 Aral×k 2008

Kabul tarihi: 28 Ocak 2010

Introduction Calabash chalk is traditionally consumed by Nigerian communities for pleasure and as a remedy for morning sickness [1]. Though it is not a conventional food, it is reported to be consumed mostly by pregnant and breast-feeding women [1,2]. This chalk is a naturally occurring mineral, which is chiefly composed of fossilized seashells. It may also be prepared from clay and mud, which may be mixed with other ingredients, including sand, wood ash and sometimes salt. The resulting product is molded and then heated to produce the final product [3]. Calabash chalk is also known as Calabar stones, La Craie or Argile in French, Nzu by the Igbos and Ndom by the Efiks/Ibibios of Nigeria, and Mabele by the Lingala of Congo. It is sold in blocks, pellets and powder forms [4]. This chalk is composed of aluminum silicate hydroxide from the kaolin clay group with the possible formula: Al2 Si2 O5 OH4. It has been tested to contain lead and arsenic [1-3,5]. The quantity of lead is reported to be approximately 40 mg/kg, with other organic pollutants being alpha lindane, endrin, endosulphan 11, and P, P1- DD [5]. Exposure to these higher levels of lead and also arsenic by pregnant and breast-feeding women poses a risk to the mental development of their unborn babies and breast-feeding infants, respectively [1,2]. Reports such as these and others, reporting cancers of the urinary bladder, lungs and skin [2], led us to investigate the effect of both salted (SCC) and non-salted calabash chalk (NSCC) on some blood parameters in adult female Wistar rats.

Material and Methods Twenty-four adult female Wistar rats with an average weight of 100 g were used for this study after acclimatization for two weeks in the Animal House of the Department of Anatomy, University of Calabar, Nigeria. The animals were equally divided into three groups as: Group 1, which served as the control and received distilled water, and Groups 2 and 3, which were treated with a solution of 40 mg/kg of NSCC and SCC, respectively. The two forms of calabash chalk were purchased as blocks from a local market in Calabar, Nigeria, and were ground into powder. One gram of each powder was dissolved in 100 ml of distilled water. Then, 40 mg/kg equivalent of the chalk solutions was administered to the experimental animals. The treatment was by oral gavage, once daily, and lasted for 14 days. On the 15th day, the animals were humanely sacrificed using chloroform anesthesia, and blood was aspirated through cardiac puncture and stored for a limited time in heparinized tubes for hematological assay [6-8]. Blood parameters assayed included hemoglobin (Hb) concentration, packed cell volume (PCV), erythrocyte sedimentation rate (ESR), and red blood cell (RBC), white blood cell (WBC) and platelet (Pl) counts. Statistical analysis One way analysis of variance (ANOVA) was applied to compare the relationship of the groups, and Dunnett post-hoc test was used to compare the

Akpantah et al. Hematological effect of calabash chalk

Turk J Hematol 2010; 27: 177-81

experimental groups and the control. All values were presented as mean±standard error of mean (SEM), and values were considered significant at p<0.05.

Results The Hb concentrations were lower in the treatment groups, and the difference was significant (p<0.01) in the 40 mg/kg NSCC group compared to the control and SCC groups. The PCV was lowest in the group treated with 40 mg/kg NSCC, but the difference was not significant (p<0.05) when compared to the control and the SCC groups. The ESR showed a significant increase (p<0.01) in the group treated with 40 mg/kg NSCC when compared to the control and SCC groups; however, there was no difference between the SCC group and the controls. The RBC count was significantly lower (p<0.05) in the NSCC group when compared to the control and SCC groups, but there was no difference between the SCC and the control groups. The WBC count revealed no significant difference among the groups. The NSCC group had a slightly higher count. The Pl count also showed no significant difference between the control and the treatment groups, though Pl count was higher in the controls than both treatment groups. These results are shown in Table 1.

Discussion The Hb concentration was lower in the group treated with 40 mg/kg NSCC, while the concentration in the SCC group was not different from the controls. Hb determines the oxygen-carrying capacity of the RBC. A decrease in Hb may be due to inability of the RBCs to incorporate iron or due to the destruction of iron already contained within the RBCs [9]. Our study is in line with previous reports [10-12], which reported decreased Hb after exposure to lead compound and arsenic. The PCV showed no significant difference among the groups, though it was lower in the group treated with 40 mg/kg NSCC compared to the others. PCV measures the percentage of red cells in total blood volume. The lack of difference between the treatment groups and the controls may be because the chalk may not have affected the total RBC volume in whole blood. This is in variance with previous reports [10-12] that showed significantly decreased PCV after treatment with lead compound. The ESR of the NSCC group was significantly higher (p<0.05) than in the control and the SCC groups. ESR is the rate at which RBCs precipitate in one hour [13]. Anemia increases ESR. Anemia may have caused the increase in ESR in the NSCC group. Red blood cell (RBC) count of the NSCC group was significantly lower (p<0.001, 0.01) than in the control and SCC groups, respectively. RBC count estimates the number of RBCs per liter of blood. A reduced RBC count may indicate anemia. This is

Table 1. The parameters measured in the control, non-salted and salted calabash chalk groups GROUPS

179

BLOOD PARAMETERS

(n=8)

Hb (g/dl)

PCV (%)

ESR (mm/hr)

RBC (X106/mm3)

WBC (X103/mm3)

Pl (X105/mm3)

1 (Control)

12.50±0.20

34.00±0.58

3.67±0.33

5.99±0.02

5.55±0.21

4.6±0.12

2 40 mg/kg of NSCC

11.25±0.20**

26.67±4.18NS

5.67±0.33**

5.51±0.80NS

5.77±0.15NS

4.33±0.09NS

3 40 mg/kg of SCC

11.99±0.26NS

34.67±1.33NS

3.67±0.33b

5.97±0.01NS

5.55±0.16NS

4.57±0.13NS

Values are presented as mean±SEM **Significantly different from control, at p<0.01, bSignificantly different from Group 2, at p<0.01, NSNot significantly different from control, at p<0.05 Hb: Hemoglobin concentration; PCV: Packed cell volume; NSCC: Non-salted calabash chalk; SCC: Salted calabash chalk; ESR: Erythrocyte sedimentation rate; RBC: Red blood cell count; WBC: White blood cell count; Pl: Platelet count

180

Akpantah et al. Hematological effect of calabash chalk

Turk J Hematol 2010; 27: 177-81

consistent with Hb in this study. It has also been reported that RBC count decreased after lead exposure [10], while arsenic-poisoned cattle showed decreased RBC count [12]. White blood cell (WBC) count estimates the total number of WBCs per liter of blood [14]. In this study, there was no significant difference between the treatment groups and the controls, though the group treated with the NSCC showed the highest value. This indicates that the NSCC has little effect on WBC count. This is in variance with a previous report in which there was decreased WBC in arsenic-poisoned cattle [12]. Our study is in line with previous reports [1012,15,16] that reported decreased RBC count, PCV and Hb after exposure to Jatropha curcas, vanadium, lead and its compound, and arsenic. However, this study is in variance with other works [12,17] that reported an increase in WBC when chlordecone was administered to male Sprague-Dawley rats [17] and when arsenic- poisoned cattle blood parameters were evaluated. Calabash chalk contains lead and arsenic [1-3,5]. This has been implicated in the disruption of the biosynthesis of Hb and anemia, increase in blood pressure, and kidney, liver and brain and other neurologic damage [9-12,18,19]. These may have been the reasons for the decreased Hb, PCV and RBC and Pl counts as seen in the group treated with 40 mg/ kg NSCC. The results obtained from the group treated with 40 mg/kg SCC showed no significant differences from the results in the control group, unlike the NSCC group. Could the addition of salt lead to some chemical reaction with the toxic substances, which may have modulated the effect of these substances in this study? This calls for a further investigation on the toxicity of these two forms of calabash chalk and the possible teratogenic effects as well since it is consumed by pregnant women. In conclusion, our investigation revealed that calabash chalk, particularly the non-salted form, is detrimental on some hematological parameters in female Wistar rats.

and/or affiliations relevant to the subject matter or materials included in this manuscript.

Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships,

16.

References 1.

10.

11.

12.

13. 14. 15.

Campbell H. Calabash chalk (Calabar stone, La Craie, Argile, Nzu, Mabele). Chief Medical Officer, CB Upper Newtownards Road, Belfast: 2002. Health Canada. Calabash chalk may pose health risk for pregnant and breastfeeding women. Advisory 2007136 in www.hc-sc.gc.ca Accessed Oct 2008. Food Standards Agency. Lead contamination of Calabash chalk category B: For Action. 2002. URL: www.food.gov.uk. Accessed December 2, 2008. Northumberland Country Council Press Release Archives. Product warning: Excess lead in Calabash chalk. Country Hall. Morpheth. Accessed December 2, 2008. Dean JR, Deary ME, Gbefa BK, Scott WC. Characterization and analysis of persistent organic pollutants and major, minor and trace elements in Calabash chalk. Chemosphere 2004;57:21-5. Mapherson RA, Pincus MR. Henryâ&#x20AC;&#x2122;s Clinical Diagnosis and Management by Laboratory Methods. Philadelphia, PA: WB Saunders, 2007. Hoffman RC, Benz EJ, Shattil SJ, Furie B, Cohen HJ. Hematology. Basic Principles and Practice. Philadelphia, PA: Churchill Livingstone, 2005. Purves WK, Sadava D, Orians GH, Heller HC. Life: The Science of Biology (7th ed). Sunderland, MA: Sinauer Associates, 2004. Lenntech. Chemical properties of lead - environmental effects of lead. URL: www.wikepedia.com. Accessed December 2, 2008. Annabi BA, Nehdi A, Hajjaji N, Gharbi N, El-Fazaa S. Antioxidant enzymes activities and bilirubin level in adult rat treated with lead. C R Biol 2007;330:581-8. Adeyemo OK, Funmilola A, Adedeji OB, Ajiboye OO. Acute toxicity of and blood profile of adult Clarias ngariepimes exposed to lead nitrate. Internet J Hematol 2008;4. Rana T, Sarkar S, Mandal TK, Batabyal S. Haematobiochemical profiles of affected cattle at arsenic prone zone in Haringhata block of Nadia District of West Bengal in India. Internet J Hematol 2008;4. Wikipedia. Erythrocyte sedimentation rate. URL: www. wikipedia.com. Accessed December 2, 2008. My Dr. Full blood count. 2006. URL: www.mydr.com. Accessed December 2, 2008. Oluwole FS, Bolarinwa AF. Jatropa curcus extract causes anaemia in rats. Phyther Res 1998;11:538-9. Scibior A, Zaporowski J, Ostrowski J. Selected haematological and biochemical parameters of blood in rats after subchronic administration of vanadium and/or

Akpantah et al. Hematological effect of calabash chalk

Turk J Hematol 2010; 27: 177-81

17.

18.

magnesium in drinking water. Arch Environ Contam Toxicol 2006;51:287-95. Chetty KN, Fantroy IL, Landau G, Ivie GW. Lack of dietary calcium effect on chlordecone increased white blood cell count, total iron, and iron-binding capacity in serum of rat. Ecotoxicol Environ Safety 1996;33:268-70. Hudson PV, Whittle DM, Wong PT, Borgmann U, Thomas RL, Chau YK, Nriagu JO, Hallet DJ. Lead con-

181

tamination of the Great Lakes. Indianapolis, IN: John Wiley & Sons, 1984. 19.

Centers for Disease Control and Prevention. Managing elevated blood lead levels among young children. Recommendations from the Advisory Committee on Childhood Lead Poisoning Prevention. Atlanta, Georgia: US Department of Health & Human Services, 2002.

182

Research Article

Role of flow cytometry in multiple myeloma and the prognostic significance of CD87 (uPAR) expression Multipl miyelomda ak×m sitometrinin rolü ve CD87 (uPAR) ekspresyonunun prognostik önemi Murat Albayrak1, Özlem ûahin Balç×k1, Simten Daùdaü2, Mesude Y×lmaz2, Funda Ceran2, Osman Yokuü3, Gülsüm Özet2 1Department of Hematology, Dr. Abdurrahman Yurtarslan Ankara Oncology Education and Research Hospital, Ankara, Turkey 2Department 3Department

of Hematology, Ankara Numune Education and Research Hospital, Ankara, Turkey of Hematology, Istanbul Göztepe Education and Research Hospital, Istanbul, Turkey

Abstract Objective: The plasminogen activator system consists of the serine protease urokinase plasminogen activator (uPA), two endogenous inhibitors of PAI-1 (plasminogen activator inhibitor-1) as well as the PAI-2 and uPA receptor (uPAR or CD87). The aim of this study was to determine the significance of flow cytometry and CD87, CD45 and CD56 expressions in the diagnosis, follow-up and prognosis of multiple myeloma (MM). Materials and Methods: Twenty-nine MM patients were included in the study. Bone marrow samples were used for flow cytometry. A panel of CD87, CD45, CD56, CD10, CD19, CD20, CD38, and CD138 was tested by flow cytometry. Results: CD87 was negative in 8 (27.5%) cases, dim positive in 9 (31.1%) and bright positive in 12 (41.4%). CD87 expression was significantly higher in CD45 (-) cases. Conclusion: Flow cytometry has a significant role in the diagnosis and prognosis of MM. Further clinical studies including large numbers of patients are needed to determine the prognostic role of CD87 in MM. (Turk J Hematol 2010; 27: 182-9)

Key words: Multiple myeloma, uPA, CD87 (uPAR), flow cytometry Received: October 5, 2009

Accepted: March 22, 2010

Özet Amaç: Plazminojen aktivatör sistemi; serin proteaz ürokinaz plazminojen aktivatörü (uPA), iki endojen inhibitörü PAI-1 (plazminojen aktivatör inhibitör-1) ve PAI-2 ile uPA reseptöründen (uPAR veya CD87) oluümaktad×r. Bu çal×ümada MM tan×, takip ve prognozunda ak×m sitometri metodunun ve CD87, CD45, CD56 ekspresyonunun önemini belirlemek amaçlanm×üt×r. Yöntem ve Gereçler: Çal×ümaya 29 MM hastas× al×nd×. Ak×m sitometri için kemik iliùi örneùi kullan×ld×. Hastalara ak×m sitometri yöntemi ile CD87, CD45, CD56, CD10, CD19, CD20, CD38, CD138’i içeren panel çal×ü×ld×. Address for Correspondence: M.D. Özlem ûahin Balç×k, Dizgi Sok. 9/6 Bas×nevleri, 06120 Ankara, Turkey Phone: +90 312 321 66 97 E-mail: drozlembalcik@yahoo.com doi:10.5152/tjh.2010.26

Albayrak et al. Flow cytometry and CD87 in myeloma

Turk J Hematol 2010; 27: 182-9

183

Bulgular: Çal×ümam×zda CD87; 8 (% 27.5) olguda negatif, 9 (% 31.1) olguda zay×f pozitif, 12 (% 41.4) olguda ise kuvvetli pozitif bulundu. CD 45 (-) olgularda CD 87 ekspresyonu anlaml× olarak yüksek bulundu. Sonuç: Ak×m sitometri MM tan×s× ve prognozu belirlemede önemli role sahiptir. MM’da CD87 ekspresyonunun prognostik rolünü belirlemek için çok say×da hasta içeren daha fazla klinik çal×ümaya ihtiyaç vard×r.

(Turk J Hematol 2010; 27: 182-9)

Anahtar kelimeler: Multipl miyelom, uPA, CD87 (uPAR), ak×m sitometri Geliü tarihi: 5 Ekim 2009

Kabul tarihi: 22 Mart 2010

Introduction Multiple myeloma (MM) is a B-cell malignancy characterized by the proliferation of malignant plasma cells in the bone marrow. It accounts for 1% of all malignancies and 10% of hematological malignancies [1]. The diagnosis of MM depends on the criteria of the ‘International Myeloma Working Group’ [2]. The International Staging System (ISS), which classifies patients into three prognostic groups according to their `2-microglobulin (`2-M) and albumin levels, is used for the staging of MM [3]. A number of prognostic factors have been defined for MM, including advanced age, low performance status, lactate dehydrogenase (LDH), albumin, C-reactive protein (CRP), `2-M levels, plasma cell labeling index (PCLI), bone marrow microvessel density (MVD), myeloma subtype, disease stage, syndecan-1 (CD138) level, soluble interleukin-6 receptor (sIL-6R) levels, and cytogenetic abnormalities including hypodiploidy and chromosome 13 deletions [1]. Studies are still ongoing to determine the role of flow cytometry in the diagnosis and prognosis of MM. The report of the European Myeloma Network suggests that the primary flow cytometric gate should depend on CD38 vs CD138 expression and that the panel including CD19, CD45, CD56, CD20, CD117, CD28 and CD27 should be used for diagnoses, differential diagnoses and follow-up [4]. The urokinase plasminogen activator (uPA) system consists of serine protease uPA, two endogenous inhibitors of PAI-1 (plasminogen activator inhibitor-1) as well as PAI-2 and uPA receptors (uPAR or CD87). uPA is a serine protease transforming plasminogen into active plasmin [5,6]. CD87 has a number of regulatory effects on cell migration, leukocyte adhesion, chemotaxis, and signal transduction. Reports have suggested that malignant plasma cells expressed uPA and uPAR and that this active proteolytic system might have an effect on the bone marrow turnover in this disease. In addition, studies suggested that CD87 had a role in the

pathogenesis of MM and was associated with the prognosis [7,8]. The studies have demonstrated that CD56 expression is determined at a rate of 72-78% in overall MM cases and is associated with poor prognosis [9-15]. CD45 is a tyrosine phosphatase expressed by hematopoietic cells. CD45 expression is demonstrated at a rate of 18% in MM cases and associated with a good prognosis [12,16-18]. Extramedullary disease is also observed in patients who are CD45-negative [10]. Therefore, CD87 expression was evaluated with CD56 and CD45 in our study. In this study, we examined the frequency of CD87, CD45, and CD56 expressions and their association with other prognostic parameters such as age, `2-M and albumin levels, genetic abnormalities, and disease stage and subtype. The main objective of the present study was to determine the significance of flow cytometry and CD87, CD45 and CD56 expressions in the diagnosis, follow-up and prognosis of MM.

Material and Methods A total of 29 MM patients (23 newly diagnosed; 6 with relapse while under follow-up) were included in the study. The MM diagnosis was based on the medical history, clinical manifestations, immune fixation tests in urine and serum, immunoglobulin levels, and morphologic examination of the bone marrow. Age, hemoglobin (Hb) concentration, numbers of leukocytes and platelets, levels of plasma calcium (Ca), creatinine (Cr), albumin, LDH, `2-M and CRP, type of plasma M protein, type of free light chain in urine, the bone marrow biopsy, and the rate of plasma cells in the bone marrow of all the cases were registered. Using these parameters, the ISS stages were established [3]. The bone marrow samples of all cases were placed in BD Vacutainers containing standard (85 IU) amount of lithium heparin, for cytogenetic evaluation. Conventional cytogenetic evaluation of urine culture was conducted through 48 hours. 100 nuclei

184

Albayrak et al. Flow cytometry and CD87 in myeloma

were assumed for each parameter and RB, IGH/ FGFR DC/DF and p53 probes were used for del 13q, t(4;14) and del 17p, respectively. Bone marrow samples were used for flow cytometry Material used in the study The acquisition and analyses of cells were carried out with the FACSCalibur device and Cell Quest program (BD Biosciences, Heidelberg, Germany) and monoclonal antibodies (BD Biosciences, Heidelberg, Germany). CD45 PER CP (peridininchlorophyll-protein), CD38 PE (phycoerythrin), CD138 FITC (fluorescein isothiocyanate), CD10 PE, CD19 PER CP, CD20 PER CP, CD56 PE, CD87 PE, cyt kappa FITC, cyt lambda PE, IgG1FITC, IgG1 Per CP, and IgG2a PE monoclonal antibodies were used. Procedure Bone marrow was directly examined by fluorescein stained (FITC, PE, PER CP) monoclonal antibodies. We used CD45/CD38/CD138/CD20, CD38/ CD138/CD19/CD87, CD45/CD38/CD19/CD56, CD38/ CD138/CD19/CD10, cytKappa/cyt Lambda/CD38/ CD19 combinations for multiparametric analysis. Following the reading of cells with the FACS CALIBUR device, they were analyzed with the CELL QUEST program. For the analysis, CD45 (-/dim+) and CD38 (+) plasma cells at the CD45/CD38 dot blot were selected. Thereafter, CD38 (+) and CD138 (+) cells at the CD38/CD138 dot blot were selected. A gate for selected CD45 (-/dim+) CD38 (+) CD138 (+) plas-

Figure 1. Selection of plasma cells by flow cytometry

Turk J Hematol 2010; 27: 182-9

ma cells was drawn from the FSC-SSC dot blot and all analyses were carried out with the cells gated in this dot blot (Figure 1). The gating strategy was optimized to exclude contamination events, particularly apoptotic cells and cellular debris. Analysis of CD38 versus CD138 expression provides the best separation of plasma cells from other leukocytes, but is also subject to contamination, with cells binding antibodies nonspecifically. This can be detected on the CD38 versus CD45 plot to the right of the plasma cell population. Thus, an initial region (R1) is set around cells expressing high level of CD38 and CD138, and a second region (R2) is set on the light scatter of gated CD38+CD138+cells. A third region (R3) was set around the cells satisfying both R1 and R2 for CD38 and CD45 expression. All samples were drawn after obtaining formal written consent from the patients, and the research was carried out in accordance with the Helsinki Declaration. Statistical Analysis Data were analyzed with the SPSS 11.5 package program. While descriptive statistics were expressed as meanÂąSD or median (min-max) for continuously measured variables, categorical variables were expressed as the number of observations and percentages. Mann-Whitney U test was used to compare two independent groups for characteristics such as age, immunoglobulin, ISS and CD87. When the number of independent groups was more than two, Kruskal-Wallis variance analysis was done for

Albayrak et al. Flow cytometry and CD87 in myeloma

Turk J Hematol 2010; 27: 182-9

comparisons. Categorical comparisons were done using chi-square and Fisher’s exact tests. A level of p<0.05 was considered to be significant.

Results A total of 29 MM patients (20 males, 9 females) who were monitored in the Hematology Clinic were examined. While 23 patients (79%) were newly diagnosed, 6 (21%) had relapsed disease. Patients who were in the plateau phase or in remission were excluded. General characteristics of the patients are listed in Table 1. A panel of CD87, CD45, CD56, CD10, CD19, CD20, CD38, and CD138 was tested by flow cytometry for each patient. Initially, samples were examined for CD38 and CD138 to support the MM diagnosis. All

patients were CD38 and CD138 (+). CD138, CD87 and CD56 positivity by flow cytometry are shown in Figures 2, 3, and 4, respectively; Figure 5 shows concurrent positivity of CD38 and CD138. Flow cytometric findings are listed in Table 2. Comparison of CD87 expression with other prognostic parameters is presented in Table 3. A significant correlation was present between CD87 and CD45 (p=0.019). While CD87 expression was low (negative or dim) in cases who were CD45 (+), it was high (bright) in those who were CD45 (-). CD87 correlated significantly with extramedullary involvement (p=0.050). CD87 expression was bright (+) in patients with extramedullary involvement (Table 3). In CD45 (+) patients, low CD87 expression (negative or dim positive) was statistically significant (p=0.019) (Table 3).

Age (years, median/range)

61.5 (36-78)

Disease status (new diagnosis/relapse)

23/6

Hb level (g/dL) ( 10/>10)

13/16

Creatinine value (mg/dL) ( 1.3/>1.3)

21/8

Lytic lesions (yes/no)

20/9

IgG/IgA/light chain

15/11/3

Free chain (kappa/lambda/none)

13/15/1 12/17

Jeta-2 microglobulin (mg/dL) ( 1.8/>1.8)

13/16

CRP (mg/L) ( 5/>5)

11/18

Sedimentation (mm/hr) (N/high)

0/29

Extramedullary involvement (yes/no)

6/23

Poor cytogenetic parameter [del13q, 17p, t(4;14)] (yes/no)

15/14

0 101

3 102 10 CD45 PerCP

Figure 2. CD138 positivity in plasma cells

Table 2. Flow cytometric findings in patients (n=29)

% (Mean) of positive cases

CD38 (-/+)

0/29

0/69.8

CD138 (- /+)

0/29

0/66.6

CD45 (-/+)

19/10

65.5/34.5

CD10 (-/+)

29/0

100/0

CD19 (-/+)

28/1

96.6/3.4

CD20 (-/+)

24/5

82.8/17.2

CD56 (-/+)

8/21

27.6/72.4

CD87 (negative-dim positive/bright positive) 17/12

58.6/41.4

CD87 PE 0 101 102 10 3 10

Hb: Hemoglobin; CRP: C-reactive protein; ISS: International Staging System

ISS (stage I/stage II-III)

15/14

Age ( 65/>65)

CD138 FITC 0 101 102 10 3

9/20

Table 1. General characteristics of patients enrolled in the study (n=29) Sex (female/male)

185

0 101

3 102 10 CD20 PerCP

Figure 3. CD87 positivity in plasma cells

186

Albayrak et al. Flow cytometry and CD87 in myeloma

Turk J Hematol 2010; 27: 182-9

There was no significant correlation between CD56 and CD87 (p=1.000) (Table 3). Similarly, CD56 and CD45 did not correlate significantly (p=0.201). CD56 positivity in plasma cells with flow cytometry is shown in Figure 4.

Discussion The surface antigens detected by flow cytometric method have begun to be used in addition to the biochemical and genetic parameters in the diagnosis, follow-up and prognosis of MM [4]. Using flow cytometry, Rigolin et al. [19] determined neoplastic cells expressing CD87 in all of the 49 MM patients included in their study. The highest CD87 expres-

sion was by CD45 (+) immature plasma cells; in addition, these cells displayed strong reactivity against CD138 and CD56, which are two important molecules for the cell adhesion process. In MM patients, CD87 expression seems to be inversely related to the maturation of malignant plasma cells. This suggests that CD87, like CD56, may be involved in the regulation of plasma cell organization and consequently in the disease pathogenesis [7,9]. We used CD38 and CD138 to diagnose MM and to differentiate malignant plasma cells. CD38 and CD138 were positive in all cases (Table 2). This finding was in accordance with the data in the literature. In a study by Plesner et al. [20] including 31 patients, the percentage of CD38 (++) cells was Table 3. Comparison of CD87 expression with prognostic parameters CD87 bright positive (n=12)

p value

CD45 (-)

0.019

CD45 (+)

CD56 (-)

CD6 (+)

CD38 PE 0 101 102 10 3 10

CD87 negative or dim positive (n=17)

0 101

102 10 CD56 FITC

1.000

EM involvement Yes

CD20 (-)

CD20 (+)

Age

0.412

Male

0.234

Female

Hb 10

>10

Creatinine value 1.3

(mg/dL)

Sex

Figure 4. CD56 positivity in plasma cells

Lytic lesions ISS `2-M (mg/dL)

>1.3 Yes

Stage I

Stage II and III

1.8

>1.8

CRP (mg/L)

0-5

between

13q14 del.

Yes

0.050 0.370

0.296 0.408 0.694 1.000 0.638 0.438 0.462

EM: Extramedullary involvement; Hb: Hemoglobin; CRP: C-reactive protein;

Figure 5. Concurrent positivity of CD38 and CD138 in plasma cells

ISS: International Staging System; `2-M: Beta-2 microglobulin

Turk J Hematol 2010; 27: 182-9

considered as the percentage of myeloma cells. Lin et al. [12] suggested that CD138 was the most specific marker for plasma cells, and that staining was 70-100%, but reported that for the initial differentiation of plasma cells, the CD38, CD138 and CD45 combination was more effective. Aberrant CD56 expression was detected in 71.7% of MM cases [12]. CD56 differentiates malignant and benign plasma cells [11]. Aggressive disease and extramedullary involvement are common in myeloma cases without CD56 expression [13]. A study by Mathew et al. [13] including 68 cases demonstrated that survival was shorter in CD56 (-) myeloma cases compared to those who were CD56 (+). In another study, 8 among 12 CD56 (-) myeloma cases were either refractory or progressive [21]. Sahara et al. [15] also examined 70 patients and found that 15 cases were CD56 (-) and 55 were CD56 (+). In this study, high `2-M level, extramedullary disease, thrombocytopenia, and renal involvement were significantly more common in patients who were CD56 (-) compared to CD56 (+) cases. Survival was significantly lower in CD56 (-) cases (22 months) than in CD 56 (+) patients (63 months) [15]. In the present study, 21 among 29 patients (72%) were CD56 (+), in accordance with the previous data. However, we could not find any correlation between CD56 and other prognostic factors. CD45 expression is variable in myeloma cells. While immature plasma cells stain CD45 bright (+), mature plasma cells stain either CD45 (-) or CD45 dim (+) [22]. Asosingh et al. [23] suggested that CD45 (+) myeloma patients had a longer survival. Moreau et al. [24] reported that while CD45 (-) myeloma patients had a poor prognosis, the prognosis of CD45 (+) cases was good. In compliance with the above-mentioned data, CD87 expression was significantly higher in CD45 (-) patients in our study. Lin et al. [12] reported that 54 among 306 cases (17.6%) expressed CD45. In our study, in 10 among 29 patients (34.5%), CD45 expression was positive; CD45 expression did not correlate significantly with other prognostic parameters. In a study by Hjertner et al. [8] including 7 cases, uPA and uPAR were expressed by myeloma cells and all patients had CD87 expression. Similarly, Rigolin et al. [19] reported CD87 expression in all of their patients (n=49). On the other hand, Plesner et al. [20] could not detect uPAR in any of their patients in a series including 10 cases. Although the reason for this difference is unclear, variations in myeloma

Albayrak et al. Flow cytometry and CD87 in myeloma

187

cell isolation were suggested to account for the difference in uPAR expression [20]. CD87 expression was bright (+) in 12 patients among 29 (41.5%) and -/dim positive in 17 (58.5%). While immature plasma cells stain CD45 bright (+), mature plasma cells stain either (-) or dim (+) [22,25,26]. The expression of CD87 by CD45 (+) immature plasma cells is 5-17 times higher than by CD45 -/dim+ mature plasma cells. The study by Hjertner et al. [8] showed that uPAR expression depended on myeloma cell maturation and that immature myeloma cells had high expression levels. Rigolin et al. [19] classified 49 patients into two groups according to CD87 expression. Patients with high CD87 expression also had high CD56, CD38, CD138 and high CD45 positivity. The studies have demonstrated that prognosis was poor in CD45 (-) myeloma patients and good in CD45 (+) cases (23,24). In our study, CD87 expression was significantly higher in CD45 (-) cases, and the rate of extramedullary involvement, which is associated with poor prognosis, was also significantly higher in this group. However, uPAR expressions are highly variable in myeloma patients. This was suggested to be associated with the translocation of uPAR stored in the cytoplasmic compartment on the cell surface after being activated, and following uPA/PAI-1 internalization and degradation in lysosomes, its return to the cell surface [8,20]. Lanza et al. [27] reported a significant correlation between high CD87 expression in acute leukemias and extramedullary involvement. This suggests an association of uPAR expression with clinical features predicting a more aggressive disease course [27]. We found a significant correlation between CD87 expression and extramedullary involvement. Patients with extramedullary involvement had higher CD87 expression. However, there was no correlation between CD87 and other prognostic parameters (Table 3). Rigolin et al. [19] determined an association between high CD87 expression and lytic bone lesions. However, we could not demonstrate such a correlation in our study. On the other hand, while Rigolin et al. could not find a correlation between circulatory soluble uPAR and uPAR (CD87), suPAR seemed to correlate with an aggressive disease course. High suPAR levels were associated with advanced disease, high Cr levels, high CD138 and high `2-M levels. However, this study was not able to demonstrate a

Albayrak et al. Flow cytometry and CD87 in myeloma

Turk J Hematol 2010; 27: 182-9

significant correlation between suPAR level and CRP and 13q14 deletion [19]. No other studies detected a significant relation between CD87 expression and 13q14 deletion in myeloma patients [7]. Similarly, in our study, CD87 expression and the 13q14 deletion did not correlate significantly. CD87 (uPAR) is expressed by myeloma cells and is likely to have a pivotal role in the biology of the disease. Moreover, serum uPAR is an independent parameter that predicts extramedullary involvement and poor prognosis in myeloma [19]. In conclusion, immunophenotyping by flow cytometry is a sensitive method that is used for the diagnosis and clinical monitoring of the disease. Flow cytometry in MM is beneficial in detecting malignant plasma cells and prognostic markers and monitoring the development and differentiation of myeloma cells. CD87, CD45 and CD56 should be tested for the prediction of prognosis in MM. However, further clinical studies including large numbers of patients are needed to determine the prognostic role of CD87. Acknowledegement Ethical committee approval was obtained in this study. Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

5. 6. 7.

10.

11.

References 1

Tricot G. Multiple myeloma and other plasma cell disorders. In: Hoffman R, Benz EJ, Shattil SJ, Furie B, Cohen HJ, Silberstein LE, McGlave P, editors. Hematology Basic Principles and Practice. 4th ed. Philadelphia: Elsevier Science, 2005:1501-21. International Myeloma Working Group. Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol 2003;121:749-57. Greipp PR, San Miguel J, Durie BG, Crowley JJ, Barlogie B, Bladé J, Boccadoro M, Child JA, Avet-Loiseau H, Kyle RA, Lahuerta JJ, Ludwig H, Morgan G, Powles R, Shimizu K, Shustik C, Sonneveld P, Tosi P, Turesson I, Westin J. International staging system for multiple myeloma. J Clin Oncol 2005;23:3412-20. Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA, Bumbea H, Dalva K, Fuhler G, Gratama

12. 13.

14.

15.

188

J, Hose D, Kovarova L, Lioznov M, Mateo G, Morilla R, Mylin AK, Omedé P, Pellat-Deceunynck C, Perez Andres M, Petrucci M, Ruggeri M, Rymkiewicz G, Schmitz A, Schreder M, Seynaeve C, Spacek M, de Tute RM, Van Valckenborgh E, Weston-Bell N, Owen RG, San Miguel JF, Sonneveld P, Johnsen HE; European Myeloma Network. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders. Haematologica 2008;93:4318. Epub 2008 Feb 11. Duffy MJ. The urokinase plasminogen activator system: role in malignancy. Curr Pharm Des 2004;10:39-49. Bu G, Warshawsky I, Schwartz AL. Cellular receptors for the plasminogen activators. Blood 1994;83:3427-36. Béné MC, Castoldi G, Knapp W, Rigolin GM, Escribano L, Lemez P, Ludwig WD, Matutes E, Orfao A, Lanza F, van't Veer M; EGIL, European Group on Immunological Classification of Leukemias. CD 87 (Urokinase-type plasminogen activator receptor), function and pathology in hematological disorders: a review. Leukemia 2004;18:394-400. Hjertner O, Qvigstad G, Hjorth-Hansen H, Seidel C, Woodliff J, Epstein J, Waage A, Sundan A, Börset M. Expression of urokinase plasminogen activator and the urokinase plasminogen activator receptor in myeloma cells. Br J Haematol 2000;109:815-22. Pellat-Deceunynck C, Bataille R, Robillard N, Harousseau JL, Rapp MJ, Juge-Morineau N, Wijdenes J, Amiot M. Expression of CD 28 and CD 40 in human myeloma cells: a comparative study with normal plasma cells. Blood 1994;84:2597-603. Pellat-Deceunynck C, Barille S, Puthier D, Rapp MJ, Harousseau JL, Bataille R, Amiot M. Adhesion molecules on human myeloma cells: significant changes in expression related to malignancy, tumor spreading, and immortalization. Cancer Res 1995;55:3647-53. Pellat-Deceunynck C, Barille S, Jego G, Puthier D, Robillard N, Pineau D, Rapp MJ, Harousseau JL, Amiot M, Bataille R. The absence of CD 56 (NCAM) on malignant plasma cells is a hallmark of plasma cell leukemia and of a special subset of multiple myeloma. Leukemia 1998;12:1977-82. Lin P, Owens R, Tricot G, Wilson CS. Flow cytometric immunophenotypic analysis of 306 cases of multiple myeloma. Am J Clin Pathol 2004;121:482-8. Dahl IM, Rasmussen T, Kauric G, Husebekk A. Differential expression of CD 56 and CD 44 in the evolution of extramedullary myeloma. Br J Haematol 2002;116:273-7. Mathew P, Ahmann GJ, Witzig TE, Roche PC, Kyle RA, Greipp PR. Clinicopathological correlates of CD 56 expression in multiple myeloma: a unique entity? Br J Haematol 1995;90:459-61. Sahara N, Takeshita A, Shigeno K, Fujisawa S, Takeshita K, Naito K, Ihara M, Ono T, Tamashima S, Nara K, Ohnishi K, Ohno R. Clinicopathological and prognostic characteristics of CD 56 negative multiple myeloma. Br J Haematol 2002;117:882-5.

Albayrak et al. Flow cytometry and CD87 in myeloma

Turk J Hematol 2010; 27: 182-9

16.

17.

18.

19.

20.

21.

Moreau P, Robillard N, Jego G, Pellat C, Le Gouill S, Thoumi S, Avet-Loiseau H, Harousseau JL, Bataille R. Lack of CD 27 in myeloma delineates different presentation and outcome. Br J Haematol 2006;132:168-70. Moreau P, Robillard N, Avet-Loiseau H, Pineau D, Morineau N, Milpied N, Harousseau JL, Bataille R. Patients with CD45 negative multiple myeloma receiving high-dose therapy have a shorter survival than those with CD45 positive multiple myeloma. Haematologica 2004;89:547-51. Kawano MM, Ishikawa H, Tsuyama N, Abroun S, Liu S, Li FJ, Otsuyama K, Zheng X. Growth mechanism of human myeloma cells by interleukin-6. Int J Hematol 2002;76(Suppl 1):329-33. Rigolin GM, Tieghi A, Ciccone M, Bragotti LZ, Cavazzini F, Della Porta M, Castagnari B, Carroccia R, Guerra G, Cuneo A, Castoldi G. Soluble urokinase-type plasminogen activator receptor (suPAR) as an independent factor predicting worse prognosis and extra-bone marrow involvement in multiple myeloma patients. Br J Haematol 2003;120:953-9. Plesner T, Behrendt N, Ploug M. Structure, function and expression on blood and bone marrow cells of the urokinase-type plasminogen activator receptor, uPAR. Stem Cells 1997;15:398-408. Van Camp B, Durie BG, Spier C, De Waele M, Van Riet I, Vela E, Frutiger Y, Richter L, Grogan TM. Plasma cells in multiple myeloma express a natural killer cell-associated antigen: CD 56 (NKH-1:leu-19). Blood 1990;76:377-82.

22.

23.

24.

25.

26.

27.

189

Schneider U, Van Lessen A, Huhn D, Serke S. Two subsets of peripheral blood plasma cells defined by differential expression of CD45 antigen. Br J Haematol 1997;97:56-64. Asosingh K, Willems A, Van Riet I, Van Camp B, Vanderkerken K. Delayed in vivo disease progression is associated with high proportions of CD 45 + myeloma cells in the 5T2 MM murine model. Cancer Res 2003;63:3019-20. Moreau P, Robillard N, Avet-Loiseau H, Pineau D, Morineau N, Milpied N, Harousseau JL, Bataille R. Patients with CD45 negative multiple myeloma receiving high-dose therapy have a shorter survival than those with CD45 positive multiple myeloma. Haematologica 2004;89:547-51. Hata H, Xiao H, Petrucci MT, Woodliff J, Chang R, Epstein J. Interleukin-6 gene expression in multiple myeloma: a characteristic of immature tumor cells. Blood 1993;81:3357-64. Joshua D, Petersen A, Brown R, Pope B, Snowdon L, Gibson J. The labelling index of primitive plasma cells determines the clinical behaviour of patients with myelomatosis. Br J Haematol 1996;94:76-81. Lanza F, Castoldi GL, Castagnari B, Todd RF 3rd, Moretti S, Spisani S, Latorraca A, Focarile E, Roberti MG, Traniello S. Expression and functional role of urokinase-type plasminogen activator receptor in normal and acute leukaemic cells. Br J Haematol 1998;103:110-23.

190

Case Report

Enzyme replacement therapy in type 1 Gaucher disease and a review of the literature Tip 1 Gaucher hastal×ù×nda enzim replasman tedavisi ve literatürün gözden geçirilmesi Gökhan Kabaçam1, Gülüah Kabaçam2, Pervin Topçuoùlu3, Iü×nsu Kuzu4, Mutlu Arat3 1Department of Gastroenterology, Ankara University Faculty of Medicine, Ankara, Turkey 2Radiology Clinic, Ankara D×ükap× Children Education and Research Hospital, Ankara, Turkey 3Department of Hematology, Ankara University Faculty of Medicine, Ankara, Turkey 4Department of Pathology, Ankara University Faculty of Medicine, Ankara, Turkey

Abstract Gaucher disease (GD) is the most common lysosomal storage disorder. Deficiency of the lysosomal enzyme glucocerebrosidase results in the intracellular accumulation of undegraded substrates in the spleen, liver and bone marrow. Enzyme replacement therapy (ERT) is a standard approach for type 1 GD. Here, we present an adult patient with hematological disorders due to type 1 GD, who markedly improved with ERT. (Turk J Hematol 2010; 27: 190-5)

Key words: Gaucher disease, glucosylceramidase, treatment Received: May 4, 2009

Accepted: July 31, 2009

Özet Gaucher Hastal×ù× (GH) en yayg×n lizozomal depo hastal×ù×d×r. Lizozomal enzim, glukoserebrosidaz eksikliùi dalak, karaciùer ve kemik iliùinde y×k×lamayan maddelerin hücre içi birikimi ile sonuçlan×r. Enzim replasman tedavisi (ERT) tip 1 GH’de standart bir yaklaü×md×r. Burada, ERT ile belirgin olarak düzelen tip bir GH nedeni ile hematolojik bozukluùu olan yetiükin bir hastay× sunmaktay×z. (Turk J Hematol 2010; 27: 190-5)

Anahtar kelimeler: Gaucher hastal×ù×, glukoserebrosidaz, tedavi Geliü tarihi: 4 May×s 2009

Kabul tarihi: 31 Temmuz 2009

Address for Correspondence: Mutlu Arat, ûisli Florence Nightingale Hospital Hematopoietic Stem Cell Transplantation Unit, Abide-i Hürriyet Cad. No:164 ûiüli, ústanbul, Turkey Phone: +90 212 315 36 44 E-mail: mutlu.arat@florence.com.tr doi:10.5152/tjh.2010.27

KabaĂ§am et al. Enzyme replacement therapy in Gaucher disease

Turk J Hematol 2010; 27: 190-5

Introduction Gaucher disease (GD) is an inherited lysosomal storage disorder (LSD). Lysosomal enzyme activity due to a mutation in the glucosylceramidase (GluCer) gene is decreased or absent, resulting in intracellular accumulation of undegraded substrates. Approximately 300 mutations, mostly autosomal recessive, have been described, and are usually seen in closed communities like in Ashkenazi Jews [1]. Though the consanguinity rate is high in the Turkish population, the disease has been observed to have a low incidence, as 0.23/100,000 live births [2]. Gaucher disease has been divided into three forms according to the clinical manifestations. Type 1 is the most common and mildest form of GD, and is essentially a monocyte/macrophage system disorder, lacking primary central nervous system involvement. It is characterized by varying degrees of splenomegaly and hepatomegaly, anemia, thrombocytopenia, bone pain, and skeletal lesions. Types 2 and 3 are both rare, with acute and fulminating (type 2), or heterogeneous and attenuated (type 3) neurological involvement accompanying visceral manifestations [3]. Quite effective treatment modalities for GD are available today, and they have raised hopes regarding the treatment of other LSDs. In this report, we present a case having severe hematological findings due to GD. The recent developments in the management of GD are also reviewed.

Case Report A 30-year-old female patient with type 1 GD was referred to our hematology clinic due to an increase in her complaints and clinical findings. She had been diagnosed with GD 10 years ago by pathological examination of the bone marrow biopsy and had been followed with supportive measurements. She suffered from abdominal fullness, early satiety and severe fatigue. On the physical examination, she was pale in appearance and had a palpable massive hepatosplenomegaly. She did not have any symptoms or signs of abnormalities of the neurological or locomotor systems. Her laboratory results on admission to our clinic are shown in Table 1. Abdominal ultrasound examination revealed hepa-

191

tomegaly (vertical height was 20 cm) and splenomegaly (100x144x230 mm), including multiple hyperechoic masses with central hypoechogenicity and distinct borders. Bone mineral densitometry monitoring with dual-energy X-ray absorptiometry (DXA) revealed total femur neck T and Z scores of -1.44 each and lumbar vertebrae T and Z scores of -2.20 and -2.19, respectively. Her bone marrow biopsy revealed diffuse Gaucher cell infiltration (Figures 1a, 1b). Glucocerebrosidase enzyme level was measured as 1.6 nmol/s/mpgr (5-13.5 nmol/s/mpgr). She was a heterozygous carrier for N370S and L444P mutations according to genetic mutation screening. The diagnosis of type 1 GD was confirmed and recombinant human GluCer (CerezymeÂŽ) replacement therapy was initiated once every three weeks intravenously at a dose of 400 IU, in addition to parenteral vitamin B12 supplementation and calcium and vitamin D treatments. After enzyme replacement therapy (ERT), her symptoms and clinical and laboratory findings significantly improved. Gaucher cells were apparently decreased in the bone marrow biopsy within one year (Figures 2a, 2b). Oral informed consent was obtained from the patient. Table 1. Laboratory findings on admission to the clinic Test

Result

Normal Range

Hemoglobin (g/dl)

9.4

11.7-15.0

Hematocrit (%)

28.1

35-45

Leukocyte count (x10e9/L)

3.9

4.5-11.0

87.0

150-400

Complete blood count

Absolute neutrophil count (x10e9/L) Platelet count (x10e9/L) Others Direct & indirect antiglobulin test

Negative

Serum IgG level (g/L)

19.4

7.0-16.0

Serum IgM level (g/L)

3.11

0.4-2.3

Serum IgA level (g/L)

3.38

0.7-4.0

Gamma globulin level in serum protein electrophoresis (%)

23 (polyclonal) 10.5-19.5

Serum immune-fixation test

Negative

Serum ferritin level (ng/ml)

263

11.0-306.8

Vitamin B12 level (pg/ml)

166

166-970

Folic acid level (ng/ml)

3.7

1.5-16.9

Abnormal values are shown in bold text

192

Kabaรงam et al. Enzyme replacement therapy in Gaucher disease

Turk J Hematol 2010; 27: 190-5

Figure 1. Bone marrow biopsy (a) and smear (b) revealed diffuse infiltration by typical Gaucher cells

Figure 2. The normal bone marrow cells became the dominant cellular components of the marrow areas after therapy. The decrease in infiltrative cells was examined either on biopsy sections (a) or smear preparations (b)

Discussion Clinically, GD has been divided into three major subtypes, namely types 1, 2 and 3, although recently there is a trend to consider GD as a continuum of disease states [3]. Despite genotype-phenotype correlations being poor, certain mutations predispose to certain disease forms, for example, homozygosity for L444P mutations results almost invariably in neuronopathic disease [4], whereas the presence of even one mutant allele for N370S normally prevents neurological and pulmonary involvement [4], as in our case. N370S is the most frequent mutation accounting for 70% of mutant alleles in Ashkenazi Jews and 25% of non-Jewish patients [5].

The clinical spectrum may range from the asymptomatic form in type 1 GD to the acute neuronopathic form in type 2 GD, characterized with brainstem and visceral involvement and eventually death in the first 2-3 years of life [6]. Type 1 GD manifests itself with organomegaly, blood cytopenias, and osteopenia, as seen in our patient, and also lytic lesions, pathologic fractures and acute bone crisis episodes, and interstitial lung disease, which are more serious clinical findings [7]. Previous splenectomy history, because of the impact of the overwhelming infections, is an important risk factor for deterioration of lung functions in lung involvement [7]. The most common initial clinical appearance, as seen in our patient, is hematological symptoms and

Turk J Hematol 2010; 27: 190-5

findings related to anemia and/or thrombocytopenia [8,9]. Cytopenia(s) is related to hypersplenism and/or infiltration of bone marrow with Gaucher cells. Leukopenia is less frequent and is usually due to hypersplenism. Though neutrophil function is defective in many patients, tendency to infection is not common because the neutrophil count is usually in normal range. Splenomegaly is almost invariably more prominent than hepatomegaly; in case of hepatomegaly being the more prominent, other common causes of hepatomegaly must be ruled out. Other causes of anemia should be sought in a more acute decrease in blood hemoglobin level, especially due to iron and vitamin B12 deficiency, autoimmune hemolytic anemia or associated hematological malignancies. In Gaucher disease, ferritin levels are generally elevated without other biochemical evidence of iron overload, consistent with anemia of chronic disease, whereas typical iron deficiency anemia is characterized by low serum iron, low transferrin saturation and low ferritin levels [9]. In a study among Ashkenazi Jews, it was reported that vitamin B12 levels tended to be lower in the diseased population and decreased in the course of ERT [10]. We gave the patient parenteral vitamin B12 supplementation due to the low level. Gaucher disease can be associated with hyperactivity of the immune system, which manifests with polyclonal hypergammaglobulinemia or monoclonal gammopathies [11]. We detected polyclonal gammopathy in the sera of our patient as well. Because of the variability in the clinical manifestations, severity and progression, a comprehensive initial assessment should be done in each patient [12]. In addition, for the diagnosis and prior to treatment, glucocerebrosidase activity should be measured. The main target in the treatment of GD is elimination of or improvement in symptoms, prevention of irreversible damage, and improvement in the overall health and quality of life [13]. There are many therapeutic approaches including ERT, Substrate Reduction Therapy (SRT), Enzyme Enhancement Therapy (EET), and Gene Therapy (GT) (14-17). Currently, the first two modalities, ERT and SRT, are available in the European and United States markets.

KabaĂ§am et al. Enzyme replacement therapy in Gaucher disease

193

The first ERT model among LSDs was recombinant human GluCer (CerezymeÂŽ), 30-120 U/kg/2-4 weeks intravenously, used in GD. Use of ERT has dramatically improved the quality of life for many patients with GD, by decreasing organ volumes, improving hematological parameters and relieving bone symptoms [16]. Enzyme replacement therapy increases the hemoglobin concentration to almost normal levels in 6-12 months. In all patients, peripheral blood platelet count increases to sufficient levels in order to prevent surgical or spontaneous bleeding in the first year of the therapy. Except for life-threatening hemorrhagic events due to severe thrombocytopenia, splenectomy should be avoided since it facilitates lung involvement and decreases pulmonary function capacity [13]. ERT prevents and also reduces enlargement of the liver and spleen within one year after initiating the therapy. No favorable effect of ERT on neural involvement in types 2 and 3 has been shown, because of poor penetration through the blood-brain barrier [17]. In the case of lung involvement, ERT reverses hepatopulmonary syndrome and improves pulmonary functional status, and thus reduces dependency on oxygen [18]. In the follow-up of patients, monitoring of complete blood count and serum levels of chitotriosidase, angiotensin converting enzyme and tartrateresistant acid phosphatase, liver and spleen volumetric computerized tomography or magnetic resonance imaging, direct X-ray of long bones, and DXA examination of femur neck and lumbar vertebrae have been suggested in the previous studies [6,19]. The cost of the treatment is one of the most important issues yet to be solved. For SRT, N-butyl deoxynojirimycin (ZavescaÂŽ) is approved as an inhibitor of glucosylceramide syntheses enzyme. It is administered orally and therefore more convenient than ERT, with no intravenous-related complications. Furthermore, it crosses the blood-brain barrier and thus may be useful for relieving symptoms and signs of neuronopathic GD. Because SRT causes many adverse effects, it is only indicated in patients in whom ERT is unsuitable or not a therapeutic option [16]. Chemical chaperones (EET) are used to stabilize or reactivate improperly formed GluCer. The preclinical studies related to the use of EET in GD are continuing.

194

Kabaçam et al. Enzyme replacement therapy in Gaucher disease

Supportive medical treatments for maintaining osteoporosis and pulmonary hypertension and bone marrow transplantation for improving hematological and neurological disturbances are suggested as other approaches with or without ERT [20]. Gene therapy is the major challenge in the future of GD therapy. Enzyme replacement therapy usually reduces liver and spleen volumes and improves hematological abnormalities within one year. In contrast, decreased bone marrow glycolipid infiltration has been reported to require up to 3-4 years of treatment [21]. However, we observed a significant decrease in Gaucher cells in the bone marrow after the first year of ERT compared to the pretreatment examination of bone marrow (Figures 2a, 2b). In conclusion, we showed a marked improvement in the clinical and pathological findings in our adult patient severely affected by GD with ERT within one year. However, evaluation and management of patients with GD is continuously and effectively changing. Novel therapeutic approaches have produced exciting results in the clinical a nd pre-clinical studies. In the near future, GD will most probably be an initial success in the LSD therapy era. Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1.

2. 3.

4. 5.

Koprivica VV, Stone DL, Park JK, Callahan M, Frisch A, Cohan IJ, Tayebi N, Sidransky E.Analysis and classification of 304 mutant alleles in patients with Type 1 and Type 3 Gaucher disease. Am J Hum Genet 2000;66:1777-86. Asuman OH, Topcu M. Sphingolipidoses in Turkey. Brain Dev 2004;26(6):363-6. Goker-Alpan O, Schiffmann R, Park JK, Stubblefield BK, Tayebi N, Sidransky E. Phenotypic continuum in neuronopathic Gaucher disease: an intermediate phenotype between type 2 and type 3. J Pediatrics 2003;143:273-6. Lachmann RH. Miglustat. Oxford Glyco Sciences/ Actelion. Curr Opin Investig Drugs 2003;4:472-9. Jmoudiak M, Futerman AH.Gaucher disease; pathological mechanisms and modern management. Br J Haemat 2005;129:178-88.

Turk J Hematol 2010; 27: 190-5

6. 7.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Grabowski GA. Phenotype, diagnosis, and treatment of Gaucher's disease. Lancet 2008;372:1263-71. Mistry PK, Sirrs S, Chan A, Pritzker MR, Duffy TP, Grace ME, Meeker DP, Goldman ME. Pulmonary hypertension in type 1 Gaucher’s disease: genetic and epigenetic determinants of phenotype and response to therapy. Mol Genet Metab 2002;77:91-8. Zimran A, Altarescu G, Rudensky B, Abrahamov A, Elstein D. Survey of hematological aspects of Gaucher disease. Hematology 2005;10:151-6. Hughes D, Cappellini MD, Berger M, Van Droogenbroeck J, de Fost M, Janic D, Marinakis T, Rosenbaum H, Villarubia J, Zhukovskaya E, Hollak C. Recommendations for the management of the haematological and oncohaematological aspects of Gaucher disease. Br J Haematol 2007;138:676-86. Gielchinsky Y, Elstein D, Abrahamov A, Green R, Miller JW, Elstein Y, Alfur N, Lahod A, Shinar E, Zimran A. High prevalence of low serum B12 in multi-ethnic Israeli population. Br J Haemat 2001;115:707-9. Shoenfeld Y, Beresovski A, Zharhary D, Tomer Y, Swissa M, Sela E, Zimran A, Zevin S, Gilburd B, Blank M. Natural autoantibodies in sera of patients with Gaucher’s disease. J Clin Immunol 1995;15:363-72. Charrow J, Esplin JA, Gribble TJ, Kaplan P, Kolodny EH, Pastores GM, Scott CR, Wappner RS, Weinreb NJ, Wisch JS. Gaucher disease: recommendations on diagnosis, evaluation, and monitoring. Arch Inter Med 1998;158:1754-60. Pastores GM, Weinreb NJ, Aerts H, Andria G, Cox TM, Giralt M, Grabowski GA, Mistry PK, Tylki-Szymanska A. Therapeutic goals in the treatment of Gaucher disease. Semin Hematol 2004;41(Suppl 5):4-14. Desnick RJ. Enzyme replacement and enhancement therapies for lysosomal storage diseases. J Inh Metab Dis 2004;27:385-410. Butters TD, Dwek RA, Platt FM. New therapeutics for the treatment of glycosphingolipid lysosomal storage diseases. Adv Exp Med Biol. 2003;535:219-26. Barton NW, Brody RO, Dambrosia JM, Di Bisceglie AM, Doppelt SH, Hill SC, Mankin HJ, Murray GJ, Parker RI, Argoff CE, Grewal RP, Yu KT. Replacement therapy for inherited enzyme deficiency-macrophage targeted glucocerebrosidase for Gaucher disease. N Engl J Med 1991;324:1464-70. Desnick RJ, Schuchman EH. Enzyme replacement and enhancement therapies: lessons from lysosomal disorders. Nat Rev Genet 2002;3:954-66. Dawson A, Elias DJ, Rubenson D, Bartz SH, Garver PR, Kay AC, Bloor CM, Beutler E. Pulmonary hypertension developing after alglucerase therapy in two patients with type 1 Gaucher disease complicated by the hepatopulmonary syndrome. Ann Int Med 1996;125:901-4. Ciana G, Addobbati R, Tamaro G, Leopaldi A, Nevyjel M, Ronfani L, Vidoni L, Pittis MG, Bembi B. Gaucher disease

Kabaรงam et al. Enzyme replacement therapy in Gaucher disease

Turk J Hematol 2010; 27: 190-5

20.

and bone: laboratory and skeletal mineral density variations during a long period of enzyme replacement therapy. J Inherit Metab Dis 2005;28:723-32. Weinreb NJ, Aggio MC, Andersson HC, Andria G, Charrow J, Clarke JT, Erikson A, Giraldo P, Goldblatt J, Hollak C, Ida H, Kaplan P, Kolodny EH, Mistry P, Pastores GM, Pires R, Prakash-Cheng A, Rosenbloom BE, Scott CR, Sobreira E, Tylki-Szymanska A, Vellodi A,

21.

195

vom Dahl S, Wappner RS, Zimran A; International Collaborative Gaucher Group (ICGG). Gaucher disease type 1: revised recommendations on evaluations and monitoring for adult patients. Semin Hematol 2004;41(Suppl 5):15-22. Grabowski GA, Leslie N, Wenstrup R. Enzyme therapy for Gaucher disease: the first 5 years. Blood Rev 1998;12:115-33.

196

Case Report

Cyclic thrombocytopenia: A case report Siklik trombositopeni: Olgu sunumu Abdullah ûumnu1, Reyhan Diz-Küçükkaya2 1Department of Internal Medicine, ústanbul University, ústanbul Medical Faculty, ústanbul, Turkey 2Division of Hematology, Department of Internal Medicine, ústanbul University, ústanbul Medical Faculty, ústanbul, Turkey

Abstract Cyclic thrombocytopenia (CTP) is a rare disorder characterized by periodic decreases and increases in platelet levels. Each cycle usually spans a period of 3-5 weeks. Clinical features are similar to those of idiopathic thrombocytopenic purpura (ITP), so patients are frequently misdiagnosed as having ITP. However, CTP usually does not respond to most treatments used in ITP such as corticosteroids, splenectomy and intravenous immunoglobulin. In this case report, we present a 33-year-old woman with CTP misdiagnosed as ITP.

(Turk J Hematol 2010; 27: 196-9)

Key words: Cyclic thrombocytopenia, idiopathic thrombocytopenic purpura, hepatitis B, case report Received: November 4, 2008

Accepted: April 8, 2009

Özet Siklik trombositopeni (ST) trombosit seviyelerinde periyodik düüüü ve yükselmeler ile karakterize olan nadir görülen bir hastal×kt×r. Bir siklusun uzunluùu genellikle 3-5 hafta aras×ndad×r. Hastal×ù×n klinik özellikleri idiyopatik trombositopenik purpura (úTP) ile benzerlik gösterdiùinden hastalara s×k olarak yanl×ül×kla úTP tan×s× koyulmaktad×r. Ancak ST genellikle kortikosteroidler, splenektomi ve intravenöz immünoglobulin gibi úTP tedavilerine yan×t vermez. Bu olgu sunusunda öncesinde úTP tan×s× ile takip edilmiü 33 yaü×nda bir kad×n hasta sunuyoruz. (Turk J Hematol 2010; 27: 196-9)

Anahtar kelimeler: Siklik trombositopeni, idiyopatik trombositopenik purpura, hepatit B, olgu sunusu Geliü tarihi: 4 Kas×m 2008

Kabul tarihi: 8 Nisan 2009

Address for Correspondence: Asst. Abdullah ûumnu, Department of Internal Medicine, Istanbul University, ústanbul Medical Faculty, ústanbul, Turkey Phone: +90 212 531 30 72 E-mail: abdullahsumnu@yahoo.com doi:10.5152/tjh.2010.28

Turk J Hematol 2010; 27: 196-9

Introduction Cyclic thrombocytopenia (CTP) is a rare disorder characterized by periodic decreases and increases in platelet levels [1]. Each cycle usually spans a period of 3-5 weeks. The platelet counts fluctuate by a large scale on a periodic basis. In the reported cases, the median nadir and peak platelet counts are 10x109/L (range 1-90 x109/L) and 330x109/L (range 72-2300 x109/L) [2]. Rebound thrombocytosis (> 500x109/L) without therapy is an important and distinctive feature for CTP. Women are affected more than men. Although some cases have been reported in myeloproliferative diseases, most cases are idiopathic [3,4]. The pathophysiology is unclear. Some potential mechanisms are autoimmune platelet destruction, megakaryocytic hypoplasia/aplasia, and infectious and hormonal etiology. Clinical presentation is not different from that of idiopathic thrombocytopenic purpura (ITP). Some patients are asymptomatic. Patients may experience easy bruising, gingival bleeding, recurrent epistaxis, menorrhagia, and hematuria. They may present with more serious bleeding like gastrointestinal or central nervous system hemorrhages. In most cases, CTP is not considered as a differential diagnosis so patients are diagnosed and treated as having ITP. CTP usually does not respond to most treatments used in ITP such as corticosteroids, splenectomy and intravenous immunoglobulin (IVIG). In this condition, a diagnosis of CTP should be considered. Close observation without treatment seem to be the best management if clinically permissible. There are some case reports with complete remission after hormonotherapy (danazol and sex hormones) [5-8].

รปumnu et al. Cyclic thrombocytopenia

197

was admitted to the hospital for the second time with purpura, she was administered 2 mg/kg/day methylprednisolone. No response was achieved. The platelet count reached 800x109/L again without therapy. Three months later, when the third purpuric attack occurred, splenectomy was suggested, and the patient was admitted to our Hematology Department. Oral informed consent was obtained from the patient. She was married, with no children, and was working in a hair salon. Her hepatitis B surface antigen was positive. Her physical examination revealed petechiae and ecchymoses on her body and hemorrhagic bullae in the oral mucosa. There was no hepatomegaly, splenomegaly or lymphadenomegaly. Signs of Cushing syndrome were seen and she was still taking 48 mg methylprednisolone per day orally. Laboratory investigations revealed: platelet: 180x109/L, hemoglobin: 10.7 g/dl, hematocrit: 32%, MCV: 74 fl, ferritin: 3 ng/ml, SGOT: 240 IU/ml, SGPT: 280 IU/ml, hepatitis B surface antigen (HBsAg): +, and HBV-DNA: +. Peripheral blood smear showed hypochromia, microcytosis, and normal leukocyte differentiation with no abnormal cells, and the platelets had normal morphology. We tapered and then stopped the corticosteroid dose, administered lamivudine for hepatitis B, and added oral iron therapy for iron deficiency anemia. We followed the patient every week with complete blood count. The

Case Report A 33-year-old woman was admitted to the hospital in April 2006 with purpura. Initial complete blood cell count showed thrombocytopenia (platelet count: 6x109/L), a mild iron deficiency anemia and normal white blood cell count. After making an initial diagnosis of ITP, the patient received 1 mg/kg/ day methylprednisolone for 1 week, 1 g/day methylprednisolone for 3 days and finally 1 g/kg IVIG therapy. There was no response. Then, the platelet count began to increase spontaneously and reached 750x109/L in the third week. After 45 days, when she

Figure 1. Platelet counts of the patient showed a cyclic pattern. Interestingly, lymphocyte and neutrophil counts also showed a cyclic pattern without neutropenia. Platelet cycles occurred in synchrony with menstruation. It has been thought that the decrease in hemoglobin level was related to hypermenorrhea

198

ûumnu et al. Cyclic thrombocytopenia

platelet count and also neutrophil and lymphocyte count showed cyclic decreases over a period of five weeks (Figure 1). Anemia as a consequence of thrombocytopenia and menorrhagia contributed to the clinic picture. The diagnosis of CTP was made. Medroxyprogesterone acetate was initiated to control hypermenorrhea after gynecological consultation. Tranexamic acid was also added to control bleeding symptoms when the patient had nadir platelet count. After we accumulated data revealing that the patient had thrombocytopenia every five weeks, we started to use oral contraceptives and tranexamic acid to manage the patient’s menstruation. With this therapy, hypermenorrhea was controlled, and the patient did not experience any major bleeding during the two-year follow-up.

Discussion In this case report, a 33-year-old woman with CTP misdiagnosed as ITP was presented. She had severe thrombocytopenia with bleeding symptoms followed by thrombocytosis. She received corticosteroids and IVIG therapy with no response. Because she had rebound thrombocytosis, we suspected the diagnosis of CTP. The observation of this patient with weekly platelet counts showed cyclic fluctuation, proving the CTP diagnosis. The pathophysiology of CTP is unclear. Some potential mechanisms are autoimmune platelet destruction, megakaryocytic hypoplasia/aplasia, and infectious and hormonal etiology. There are some reported cases that respond well to hormonotherapy, especially to danazol. It has been shown that most female CTP patients had low platelet counts during menstruation. It has also been shown that hysterectomy and bilateral salpingo-oophorectomy did not affect platelet fluctuation in those patients [2]. In our case, platelet cycles occurred in synchrony with menstruation (Figure 1). Because our patient had hypermenorrhea causing anemia, we used oral contraceptives for controlling menstruation. Although the oral contraceptive drug did not affect platelet fluctuations, it helped to manage the time of menstruation. We also used tranexamic acid to decrease bleeding symptoms. The association between CTP and infections has not been described. To date, CTP had been report-

Turk J Hematol 2010; 27: 196-9

ed only in dogs via Ehrlichia platys [9,10] and it was documented to resolve after antibiotherapy [11]. However, there is not yet any data about human ehrlichiosis causing CTP. Considering that HBsAg in our patient was positive, the association between CTP and hepatitis B needs to be studied. There are some case reports about isolated thrombocytopenia in the course of acute or chronic hepatitis B [12,13]; however, a CTP-hepatitis B association is unknown. In conclusion, CTP is a rare disorder, but in patients with ITP who have not responded to several treatments and have rebound thrombocytosis, a diagnosis of CTP should be considered. Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1.

2. 3.

Balduini CL, Carlo Stella C, Rosti V, Bertolino G, Nons P, Ascari E. Acquired cyclic thrombocytopenia-thrombocytosis with periodic defect of platelet function. Br J Haematol 1993;85:718-22. Go RS. Idiopathic cyclic thrombocytopenia. Blood Rev 2005;19:53-9. Steensma DP, Harrison CN, Tefferi A. Hydroxyureaassociated platelet count oscillations in polycythemia vera: a report of four new cases and a review. Leuk Lymphoma 2001;42:1243-53. Abe Y, Hirase N, Muta K, Okada Y, Kimura T, Umemura T, Nishimura J, Nawata H. Adult onset cyclic hematopoiesis in a patient with myelodysplastic syndrome. Int J Hematol 2000;71:40–5. Tomer A, Schreiber AD, McMillan R, Cines DB, Burstein SA, Thiessen AR, Harker LA. Menstrual cyclic thrombocytopenia. Br J Haematol 1989;71:519-24. Rocha R, Horstman L, Ahn YS, Mylvaganam R, Harrington WJ. Danazol therapy for cyclic thrombocytopenia. Am J Haematol 1991;77:140-3. Kashyap R, Choudhry VP, Pati HP. Danazol therapy in cyclic acquired amegakaryocytic thrombocytopenic purpura: a case report. Am J Haematol 1999;60:225-8. Kuayama J, Take H, Matsumoto S, Nakajima T, Sugase T, Hata A, Hanada M, Nakagawa K. Synchronous fluctuation of interleukin-6 and platelet count in cyclic thrombocytopenia and thrombocytosis. Intern Med 1995;34:636-9.

รปumnu et al. Cyclic thrombocytopenia

Turk J Hematol 2010; 27: 196-9

10.

Waner T, Leykin I, Shinitzky M, Sharabani E, Buch H, Keysary A, Bark H, Harrus S. Detection of plateletbound antibodies in beagle dogs after artificial infection with Ehrlichia canis. Vet Immunol Immunopathol 2000;77:145-50. Wong SJ, Thomas JA. Cytoplasmic, nuclear, and platelet autoantibodies in human granulocytic ehrlichiosis patients. J Clin Microbiol 1998;36:1959-63.

11.

12.

13.

199

Chang WL, Su WL, Pan MJ. Two-step PCR in the evaluation of antibiotic treatment for Ehrlichia platys infection. J Vet Med Sci 1997;59:849-51. Ozaras R, Celik AD, Kisacik B, Mert A, Aki H, Ozturk R, Tabak F. Acute hepatitis B and isolated thrombocytopenia. J Clin Gastroenterol 2003;37:87-8. Romero R, Kleinman R. Thrombocytopenia associated with acute hepatitis B infection. Pediatrics 1993;91:150-2.

200

Case Report

HbQ-India associated with microcytosis: An uncommon hemoglobin variant associated with a common hematologic condition Mikrositoz ile iliükili HbQ-India: Yayg×n bir hematolojik durumla iliükilendirilen yayg×n olmayan bir hemoglobin varyant× Amit Kumar Yadav, Usha Rusia Department of Pathology, University College of Medical Sciences, New Delhi, India

Abstract HbQ-India is a rare alpha chain variant that usually presents in the heterozygous state. Normally, HbQ-India is clinically silent. It becomes symptomatic when present in association with other conditions. We report a case of HbQ-India with concomitant presence of iron deficiency anemia. A 16-year-old female presented with weakness and pallor intermittently for six years. Complete blood count showed severe microcytic hypochromic anemia. Hemoglobin electrophoresis showed a prominent band in the S,D,G region. Tests for sickling were negative. High performance liquid chromatography (HPLC) showed a peak in the unknown window (4.70-4.90 min) suggestive of HbQ-India. Serum iron profile was suggestive of iron deficiency anemia. Based on the above findings, a diagnosis of coexistent HbQ-India–iron deficiency anemia was made. A family study revealed the father as having moderate anemia with similar findings while the mother was normal. Abnormal hemoglobin in the patient was confirmed by molecular diagnosis. HbQ variants are the alpha globin chain variants due to structural mutations (_64 AspAHis) inherited in autosomal dominant fashion. Three molecular variant types have been documented, namely HbQ-India, HbQ-Thailand and HbQ-Iran. Normally, HbQ is clinically silent. Therefore, careful screening of the samples using routine techniques like Hb electrophoresis and HPLC are needed for identification of such abnormal hemoglobin variants like HbQ-India. (Turk J Hematol 2010; 27: 200-3) Key words: HbQ-India, HbQ, hemoglobin variants Received: March 12, 2008

Accepted: April 8, 2009

Özet Hb Q-India genelde heterozigoz durumda bulunan nadir bir alfa zincir varyant×d×r. Normalde HbQIndia klinik olarak sessizdir. Diùer koüullar×n varl×ù× ile birlikte semptomatik hale gelir. Biz burada, demir eksikliùi anemisi ile eüzamanl× bir HbQ-India vakas×n× bildiriyoruz. Alt× yaü×ndan beri halsizlik ve gidip gelen solgunluùa sahip 16 yaü×nda bir kad×n sunulmuütur. Tam kan say×m× üiddetli derecede mikrositik hipokromik anemi göstermiütir. Hemoglobin elektroforezi S, D, G Address for Correspondence: M. D. Amit Kumar Yadav, Department of Pathology, University College of Medical Sciences, Dilshad Garden 110095 New Delhi, India Phone: 011-22586262 E-mail: amityadav2@indiatimes.com doi:10.5152/tjh.2010.29

Yadav et al. HbQ-India and microcytosis

Turk J Hematol 2010; 27: 200-3

201

bölgesinde belirgin bant göstermiütir. Oraklaüma testleri negatiftir. High performance liquid chromatography (HPLC) bilinmeyen pencerede (4,70-4,90 dakika) yapt×ù× pik ile HbQ-India öne sürmüütür. Serum demir profili ise demir eksikliùi anemisine iüaret etmiütir. Yukar×daki bulgular temel al×narak eülik eden HbQ-India-Demir eksikliùi anemisi tan×s× konmuütur. Aile çal×ümas× annenin normal olmas×na karü×n baban×n benzer bulgularla orta üiddette anemisi olduùunu ortaya ç×karm×üt×r. Hastada anormal hemoglobin moleküler teühis ile onaylanm×üt×r. Hb Q varyantlar× otozomal dominant üekilde kal×t×lan yap×sal mutasyonlardan ötürü (_64 AspAHis) alfa globin zincir varyantlar×d×r. HbQ-India, HbQ-Thailand ve HbQ-Iran olmak üzere üç moleküler varyant türü belgelenmiütir. Normalde HbQ klinik olarak sessizdir. Bu nedenle Hb elektroforezi & HPLC gibi rutin tekniklerden yararlan×larak numunelerin dikkatle izlenmesi HbQ-India gibi anormal hemoglobin varyantlar×n×n tan×mlanabilmesi için gereklidir. (Turk J Hematol 2010; 27: 200-3) Anahtar kelimeler: Hb Q India, Hb Q, Hemoglobin varyantlar× Geliü tarihi:12 Mart 2008

Kabul tarihi: 8 Nisan 2009

Introduction HbQ-India is a rare alpha globin chain variant and usually presents in the heterozygous state. Normally, HbQ-India is clinically silent. It becomes symptomatic when present in association with other conditions like beta-thalassemia, alphathalassemia, HbE and HbH. We determined one such case in a Punjabi family. The hematological findings of the case along with the family study are presented.

Case Report An 18-year-old female presented with complaints of weakness and pallor intermittently for six years. Physical examination showed severe pallor. No other significant finding was noted. The findings of the

complete blood count (CBC) with erythrocyte sedimentation rate (ESR) are summarized in Table 1. Peripheral smear showed severe microcytic hypochromic anemia. Hb electrophoresis (TEB Buffer pH 8.6) showed a prominent band in the S, D,G region in the patient and father while that of the mother showed a normal pattern. Test for sickling and solubility test were negative in both the patient and her father. Serum iron profile of the patient was: serum iron: 41 mg/dl, total iron-binding capacity (TIBC): 556 mg/dl, and transferrin saturation: 7.4%. High performance liquid chromatography (HPLC) showed a prominent peak in the unknown window (4.70-4.90 min) in both the patient (Figure 1) and her father. The exact percentages of various fractions are summarized in Table 1. HbQ-India was found to be 19.5% in the patient and 22.5% in the father. Serum iron profile of father was: serum iron:

Table 1. Results of complete blood count and percentages of various hemoglobin fractions obtained by HPLC Hb g/dl

RBC 1012/L

Hct %

MCV fl

MCH pg

MCHC g/dl

Retic. %

HbA2

HbA

HbQIndia

HbF

Patient

5.8

3.52

18.8

53.3

16.4

30.7

0.9

72.8

19.5

Father

9.5

5.07

32.9

18.7

26.8

0.9

64.2

22.5

Mother

11.3

4.03

33.8

83.9

33.3

Patient (Post iron therapy at 3 months)

9.7

4.52

29.8

22.1

3.5

Patient (Post iron therapy at 6 months)

12.9

4.65

38.9

84.1

27.8

33.1

1.5

Father (Post iron therapy at 3 months)

11.8

5.12

36.2

71.3

23.5

32.8

Father (Post iron therapy at 6 months)

14.2

5.02

47.1

93.6

28.6

31.2

202

Yadav et al. HbQ-India and microcytosis

38 mg/dl, TIBC: 489 mg/dl and transferrin saturation: 8.7%, thereby explaining the significant anemia. Molecular diagnosis of the abnormal hemoglobin was confirmed using a method described previously [2]. The characteristic 370 bp fragment (Figure 2) was observed in the agarose gel electrophoresis. Based on the above findings, a diagnosis of coexistent HbQ India-iron deficiency anemia was made. Following the diagnosis, the patient was placed on iron therapy and she showed improvement as shown in Table 1. At the six-month follow-up, the hematological parameters had returned to normal range. The father, who was initially reluctant to receive iron therapy, was counseled and he eventu-

Figure 1. HPLC of the patient showing a peak in the unknown window (4.70-4.90 min)

Figure 2. Agarose gel showing the characteristic 370 bp fragment (yellow arrow) in lane 4. Lane 3 - control showing the normal 766 bp fragment (pink arrow), lane 1 - X174 DNA marker fragments and lane 2 - water blank

Turk J Hematol 2010; 27: 200-3

ally agreed. At the six-month follow-up, his hematological parameters had returned to normal range. Oral informed of consent was ontained from the patient.

Discussion HbQ variants are the alpha globin chain variants due to structural mutations. Three molecular variant types have been documented, namely HbQIndia [1] (alpha 64 Asp to His), HbQ-Thailand (alpha 74 Asp to His) and HbQ-Iran (alpha 75 Asp to His). Normally, HbQ-India is clinically silent. This is because the residue involved, _64 (E 13), is on the surface of the hemoglobin tetramer. However, charge changes at these positions do not affect the properties of the hemoglobin molecule [2]. The majority of the cases reported in the literature are in association with beta- thalassemia [3]. The quantity of the HbQ variant is determined by the ratio of alpha A, alpha Q and beta A globin chains. HbQ-India is also known to be affected by the presence of other hemoglobinopathies. Presence of alpha-thalassemia favors the formation of HbQ, whereas beta-thalassemia reduces the formation of HbQ. This has been explained to be due to a post-translational control mechanism [4]. In the present case, the patient and her father improved remarkably on iron therapy. This rules out the possibility of any other associated hemoglobinopathy like alpha-thalassemia. The definitive method for the identification of HbQ-India is DNA sequencing of the alpha globin and beta globin genes. However, it is an expensive technique and not practical for routine use. Amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) [2], which is a relatively inexpensive technique, has been adapted to enable the quick identification of HbQ-India. In conclusion, awareness about HbQ-India is important as it may be confused with HbS and HbD. HPLC and molecular techniques should be performed in cases with band in the S,D,G region and negative tests for sickling in order to exclude HbQ-India. Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

Yadav et al. HbQ-India and microcytosis

Turk J Hematol 2010; 27: 200-3

References 1.

Sukumaran PK, Merchant SM, Desai MP, Wiltshire BG, Lehmann H. Hemoglobin Q India (alpha 64(E13) aspartic acid to histidine) associated with beta-thalassemia observed in three Sindhi families. J Med Genet 1972;9:436-42. Abraham R, Thomas M, Britt R, Fisher C, Old J. Hb Q-India: an uncommon variant diagnosed in three

3. 4.

203

Punjabi patients with diabetes is identified by a novel DNA analysis test. J Clin Pathol 2003;56:296-9. Dash S, Huisman TH. Hemoglobin-Q-India (64 (E13) Asp-His) and beta thalassemia: a case report from Punjab (North India). Eur J Haematol 1988;40:281. Qin WB, Baysal E, Wong KF, Molchanova TP, Pobedimskaya DD, Sharma S, Wilson JB, Huisman TH. Quantities of alpha Q chain variants in heterozygotes with and without a concomitant beta-thalassemia trait. Am J Hematol 1994;45:91-3.

204

Case Report

Severe iron overload and hyporegenerative anemia in a case with rhesus hemolytic disease: therapeutic approach to rare complications Rhesus hemolitik hastal×kl× bir vakada hiporejeneratif anemi ve aü×r× demir yüklenmesi: Nadir geliüen komplikasyona terapötik yaklaü×m Fatih Demircioùlu1, ûule Çaùlayan Sözmen3, ûebnem Y×lmaz2, Hale Ören2, Nur Arslan4, Abdullah Kumral5, Erdener Özer6, Gülersu úrken2 1Dörtçelik Children’s Hospital, Bursa, Turkey 2Department of Pediatric Hematology, Dokuz Eylül University Faculty of Medicine, úzmir, Turkey 3Department of Pediatrics, Dokuz Eylül University Faculty of Medicine, úzmir, Turkey 4Department of Pediatric Gastroenterology, Dokuz Eylül University Faculty of Medicine, úzmir, Turkey 5Department of Neonatology, Dokuz Eylül University Faculty of Medicine, úzmir, Turkey 6Department of Pathology, Dokuz Eylül University Faculty of Medicine, úzmir, Turkey

Abstract A 33 weeks’ gestation, a baby with rhesus hemolytic disease (RHD), who had received intrauterine transfusions twice, developed cholestatic hepatic disease and late hyporegenerative anemia. Her serum ferritin and bilirubin levels increased to 8842 ng/ml and 17.9 mg/dl, respectively. Liver biopsy showed cholestasis and severe iron overload. Treatment with recombinant erythropoietin (rHuEPO) decreased the transfusion need, and intravenous deferoxamine resulted in a marked decreased in serum ferritin levels and normalization of liver function. In patients who have undergone intrauterine transfusions due to RHD, hyperferritinemia and late hyporegenerative anemia should be kept in mind. Chelation therapy in cases with symptomatic hyperferritinemia and rHuEPO treatment in cases with severe hyporegenerative anemia should be considered. (Turk J Hematol 2010; 27: 204-8) Key words: Rhesus hemolytic disease, late hyporegenerative anemia, transfusion-related hepatic iron overload, chelation therapy Received: May 10, 2009

Accepted: December 10, 2009

Özet Rhesus hemolitik hastal×ù× (RHD) tan×s×yla izlenen, iki kez intrauterin transfüzyon alan ve 33. gestasyonel haftada doùan hastada takipte geç hiporejeneratif anemi ve kolestatik karaciùer hastal×ù× geliüti. Ferritin 8842 ng/mL ve bilirubin 17.9 mg/dL’ye kadar yükseldi. Karaciùer biyopsisinde kolestatik Address for Correspondence: M.D. Fatih Demircioùlu, Dörtçelik Children’s Hospital, Bursa, Turkey Phone: +90 505 525 03 47 E-mail: fatih_demircioglu@yahoo.com doi:10.5152/tjh.2010.30

Turk J Hematol 2010; 27: 204-8

Demircioùlu et al. Rhesus hemolytic disease and transfusion related severe iron overload

205

hepatit ve aü×r× demir birikimi saptand×. Rekombinan erythropoietin (rHuEPO) ile transfüzyon ihtiyac× azald×. úntravenöz desferoxamine ile karaciùer fonksiyon testleri ve serum ferritin deùerleri normale döndü. Sonuç olarak, RHD nedeniyle intrauterin transfüzyon tedavisi yap×lan hastalarda hiporejeneratif anemi ve hiperferritinemi geliüebilir. Semptomatik hiperferritinemi durumunda üelasyon tedavisi, hiporejeneratif anemi durumunda da rHuEPO tedavisi göz önünde bulundurulmal×d×r. (Turk J Hematol 2010; 27: 204-8)

Anahtar kelimeler: Rh hemolitik hastal×ù×, geç hiporejeneratif anemi, transfüzyona ikincil karaciùer demir birikimi, üelasyon tedavisi Geliü tarihi:10 May×s 2009

Kabul tarihi: 10 Aral×k 2009

Introduction The incidence of rhesus hemolytic disease (RHD) has significantly declined all over the world with the use of prophylactic anti-D immunoglobulin [1]. However, in developing countries like Turkey, RHD remains a major cause of severe mortality and morbidity since prophylactic anti-D immunoglobulin may not be administered appropriately or because RHD may develop despite anti-D. Intrauterine transfusions (IUTs) are performed as part of the management of neonates who have developed fetal anemia due to RHD. Along with technological improvements, IUT can be easily applied to babies with RHD and mortality and morbidity ratios may decrease distinctly [1,2]. Although there are many detailed reports in the literature about anemia and hyperbilirubinemia development in RHD, the data about complications such as iron overload secondary to IUT and hyporegenerative anemia are limited; the postnatal management of severely affected infants is still challenging. In RHD, the disease itself and IUT increase the risk of iron overload and late hyporegenerative anemia [3-8]. The new transfusions, which are done postnatally after hyporegenerative anemia, usually raise the iron overload and affect the prognosis poorly. In this case report, a neonate with cholestatic hepatic disease and late hyporegenerative anemia due to RHD is reported to discuss the therapeutic approach to these complications in neonates with RHD.

Case Report A female infant weighing 2335 g was born at 33 weeks’ gestation to a 22-year-old gravida 3, para 2, blood group A Rh-negative woman. The father was Rh-positive and the mother had not received any anti-D after her deliveries. She had one healthy Rh-positive child from her first pregnancy. A second baby with hydrops fetalis died intrauterinely. In the

present pregnancy, indirect Coombs test was found positive (anti-D antibody titer 1:256) and the fetus received intrauterine 120 ml and 240 ml O Rh-negative red cell concentrate transfusions in the 29th and 31st weeks, respectively. Cord blood hemoglobins (Hb) were 8.6 g/dl and 6.4 g/dl before the IUTs, respectively. At gestational week 33, the mother presented to the obstetric unit of our hospital with uterine contractions. An emergency cesarean section was performed and a baby girl was born with Apgar scores of 8 and 10 at 1 and 5 minutes, respectively. On initial physical examination, the infant was not hydropic and was transferred to the neonatal intensive care unit. Cord blood typing showed O Rh-negative blood. Direct Coombs test was negative. A complete blood count at birth revealed a Hb of 10.1 g/dl and hematocrit (Hct) of 29.7%. Total bilirubin was 5.2 mg/dl, with a conjugated fraction of 0.6 mg/dl. A transfusion of 15 cc/kg of packed red cell raised the Hct to 43%. Bilirubin levels reached a peak of 8.3 mg/dl by the first day of life and phototherapy was given according to standard neonatology protocols. The patient was discharged home at the fifth day of age, with a Hct value of 42%. At four weeks of age, she was hospitalized for anemia with a Hct of 17.6% and reticulocyte count of 0%. Total serum bilirubin level was 14.7 mg/dl and conjugated bilirubin level was 3.6 mg/dl. Other hematological findings and liver enzymes were normal. On her physical examination, she was icteric and tachycardic. Hepatosplenomegaly was not present. She received packed red cell transfusion. Bone marrow examination showed erythroid hypoplasia. Thirteen days later another transfusion was necessary (Hct was 16.6% and reticulocyte 0.1%). Hyporegenerative anemia secondary to RHD was diagnosed, and treatment with recombinant human erythropoietin (rHuEPO) (250 U/kg by subcutaneous injection, 3 times a week) was initiated on day 42 of life. After initiation of rHuEPO, reticulocytopenia and anemia

206

DemircioĂšlu et al. Rhesus hemolytic disease and transfusion related severe iron overload

continued for two weeks and she had to be transfused again (Figure 1). By the end of the third week, reticulocyte count increased and Hb level stabilized. rHuEPO was discontinued after six weeks of treatment; Hb was 12.6 g/dl and reticulocyte count was 13%. Paralleling the increase in conjugated bilirubin from hemolysis, the conjugated bilirubin fraction rose to 3.6 mg/dl on the postnatal 30th day. From the time of her rehospitalization, liver transaminases increased progressively and reached peak aspartate aminotransferase (AST) of 105 U/L (normal ranges, 1 to 32 U/L) and alanine aminotransferase (ALT) of 142 U/L (normal ranges, 1 to 31 U/L) on the 40th day. The tests for final and differential diagnosis of conjugated bilirubinemia were done in order to determine the underlying pathology. TANDEM mass spectrometry, urine-blood amino acid chromatography, sweat test, and _1-antitrypsin test were normal. Serological test results for TORCH, hepatitis A, B, and C, Epstein-Barr virus, and parvovirus were negative. The ferritin level was found to be 4620 ng/ml on day 40. A liver biopsy performed at 7 weeks of age showed histological features consistent with iron overload with hepatocytes with hemosiderin pigment, fibrosis of the portal tracts, and canalicular cholestasis (Figure 2). Liver iron concentration was 6320 ug Fe/g dry weight (normal ranges 200 to 2400 ug Fe/g dry weight). After biopsy, she had to be transfused and both conjugated and unconjugated bilirubin levels increased (at the 60th day, unconjugated bilirubin: 17.9 mg/dl and conjugated bilirubin: 12.4 mg/dl). Ferritin levels also increased to 8842 ng/ml. The oral mucosa biopsy that was performed for the neonatal hemochromatosis differential diagnosis was normal. Deferoxamine (30 mg/kg/daily intravenous, 5 times per week) was started with these clinical and laboratory features. No side effect was noted during chelation therapy. On follow up, bilirubin levels decreased after the first week of iron chelation and normalized at the 10th week of iron chelation therapy (Figure 3). The serum ferritin level decreased to 684 ng/ml and chelation therapy was stopped (Figure 4). The infant was discharged from the hospital in good clinical condition. When she was six months old, physical examination findings and complete blood cell count were in normal ranges and the reticulocyte count was 3.2%. Her blood group was A Rh-positive and direct Coombs test was negative. Liver function tests showed a significant improvement with total bilirubin 0.4 mg/dl, conjugated bilirubin 0.2 mg/dl,

Turk J Hematol 2010; 27: 204-8

Figure 1. Need for erythrocyte transfusion was decreased with recombinant erythropoietin treatment

Figure 2A and 2B. Histological appearance of the liver biopsy. Note the brownish pigment in the hepatocytes in Figure A (H&E staining, x100) consistent with bluish-stained iron in Figure B (Prussia staining, x100)

Figure 3. The bilirubin level was normalized with chelation therapy

ALT 64 U/L, AST 45 U/L, and serum ferritin level 249 ng/ml. Liver biopsy was not repeated. Oral informed of consent was ontained from the patient.

Discussion Intrauterine transfusions (IUTs) have markedly decreased the risk of hydrops fetalis and stillbirth of infants with RHD, but it can be a rare cause of complications like hyporegenerative anemia and symp-

Turk J Hematol 2010; 27: 204-8

DemircioĂšlu et al. Rhesus hemolytic disease and transfusion related severe iron overload

Figure 4. Ferritin values were successfully decreased with chelation therapy

tomatic iron overload [1]. Hyporegenerative anemia is characterized by depressed erythropoiesis with reduction in circulating reticulocyte count and low or undetectable numbers of erythrocytes containing fetal hemoglobin. It is seen between 2 to 6 weeks after birth and the pathogenesis is still unclear. The possible explanations of anemia are intramedullary destruction of normoblasts with Rh antibodies and low erythropoietin levels due to suppression of the bone marrow by IUT [1,6,9]. These patients require erythrocyte transfusions in the postnatal period, and the use of rHuEPO may reduce the need for postnatal transfusions [10-13]. Even though rHuEPO administration is highly effective in many patients, treatment of hyporegenerative anemia with rHuEPO may be ineffective when anti-D titers are high [14]. Anemia of prematurity (our patient was born at 33 weeksâ&#x20AC;&#x2122; gestation) is a multifactorial disease associated with relatively low plasma erythropoietin levels and insufficient erythropoiesis. Other complicating variables including small circulating blood volume, iatrogenic blood loss, hemorrhage, hemolysis, and shortened red blood cell survival also contribute to anemia in premature newborns. It occurs in 1-3 months after birth, and is associated with hemoglobin levels below 7-10 g/dl. Traditionally, anemia of prematurity has been treated with frequent packed erythrocyte transfusions [15]. We evaluated our patient as late hyporegenerative anemia because of reticulocytopenia and significant erythroid hypoplasia. History of IUT secondary to RHD is an important point for the differential diagnosis of anemia of prematurity. Our patient showed good response to rHuEPO and required no further transfusions after the second week of therapy. Iron metabolism is dynamic in the fetal and neonatal periods. In the intrauterine period, iron is taken

207

up as transferrin from maternal plasma by the placental trophoblasts. Ferritin levels rise during pregnancy and iron load is much more than adults in the neonatal period. These iron depots progressively decrease to normal limits in the first year of life [1618]. Iron load decreases due to decreased placental iron transport in patients with intrauterine growth retardation, preeclampsia, hypertension, and diabetes mellitus, whereas it increases with delayed clamping of the umbilical cord, twin to twin transfusion and intrauterine hemolysis with RHD [3,5,16,17]. Iron deposits can be seen during non-alcoholic liver disease and chronic hepatitis C infection. Insulin resistance and the influence of iron metabolism can lead to iron deposits in these patients. In the case of insulin resistance, increased insulin and proinflammatory cytokines and decreased hemojevulin and ferroportin-1 from hepatocytes are attributed to iron deposits due to decreased iron excretion from hepatocytes. Furthermore, in chronic hepatitis C infection, decreased hepcidin also leads to iron deposits as well as insulin resistance [18,19]. In our patient, overload iron deposits could have caused the liver damage, or on the other hand, liver damage could have led to iron deposits via its effect on hepcidin, hemojevulin and ferroportin-1 metabolism. Furthermore, intrauterine hypoxia due to anemia caused by Rh immunization can contribute to increased serum ferritin levels and liver function tests. Hypoxia-induced inflammation can cause the increase in hepcidin and ferritin synthesis. Hepcidin is a peptide hormone that inhibits intestinal iron absorption, iron recycling by macrophages, and iron mobilization from hepatic stores, and synthesis of hepcidin is increased by iron loading [1,20,21]. The clinical course of RHD patients with excess iron load is variable; while most cases have no clinical findings, chelation therapy can be necessary in some cases with severe organ damage [22-25]. Ferritin and hepatic iron content are extremely variable in neonates such that there are no definite criteria for chelation therapy. Chelation therapy is recommended for patients with clinical and histopathologic evidence of iron overload, as in our patient [23,25,26]. Our patient had hyperferritinemia, cholestatic liver disease, and iron overload at liver biopsy. Chelation therapy resulted in rapid reduction in ferritin levels. In the literature, chelation therapy was required for 6-12 weeks [23,24]. In our case, chelation therapy was given for 10 weeks. In conclusion, in patients who have undergone IUT due to RHD, hyperferritinemia and late hypore-

208

Demircioùlu et al. Rhesus hemolytic disease and transfusion related severe iron overload

generative anemia should be kept in mind. Chelation therapy in cases with symptomatic hyperferritinemia and rHuEPO treatment in cases with severe hyporegenerative anemia should be considered. Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1. 2.

3. 4.

7. 8. 9. 10.

11.

12.

Greenough A. Rhesus disease: postnatal management and outcome. Eur J Pediatr 1999;158:689-93. Oepkes D, van Scheltema PA. Intrauterine fetal transfusions in the management of fetal anemia and fetal thrombocytopenia. Semin Fetal Neonatal Med 2007;12:432-8. Aygun C, Tekinalp G, Gurgey A. Increased fetal iron load in rhesus hemolytic disease. Pediatr Hematol Oncol 2004;21:329-33. Berger HM, Lindeman JH, van Zoeren-Grobben D, Houdkamp E, Schrijver J, Kanhai HH. Iron overload, free radical damage, and rhesus haemolytic disease. Lancet 1990;335:933-6. Nasrat HA, Nicolini U, Nicolaidis P, Letsky EA, Gau G, Rodeck CH. The effect of intrauterine intravascular blood transfusion on iron metabolism in fetuses with Rh alloimmunization. Obstet Gynecol 1991;77:558-62. Thorp JA, O'Connor T, Callenbach J, Cohen GR, Yeast JD, Ablin J, Plapp F. Hyporegenerative anemia associated with intrauterine transfusion in rhesus hemolytic disease. Am J Obstet Gynecol 1991;165:79-81. al-Alaiyan S, al Omran A. Late hyporegenerative anemia in neonates with rhesus hemolytic disease. J Perinat Med 1999;27:112-5. Ovali F. Late anaemia in Rh haemolytic disease. Arch Dis Child Fetal Neonatal Ed 2003;88:F444. Koenig JM, Ashton RD, De Vore GR, Christensen RD. Late hyporegenerative anemia in Rh hemolytic disease. J Pediatr 1989;115:315-8. Ohls RK, Wirkus PE, Christensen RD. Recombinant erythropoietin as treatment for the late hyporegenerative anemia of Rh hemolytic disease. Pediatrics 1992;90:678-80. Ovali F, Samanci N, Daùoùlu T. Management of late anemia in Rhesus hemolytic disease: use of recombinant human erythropoietin (a pilot study). Pediatr Res 1996;39:831-4. Zuppa AA, Maragliano G, Scapillati ME, Florio MG, Girlando P, Noia G, De Santis M, Cavaliere AF, Romagnoli

13.

14.

15. 16. 17. 18.

19.

20. 21. 22.

23.

24.

25.

26.

Turk J Hematol 2010; 27: 204-8

C, Tortorolo G. Recombinant erythropoietin in the prevention of late anaemia in intrauterine transfused neonates with Rh-haemolytic disease. Fetal Diagn Ther 1999;14:270-4. Nicaise C, Gire C, Casha P, d'Ercole C, Chau C, Palix C. Erythropoietin as treatment for late hyporegenerative anemia in neonates with Rh hemolytic disease after in utero exchange transfusion. Fetal Diagn Ther 2002;17:22-4. Pessler F, Hart D. Hyporegenerative anemia associated with Rh hemolytic disease: treatment failure of recombinant erythropoietin. J Pediatr Hematol Oncol 2002;24:689-93. Kling PJ, Winzerling JJ. Iron status and the treatment of the anemia of prematurity. Clin Perinatol 2002;29:283-94. Rao R, Georgieff MK. Neonatal iron nutrition. Semin Neonatol 2001;6:425-35. Rao R, Georgieff MK. Iron in fetal and neonatal nutrition. Semin Fetal Neonatal Med 2007;12:54-63. Lecube A, Hernández C, Simó R. Glucose abnormalities in non-alcoholic fatty liver disease and chronic hepatitis C virus infection: the role of iron overload. Diabetes Metab Res Rev 2009;25:403-10. Licata A, Nebbia ME, Cabibbo G, Iacono GL, Barbaria F, Brucato V, Alessi N, Porrovecchio S, Di Marco V, Craxì A, Cammà C. Hyperferritinemia is a risk factor for steatosis in chronic liver disease. World J Gastroenterol 2009;15:2132-8. Nicolas G, Viatte L, Bennoun M, Beaumont C, Kahn A, Vaulont S. Hepcidin, a new iron regulatory peptide. Blood Cells Mol Dis 2002;29:327-35. Robson KJ. Hepcidin and its role in iron absorption. Gut 2004;53:617-9. Carpani G, Marini F, Ghisoni L, Buscaglia M, Sinigaglia E, Moroni G. Red cell and plasma ferritin in a group of normal fetuses at different ages of gestation. Eur J Haematol 1992;49:260-2. Sreenan C, Idikio HA, Osiovich H. Successful chelation therapy in a case of neonatal iron overload following intravascular intrauterine transfusion. J Perinatol 2000;20:509-12. Yilmaz S, Duman N, Ozer E, Kavas N, Oren H, Demircioùlu F, Kumral A, Ozkan H, Irken G, Ozer E. A case of rhesus hemolytic disease with hemophagocytosis and severe iron overload due to multiple transfusions. J Pediatr Hematol Oncol 2006;28:290-2. Ng PC, Lam CW, Lee CH, To KF, Fok TF, Chan IH, Wong E. Hepatic iron storage in very low birthweight infants after multiple blood transfusions. Arch Dis Child Fetal Neonatal Ed 2001;84:F101-5. Faa G, Sciot R, Farci AM, Callea F, Ambu R, Congiu T, et al. Iron concentration and distribution in the newborn liver. Liver 1994;14:193-9.

Letter to the Editor

209

Idiopathic thrombocytopenic purpura with venous thrombosis: A case report Venöz trombozlu idiopatik trombositopenik purpura: Bir olgu sunumu

Gül úlhan, Selami K. Toprak, Neslihan And×ç, Sema Karakuü Department of Hematology, Baükent University School of Medicine, Ankara, Turkey

To the Editor, We report a case with idiopathic thrombocytopenic purpura (ITP) who had venous thrombosis. A 59-year-old man with ITP was referred to our hospital because of chest pain. We planned splenectomy after intravenous immunoglobulin (IVIG) treatment because he showed no response to corticosteroid. During IVIG treatment and after splenectomy, venous thrombosis occurred. He was found heterozygous for factor V Leiden, prothrombin (PT) 20210 G>A and methyltetrahydrofolate reductase (MTHFR) C677T. Idiopathic thrombocytopenic purpura (ITP) is an acquired autoimmune disorder characterized by thrombocytopenia and mucocutaneous bleeding and it may occur acutely, chronically or recurrently. While the acute form of the disease is seen in childhood, the chronic form is generally seen in adults. Splenectomy is recommended for patients who do not respond to primary therapy (glucocorticoids, IVIG and/or anti-D) and who have clinical symptoms of bleeding when there is no contraindication for surgery. Thromboembolic complications after splenectomy are described in 10% of patients with ITP and may be seen as portal vein thrombosis, pulmonary embolism or crural deep vein thrombosis [1].

A 59-year-old man was referred to our hospital because of chest pain. No peripheral lymphadenopathy or petechial or purpuric lesions were determined on his physical examination. Routine laboratory tests showed thrombocytopenia (4000/mm3) confirmed by peripheral blood smear. Etiologic tests were as follows: HBs Ag: (-), antiHBs: (-), antiHCV: (-), antiHIV: (-), antinuclear antibody (ANA): (-), anticardiolipin IgM: 3.1 (0-7) MPL U/ml, and anticardiolipin IgG: 3.2 (0-10) GPL U/ml. Abdominal ultrasonography showed no organomegaly or lymphadenopathy. Bone marrow aspiration smear analysis showed that development of myeloid and erythroid series was normal, both young and mature forms of megakaryocytes were seen, and counts were elevated. Platelet release was decreased. We accepted this case as ITP. Since his platelet count showed no response to oral prednisolone treatment, pulse steroid was administered at a dose of 1 g/day for 3 days, but again there was no response in platelet count, and splenectomy was planned. In the preoperative period just after IVIG treatment, pain and hyperemia associated with the venous catheter line developed. Upper extremity Doppler ultrasonography showed acute thrombosis in the left brachial and axillary veins. At this time, his

Address for Correspondence: M.D. Selami K. Toprak, 1. Cadde No: 117/14 P×nar Apt. Bahçelievler, 06490 Ankara, Turkey Phone: +90 532 656 02 06 E-mail: sktoprak@yahoo.com doi:10.5152/tjh.2010.31

210

Ăşlhan et al. Idiopathic thrombocytopenic purpura with venous thrombosis

thrombocyte count was 18,000/mm3. Although IVIG can rarely cause deep vein thrombosis, we studied all thrombophilic tests. Results of these tests were as follows: protein S: 82 (6-140%), protein C: 122 (70-140%), antithrombin III (ATIII): 139 (80-120), lupus anticoagulant: 34.2 (20-60), and fasting homocysteine: 10.36 (4.5-15) umol/L. At the one-month follow-up after splenectomy, he complained of swelling and pain on his right leg. Doppler ultrasonography showed acute-subacute thrombosis in the right popliteal and left saphenous veins. At this time, his platelet count was 230,000/mm3. He was found heterozygous for factor V Leiden, PT 20210 G>A and MTHFR C677T genes. We planned anticoagulation with lifelong warfarin. In patients presenting with thrombocytopenia, diagnosis of ITP is made by exclusion of conditions that may cause thrombocytopenia, which include: drug use (generally heparin, alcohol, quinine/quinidine, sulphonamides), bacterial infections, viral infections (HIV, hepatitis, cytomegalovirus, EpsteinBarr virus), rickettsia infections, mycoplasma infections, lymphoproliferative diseases (chronic lymphocytic leukemia, large granular lymphocytic leukemia, lymphoma), autoimmune diseases (especially systemic lupus erythematosus, antiphospholipid antibody (APA) syndrome), disseminated intravascular coagulopathy, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, preeclampsia/eclampsia, HELLP syndrome, gestational thrombocytopenia, and hypersplenism. Patients who develop thrombosis after presenting with thrombocytopenia should be investigated for APA syndrome. This syndrome is characterized by thrombosis in the arterial and venous systems and persistently high levels of antiphospholipid antibodies. APA syndrome is a multisystem disease that may be accompanied by thrombocytopenia and recurrent fetal losses. In our case, both anticardiolipin antibodies and lupus anticoagulant levels were normal at the time of diagnosis. Our patient was taking IVIG when the first thrombosis attack occurred. Especially thromboembolic events may be seen in patients with advanced age, diabetes mellitus, hypertension, and dyslipidemia or in patients given high doses of IVIG rapidly [2]. Thromboembolic events have been

Turk J Hematol 2010; 27: 209-10

reported in 10% of patients undergoing splenectomy for hematologic diseases. Causes of thrombosis seen in these patients have not been clarified yet. The reported etiologic factors are damage to the portal vein and mesenteric veins during operation, hypercoagulability (protein C, protein S, ATIII deficiency, etc.) and postsplenectomy thrombocytosis. In addition, according to some studies, hematologic disease and spleen size may play a role in thrombosis development. Postsplenectomy portal vein and mesenteric thrombosis is most often seen following splenectomy for myeloproliferative disorders and almost never after trauma [3-5]. Because our patient had thrombosis attacks in the upper and lower extremities, although he was thrombocytopenic when the first attack occurred, we investigated hereditary thrombophilia and found three hereditary thrombophilic risk factors. Thus, anticoagulant therapy was planned despite his advanced age. Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript. Oral informed consent was obtained from the patient.

References 1.

3. 4.

Mohren M, Markmann I, Dworschak U, Franke A, Maas C, Mewes S, Weiss G, Jentsch-Ullrich K. Thromboembolic complications after splenectomy for hematologic diseases. Am J Hematol 2004;76:143-7. Orbach H, Katz U, Sherer Y, Shoenfeld Y. Intravenous immunoglobulin: adverse effects and safe administration. Clin Rev Allergy Immunol 2005;29:173-84. Solson MM, Ilada PB, Alpergen KN. Portal vein thrombosis. Surg Endosc 2003;17:1322. Silberstein E, Smolikov A, Levi I. Portal and superior mesenteric vein thrombosis after splenectomy for idiopathic thrombocytopenic purpura. Harefuah 1999;136:364-6, 418. Valeri A, Venneri F, Presenti L, Nardi F, Grossi A, Borrelli D. Portal thrombosis. A rare complication of laparoscopic splenectomy. Surg Endosc 1998;12:1173-6.

Letter to the Editor

211

The prevalence of factor V 1691G-A mutation in Van region of Turkey Türkiye'de Van bölgesinde Faktör V 1691G-A mutasyonunun prevalans×

Sinan Akbayram1, Cihangir Akgün2, Murat Doùan2, Mehmet Selçuk Bektaü2, Ahmet Faik Öner1 1Department of Pediatric Hematology, Yüzüncü Y×l University Faculty of Medicine, Van, Turkey 2Department of Pediatrics, Yüzüncü Y×l University Faculty of Medicine, Van, Turkey

To the Editor, The prevalence of hereditary thrombotic risk factors shows marked ethnic and geographic variations. Hereditary disorders predisposing for thrombosis are not only antithrombin, protein C and protein S deficiencies, but also hereditary defects such as factor V 1691 G-A (Leiden) (FVL) and prothrombin 20210 G - A mutations [1,2]. FVL causes activated protein C resistance and is the most common thrombophilic mutation worldwide [3]. Guanine to adenine change leads to a replacement of glutamine with arginine at amino acid position 506. It is most prevalent among Caucasians but not found in the Japanese and Africans [1-3]. FVL frequency in the Turkish population has been reported from different parts of Turkey. Overall frequency was reported to be 8% [4]. FVL is believed to have originated somewhere close to Anatolia approximately 20,000 years ago, and the existence of mutation in this geography is expected to be high [5]. Urartu, one of the ancient civilizations in Anatolia, was an Iron Age kingdom, centered on Van Lake in Eastern Turkey. In the study of Dogan Alakoc et al. [5], 60 teeth from the excavations of Van

Yoncatepe fortress were subjected to DNA analysis. Of the total 60 samples, one male sample demonstrated a positive amplification result for FV gene and was found to be heterozygous for FVL. As there is no report on the prevalence of FVL in the population in the Van region of Turkey, we aimed to determine the prevalence in that province. A total of 100 healthy children were studied. The mean age of the 100 subjects was 9.08±5.94 years, and 49 (49%) were males and 51 (51%) females. A written consent was obtained from their parents. The frequency of FVL was determined as 9%. FVL frequency is about 3% to 10% in Europeans, but the FVL mutation is rare in East and/or Southeast Asian populations; however, it has been determined at high frequencies in Azerbaijan and Kyrgyzstan populations [6-9]. Wide variations in the frequency of FVL have been reported in different surveys carried out in many countries, depending on the geographical location and the ethnic background of the population. In Caucasian populations, the carrier frequency of FVL was reported to be 2–15% [4]. The highest prevalence rates were obtained in Mediterranean countries [3-7].

Address for Correspondence: Asst. Prof. Sinan Akbayram, Yüzüncü Y×l Üniversitesi T×p Fakültesi, Çocuk Servisi 65200 Van, Turkey Phone: +90 505 284 08 49 E-mail: drsinanakbayram@gmail.com doi:10.5152/tjh.2010.32

212

Akbayram et al. Factor V 1691G-A mutation

The rates from different regions of Turkey and our results showed similar frequencies. The high prevalence of the mutation in the Middle East leads to the possibility that the founders of the FVL mutation lived around this region [10]. Another study on population genetics of the mutation showed that during the Neolithic period, FVL in Europe had probably expanded from Turkey, Anatolia [6]. One more clue to this statement is the presence of FVL in Urartians [5]. In conclusion, according to the results of this study, FVL prevalence is quite high and not rare in the population of the Van region of Turkey. Acknowledgement We are grateful to Nejat Akar and Yonca Eùin (Ankara University, Pediatric Genetics Department) for the analysis of the samples. Informed consent was obtained from the patient. Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1.

Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma

Turk J Hematol 2010; 27: 211-2

3. 4. 5.

6. 7.

10.

PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64-7. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3v-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;88:3698-703. Rees DC, Cox M, Clegg JB. World distribution of factor V Leiden. Lancet 1995;346:1133-4. Akar N. Factor V 1691 G-A mutation distribution in a healthy Turkish population. Turk J Hematol 2009;26:9-11. Dogan Alakoc Y, Aka S, Egin Y, Akar N. Factor V Leiden in an Urartian, dating back to 1000 BC. Clin Appl Thromb Hemost 2009 Jul 17. [Epub ahead of print] Lucotte G, Mercier G. Population genetics of factor V Leiden in Europe. Blood Cells Mol Dis 2001;27:362-7. Herrmann FH, Koesling M, Schröder W, Altman R, Jiménez Bonilla R, Lopaciuk S, Perez-Requejo JL, Singh JR. Prevalence of factor V Leiden mutation in various populations. Genet Epidemiol 1997;14:403-11. Gurgey A, Rustemov R, Parlak H, Balta G. Prevalence of factor V Leiden and methylenetetrahydrofolate reductase C677T mutations in Azerbaijan. Thromb Haemost 1998;80:520-1. Gurgey A, Kudayarov DK, Tuncer M, Parlak H, Altay C. The factor V Leiden and prothrombin G20210A mutations in Kirghiz population. Thromb Haemost 2000;84:356. Akar N, Akar E, Dalgin G, Sözüöz A, Omürlü K, Cin S. Frequency of factor V (1691 G --> A) mutation in Turkish population. Thromb Haemost 1997;78:1527-8.

Letter to the Editor

213

Portal vein thrombosis secondary to Klebsiella oxytoca bacteriemia Klebsiella oxytoca bakteriyemisine sekonder portal ven trombozu Ebru Uz1, Al×c× Özlem2, Özlem ûahin Balç×k3, Mehmet Kanbay1, Ayüe Iü×k4, Burak Uz4, Arif Kaya4, Ali Koüar3 1Department of Nephrology, Fatih University School of Medicine, Ankara, Turkey 2Department of Infectious Diseases, Fatih University School of Medicine, Ankara, Turkey 3Department of Hematology, Fatih University School of Medicine, Ankara, Turkey 4Department of Internal Medicine, Fatih University School of Medicine, Ankara, Turkey

To the Editor, Pylephlebitis, also called septic thrombophlebitis of the portal vein, is a life-threatening complication of intra-abdominal infection. It usually develops secondary to infection in the drainage area of the portal venous system [1,2]. Klebsiella spp are opportunistic pathogens that cause a wide spectrum of severe diseases such as septicemia, pneumonia, urinary tract infection, and soft tissue infection. Klebsiella oxytoca (K. oxytoca) has been isolated from human clinical specimens to a much lesser degree when compared to the other members of the genus Klebsiella [3]. Herein, we report a rare case of pylephlebitis secondary to K. oxytoca bacteriemia related with urinary infection, without any other pathology. A 54-year-old male patient was hospitalized with hyperpyrexia, chills, general weakness, and dysuria for one week. He had been taking ampicillin-sulbactam orally for five days for a urinary system infection. The other medical history included essen-

tial hypertension and coronary artery disease. Family history was unremarkable. On examination, he appeared unwell, body temperature was 39.5°C, blood pressure 120/70 mmHg, and pulse regular at 100/min. His liver was palpable 3 cm below the costal margin without tenderness. There was no jaundice, ascites or stigmata of chronic liver disease. No other abnormalities could be found. Urea nitrogen and creatinine, partial thromboplastin time and prothrombin time were all within normal limits. Chest radiograph showed no abnormalities. There were 10-12/hpf (high power field) white blood cells, 3/hpf red blood cells and no bacteria in the urinalysis. Other laboratory test results are shown in Table 1. After blood and urine cultures were performed, intravenous (i.v.) ampicillin-sulbactam 4 g/d was started. On day 3, K. oxytoca was isolated in the blood culture of the sample drawn at admission. In the antibiotic sensitivity testing, the microorganism was sensitive to ampicillin-sulbactam, so his treatment was continued. Urine culture was negative.

Address for Correspondence: M.D. Özlem ûahin Balç×k, Dizgi Sok. 9/6 Bas×nevleri, 06120 Ankara, Turkey Phone: +90 312 321 66 97 E-mail: drozlembalcik@yahoo.com doi:10.5152/tjh.2010.33

214

Uz et al. Portal vein thrombosis secondary to bacteriemia

Table 1. Laboratory findings

WBC (x109/L)

Lab. Findings

Normal Range

10.9

(4.4-11.3)

Neutrophil (%)

(37-73)

Platelet (x109/L)

363

(150-450)

Hb (g/dl)

12.9

(14-17.5)

CRP (mg/L)

314

(0-5)

AST (U/L)

(10-34)

ALT (U/L)

(7-33)

ALP (U/L)

405

(15-250)

GGT (U/L)

244

(8-61)

T. bilirubin (mg/dl)

3.03

0.2-1.3

HBsAg, Anti-HIV, Anti-HCV

(-)

Anti CMV Ig M (IU/ml)

(-)

Anti-EBV (VCA, EBNA) Ig M (IU/ml)

(-)

Anti-CMV Ig M (AU/ml)

(-)

ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CMV: Cytomegalovirus; CRP: C-reactive protein; EBV: Epstein-Barr virus; GGT: Gamma-glutamyl transpeptidase; HCV: Hepatitis C virus; HIV: Human immunodeficiency virus

On day 10, the patient had persistent fever, and echocardiography was performed, but no vegetation was shown. Abdominal ultrasound scan showed hepatomegaly, but a normal biliary tree with no focal liver abnormality and no evidence of intra-abdominal abscess. Due to worsening of his symptoms, gentamicin was added. Five days later, the patient deteriorated; ampicillin-sulbactam and gentamicin were stopped and meropenem (1 g i.v. every 8 hours) was started. Computed tomography (CT) scan of the abdomen, pelvis and thorax was performed. Although thorax CT appeared normal, abdominal CT revealed complete thrombosis of the main stem of the portal vein with a total occlusion, which was confirmed by color Doppler ultrasound. Small bowel and colon were normal in appearance, and there was no evidence of appendicitis or diverticulitis in the pelvis. Endoscopic study of the gastrointestinal tract showed no tumor, varices or diverticulum. Coagulation tests including for lupus anticoagulant, dysfibrinogenemia, resistance to activated protein C, deficiencies of protein S, C or antithrombin, and mutations of factor V were all detected as normal. Antiphospholipid and anticardiolipin antibodies were negative and the plasma homocysteine level was normal (normal value: 5-14 Â&#x2014;mol/L). The patient was treated with meropenem for two weeks and heparin for 10 days (continuous

Turk J Hematol 2010; 27: 213-5

infusion of 25,000 IU/24 h after a bolus of 5000 IU), followed by oral anticoagulation. Within 10 days, duplex ultrasound showed partial recanalization of the thrombus. The fever did not recur. The patient made a complete recovery and was discharged home on day 26, at which time liver enzymes and C-reactive protein had normalized; he had been afebrile for three days. Two months later, follow-up Doppler ultrasound showed complete recanalization of the thrombus. He remained asymptomatic after discharge, with no abnormal findings on clinical examination, and warfarin was discontinued six months after diagnosis. Portal vein thrombosis (PVT) is an infrequent but serious complication occurring in several diseases such as abdominal malignancy, infections or after surgical intervention. It is a complex situation that is associated with several risk factors including mostly acquired and inherited local precipitating factors. Approximately 8%-15% of cases are reported as idiopathic [4-6]. Intra-abdominal inflammatory conditions include bowel inflammatory disease, cholecystitis, pancreatitis, or intra-abdominal sepsis [7]. Our patient did not have an intra-abdominal infection but he had urinary infection complicated with bacteriemia. To the best of our knowledge, this is only the second case of pylephlebitis secondary to urinary infection in the literature; however, this is the first report in association with K. oxytoca bacteriemia. Clinical manifestations of PVT have a wide spectrum ranging from no symptoms to acute massive hematemesis [8]. Our patient had a symptomless PVT, most probably of recent origin. As there are no specific symptoms of PVT, imaging studies should be performed to confirm the presence of thrombus if PVT is suspected. Imaging studies include color Doppler-ultrasonography (CDU), CT, magnetic resonance imaging (MRI), and angiography. Fresh thrombus can go undetected by sonography because of the low echogenicity but can be recognized by (CDU) [9]. However, there were no pathologic findings in our patientâ&#x20AC;&#x2122;s abdominal ultrasound, while his abdominal CT revealed complete thrombosis of the main stem of the portal vein, which was confirmed by (CDU). Spontaneous repermeation of PVT is possible but uncommon. Anticoagulation is recommended in acute PVT as complete or partial repermeation can be achieved in up to 80% of patients. The reported recurrence rate of PVT ranges from 6% to 40%, so some researchers recommend anticoagu-

Uz et al. Portal vein thrombosis secondary to bacteriemia

Turk J Hematol 2010; 27: 213-5

lant therapy be continued for at least six months [10]. No widely-accepted guidelines for anticoagulant therapy are available. To prevent portal hypertension with long-term complications and to accelerate resolution of the thrombus, our patient was treated with anticoagulants. In summary, although rare, pylephlebitis remains a less-recognized entity with a high rate of mortality. We report a patient with urinary infection of K. oxytoca complicated by PVT. We emphasize the importance of clinical suspicion and early radiology for an early diagnosis of this entity, which makes it possible to establish an efficient treatment. Eradication of infectious foci and judicious administration of antimicrobials are essential to reduce the catastrophic morbidity and mortality of pylephlebitis. Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1.

Chirinos JA, Garcia J, Alcaide ML, Toledo G, Baracco GJ, Lichtstein DM. Septic thrombophlebitis: diagnosis and management. Am J Cardiovasc Drugs 2006;6:9-14.

2. 3.

4. 5.

10.

215

Wang JT, Zhao HY, Liu YL. Portal vein thrombosis. Hepatobiliary Pancreat Dis Int 2005;4:515-8. Kim BN, Ryu J, Kim YS, Woo JH. Retrospective analysis of clinical and microbiological aspects of Klebsiella oxytoca bacteremia over a 10-year period. Eur J Clin Microbiol Infect Dis 2002;21:419-26. Walker AP. Portal vein thrombosis: what is the role of genetics? Eur J Gastroenterol Hepatol 2005;17:705-7. Amitrano L, Guardascione MA, Brancaccio V, Margaglione M, Manguso F, Iannaccone L, Grandone E, Balzano A. Risk factors and clinical presentation of portal vein thrombosis in patients with liver cirrhosis. J Hepatol 2004;40:736-41. Chu G, Farrell GC. Portal vein thrombosis associated with prolonged ingestion of oral contraceptive steroids. J Gastroenterol Hepatol 1993;8:390-3. Cheung DY, Kim JK, Jo DH, Oh HJ, Kim TH, Lee SY, Park SH, Han JY, Chung KW, Sun HS. A case of portal vein thrombosis associated with acute pancreatitis and cholangitis. Korean J Gastroenterol 2005;46:60-5. De Cleva R, Herman P, Saad WA, Pugliese V, Zilberstein B, Rodrigues JJ, Laudanna AA. Postoperative portal vein thrombosis in patients with hepatosplenic mansonic schistosomiasis: relationship with intraoperative portal pressure and flow. A prospective study. Hepatogastroenterology 2005;52:1529-33. Hidajat N, Stobbe H, Griesshaber V, Schroder RJ, Felix R. Portal vein thrombosis: etiology, diagnostic strategy, therapy and management. Vasa 2005;34:81-92. Condat B, Valla D. Portal vein thrombosis. Presse Med 2003;32:1460-5.

216

Letter to the Editor

Late onset of isovaleric acidemia presenting with bicytopenia Bisitopeni ile birliktelik gösteren geç baülang×çl× izovalerik asidemi Bar×ü Malbora1, Zekai Avc×1, Alev Hasanoùlu2, Füsun Alehan3, Nam×k Özbek1 1Department of Pediatric Hematology, Baükent University Faculty of Medicine, Ankara, Turkey 2Department of Pediatric Metabolism, Gazi University Faculty of Medicine, Ankara, Turkey 3Department of Pediatric Neurology, Baükent University Faculty of Medicine, Ankara, Turkey

To the Editor, Isovaleric academia (IVA) is an autosomal recessive inborn error of leucine metabolism caused by a deficiency of isovaleryl-CoA dehydrogenase. In the acute phase of this disease, thrombocytopenia, neutropenia or pancytopenia may be common features [1-4]. However, the physiopathology of the bone marrow suppression in this disease is still unclear. Here, we report the case of an IVA patient who presented with thrombocytopenia and neutropenia at the age of 31 months. A 31-month-old male patient had been admitted to our hospital with fever, vomiting, fatigue, and loss of appetite. There was no history of any drug use. On physical examination, the patient was lethargic. Vital signs were normal except for mild tachycardia and tachypnea. Skin turgor and tonus were found to be decreased. The results of a complete blood count revealed the following: white blood cells (WBC) count 1.4x109/L; absolute neutrophil count 0.92x109/L; hemoglobin 11 g/dl; and platelet count 57.5x109/L. The bone marrow aspiration showed normal maturation of three lineages without any hemophagocy-

tosis, megaloblastic cells, extramyeloid cells, fatty changes, or myelodysplasia. There were numerous necrotic cells. The number and the morphology of megakaryocytes were normal. In the aspiration smear, 18% myelocyte, 35% metamyelocyte, 11% polymorphonuclear leukocyte, 28% lymphocyte, 2% normoblast, 1% monocyte, 1% eosinophil, and 4% lymphoblast-like cells were present. Bone marrow flow cytometry did not reveal lymphoblasts. Biochemical analyses were within normal limits except for hypocalcemia, hyperammonemia, and mildly increased aspartate aminotransferase and lactate dehydrogenase. Serum C-reactive protein and erythrocyte sedimentation rate were in normal range. Ketonuria was determined. The results of blood, throat, urine, stool, and cerebrospinal fluid cultures were negative. Viral serology of the cerebrospinal fluid and serum were also negative. Serum Brucella agglutination and Salmonella agglutination were negative. Serum lactate and pyruvate levels were 4 mmol/L (normal range: 0.7-2.1 mmol/L), and 2.74 mg/dl (normal range: 0.3-1 mg/ dl), respectively. The urine organic acid examination showed that the excretions of isovaleryl glycine and methyl malonic acid were significantly

Address for Correspondence: M.D. Bar×ü Malbora, Baükent University Faculty of Medicine, Department of Pediatric Hematology, 6. Cadde, No: 72/3, Bahcelievler 06490 Ankara, Turkey Phone: +90 312 212 68 68 E-mail: barismalbora@gmail.com doi:10.5152/tjh.2010.34

Malbora et al. Hematologic problems in isovaleric acidemia

Turk J Hematol 2010; 27: 216-8

217

Figure 1. The hematologic parameters of the patient during isovaleric acidemia episode. A) WBC (white blood cell) and ANC (absolute neutrophil count), B) Platelet count

increased: 1299 mmol/mol creatinine (normal: 0 mmol/mol creatinine) and 42.7 mmol/mol creatinine (normal: 0 mmol/mol creatinine), respectively. Tandem mass spectrometry of spot serum, free carnitine and amino acid profiles were normal. There was an increase in the isovaleryl and 3-OH butyryl carnitine levels, whereas there was a decrease in the levels of acetyl, propionyl, and palmitoylcarnitine. Lymphocyte isovaleryl-CoA dehydrogenase activity was 0.02 nmol/min.mg [controls mean±SD: 1.51±0.31 nmol/min.mg]. We initiated antibiotic therapy and protein-poor diet. Carnitine and L-glycine were added to the treatment. During this period, his laboratory examination revealed WBC count of 0.64x109/L (absolute neutrophil count, 0.43x109/L); a hemoglobin level of 10.7 g/ dl; and a platelet count of 31.8x109/L. On the fifth day of antibiotics, his fever was controlled, and the WBC number and platelet count began to increase. His blood count was completely normal one week after the end of antibiotic treatment (Figure 1a, b). Hematologic problems can be seen in patients with inborn errors of branched-chain amino acid metabolism. Various cytopenias have been reported in IVA [5]. However, the physiopathology of myelosuppression in this disease is not well known. Some infections may also result in myelosuppression, but in our patient, we did not determine any serologic or microbiologic evidence of infection. There is also a report of a patient with IVA with promyelocytic myeloproliferative syndrome [6,7]. In patients with IVA, hematologic problems are usually reported in early infancy [1,2,6]. Compatible with this, no patients have been reported in the English literature with IVA diagnosed later in childhood with neutropenia, thrombocytopenia or pancytopenia.

In some patients, this disorder may be diagnosed as late as 5 years [8]. However, hematologic problems are not observed at that age. Another interesting finding in our patient was the lymphoblast-like cells observed in the bone marrow. We believe that the lymphoblast-like cells observed in our patient’s bone marrow were due to maturation arrest in the normal lymphoblastic series caused by IVA. Although both forms of IVA often occur in the first year of life, IVA may be observed later in childhood. Hematologic problems frequently observed in the acute form of the disease can be seen in the chronic intermittent form, as was the case in our patient. Thus, in such cytopenias, inborn errors of branched -chain amino acid metabolism, like IVA, should be kept in mind. Acknowledgement This study conforms to the principles outlined in the Declaration of Helsinki (1975) and later revisions, and was approved by Ethical Committee of Baskent University Faculty of Medicine, Ankara, Turkey. Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1. 2. 3.

Newman CG, Wilson BD, Callaghan P, Young L. Neonatal death associated with isovaleric acidaemia. Lancet 1967;2:439-42. Fischer AQ, Challa VR, Burton BK, McLean WT. Cerebellar hemorrhage complicating isovaleric acidemia: a case report. Neurology 1981;31:746-8. Hou JW, Wang TR. Isovaleric acidemia: report of one case. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi 1990;31:262-5.

218 4.

Malbora et al. Hematologic problems in isovaleric acidemia

Kelleher JF Jr, Yudkoff M, Hutchinson R, August CS, Cohn RM. The pancytopenia of isovaleric acidemia. Pediatrics 1980;65:1023-7. Guerra-Moreno J, Barrios N, Santiago-Borrero PJ. Severe neutropenia in an infant with methylmalonic acidemia. Bol Asoc Med P R 2003;95:17-20. Gilbert-Barness E, Barness LA. Isovaleric acidemia with promyelocytic myeloproliferative syndrome. Pediatr Dev Pathol 1999;2:286-91.

Turk J Hematol 2010; 27: 216-8

Hutchinson RJ, Bunnell K, Thoene JG. Suppression of granulopoietic progenitor cell proliferation by metabolites of the branched-chain amino acids. J Pediatr 1985;106:62-5. Berry GT. Inborn errors of carbohydrate, ammonia, amino acid, and organic acid metabolism. In: Ballard RA, Berry GT, editors. Averyâ&#x20AC;&#x2122;s Diseases of the Newborn. 8th ed. Philadelphia: Elsevier Saunders, 2005:227-57.

Letter to the Editor

219

Primary uterine lymphoma of the uterine cervix in advanced age úleri yaüta uterus serviksinde oluüan primer uterus lenfomas× M. Murat Naki1, Hasniye Çelik1, Oluü Api1, Nagehan Özdemir2, Sadullah Bulut1, Orhan Ünal1 1Department of Obstetrics and Gynecology, Dr. Lütfi K×rdar Kartal Research and Training Hospital, ústanbul, Turkey 2Department of Pathology, Dr. Lütfi K×rdar Kartal Research and Training Hospital, ústanbul, Turkey

To the Editor, Vaginal bleeding (70%), perineal discomfort (40%), and persistent vaginal discharge (20%) [1] are the most common clinical symptoms of primary malignant lymphoma of the uterine cervix, which is an extremely rare malignancy of the female genital tract occurring at a median of 40 years of age [2]. An 82-year-old parous woman presented with persistent vaginal discharge for one year despite several antifungal and antibiotic combination regimens. Her past history and systemic examination were unremarkable. Papanicolaou smear revealed normal findings. Detection of a 15x15 mm nodular lesion in chest X-ray was confirmed to be a 35x30 mm lesion in the middle lobe and a 20 mm lesion in the lower part of the right lung with thoracic computerized tomography (CT). Serum lactate dehydrogenase (S-LDH) level was 402 U/ml. Colposcopically directed punch biopsies showed inflammation and necrosis while punch biopsy of the bladder revealed chronic cystitis. Inflammation was confirmed on repeated punch and fine needle aspiration biopsies (FNAB). Magnetic resonance imaging (MRI) scan showed a 52x40x45 mm cervical mass in the cervix uteri. Despite the lack of histological evidence, cer-

vical malignancy was suspected based on clinical and radiological findings. Total abdominal hysterectomy and bilateral salpingo-oophorectomy were performed together with intraoperative frozen section procedure. The operative finding was a hard mass that infiltrated the uterus and cervix with pelvic wall extension. Macroscopic examination of the hysterectomy specimen revealed a cervical mass measuring 50x40x15 mm and pathological examination showed non-Hodgkin’s lymphoma (NHL) and diffuse large B cells positive for LCA and CD20 and negative for CD3 and cytokeratin (Figure 1). Lymph nodes and appendectomy specimens were negative for tumor metastasis. Bronchoscopic biopsy following thoracic CT revealed no sign of malignancy. The patient was classified as stage IE according to the Ann-Arbor classification and received three cycles of postsurgical chemotherapy with R-CHOP protocol. She remains under follow-up and is disease-free. Suspicion of cervical malignancy with respect to radiological findings in our case seems to be in accordance with past studies concerning the benefits of preoperative imaging studies in the diagnosis of cervical lymphomas [3,4]. Given that the patient was in stage IE with better prognosis [5] and

Address for Correspondence: M.D. M. Murat Naki, Cihat Saran Sok. Çaùdaü Apt. A Blok No: 11/3 Küçükyal×, 34841 ústanbul, Turkey Phone: +90 216 518 28 03 E-mail: mmuratnaki@yahoo.com doi:10.5152/tjh.2010.35

220

Naki et al. Primary lymphoma of the uterine cervix

Turk J Hematol 2010; 27: 219-20

in the management of primary uterine lymphoma in patients with advanced age. Acknowledgement Informed consent was obtained from the patient. Conflict of Interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1. Figure 1. CD20-positive immunoreactivity in neoplastic cells (X40)

was free of fertility-related surgical limitations, complete surgical resection followed by three cycles of chemotherapy according to R-CHOP protocol seemed to be quite effective in this case. In conclusion, while factors such as rarity of the disease, absence of specific gynecological as well as systemic symptoms and limited value of cervical smears are likely to complicate the diagnosis of primary uterine NHL, MRI findings seem to be directive in construction of a frozen section-based definitive diagnosis. Associated with good prognosis, complete surgical resection followed by chemotherapy according to R-CHOP protocol is beneficial

2. 3.

4. 5.

Garavaglia E, Taccagni G, Montoli S, Panacci N, Ponzoni M, Frigerio L, Mangili G. Primary stage I-IIE non-Hodgkin's lymphoma of uterine cervix and upper vagina: evidence for a conservative approach in a study on three patients. Gynecol Oncol 2005;97:214-8. Muntz HG, Ferry JA, Flynn D, Fuller AF, Tarraza HM. Stage IE primary malignant lymphomas of the uterine cervix. Cancer 1991;68:2023-32. Perren T, Farrant M, McCarthy K, Harper P, Witshaw E. Lymphomas of the cervix and upper vagina: a report of five cases and review of the literature. Gynecol Oncol 1992;44:87-95. Marin C, Seoane JM, Sanchez M, Ruiz Y, Garcia JA. Magnetic resonance imaging of primary lymphoma of the cervix. Eur Radiol 2002;12:1541-5. Hariprasad R, Kumar L, Bhatla DM, Kukreja M, Papaiah S. Primary uterine lymphoma: report of 2 cases and review of literature. Am J Obstet Gynecol 2006;195:308-13.

Images in Hematology

221

Leishman-Donovan (LD) bodies in bone marrow biopsy of an adult male with AIDS AIDS hastas× yetiükin erkeùin kemik iliùi biyopsisinde Leishmania Donovan (LD) cisimciùi Abbas Hashim Abdulsalam1, Fatin Al-Yassin2 1Hematology Unit, Lab Department, Al-Yarmouk Teaching Hospital, Baghdad, Iraq 2Department of Hematology, Teaching Laboratories, Medical City, Baghdad, Iraq

We report the case of Leishman-Donovan (LD) bodies in the bone marrow biopsy of an Iraqi adult male with acquired immunodeficiency syndrome (AIDS). The patient was working in a United States military camp in Baghdad, Iraq. He was diagnosed as having human immunodeficiency virus (HIV) infection within only a few days of diagnosis of visceral leishmaniasis (kala-azar). Visceral leishmani-

asis is one of the opportunistic infections in AIDS patients. The interesting point of this case is that the finding of LD bodies in the bone marrow biopsy is rare. In this case, the bone marrow aspirate was diluted, and this caused us to miss the diagnosis of visceral leishmaniasis. However, when we examined the biopsy slides, the diagnosis was clear. A

Figure 1. Bone marrow trephine biopsy, H&E stain X100, parrafin embedded: Patient with AIDS and Visceral leishmaniasis.

Figure 2. Bone marrow trephine biopsy, H&E stain X100, parrafin embedded: Many Leishman-Donovan bodies seen.

Address for Correspondence: M.D. Abbas Hashim Abdulsalam, Iraq-Baghdad-Al-Yarmouk Teaching Hospital 964 Baghdad, Iraq Phone: 964 7904 188690 E-mail: dr.abbas77@yahoo.com doi:10.5152/tjh.2010.36

222

Abdulsalam et al. LD bodies in bone marrow biopsy

Turk J Hematol 2010; 27: 221-2

Figure 3. Bone marrow trephine biopsy, H&E stain X100, parrafin embedded: Leishman-Donovan bodies, nucleus and paranuclear kinetoplast seen, few are with the typical "double-dot" appearance.

Figure 4. Bone marrow trephine biopsy, H&E stain X100, parrafin embedded: The tissue form of the protozoal pathogen producing Visceral leishmaniasis is the Leishman-Donovan body which is an intracellular amastigote.

retrospective reexamination of the aspirate slides revealed only a few LD bodies.

including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

Acknowledgement Informed consent was obtained from the patient. Conflict of Interest No author of this paper has a conflict of interest,

References 1.

Hoffbrand AV, Pettit JE. Color Atlas of Clinical Hematology. 3rd ed. St. Louis: Mosby, 2006.

Erratum

Please kindly be informed that institutional info of the authors and figure 1 in the article "Enhanced platelet adhesion in essential thrombocythemia after in vitro activation" published in volume 27

223

issue June 2010, Page 82-90 has been published incorrect. The corrected info and the figure 1 is given below. Hereby we are amending the incorrect info and offering our excuses for inconvenience.

Andreas C. Eriksson1, Kourosh Lotfi2, Per A. Whiss1 1Division of Drug Research/Pharmacology, Department of Medical and Health Sciences, Linköping University, Sweden 2Division of Drug Research/Clinical Pharmacology, Department of Medical and Health Sciences, Linköping University, Sweden

and Department of Hematology, University Hospital, Linköping, Sweden

Figure 1. Platelet adhesion to albumin (a), collagen (b), fibrinogen (c), and fibronectin (d) was significantly increased by ADP (1 and 10 mol/L) for both controls and ET-platelets. A dose-response relationship was seen on all surfaces for ET-platelets, but only on albumin and fibronectin for controls. Differences in basal adhesion (solvent) between the three groups were not significant. Data are presented as mean+SD. Filled bars = Controls (n = 11 except for fibronectin where n = 10, age range: 31-63 years), open bars = Age-matched patients (n = 14, age range: 30-66 years), patterned bars = Patients 67 years (n = 16, age range: 67-87 years). ns = not significant, **p<0.01, ***p<0.001

Advisory Board of This Issue (September 2010) Ahmet Öztürk, Turkey

Fatma Gümrük, Turkey

Mustafa Nuri Yenerel, Turkey

Akif Yeüilipek, Turkey

Feride Duru, Turkey

Ali Ünal, Turkey

Fevzi Altuntaü, Turkey

Alpay Azap, Turkey

Hale Ören, Turkey

Aytemiz Gürgey, Turkey

Hamdi Akan, Turkey

Betül Tavil, Turkey

Hayri Özsan, Turkey

Burhan Ferhanoùlu, Turkey

únci Alacac×oùlu, Turkey

Cengiz Beyan, Turkey

Kaan Kavakl×, Turkey

Duran Canatan, Turkey

Klara Dalva, Turkey

Ebru Koca, Turkey

Lale Olcay, Turkey

Elif Ünal, Turkey

Mehmet Ali Özcan, Turkey

Erol Erduran, Turkey

Mehmet Ertem, Turkey

Emre Tekgündüz, Turkey

Meliha Nalçac×, Turkey

Evren Özdemir, Turkey

Meral Beksaç, Turkey

Fahir Özkalemkaü, Turkey

Mine Hekimgil, Turkey

Fatih Demirkan, Turkey

Murat Akova, Turkey

Muzaffer Demir, Turkey Nam×k Özbek, Turkey Neüe Yaral×, Turkey Özcan Çeneli, Turkey Paul Imbach, USA Pervin Topçuoùlu, Turkey Reyhan Diz Küçükkaya, Turkey ûule Mine Bakanay, Turkey ûule Ünal, Turkey Tunç F×üg×n, Turkey Türkan Pat×roùlu, Turkey Türkiz Gürsel, Turkey Ülker Koçak, Turkey Yeüim Ayd×nok, Turkey Yusuf Baran, Turkey Zahit Bolaman, Turkey

Announcements 10-13 October 2010 The 33rd World Congress of the International Society of Hematology (ISH 2010) Jerusalem, Israel 21-23 October 2010 Lymphoma & Myeloma 2010: An International Congress on Hematologic Malignancies New York, NY, United States 3-6 November 2010 36. Turkish National Hematology Congress Antalya, Turkey

4-7 December 2010 52. ASH Annual Meeting and Exposition Orlanda, Florida, USA 11-14 May 2011 3. International Congress on Leukemia Lymphoma Myeloma, Ăşstanbul, Turkey

3 International Congress rd

Leukemia Lymphoma Myeloma 11-14 May 2011 Hilton Otel

Ista nbul, TURKEY

www.icllm2011.org

Turkish Society of Hematology

ress Sancak ahallesi Turan ne ul ar Sok ankaya nkara T hone a e th org tr mail secretary th org tr

Serenas Tourism Congress & Organisation ress Turan

Ca e

e o l ankaya T hone a serenas com tr mail gokce onay serenas com tr