tjh-2009-4

Issue 4

December 2009

40 TL

ISSN 1300-7777

Volume 26

TURKISH JOURNAL OF HEMATOLOGY • VOL.: 26 ISSUE: 4 December 2009

Crimean-Congo hemorrhagic fever Feride Duru et al.; Samsun, Turkey

Cap+1 mutation Muhammad Usman Abdul Karim et al.; Karachi, Pakistan

HIT in medical patients Muzaffer Demir et al.; Edirne, Turkey

Hyperbaric oxygen in hemorrhagic cystitis Mustafa Nuri Yenerel et al.; Istanbul, Turkey

Electromagnetic field effects in mice Mohamed Abd El-azim Hashem et al.; Zagazig, Egypt

Invasive fungal infections Birol Baytan et al.; Bursa, Turkey

Megaloblastic anemia and nitric oxide Mehmet Ali Erkurt et al.; Malatya, Konya, Turkey

Cover Picture : Ali UĂşur Ural, 2009 Father and Son

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Editor-in-Chief

International Review Board Nejat Akar (Turkey) Görgün Akpek (USA) Serhan Alkan (USA) Çiúdem Altay (Turkey) Ayhan Çavdar (Turkey) M.SÕraç Dilber (Sweden) Ahmet Doúan (USA) Jawed Fareed (USA) Gösta Gahrton (Sweden) Dieter Hoelzer (Germany) Marilyn Manco-Johnson (USA) Andreas Josting (Germany) Emin Kansu (Turkey)

Aytemiz Gürgey Associate Editors

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

Erich Frank Orhan Ulutin Hamdi Akan

Contact Information

Editorial Correspondence should be addressed to Prof.Dr.Aytemiz Gürgey Editor-in-Chief Hacettepe University Faculty of Medicine Pediatric Hematology Department 06100 SÕhhiye, Ankara/TURKEY Tel No : + 90 312 305 41 17 Fax No : + 90 312 305 41 17 E-mail : agurgey@hacettepe.edu.tr

Senior Advisory Board

Orhan Ulutin Yücel Tangün Osman ûlhan

Language Editor

Corinne Can

Statistic Editor

Mutlu Hayran

Turkish Society of Hematology

Editorial Secretary

ûpek Durusu

Cover Picture: Ali Uúur Ural was born in 1960,Ankara, Turkey.

He is working at Gülhane Military Hospital Hematology Department. He is the first prize winner of the “35. Turkish National Hematology Congress Competition of Photography” in October 2009.

Muhit Özcan, President Mutlu Arat, General Secretary Hale Ören, Vice President Muzaffer Demir, Research Secretary Teoman Soysal, Treasurer Fahir Özkalemkaý, Member Mehmet Sönmez, Member Türk Hematoloji Derneúi, 07.10.2008 tarihli 6 no’lu kararÕ ile Turkish Journal of Hematology’nin Türk Hematoloji Derneúi ûkdisadi ûýletmesi tarafÕndan yayÕnlanmasÕna karar vermiýtir.

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) All other inquiries should be adressed to

TURKISH JOURNAL OF HEMATOLOGY Sancak Mahallesi , Turan Güneý BulvarÕ 613. Sk. No:8 06550 Çankaya, Ankara/Turkey Tel. : +90 312 490 98 97 Fax : +90 312 490 98 68 E-mail: info@tjh.com.tr ISSN: 1300-7777

Web page

www.tjh.com.tr

Sahibi

Türk Hematoloji Derneúi adÕna Muhit Özcan

Sorumlu YazÕ ûýleri Müdürü Aytemiz Gürgey

YayÕn ve Yönetim Yeri Türk Hematoloji Derneúi Türk OcaúÕ Cad. 17/6 Caúaloúlu-Eminönü-ûstanbul Üç ayda bir yayÕnlanan yerel bilimsel dergidir.

Publisher: Aves YayÕncÕlÕk Address: KÕzÕlelma cad. 5/3 34096 FÕndÕkzade-ûstanbul-Turkey Phone: +90 212 589 00 53 Fax: +90 212 589 00 94 E-mail: info@avesyayincilik.com Place of printing: Özgün Ofset Tic.Ltd.üti.

Date of printing: December 2009 Broadcast as: Local periodical

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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 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 - EBSCO - DOAJ - 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: Sancak mah. Turan Güne¿ 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: press@thd.org.tr Permissions Requests for permission to reproduce published material should be sent to the editorial office.

Editor: Prof.Dr. Aytemiz Gürgey Adress: Sancak mah. Turan Güne¿ Bulvar› 613. sok. No: 8 Çankaya-Ankara, Turkey Telephone: +90 312 490 98 97 Fax: +90 312 490 98 68 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.

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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.

Original research articles Regular Articles Maximum length for a Regular Article is 4,000 words of text. Abstracts must not exceed 150 words and should be a single psaragraph with no subheadings.

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 format of the articles should comply with the Uniform Requirements for Manuscripts Submitted to Biomedical Journals: Writing and Editing for Biomedical Publication (http://www.icmje.org) 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 150 words.

Review Articles Review articles should not exceed 4,000 words in length, must include an abstract of 150 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.

Images in Haematology 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.

Preparation of Manuscript 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

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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.

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 150 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, 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.

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.

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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, fonts and other formats are given in Table 1.

Table 1.

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

Supported fonts Arial Times Helvetica Times New Roman Courier

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 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 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.

Supported images .bmp .jpg .jpeg .gif .tif

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 161

Hematological aspects of Crimean-Congo hemorrhagic fever Feride Duru, Tunç FÕýgÕn, Samsun, Turkey

Research Articles 167

Cap +1 mutation; an unsuspected cause of beta thalassaemia transmission in Pakistan

171

Incidence of anti-heparin/platelet factor 4 antibodies and heparin-induced thrombocytopenia in medical patients

176

Hyperbaric oxygen: an important treatment modality in severe hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation

Muhammad Usman Abdul Karim, Moinuddin Moinuddin, Sadia Usman Babar, Karachi, Pakistan

Muzaffer Demir, Emre Tekgündüz, Mustafa Edis, Enver Duran, Turan Kürüm, Ömer YiúitbaýÕ, Mahmut Yüksel, Edirne, Turkey

Mustafa Nuri Yenerel, Sevgi Kalayoúlu-BeýÕýÕk, Oktay Perdeci, Nuray Gürses, Murat Tunç, Deniz SargÕn, ûstanbul, Turkey

181

The effects of low electromagnetic field and lead acetate combination on some hemato-biochemical and immunotoxicological parameters in mice Mohamed Abd El-azim Hashem, Nabela Imam El-sharkawy, Sharkia Province, Egypt

190

Invasive fungal diseases in children with hematologic disorders

197

The levels of nitric oxide in megaloblastic anemia

Birol Baytan, Adalet Meral Güneý, Solmaz Çelebi, Ünsal Günay, Bursa, Turkey Mehmet Ali Erkurt, ûsmet Aydoúdu, Nihayet Bayraktar, ûrfan Kuku, Emin Kaya, Malatya, Konya, Turkey

Case Reports 201

Simultaneous occurrence of Kaposi’s sarcoma and nodular lymphocyte predominant subtype of Hodgkin’s lymphoma in the same lymph node Duygu Kankaya, Gülýah Kaygusuz, IýÕnsu Kuzu, Berna Savaý, üule Mine Bakanay, Muhit Özcan, Ankara, Turkey

204

Acute massive myelofibrosis with acute lymphoblastic leukemia

207

Intestinal mucus accumulation in a child with acute myeloblastic leukemia

Zekai AvcÕ, BarÕý Malbora, Meltem Gülýan, Feride ûffet üahin, Bülent Celasun, NamÕk Özbek, Ankara, Turkey BarÕý Malbora, Zekai AvcÕ, Deniz Anuk ûnce, Ünser ArÕkan, NamÕk Özbek, Ankara, Turkey

Images in Hematology 210

Recurrent bruising of lower eyelids: a presenting symptom of neuroblastoma in a child Mustafa BüyükavcÕ, Zühal Keskin YÕldÕrÕm, Erzurum, Turkey

Letters to the Editor 211

Treatment of primary myelofibrosis

213

Megadose methylprednisolone for granulocytic sarcoma

214

Premarital screening in Muúla Region of Turkey

216

Chemotherapeutic trial for acute leukemia in Iraq

üinasi Özsoylu, Ankara, Turkey üinasi Özsoylu, Ankara, Turkey

Sibel Özdemir, ûsmail HakkÕ Timur, ûskender Gencer, Nejat Akar, Muúla, Ankara, Turkey Abbas Hashim Abdulsalam, Baghdad,Iraq

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Review

161

Hematological aspects of Crimean-Congo hemorrhagic fever KÕrÕm-Kongo kanamalÕ ateýinin hematolojik yönleri

Feride Duru, Tunç FÕýgÕn

Department of Pediatric Hematology, Ondokuz MayÕs University, Faculty of Medicine, Samsun, Turkey

Abstract Crimean-Congo hemorrhagic fever (CCHF) is an acute tick-borne viral disease transmitted to humans by Hyalomma ticks or by direct contact with the blood of infected humans or domestic animals. In certain areas of the world, including Africa, Asia, South East Europe and Middle East, sporadic cases or outbreaks of CCHF have been reported. During the last sixyear period from 2003 to 2009, CCHF has also occurred endemically in Turkey, particularly during spring and summer, with a case-fatality rate of approximately 5%. The disease is characterized by acute fever, nausea, vomiting, headache, myalgia, elevated liver enzymes and hemorrhagic manifestations ranging from mucocutaneous bleeding to life-threatening massive hemorrhage with disseminated intravascular coagulation (DIC) and hemophagocytosis. As with other viral hemorrhagic diseases, activation of lymphocytes, monocytes, macrophages and oversecretion of cytokines play a pivotal role in the pathogenesis and prognosis of CCHF. Recently an increasing number of publications on CCHF have been emerging in the literature, majority of which have been written by infection specialists. In this article, recent literature on CCHF has been reviewed, with particular emphasis on hematological manifestations, pathogenesis and therapeutic approaches in CCHF from the hematologist’s point of view. (Turk J Hematol 2009; 26: 161-6) Key words: Crimean-Congo hemorrhagic fever, hematological manifestation, hemophagocytosis Received: May 11, 2009

Accepted: August 4, 2009

Özet KÕrÕm-Kongo kanamalÕ ateýi (KKKA) insanlara Hiyalom keneleri aracÕlÕúÕyla ya da enfekte veya evcil hayvanlarÕn kanlarÕna doúrudan temas aracÕlÕúÕyla insanlara geçen akut bir kene yoluyla bulaýan viral hastalÕktÕr. Afrika, Asya, Güney Doúu Avrupa ve Orta Doúu da dahil dünyanÕn belirli bölgelerinde KKKA’ya ait sporadik vakalar yada salgÕnlar rapor edilmiýtir. 2003’ten 2009 yÕlÕna kadar geçen altÕ yÕllÕk süre boyunca özellikle bahar ve yaz aylarÕnda yaklaýÕk %5’lik bir vaka-ölüm oranÕyla endemik olarak Türkiye'de de görülmüýtür. HastalÕk akut ateý, bulantÕ, kusma, baý aúrÕsÕ, miyalji, karaciúer enzimlerinin yükselmesi ve mukokütanöz kanamadan yaýamÕ tehdit eden yaygÕn damariçi intravasküler koagülasyon (DIC) ve hemofagositozla birlikte masif kanamalara kadar bir yelpazede hemorajik manifestasyonlar ile karakterizedir. Diúer viral hemorajik hastalÕklarda olduúu gibi, lenfositlerin, monositlerin, makrofajlarÕn aktivasyonu ve sitokinlerin aýÕrÕ sekresyonu KKKA’nÕn patojenez ve prognozunda kilit bir rol oynar. Son zamanlarda literatürde KKKA ile ilgili olarak daha çok enfeksiyon uzmanlarÕ Address for Correspondence: Prof.Dr. Feride Duru, Ondokuz MayÕs University Faculty of Medicine, Department of Pediatric Hematology, Kurupelit 55200 Samsun, Turkey Phone: +90 362 312 19 19 / 30 99 E-mail: ferideduru@superonline.com

162

Duru et al.

Crimean-Congo hemorrhagic fever

Turk J Hematol 2009; 26: 161-6

tarafÕndan yazÕlan yayÕnlarda artma olduúu görülmektedir. Bu makalede hematologun bakÕý açÕsÕyla özellikle KKKA’daki hematolojik belirtiler, patojenez ve tedavi yaklaýÕmlarÕ üzerine vurgu yapÕlarak KKKA hakkÕnda yayÕnlar gözden geçirilmiýtir.

(Turk J Hematol 2009; 26: 161-6) Anahtar kelimeler: KÕrÕm-Kongo hemarojik ateýi, hematolojik belirtiler, hemafagositoz Geliý tarihi: 11 MayÕs 2009

Kabul tarihi: 4 Aúustos 2009

Epidemiology

Crimean-Congo hemorrhagic fever (CCHF) is an acute tickborne viral disease that is transmitted to humans by Hyalomma ticks or by direct contact with the blood of infected humans or domestic animals. Nosocomial spread of infection mostly among healthcare personnel has also been reported. The causative virus is a member of the Nairovirus genus of the family Bunyaviridae and has a viral envelope [1-3]. Sporadic or endemic occurrence of the disease has been reported in some regions of Africa, Asia, southeastern Europe, and the Middle East. The estimated fatality rate is between 5% and 50%, reaching up to 72.2% in an outbreak in the United Arab Emirates [4-7]. Turkey is one of the countries where CCHF has occurred endemically in the spring and summer seasons of the past six years. The majority of cases were from the central northern and eastern regions of Turkey [8-15]. Between 2002 and 2007, 1820 cases with a confirmed diagnosis by enzymelinked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) were identified, and 92 of these died of the disease [8]. According to the most recent data published by the Turkish Ministry of Health for the period between 2002 and 2008, a total of 3135 cases were diagnosed with CCHF, and 155 (5%) of these died [9,10].

As its name implies, CCHF virus often results in severe hemorrhagic disease in humans, although not all patients develop the classic form of hemorrhagic disease. Viral load, acquisition route, and nature of host defense may play a major role in disease presentation. Nosocomial spread of infection carries higher risk than exposure through tick bite [5,6,16,17].

Clinical and Laboratory Findings

Clinically, CCHF consists of four phases, i.e. incubation, prehemorrhagic, hemorrhagic, and convalescence phases (Figure 1). The duration of the incubation period usually depends on the route of acquisition and extent of virus contamination. Following an incubation period of 3-7 days, the prehemorrhagic phase starts with acute-onset fever and nonspecific symptoms such as headache, photophobia, back and abdominal pain, myalgia, arthralgia, nausea, vomiting, and diarrhea. Fever generally subsides within 4-5 days but may persist up to 12 days. The hemorrhagic phase usually begins on the 4th-6th days of the disease, and during this phase, patients show signs of hemorrhagic diathesis ranging from mucocutaneous bleeding to fatal massive hemorrhage and disseminated intravascular coagulation (DIC). Bleeding symptoms may occur in 34 to 90% of the patients with CCHF. In addition to massive hemorrhage and hepatorenal failure, myocardial infarction, cardiorespiratory arrest and central nervous system dysfunction may develop with a fatal outcome.

Laboratory findings of the disease include cytopenias (anemia

and/or leukopenia and/or thrombocytopenia); elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatinine phosphokinase (CPK), ferritin, bilirubin and fibrin degradation products; prolonged activated partial thromboplastin time (aPTT) and prothrombin time (PT); and decreased fibrinogen. It is noteworthy to mention that fibrinogen levels do not decrease in all cases since it is an acute phase reactant. For surviving patients, the convalescence period begins 15-20 days after the onset of disease. Weakness, weak pulse, sweating, dizziness and poor appetite have been reported in this period [2,5,6,12-18]. Hair, hearing or memory loss has also been reported in some patients [2,5,6]. Clinical and laboratory findings of CCHF are shown in Table 1.

Hematological Manifestations of CCHF Virus Infection

Epistaxis, gingival bleeding, petechiae, large ecchymoses, hematemesis, melena, hemoptysis with alveolar hemorrhage, hematuria, vaginal bleeding, and bleeding from venipuncture sites may occur due to thrombocytopenia and prolonged PT and aPTT in the hemorrhagic phase of CCHF [2,5,6,18-20].

Ocular findings with subconjunctival and retinal hemorrhage and without visual symptoms have also been reported in 73.7% of 19 patients [21]. Remarkably, a single patient was operated on the suspicion of acute appendicitis, but during the operation, hemorrhages in the oblique muscles and cecum were identified without any evidence of appendiceal pathology [22]. In severe cases with massive hemorrhage, DIC, intracranial bleeding, massive liver necrosis, and irreversible shock may develop, ultimately leading to death. The reported frequencies of hemorrhagic symptoms in fatal cases were 85% and 81.8% in the studies by Mardani et al. and Cevik et al., respectively [18,23]. Particularly, bleeding in the gastrointestinal and central

Figure 1. Clinical Stages of CCHF (Ergönül et al.)

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Table 1. Clinical and laboratory findings of CCHF (1,5,6,12,14,1825,28-30,37) Clinical Findings

Laboratory Findings

Cardiovascular System

Biochemical

Bradycardia

Elevated AST, ALT

Low blood pressure

Elevated lactate dehydrogenase

Heart murmur

Elevated creatine kinase

Myocardial infarction

Hyperferritinemia

Cardiorespiratory arrest

Hypertriglyceridemia

Respiratory System

Hematological

Cough, rales, tachypnea

Anemia

Pleural effusion

Leukopenia, leukocytosis

Acute respiratory distress syndrome

Thrombocytopenia

Diffuse alveolar hemorrhage

Prolonged PT, aPTT Decreased fibrinogen Elevated D-dimer

Skin Jaundice, hair loss

Immunological Increased levels of cytokines (TNF_, IL-1,IL-6, IL-10) Increased serum neopterin Increased absolute number of peripheral blood natural killer cells

Gastrointestinal System Hepatomegaly, splenomegaly Abdominal tenderness Central Nervous System Polyneuritis, headache Somnolence, lethargy Loss of hearing, poor vision Loss of memory Hematological Findings and Bleeding Symptoms: Petechia, ecchymoses Hematemesis, melena Gingival bleeding, epistaxis Bloody otorrhea Subconjunctival and/or retinal hemorrhage Hematuria Vaginal bleeding Hemoptysis Diffuse alveolar hemorrhage Intracerebral hemorrhage Hemophagocytosis

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nervous system is associated with a poor prognosis [23,24].

Hepatosplenomegaly was detected in approximately one-third of the patients with CCHF [2,3,5]. Hemophagocytosis was first reported in the study by Karti et al. [12] from Turkey, where half of the patients had this condition. Subsequently, other investigators also reported this important finding in CCHF [25,26], which is now considered to be associated with the development of cytopenia and poor prognosis [12,25]. In our view, routine use of bone marrow aspiration might yield a higher frequency of hemophagocytosis than the one reported in patients with CCHF. Cevik et al. [23] reported melena and hematemesis as the most common bleeding symptoms among fatal cases, with a particular emphasis on prolonged PTT (>60 sec) and low platelet count (<20000/mm3) as the most important risk factors for mortality. In addition, profound thrombocytopenia has been recognized as a poor prognostic sign by other investigators [2,5,23,24,27,28], and Tasdelen Fisgin et al. [27] found that 42% of patients with CCHF required transfusion of platelet suspensions. In a recent study by Ergonul et al. [24], hematemesis, melena, somnolence, low platelet count, prolonged PT and aPTT, and higher AST, ALT and fibrinogen levels were significantly more common among fatal cases compared to survivors. Similarly, Joubert et al. [16] found higher DIC scores in fatal cases. Decreased activity of thrombin- activatable fibrinolysis inhibitor (TAFI) was present in patients with CCHF and was attributed to the imbalance in fibrinolysis and DIC [29]. In another study from Turkey, increased natural killer (NK) cell counts were observed in CCHF patients, and this finding was correlated with abnormalities in AST/ALT and aPTT [30]. In another study, cytotoxic T lymphocyte (CD8) counts were increased [31].

Pathogenesis

The basic pathogenesis of CCHF at the molecular level is complex and not well defined. Endothelial cells, immune response, viral load, and coagulation cascade play important roles in the disease pathogenesis. Blood and endothelium appear to be the target tissues of the disease. Marked viral replication results in severe viremia during the first two phases of the disease. On the other hand, inappropriately activated T helper 1 (CD4 Th1) lymphocytes produce tumor necrosis factor-_ (TNF-_) and interferon gamma (IFNa), which promote macrophage co-activation and oversecretion of interleukin-1 (IL1) and IL-6. The endothelium is directly infected by the virus and/ or damaged by secreted cytokines that stimulate the production of some vasodilator substances, platelet aggregation, and activation of coagulant proteins. Activation of coagulation may contribute to the development of DIC and multiorgan failure [1,5,6,12,15,18,23,24,28]. Following macrophage activation and profound cytokine secretion, hemophagocytosis may occur in various infectious diseases including CCHF. Hemophagocytosis is an important clinical entity, which is presumed to be associated with cytopenia and increased risk of mortality [12,25,27,28,32-34]. Papa et al. [35] and Ergonul et al. [36] reported increased levels of TNF-_, IL-1, and IL-6 in patients with CCHF. They also found significantly higher TNF-_ and IL-6 levels in fatal cases as compared to mild cases, emphasizing the pivotal role of the cytokine storm in the pathogenesis of CCHF

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[35-38]. Ongoru et al. [39] showed high serum neopterin levels in CCHF and its increment correlated with mortality from the disease. Since neopterin is produced by activated macrophages upon secretion of IFNa, their findings may be considered as a further piece of evidence supporting the important role of cytokine secretion and macrophage activation in the pathogenesis of CCHF. Oversecretion of IL-18-induced Fas ligand expression on lymphocytes may contribute to the cytotoxicity of Fas-expressing hepatocytes, which might be responsible for hepatic failure and bleeding in secondary hemophagocytosis [40,41]. A similar mechanism might also be relevant in patients with CCHF. Extremely increased ferritin levels were reported in patients with CCHF virus infection [27,28], and this has been accepted among the diagnostic criteria for primary hemophagocytic lymphohistiocytosis (HLH) [42].

Our Experience

The facilities of our university are located in the Black Sea region of Turkey, which is close to the area endemic for CCHF. To date, 164 cases, most of whom were adults, have been diagnosed and followed in our hospital [14,25,27,47]. Between 2004 and 2006, we diagnosed and followed nine children with CCHF. The age rage was between 2 and 16 years, and their initial clinical and laboratory findings were as follows: fever and fatigue were present in all nine patients; nausea, vomiting, and petechial rash were present in eight, hematemesis and melena in five, and epistaxis in two. One patient presented with hemoptysis, somnolence and convulsions. Two patients had hepatomegaly and one had splenomegaly. Laboratory findings included anemia (Hb: 4.5-10 g/dl) and leukopenia (WBC counts: 1400-3900/mm3) in five and thrombocytopenia (platelet count: 5000-36000/mm3) in seven children. Elevated levels of AST (105-2852 U/L), ALT (372-3100 U/L), LDH (912-5100 U/L) and CPK (297-2900 U/L) were present in eight, seven, eight, and six patients, respectively. Prolonged PT (16-32 sec) and aPTT (54-140 sec) were detected in four and seven cases, respectively. Increased D-dimer (609-1430 U/L) was found in seven children. Bone marrow aspiration was performed in three children and significant hemophagocytosis was determined in all of these cases. With a presumptive diagnosis of CCHF, ribavirin therapy and supportive measures including the administration of fresh frozen plasma (FFP) and/or packed red cells and/or platelet suspensions were initiated until the diagnosis was confirmed with ELISA or PCR. Full recovery was obtained in all children without any complication [25,43].

Diagnosis and Treatment

Early diagnosis is very important with regard to the outcome in CCHF. Patients with clinical and laboratory findings of CCHF and history of tick bite or exposure to infection should be tested for CCHF. Laboratory methods include virus isolation and serologic and molecular assays with reverse transcription PCR or real time PCR. Antibodies are detectable in the serum

by ELISA or immunofluorescence assay (IFA) about seven days after the onset of disease. Antibodies are not detectable in fatal cases who die within the first days of illness. Molecular studies are necessary for confirmation of the disease in such cases [1,2,5,44,45].

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There are three principal and equally important objectives in the treatment of CCHF: close monitoring of all coagulation parameters, supportive treatment with FFP, erythrocyte and platelet suspension, and early antiviral treatment [1,5,6,46].

Continuous evaluation of clinical status, including vital organ functions and observation of bleeding, are mandatory for patients with CCHF. Supportive treatment should be provided with FFP and erythrocyte and platelet suspensions. Close monitoring of platelet count, fibrinogen and D-dimer levels is important for early detection of DIC, which also allows early correction of coagulation parameters. Ergonul et al. [1] reported a higher requirement for platelet suspensions in cases with a fatal outcome. If available, apheresis of platelet suspensions that provide a large amount of platelet content (3x1011/per apheresis platelet suspension) should be preferred.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit blood clotting and cause an increased risk of bleeding; therefore, they should not be used in CCHF. FFP transfusion is indicated for the correction of coagulopathy in bleeding or for the prevention of bleeding in CCHF. When PT and aPTT are at least 1.5 times greater than the upper limit of the normal range or international normalized ratio (INR) is >1.6, FFP (15-20 ml/ kg) should be given, and this treatment should be repeated every 8-12 hours, if necessary. In addition to these supportive measures, unfractionated or low molecular weight heparin, recombinant human activated protein C, recombinant activated factor VII, and antifibrinolytic agents should also be considered in CCHF patients with uncontrolled bleeding and DIC [1,5,6,12,14,18,46].

Although the effect of ribavirin treatment in CCHF virus infection has not been proven in randomized prospective clinical trials, and there are some conflicting results in the clinical reports, however majority of them indicate its beneficial effects (11,15,46-49). Based on these reports, ribavirin seems to be widely accepted in the treatment of CCHF and its use is recommended especially in the early phase of the disease [11,15,46-48].

Secondary hemophagocytosis may resolve upon the treatment of underlying disease, although it may be as fatal as primary HLH [32,33]. A genetic mutation is responsible for the impaired antiviral defense, dysregulated immune response and insufficient lymphocytic apoptosis in primary HLH [32,33,42,50].

A protocol consisting of dexamethasone, cyclosporine A and etoposide has been widely accepted in the treatment of patients with primary HLH [42]. This was also shown to be beneficial in the treatment of Epstein-Barr virus-associated HLH [51,52].

Although a genetically transmitted dysregulated immune response occurs in the above-mentioned HLHs, which are completely distinct from secondary hemophagocytoses including CCHF, these entities share some common clinical and laboratory features, most of which are mediated via the same mechanisms. Uncontrolled CD4 Th1 activation and overproduction of proinflammatory cytokines with macrophage coactivation-induced hemophagocytosis are well established in all types of HLH [32,33,42,50-52]. Pathogen-directed therapy might not always be sufficient to control cytokine storm in secondary hemophagocytoses including the one associated with CCHF. Although immunosuppression is known to exacerbate the spread of infections, use of immunosuppressive

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therapy along with ribavirin might be considered in the treatment of the patients with CCHF who have uncontrolled hemophagocytosis and severe bleeding symptoms. Jabbari et al. [53] reported a complete recovery in six patients with CCHF who received corticosteroids combined with ribavirin in the early phase of the disease. Similarly, cyclosporin and/or etoposide with or without steroids successfully controlled excessive activation of lymphocytes/monocytes and oversecretion of cytokines in various infection-associated HLHs [52-59].

New treatment strategies such as IFN or antibodies against TNF or IL-6 could prove to be valuable in CCHF [38], but the experience is limited, and further studies are warranted.

Obviously, the timing of these therapeutic approaches should also carry a significant importance.

It is our hope that an effective immunization strategy with a safe vaccine and/or specific serum against CCHF virus will soon be in use in order to overcome this frequently fatal disease.

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.

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Research Article

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Cap +1 mutation; an unsuspected cause of beta thalassaemia transmission in Pakistan Cap +1 mutasyon; Pakistan’da beta talasemi taýÕyÕcÕlÕúÕnÕn ýüphe götürmez bir nedeni

Muhammad Usman Abdul Karim, Moinuddin Moinuddin, Sadia Usman Babar Institute of Hematology Baqai Medical University, Karachi, Pakistan

Abstract Objective: Thalassemia is one of the most common genetic disorders worldwide. Cap +1 mutation which causes ‘silent beta thalassemia’ is present around all ethnic groups of Pakistan. This study was designed to detect the frequency of Cap+1 mutation in Pakistani Population.

Materials and Methods: Molecular genetic for Cap+1 beta thalassemic mutation was done by extracting DNA from whole blood by using Genomic DNA Purification Kit (Gentra system USA). Amplification Refractory Mutation System (ARMS) primers were designed for detection of normal and mutant DNA. Basic hematological parameters were performed by using automated analyzer (Sysmex KX-21). Cellulose acetate hemoglobin electrophoresis was done by using semi-automated technique (INTERLAB Roma Microtech Series Electrophoresis system 4.23).

Results: The frequency of Cap+1 mutation was observed 5% (10/200) in targeted thalassemic families (having patients with beta-thalassemia intermedia) while its frequency was observed 2% (12/600) in total thalassemic genes in Pakistani population.

Conclusion: Cap+1 (A-C) is a silent mutation and it has very minimum effect on beta globin synthesis because of which it produces very less clinical severity and certain important laboratory diagnostic tests like basic hematological parameters and Hb A2 levels are also remain in normal range. Therefore individuals with Cap+1 mutation may produce children with beta-thalassemia intermedia if they marry an individual with beta-thalassemia minor. Cap+1 (A-C) mutation is an unsuspected cause of beta thalassemia transmission in Pakistani population. This mutation can identify at molecular level. As this molecular defect is difficult to diagnose in Laboratory with routine laboratory tests because of that it has become a serious hindrance for thalassemia prevention program in Pakistan. (Turk J Hematol 2009; 26: 167-70) Key words: Cap+1(A-C) mutation, Silent beta thalassaemia, Polymerase chain reaction (PCR) Received: April 26, 2009

Accepted: September 1, 2009

Özet Amaç: Talasemi dünya genelinde en yaygÕn genetik bozukluklardan biridir. ‘Sessiz beta talasemi’ye neden olan Cap +1 mutasyonu Pakistan’daki tüm etnik gruplarda görülmektedir. Bu çalÕýma Pakistan nüfusu içinde Cap +1 mutasyonun sÕklÕúÕnÕ belirlemek için tasarlanmÕýtÕr.

Address for Correspondence: Asst. Prof. Muhammad Usman Abdul Karim, Super High Way Near Toll Plaza Karachi, Pakistan Karachi, Pakistan Phone: +92-322-3561923 E-mail: staytune1@hotmail.com

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Cap+1 mutation

Turk J Hematol 2009; 26: 167-70

Yöntem ve Gereçler: Cap +1 beta talasemik mutasyon tayini Genomik DNA Purifikasyon Kiti (Gentra system ABD) kullanÕlarak kandan ekstre edildi. Normal ve mutant DNA tespiti için Amplifikasyon Refrakter Mutasyon Sistemi (ARMS) primerleri tasarlandÕ.Temel hematolojik parametreler otomatik analizör (Sysmex KX-21) kullanÕlarak çalÕýÕldÕ. Selüloz asetat hemoglobin elektroforezi yarÕ-otomatik teknik (INTERLAB Roma Microtech Series Electrophoresis system 4.23) kullanÕlarak yapÕldÕ.

Bulgular: Cap+1 mutasyon sÕklÕúÕ, hedef talasemik ailelerde (beta-talasemi intermedia hastalarÕ) %5 (10/200) olarak gözlemlenirken bu sÕklÕk Pakistan popülasyonu içindeki total talasemik genlerde %2 (12/600) olarak bulundu.

Sonuç: Cap+1 (A-C) sessiz bir mutasyondur ve beta globin sentezi üzerinde minimum bir etkiye sahiptir. Bu yüzden klinik önemi azdÕr. Hematolojik parametreler ve Hb A2 seviyeleri gibi önemli laboratuvar tanÕ testleri de normal düzeyde kalmaktadÕr. Bu nedenle Cap+1 mutasyonu taýÕyan bireyler eúer beta-talasemi minör bulunan bir bireyle evlenirlerse betatalasemi intermediyalÕ çocuúa sahip olabilir. Pakistan popülasyonunda Cap+1 (A-C) mutasyon taýÕyÕcÕlÕúÕ, beta talasemi taýÕnmasÕnÕn nadir ve ýüphe duyulmayan bir nedenidir. Bu mutasyon moleküler düzeyde belirlenebilir. Bu moleküler bozukluk Polimeraz Zincir Reaksiyon (PCR) yardÕmÕ olmadan laboratuvarda zor teýhis edilebileceúi için Pakistan’daki talasemi önleme programÕ için ciddi bir engel haline gelmiýtir. (Turk J Hematol 2009; 26: 167-70) Anahtar kelimeler: Cap+1(A-C) mutasyon, sessiz beta talasemi, polimeraz zincir reaksiyonu (PCR) Geliý tarihi: 26 Nisan 2009

Kabul tarihi: 1 Eylül 2009

Introduction Thalassemia is the most common genetic disorder across the globe [1,2]. The term thalassemia comes from a Greek word “thalas” which means the sea, and “emia” that stands for blood [3,4]. The composite word came into use because this type of anemia was originally described only in countries bordering the Mediterranean Sea. Thalassemia was not recognized as a clinical entity until 1925, when Cooley and Lee, described a syndrome occurring early in life that was associated with splenomegaly and bony deformities [4].

Beta thalassemia is the most common genetic disorder in Pakistan [5]. Pakistan has a population of 160 million people [6]. The annual rate of population growth is 3% and almost 40% of the population is below 15 years of age [7,8]. There are five major ethnic groups Punjabi, Pathan, Sindhi, Baluchi and Urdu speaking. Each ethnic group is subdivided into Casts or ‘Biradris’ of people ranging from a few thousands to a few millions. There is a very strong tradition for people to marry within their Biradris. Among very common custom is marriage between close relatives, especially the first cousins [8].

A number of studies have been done worldwide at molecular level to describe mutations, deletions and substitutions in beta globin gene causing beta thalassemia with different clinical severity. More than 200 causative molecular defects have so far been described in the ß-globin gene causing beta thalassemia [9]. Thirteen mutations are commonly reported in Pakistani population in which five are the most common mutations; these include IVS-1-5 (G-C), Frameshift (Fs) 8/9 (+G), Fs 41/42 (-TTCT), IVS-1-1 (G-T) and Del 619.

Other mutations that were reported in Pakistani population are a few uncommon and some rare mutations [8,10]. Cap +1 (A-C) mutation is a silent mutation causes ‘silent beta thalassemia’ [10,11]. This mutation is present in all ethnic groups of Pakistan.

This study was designed to detect the prevalence of Cap+1 (A-C) mutation in Pakistan, to determine its effect on basic hematological parameters and Hb-A2 levels and to reveal the role of this mutation in propagation of beta thalassemia gene in Pakistani population.

Material and Methods This molecular study was performed at Baqai Institute of hematology, Baqai Medical University, Karachi from March 2004 to April 2009. Venous blood samples from 610 patients with beta thalassemia major, beta thalassemia intermedia and beta thalassemia minor were collected in EDTA. Test population was divided into two groups; one group comprised of targeted families (having children with thalassemia intermedia) while the second group included random samples from patients with beta thalassemia major and minor.

All samples were tested by the modified method of Amplification of Refractory Mutation System (ARMS) for Cap+1 (A-C) mutation. Basic hematological parameters and Hb-A2 levels were also done on blood samples that had Cap+1 mutation.

Basic hematological parameters were determined by automated analyzer (Sysmex KX-21). Cellulose acetate hemoglobin electrophoresis was done by semi-automated technique (INTERLAB Roma Microtech Series Electrophoresis system 4.23).

DNA was extracted from whole blood by using Genomic DNA Purification Kit (Gentra system USA). Amplification refractory mutation system (ARMS) primers were designed for detection of normal and mutant DNA. A control pair of primers was included in each assay. Control primers A, B and C were amplified at 861 bp fragments from 3’ end of the ß globin gene [12,13].

PCR was conducted with modified method in a mixture of 10 mmol/l tris (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl2. 500 M each dNTP’s, 0.2 mol/l of each primer, 0.5 units of Thermus aqaticus (Taq) 0.5 to 1 pg of genomic DNA was added to the PCR mixture in a total volume of 20 l. The modified cycling reaction (DNA thermal cycle; Perkin-Elmer/ Cetus) was programmed at 94oC for 1 minute (denature), 65oC for 1 minute (anneal) and 72oC for 1.5 minutes (extend). After 25 cycles, the samples were incubated for an additional 3 minutes at 66oC [12,13].

The amplified PCR products were observed using agarose gel electrophoresis and mutation was characterized with 100 bp or 50 bp ladder.

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Table 1. Basic hematological parameters and Hb-A2 levels of beta thalassemia minor with Cap+1 mutation Group

No. of

Hb

RBC

MCV

MCH

MCHC

Hb-A2

samples

(gm/dl)

(millions/ul)

(fl)

(pg)

(gm %)

(%)

Male

12

13.6 (±0.37)

4.8 (±0.34)

84.8 (±5.2)

29 (±1.37)

32.7 (±1.4)

3.0 (±0.12)

Female

10

12.0 (±0.71)

4.0 (±0.7)

80.4 (±3.1)

28.4 (±0.78)

31.4 (±1.8)

3 (±0.19)

Total no. of samples

22

Control 861 bp Cap+1 567 bp

Control 861 bp Cap+1 567 bp

Figure 1. Analysis of DNA sample with mutation primers Cap+1 (A-C) using 100 bp ladder

Statistical analysis Results were analyzed using SPSS statistical software version 13.

Results Result of the first group which included 160 individuals from targeted families (having children with thalassemia intermedia) identified 200 beta thalassemia genes in which frequency of Cap+1(A-C) mutation was observed at 5% (10/200). While result of the second group which included 450 random samples from patients with beta thalassemia major and minor revealed 600 beta thalassemia genes in which frequency of Cap+1(A-C) mutation was 2% (12/600) in Pakistani population.

Prevalence of this mutation in five major ethnic groups was observed with a frequency of 2.1% in Punjabi’s, 1.7% in Pathan’s, 1.7% in Sindhis, 2% in Baluchi’s and 2.5% in Urdu speaking population of Pakistan.

Discussion Result of this study revealed that Cap+1(A-C) is one of the common mutation which is present in all major ethnic groups of Pakistan. Frequency of Cap+1 (A-C) mutation was observed at 5% (10/200) in targeted beta thalassemic families (having patients with beta thalassemia intermedia) while its frequency was 2% (12/600) in total beta thalassemic genes in Pakistani population. Prevalence of this mutation in five major ethnic groups was observed with a frequency of 2.1% in Punjabi’s, 1.7% in Pathan’s, 1.7% in Sindhis, 2% in Baluchi’s and 2.5% in Urdu speaking population of Pakistan.

Cap+1(A-C) mutation is also reported in Indian population and U.A.E (United Arab Emirates) nationals [14-18]. This mutation is also identified in Arab population (Jordan, Egypt, Syria, Lebanon, Yemen and Saudi Arabia) [19]. Cap+1 mutation is also reported in Malaysian population [20].

This study revealed that Cap+1 (A-C) is a silent mutation and it has only minimal effect on beta globin synthesis.

Because of this it produces very few clinical manifestations.

Also various basic hematological parameters (Hb, RBC, MCV, MCH, MCHC) and Hb A2 levels remain in normal range (Table 1). Commonly prevalent beta thalassemia mutations other than Cap+1(A-C) mutation present in Pakistani population show characteristic hematological variation in the form of low Hb, increased RBC count, decreased MCV, decreased MCH, normal MCHC and increased level of Hb-A2. Because of the negligible effect of Cap+1 mutation on these hematological parameters and Hb-A2 levels it is difficult to diagnose beta thalassemia minor caused by Cap+1(A-C) mutation. Molecular techniques like PCR are essential for the diagnosis of beta thalassemia minor associated with Cap+1 mutation.

It is therefore important to accurately diagnose Cap+1 mutation lest it propagates beta thalassemia major as a result of marriage between a known case of beta thalassemia minor and a “normal” individual for beta thalassemia minor who indeed is a carrier of Cap+1 mutation which remain undetected on routine hematological parameters including Hb-electrophoresis for Hb-A2 level.

This mutation was not observed in homozygous state during this study and no case of beta thalassemia major was identified having Cap+1 (A-C) mutation. This mutation was observed as one of the most important molecular defects causing beta thalassemia intermedia if inherited with other major beta thalassemic genes reported in Pakistani population.

Therefore individuals with Cap+1 mutation may produce children with beta thalassemia intermedia if they marry an individual with beta-thalassemia minor. Cap+1 (A-C) mutation is an uncommon and unsuspected cause of beta thalassemia transmission in Pakistani population. Population study at a larger scale should be conducted to determine the frequency of CAP+1 mutation in Pakistani population; this will stop the propagation of thalassemia genes in homozygous state and prevent the birth of children with beta thalassemia intermedia.

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.

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7. Baig SM, Azhar A, Hassan H, Baig JM, Aslam M, Ud Din MA, Qureshi JA, Zaman T. Prenatal diagnosis of beta-thalassemia in Southern Punjab, Pakistan. Prenat Diagn. 2006;10:903-5.

8. Ahmed S, Petrou M, Saleem M. Molecular genetics of beta thalassaemia in Pakistan: a basis for prenatal diagnosis. Br J Haematol 1996;94:476-82.

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11. Garewal G, Das R, Awasthi A, Ahluwalia J, Marwaha RK. The clinical significance of the spectrum of interactions of CAP+1 (A-->C), a silent beta-globin gene mutation, with other betathalassemia mutations and globin gene modifiers in north Indians. Eur J Haematol 2007;79:417-21.

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12. El-Gawhary S, El-Shafie S, Niazi M, Aziz M, El-Beshlawy A. Study of beta thalassemia mutations using the polymerase chain reaction-amplification refractory mutation system (ARMS) and direct DNA sequencing techniques in a group of Egyptian thalassemia patients. Hemoglobin 2007;31:63-9.

13. Najmabadi H, Teimourian S, Khatibi T, Neishabury M, Pourfarzad F, Jalil-Nejad S, Azad M, Oberkanins C, Krugluger W. Amplification Refractory Mutation System (Arms) and Reverse Hybridization in the detection of Beta-thalassemia mutations. Archives of Iranian Medicine 2001;4:165-70.

14. Vaz FE Thakur CB, Banerjee MK, Gangal SG. Distribution of beta-thalassemia mutations in the Indian population referred to a diagnostic center. Hemoglobin 2000;24:181-94.

15. Gupta A, Hattori Y, Gupta UR, Sarwai S, Nigam N, Singhal P, Agarwal S. Molecular genetic testing of beta-thalassemia patients of Indian origin and a novel 8-bp deletion mutation at codons 36/37/38/39. Genet Test 2003;7:163-8.

16. Colah R, Nadkarni A, Gorakshakar A, Phanasgaonkar S, Surve R, Subramaniam PG, Bondge N, Pujari, K, Ghosh K, Mohanty D. Impact of beta globin gene mutations on the clinical phenotype of beta thalassemia in India. Blood Cells Mol Dis 2004;33:153-7.

17. Chakrabarti P, Gupta R, Mishra A, Rai M, Singh VP, Dash D. Spectrum of beta-thalassemia mutations in North Indian states: a beta-thalassemia trait with two mutations in cis. Clin Biochem 2005; 38:576-8.

18. Quaife R, al-Gazali L, Abbes S, Fitzgerald P, Fitches A, Valler D, Old JM. The spectrum of beta thalassaemia mutations in the UAE national population. J Med Genet 1994; 31:59-61.

19. El-Hazmi MA, Warsy AS, al-Swailem AR. The frequency of 14 beta-thalassemia mutations in the Arab populations. Hemoglobin 1995; 19:353-60.

20. Chan Yoke Fan, Tan Kim Lian, Wong Yean Ching, Wee Yong Chui, Yap Sook Fan, Tan Jin Ai Mary Anne. The use of the amplification refractory mutation system (arms) in the detection of rare betathalassemia mutations in the Malays and Chinese in Malaysia.

Southeast Asian J Trop Med Public Health 2001;32:872-9.

Research Article

171

Incidence of anti-heparin/platelet factor 4 antibodies and heparin-induced thrombocytopenia in medical patients Medikal hastalarda anti-heparin trombosit faktör 4 antikor ve heparine baúlÕ trombositopeni sÕklÕúÕ Muzaffer Demir1, Emre Tekgündüz1, Mustafa Edis2, Enver Duran2, Turan Kürüm3, Ömer YiúitbaýÕ4, Mahmut Yüksel4 1Trakya

University, University, 3Trakya University, 4Trakya University, 2Trakya

Faculty Faculty Faculty Faculty

of of of of

Medicine, Medicine, Medicine, Medicine,

Department Department Department Department

of of of of

Medicine, Division of Hematology, Edirne, Turkey Cardiovascular Surgery, Edirne, Turkey Cardiology, Edirne, Turkey Nuclear Medicine, Edirne, Turkey

Abstract Objective: Heparin-induced thrombocytopenia (HIT) is a life threatening complication of heparin therapy, causing thrombosis. The aim of our study was to find out the frequencies of HIT antibody seroconversion and clinical HIT in Turkish medical patients on different forms of heparins.

Materials and Methods: Our study included 61 patients who were on unfractionated heparin (UFH) (n: 37) and low molecular weight heparin (LMWH) (n: 24) therapies. The frequency of HIT antibody formation was determined by means of antigenic (ELISA), and functional assays (serotonin release assay-SRA).

Results: The seroconversion rates in UFH and LMWH groups were found to be 18.9% and 4.1% (ELISA), and 8.1% and 4.1% (SRA), respectively. One patient (2.1%) on UFH therapy developed deep vein thrombosis. No thromboembolic event was observed in patients taking LMWH. Conclusion: Seroconversion rates by means of antigenic and functional assays and clinical HIT were more common in patients on UFH than patients on LMWH therapy. (Turk J Hematol 2009; 26: 171-5) Key words: Heparin-induced thrombocytopenia, medical patients, Turkish cohort, unfractionated heparin, low molecular weight heparin.

Received: May 28, 2009

Accepted: September 29, 2009

Özet Amaç: Heparine baúlÕ trombositopeni (HûT) heparin tedavisinin tromboza neden olan, hayatÕ tehdit eden bir komplikasyonudur. ÇalÕýmamÕzÕn amacÕ farklÕ heparin formlarÕ kullanan dahili Türk hastalarda HûT antikor serokonversiyonu ve klinik HûT sÕklÕúÕnÕn saptanmasÕydÕ.

Yöntem ve Gereçler: ÇalÕýmamÕza anfraksiyone heparin (AFH) (n: 37) ve düýük molekül aúÕrlÕklÕ heparin (DMAH) (n: 24) tedavisi alan 61 hasta katÕldÕ. HIT antikor oluýumu antijenik (ELISA) ve fonksiyonel (serotonin salÕnÕm testi-SRA) testler ile deúerlendirildi.

Address for Correspondence: Prof. Muzaffer Demir, Trakya University, School of Medicine, Division of Hematology, 22030 Edirne, Turkey Phone: +90 284 235 76 42 – +90 284 235 10 41 E-mail: muzaffer@tnn.net

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Bulgular: AFH ve DMAH gruplarÕnda serokonversiyon oranlarÕ sÕrasÕyla %18,9-%4,1 (ELISA) ve %8,1-%4,1 (SRA) saptandÕ.

AFH kullanan bir hastada (%2,1) derin ven trombozu geliýti. DMh kullanan hastalarda tromboembolik olay izlenmedi.

Sonuç: aFH kullanan hastalarda dmah kullananlara göre antijenik ve fonksiyonel yöntemlerle serokonversiyon ve klinik HIT oranlarÕ daha yüksek saptandÕ. (Turk J Hematol 2009; 26: 171-5) Anahtar kelimeler: Heparine baúlÕ trombositopeni, medikal hastalar, Türk hasta grubu, fraksiyone olmayan heparin, düýük molekül aúÕrlÕklÕ heparin Geliý tarihi: 28 MayÕs 2009

Kabul tarihi: 29 Eylül 2009

Introduction Heparin is an important anticoagulant drug which is widely used in almost every discipline of medicine and has saved thousands of lives for over 50 years. The most feared complications of heparin therapy are hemorrhage and heparininduced thrombocytopenia (HIT). HIT is an acquired, transient, prothrombotic disorder and a life-threatening complication of unfractionated (UFH) and low molecular weight heparin (LMWH) therapy presenting with thrombocytopenia and/or complicating venous or arterial thromboembolism that is associated with increased in vivo thrombin generation [1]. HIT is a clinicopathologic condition and adverse drug reaction caused by platelet-activating antibodies of mostly pathogenic IgG class which are directed against a molecular complex formed by heparin and platelet _-granule protein, platelet factor 4 (PF4) [2]. The frequency of HIT is dependent on four factors: duration of heparin use (>1 week versus<1 day), type of heparin (UFH>LMWH>fondaparinux which is synthetic heparin pentasaccharides), type of patient population (surgery> medical>pregnancy), and patient gender (female>male) [3]. HIT occurs in 3% to 5% and 0.5% of patients receiving UFH and LMWH, respectively [4]. In the absence of alternative anticoagulation, the risk of thrombosis is ~ 5% to 10% per day in the first few days after cessation of heparin [5] and mortality from HIT ranges from 18-50% [6]. In medical conditions, the frequency of both antibody formation and thrombocytopenia or/and thrombosis are much less than in surgical settings [7,8].

The confirmation of HIT by laboratory methods is complex and difficult but is a sine qua non of HIT diagnosis. To date, there are two types of assays used to measure the antibody formation (immunologic and functional assays) [9-11]. The frequency of AHPF4 formation is much greater than the risk of HIT and only minority of antibodies detected with EIA are able to activate platelets in vivo. The interrelationship between antibody formation and clinical HIT has been demonstrated with an iceberg model [11]. We recently reported the frequency of AHPF4 antibody formation and clinical HIT in Turkish patients undergoing cardiac surgery [12]. Our study was designed to establish the laboratory methods of HIT and to determine the frequency of antibody generation and clinical HIT events in medical patients in Turkey.

Materials and Methods Patients Selection: This prospective study enrolled a total of 61 consecutive medical patients who were treated at a single institution (Trakya University Hospital) during 2004-2005 and no history of recent (< 100 days) heparin/LMWH exposure.

37 patients with acute coronary syndrome (ACS) and venous thromboembolism (VTE) (deep vein thrombosis (DVT) and/or pulmonary embolism (PE)) who were treated with UFH for at least 5±2 days, and 24 DVT patients who were on therapeutic dose LMWH therapy for at least 6±2 days were included.

Patients of LMWH therapy arm were treated with nadroparin (n=5), dalteparin (n=5), and enoxaparin (n=14). Written informed consent was obtained from all patients. The study was conducted according to the Declaration of Helsinki.

Sample collections: Blood samples were taken on 10±2. days of heparin therapy. Daily and alternate day platelet counts were performed on patients belonging to UFH and LMWH therapy arm, respectively. Thromboembolic attacks were evaluated on clinical grounds. In suspected cases radiological (Doppler ultrasonography and/or spiral computerized tomography), and scintigraphic (ventilation-perfusion scan) confirmations were made. The first day of heparin exposure was considered day 0. Ten ml of blood without anticoagulant was taken and allowed to incubate at room temperature for 1 hour to assure blood clot formation. The samples were centrifuged at 3000/min for 15 minutes. All serum samples were inactivated by heat inactivation at 56 ºC for 45 minutes and stored at -85 ºC until tested.

Laboratory assays: For determining the antibodies, both antigenic and activation assays were carried out. The determination of anti-heparin/platelet factor 4 antibodies (HPF4-A) was performed as previously described by using a commercially available ELISA kit (Asserachrome HPIA; Diagnostica Stago, France) [11]. The positive results were defined as an absorbance value of equal or greater than 0.5 optical density units at A492 nm. This ELISA kit detected two other anti-human immunoglobulin (Ig) classes (Ig A and Ig M) besides Ig G. Heparin-PF4 dependent platelet reactive antibodies were detected with two functional assays, the platelet aggregation test (PAT) and the serotonin release assay (SRA). The patient samples were tested in duplicate with different platelet donors.

A pool of ten normal-healthy-previously tested donors’ platelets were used in these activation assays. PAT was performed as previously reported [9,13]. In this method, platelet rich citrated plasma was used and the samples were considered positive if 20% maximum aggregation was met at 0.1 to 1.0 U/ml heparin concentrations. Aggregation response was monitored for 20 min. SRA was performed as previously described [9,14].

For the SRA, the samples were considered positive if all the following criteria were met: 1) 20% serotonin release at 0.1U/ ml heparin and 2) inhibition of platelet activation at high concentration of heparin (100 U/ml) 3) appropriate activation profiles observed with positive and negative control samples. If

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a patient sample was negative by PAT or SRA, it was repeated in duplicate with a different platelet donor.

Definition of HIT: HIT was defined according to the following criteria. 1) unexplained absolute or relative thrombocytopenia with a decrease of 50% from baseline platelet counts after day 5 of heparin therapy for no other reason except heparin treatment. 2) new thromboembolic complications such as venous or arterial thrombosis associated with heparin exposure and not due to surgery, thrombophilia, DIC, etc. 3) laboratory evidence of anti-heparin-platelet factor 4 antibodies.

Statistical analysis: All statistical calculations were performed by using Microsoft Excel software. Results were expressed as mean ± standard deviation. The positive results were given as percent value of total patient number. Chisquare test was used to compare seroconversion rates among groups. The p value if equal or lower than 0.05 was considered as significant.

Results The study cohort included 37 patients (11 female, 26 male) with acute coronary syndrome (n:29) and VTE (n:8) (DVT and/ or PE), and 24 DVT patients ( 12 female, 12 male). Mean age of patients on UFH and LMWH therapy were 65±9.5 years and 61±7.2 years, respectively. The frequency of HIT antibody generation measured by ELISA in patients taking UFH and LMWH were found to be 18.9% and 4.1%, respectively. SRA detected HIT antibodies in 8.1% of patients on UFH and 4.1% of patients on LMWH therapy. PAT was positive in 8 patients (21.6%) on UFH and 1 (4.1%) patient on LMWH therapy. One patient (2.1%) with ACS who was on UFH therapy developed

173

DVT. No thromboembolic attack was observed in patients taking LMWH (Figure 1 and 2). Seroconversion rates by means of ELISA (p: 0.01), PAT (p: 0.02) and SRA (p: 0.04) and clinical HIT were more common in patients on UFH than patients on LMWH therapy (Figure 3).

Discussion HIT is a very strong risk factor for arterial and venous thromboembolism, both in relative (odds ratio for thrombosis = 20-40) and absolute (thrombosis risk 30-45%) terms, depending on the patient population affected [15]. As HIT is a clinicopathologic syndrome its diagnosis depends on any clinical event related to heparin exposure like thrombocytopenia, thrombosis, anaphlaxis and demonstration of HIT antibody serocoversion by serologic and functional assays. Only a minority of patients with antibody seroconversion develops HIT. In one third to one half of the cases isolated HIT-defined as HIT presented with thrombocytopenia but without thrombosis- is subsequently complicated by thrombosis [16].

Therefore simply discontinuing heparin is not enough to prevent thromboembolic complications and an alternative nonheparin anticoagulant should be commenced immediately in case of highly probable or confirmed HIT. In order to prevent HIT overdiagnosis a clinical scoring system (4Ts) is very useful for predicting pretest probability of HIT [17].

In general both seroconversion, thrombocytopenia and thrombosis rates are lower among medical patients compared to surgical patients. Because we did not perform baseline search for HIT antibodies, we cannot exclude that some patients were already seropositive before study entry. In three different reports on ACS patients who were treated with UFH

HITT (2.1%)

Platelet activating antibody formation SRA (8.1%) PAT (21.6%)

Seroconversion rate by means of ELISA (18.9%)

No measurable antibodies against heparin (81%)

Figure 1. Data on the frequency of antibody generation and clinical HIT in medical patients on UFH therapy are presented as an iceberg model. SRA: Serotonin release assay; PAT: Platelet aggregation assay; HITT: Heparin induced thrombocytopenia thrombosis

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HITT (0%)

Platelet activating antibody formation SRA (4.1%) PAT (4.1%)

Seroconversion rate by means of ELISA (4.1%)

No measurable antibodies against heparin (95.9%)

Figure 2. Data on the frequency of antibody generation and clinical HIT in medical patients on LMWH therapy are presented as an iceberg model. SRA: Serotonin release assay; PAT: Platelet aggregation assay; HITT: Heparin induced thrombocytopenia thrombosis Table 1. The clinical and laboratory results of SRA positive samples Age /Sex

Heparin

ELISA

SRA

Platelet count

Minimum platelet

Thromboembolic

type

(OD)

(% release)

at study entry

count during

event

(mm3)

study period

(mm3)

77/F

UFH

0,870

74

222000

192000

-

75/M

UFH

2,870

85

267000

212000

DVT

66/M

UFH

0,807

65

288000

292000

-

62/F

LMWH

0,555

59

198000

188000

-

F: Female; M: Male; OD: Optical density; SRA: Serotonin release assay; DVT: Deep vein thrombosis

Results of both antigenic and activation assays

Percentage of positive results

25 20

p:0.01

p:0.02 21,6

UFH

18,9

LMWH

15

p:0.04 10 5 0

8,1 4,1

ELISA

4,1

PAT

4,1

SRA

Figure 3. Results of serologic and functional assays on heparin and LMWH

seroconversion rates were found to be 8.7%, 10.6% and 30%, respectively [11,18,19]. There is a trend for increased seropositivity with time both in surgical and medical patients on heparin and LMWH therapy [12,20]. In their multicenter study Lindhoff-Last E et al. [20] evaluated a total of 1137 patients with DVT who were randomly assigned to UFH or reviparin for 5-7 days. On day 5-7 the incidence of AHPF4 were found to be 9.1% and 3.7%, who were on UFH and LMWH therapy, respectively. These values were 20.7% (UFH) and 7.5% (LMWH) on day 21. In our study seroconversion rates were 18.9% and 4.1% for patients on UFH and LMWH, respectively.

As we did not perform serologic assays on different time periods we were not able to demonstrate this phenomenon. Ig A and IgM antibodies cannot activate platelets by way of Fca (IgG) receptors and therefore not pathogenic. As our ELISA kit detected both IgA and IgM antibodies besides IgG, our results could be an overestimation of true seroconversion rates. The incidence of HIT associated thrombosis among medical patients varies between 0.8%-1.6% and 0.53%-0.8% in

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HIT in medical patients

Turk J Hematol 2009; 26: 171-5

patients on UFH and LMWH treatment, respectively [7,8,18,20].

Seroconversion rates by means of antigenic and functional assays and clinical thrombosis on our patient cohort are compatible with the vast majority of reports on HIT in the literature.

In conclusion, seroconversion rates and clinical HIT were more common in patients on UFH than patients on LMWH therapy. The small patient number, the most important shortcoming of our study, make it difficult to draw any firm conclusions. Nevertheless, as far as we know this is the first effort evaluating the frequency of HIT among Turkish medical patients on different heparin preparations.

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.

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5.

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Warkentin TE. Think of HIT. Hematology (Am Soc Hematol Educ Prog) 2006;408-14.

Warkentin TE. Heparin-induced thrombocytopenia: a clinicopathologic syndrome. Thromb Haemost.1999; 82: 439-47.

Warkentin TE. Heparin-induced thrombocytopenia. Hematol Oncol Clin N Am 2007;21:589-607.

Rice L. Heparin-induced thrombocytopenia. Myths and misconceptions (that will cause trouble for you and your patient).

Arch Intern Med. 2004;164:1961-4.

Greinacher A, Eichler P, Lubenow N, Kwasyn H, Luz M. Heparininduced thrombocytopenia with thromboembolic complications: metaanalysis of 2 prospective trials to assess the value of parenteral treatment with lepirudin and its therapeutic aPTT range. Blood 2000;96:846-51.

Kuo KHM, Kovacs MJ. Fondaparinux: A potential new therapy for HIT. Hematology 2005;10:271-75.

Girolami B, Prandoni P, Stefani PM, Tandou C, Sabbion P, Eichler P, Ramon R, Baggio G, Fabris F, Girolami A. The incidence of heparin-induced thrombocytopenia in hospitalized medical patients treated with subcutaneous unfractionated heparin: a prospective cohort study. Blood 2003;101:2955-9.

Prandoni P, Siragusa S, Girolami B, Fabris F. BELZONI Investigators Group. The incidence of heparin-induced thrombocytopenia in medical patients treated with low-

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molecular-weight heparin: a prospective cohort study. Blood 2005;106:3049-54.

Walenga JM, Jeske WP, Fasanella AR, Wood JJ, Bakhos M. Laboratory tests for the diagnosis of heparin-induced thrombocytopenia. Semin Thromb Hemost 1999; 25 (Suppl 1): S43-9.

Demir M, Ahmad S, Walenga JM, Fareed J. Laboratory diagnosis of heparin-induced thrombocytopenia. Turk J Haematol 2000;17(3 Suppl 3):181-90.

Warkentin T and Sheppard JAI. Testing for HIT antibodies.

Transfusion Medicine Reviews 2006; 20: 259-72.

Demir M, Duran E, Yigitbasi O, Vural O, Kurum T, Yuksel M, Turgut B, Walenga JM, Fareed J. Incidence of antiheparin-platelet factor 4 antibodies and heparin-induced thrombocytopenia in Turkish patients undergoing cardiac surgery. Clin Appl Thromb Hemost 2007;13:279-84.

Chong BH, Burgess J, Ismail F. The clinical usefulness of the platelet agregation test for the diagnosis of heparin-induced thrombocytopenia. Thromb Haemost. 1993;69:344-50.

Sheridan D, Carter C, Kelton JG. A diagnostic test for heparininduced thrombocytopenia. Blood 1986;67:27-30.

Warkentin TE, Greinacher A, Koster A and Lincoff AM. Treatment and prevention of heparin-induced thrombocytopenia. The 8th ACCP conference on antithrombotic and thrombolytic therapy.

Chest 2008; 133:340S-380S. Warkentin TE, Greinacher A. Heparin-induced thrombocytopenia: recognition, treatment and prevention. The 7th ACCP conference on antithrombotic and thrombolytic therapy. Chest 2004;126: 311S-337S. Keeling D, Davidson S, Watson H. Management of heparininduced thrombocytopenia. Br J Haematol. 2006; 133: 259-69.

Matsuo T, Tomaru T, Kario K, Hirokawa T and on behalf of HIT Research Group of Japan. Incidence of heparin-PF4 complex antibody formation and HIT in ACS. Thrombosis Research 2005; 115: 475-81.

Mascelli MA, Macselli MA, Deliargyris EN, Damaraju LV, Barnathran ES, Sane DC. Role of anti-PF4/heparin antibodies in recurrent thrombotic events after ACS. Seminars in Thrombosis and Hemostasis 2004; 30: 347-50.

Lindhoff-Last E, Nakov R, Misselwitz F, Breddin HK, Bauersachs R. Incidence and clinical relevance of heparin-induced antibodies in patients with deep vein thrombosis treated with unfractionated heparin or low-molecular-weight heparin. Br J Haematol 2002; 118: 1137-42.

Research Article

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Hyperbaric oxygen: an important treatment modality in severe hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation Hiperbarik oksijen: Allojeneik hematopoietik kök hücre nakli sonrasÕ geliýen aúÕr hemorajik sistit olgularÕnda önemli bir tedavi seçeneúi

Mustafa Nuri Yenerel, Sevgi Kalayoúlu-BeýÕýÕk, Oktay Perdeci, Nuray Gürses, Murat Tunç, Deniz SargÕn

ûstanbul University, ûstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, ûstanbul, Turkey

Abstract Objective: Hemorrhagic cystitis (HC) is a generally self-limited complication of hematopoietic stem cell transplantation (HSCT). It may occur in the early or late posttransplant period and can promote sometimes severe morbidity. We analyzed our data regarding HC in allogeneic HSCT patients in order to establish the efficacy of hyperbaric oxygen (HBO) therapy in severe HC and to document the main problems during its use.

Material and Methods: Between March 1993 and August 2006, 161 patients received allogeneic HSCT. Mesna, hyperhydration and forced diuresis were used as early HC prophylaxis of cyclophosphamide-induced HC. However, HC was diagnosed in 49 of the 161 recipients and 17 of them were considered as severe HC. We analyzed their data retrospectively.

Results: Forced diuresis with hyperhydration (up to 8 L/day) and transfusion support to maintain a platelet count above 30x109/L were sufficient in 10 of the 17 patients with severe HC. Alternative therapies used included intravesical irrigation with formalin and prostaglandin (PG)F2 alpha and HBO, and HBO appeared to be the most useful among them.

Conclusion: We conclude that HBO offers a noninvasive therapeutic alternative in the management of intractable HC in the HSCT setting. (Turk J Hematol 2009; 26: 176-80) Key words: Hyperbaric oxygen, hemorrhagic cystitis, stem cell transplantation Received: March 17, 2009

Accepted: September 1, 2009

Özet Amaç: Hemorajik sistit, hematopoetik kök hücre naklinin genellikle kendiliúinden düzelen bir yan etkisidir. Nakil sonrasÕ erken ve geç dönemlerde geliýebilen bu komplikasyon bazen oldukça aúÕr geçebilmektedir. Biz allojeneik hematopoetik kök hücre nakli sonrasÕ hemorajik sistit geliýen olgularÕmÕzÕ, özellikle hiperbarik oksijen tedavisinin bu olgulardaki etkinliúini ve kullanÕmda dikkat edilmesi gereken özellikler açÕsÕndan deúerlendirdik.

Address for Correspondence: Assoc.Prof. Mustafa Nuri Yenerel, ûstanbul University, ûstanbul Medical Faculty, Department of Internal Medicine, Hematology Field, Çapa, ûstanbul, Turkey Phone: +90 212 414 20 00-31613 E-mail: mnyenerel@gmail.com

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177

Yöntem ve Gereçler: Mart 1993 ve Aúustos 2006 yÕllarÕ arasÕnda 161 hastaya allojeneik hematopoetik kök hücre nakli uygulandÕ. Siklofosfamide baúlÕ hemorajik sistit geliýimi engelleyebilmek amacÕyla profilaktik olarak Mesna, hiperhidrasyon ve zorlu diürez uygulandÕ. Buna raúmen 161 olgunun 17’si aúÕr olmak üzere 49’unda hemorajik sistit geliýti. Tedavi yaklaýÕmÕ ve bulgular geriye dönük olarak deúerlendirildi.

Bulgular: Zorlu diürez ve günde 8 litreye varan hiperhidrasyon tedavisi ile trombosit sayÕsÕnÕ 30.000/mm3 üzerinde tutacak ýekilde trombosit transfüzyonlarÕ aúÕr hemorajik sistit olan olgularÕn 10’unda yeterli oldu. Alternatif tedavi yaklaýÕmlarÕ olarak mesane içinin formalin ve prostaglandin F2 alpha ile yÕkanmasÕ ile hiperbarik oksijen tedavisi kullanÕldÕ. Bu tedavilerden en yararlÕsÕnÕn hiperbarik oksijen olduúu gözlendi.

Sonuç: Bu nedenle özellikle hematopoetik kök hücre nakilleri sonrasÕ geliýen inatçÕ hemorajik sistit olgularÕnÕn tedavisinde hiperbarik oksijen uygulamasÕnÕn önemli bir tedavi yaklaýÕmÕ olduúunu düýünüyoruz. (Turk J Hematol 2009; 2009; 26: 176-80) Anahtar kelimeler: Hiperbarik oksijen, hemorajik sistit, kök hücre nakli Geliý tarihi: 17 Mart 2009

Kabul tarihi: 1 Eylül 2009

Introduction Hemorrhagic cystitis (HC) is a frequent complication of hematopoietic stem cell transplantation (HSCT). The reported frequency changes mainly with the definition (according to inclusion or exclusion of microscopic hematuria) and preventive manipulations. It may occur in the early or late posttransplant period and can promote sometimes severe morbidity. Several risk factors are reported to date, which include drugs, chemical toxins, infection, and pelvic irradiation [1,2]. It is mostly selflimited, and supportive treatment with hyperhydration and increased transfusion can be sufficient [3]. To facilitate the urinary flow, placement of urinary catheter is sometimes needed for intermittent or continuous bladder irrigation.

Instillation of agents such as alum, silver nitrate, prostaglandin (PG) or formalin is the therapeutic intervention used to halt hemorrhage in severe HC [4-7].

Such agents are cumbersome for both patients and physicians and are seldom selected. Hyperbaric oxygen (HBO) therapy is a relatively new treatment modality for HC. The role of HBO therapy in HC was reported firstly in radiation-induced cystitis, but in recent years HBO has also been used in cyclophosphamide (CY)-induced HC [8-13]. It decreases tissue edema and ensures the necessary oxygen gradients required to stimulate continued angiogenesis, fibroblast proliferation, collagen formation, and leukocyte activation, which are required for tissue healing and repair [9]. We analyzed our data regarding HC in allogeneic (allo)HSCT patients in order to establish the efficacy of HBO therapy in severe HC and to document the main problems occurring during its use.

Materials and Methods Between March 1993 and August 2006, 161 patients underwent alloHSCT (bone marrow transplantation (BMT): n=91; peripheral blood (PB) stem cell transplantation: n=70).

All of the patients had hematological malignancies except three (aplastic anemia: n=2; paroxysmal nocturnal hemoglobinuria: n=1). The donors were all HLA-matched siblings. One hundred fifty-three of the patients received CY (60 mg/kg per day-2) as part of the conditioning regimen combined with busulfan (in 136 patients) (4 mg/kg per day divided in 4 doses-4) or total

body irradiation (TBI) (in 17 patients) (1200 cGy). Mesna (6 mercapto-ethane sodium sulphonate, a sulfhydryl-containing compound) (1/2 of the CY total dose x 6), hyperhydration (3 L/ m2/day) and forced diuresis were used as early HC prophylaxis of CY-induced HC. The first dose of Mesna was given 30 minutes before CY infusion, and was followed by subsequent infusions every four hours until the day following the last dose of CY. Cyclosporine (CsA, 12.5 mg/kg per day divided in 2 doses) and a short-course of methotrexate (15 mg/m2 per day on day-1 and 10 mg/m2 per day on days-3,-6 and-11) were given as graft-versus-host disease (GVHD) prophylaxis. Written informed consent was obtained from all patients.

Heme dipstick testing was used as a method for hematuria investigation and was performed on a daily basis beginning with conditioning regimen until the end of hospitalization. When heme was found to be positive, the urine specimen was sent for microscopic analysis and culture. Diagnosis of HC was based on the appearance of microscopic hematuria with negative bacterial and fungal culture. HC was graded according to the following criteria: grade I, microscopic hematuria; grade II, macroscopic hematuria without clots; grade III, macroscopic hematuria with clots; and grade IV, gross hematuria and clinical complications secondary to urinary outflow obstruction.

Patients with grade III and grade IV were accepted as having severe HC. Urine culture for adenovirus could be performed in only five patients. Cytomegalovirus (CMV) antigen was screened in both PB and urine samples in all the patients with HC. We did not have the opportunity to screen the urine for BK virus in our patients with HC. When severe HC is diagnosed in our HSCT unit, forced diuresis with hyperhydration (3 L/m2 up to 8 L/day) and transfusion support to maintain a platelet count above 30x109/L are the first-line treatment methods employed. Pelvic ultrasonographic examination is also performed in those patients to determine any obstruction in the vesicle and, if present, continuous bladder irrigation at 6-7 L/day through a tri-lumen irrigation catheter is also started. If severe HC is refractory to the first-line management, we choose alternative therapies such as intravesical instillation of PG (200 micrograms in 50cc isotonic sodium chloride solution), formalin and/or HBO therapy. We could not perform alum irrigation in our patients.

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HBO therapy: Patients were referred to the HBO center. All patients received HBO therapy in a single room. They received 100% oxygen at 2.5 atmospheres chamber pressure for 120 minutes daily at least one month but continued until the symptoms subsided and bleeding completely disappeared. All calculations were performed using the SPSS software package, version 14 (SPSS Inc, Chicago, IL, USA)

Results Hemorrhagic cystitis was diagnosed in 49 of 161 (30%) HSCT recipients at a median +39 days (range: from -2 to +241 days). HC occurred in 25 of the 70 alloPBSCT group (36%) and in 24 of the 91 BMT group (26%), and the difference was not statistically significant (p=0.228).

Seventeen of the 49 HC cases (8 BMT, 9 PBSCT) were described as severe HC (>grade II). Although most of our patients with HC responded to the first-line therapies, HC affected the morbidity in a median of 12 days (range: 4- 90 days). Nine of the patients died before resolution of HC with complications of severe GVHD. Four of the HC cases (8.1%) developed in the first week of the transplantation and preparative regimens in these cases included CY and busulfan. Urine culture for adenovirus was found to be positive in one case. CMV antigenemia was also observed in six of the patients with HC. The results of all patients with HC: Forced diuresis with hyperhydration up to 8 L/day and transfusion support to maintain a platelet count above 30x109/L were sufficient in all the patients with mild HC and in eight of the 17 patients with severe HC. Cystoscopy was needed in 14 of the patients and evacuations of the hematoma were performed before increasing

the irrigation solutions up to 30-40 L/day. Vesical sarcoma was diagnosed in one case with cystoscopy and the patient underwent radical cystectomy. Alternative therapies were applied in eight of the patients. The first patient was successfully treated with intravesical formalin irrigation therapy. The next patient did not respond to formalin and then intravesical PGF2 alpha irrigation and was successfully treated with HBO 2.4 atmospheres absolute (30 minutes 3 times daily for 8 days).

The results of HBO therapy: We performed HBO as a firstchoice alternative therapy in another six patients. All results for these seven cases are summarized in Table 1. We found HBO therapy quite useful in our first four cases. We followed our patients with weekly CMV antigenemia and noted CMV reactivation in our fifth patient after three days of HBO and stopped the therapy. This patient died with multiorgan failure after three months follow-up and HC never resolved. We only used three and four days of HBO therapy, respectively, in our last two patients and they also died with severe GVHD and severe HC in a short period of two weeks.

Discussion Hemorrhagic cystitis can occur at any time after HSCT and may affect the morbidity of the procedure. The incidence of HC varies considerably according to the preparative regimen, the preventative measures employed and perhaps the incidence of GVHD. HC occurring during or shortly after the conditioning regimen of high-dose chemotherapy or chemoradiotherapy is referred to as early-onset [14]. Oxazaphosphorine drugs such as CY or ifosfamide have been widely used as preparative regimen agents. The acrolein formed as a urinary byproduct of the metabolism of these drugs is thought to be responsible for

Table 1. Clinical findings and therapy results of 7 patients with intractable hemorrhagic cystitis (HC) Case 1

Case 2

Case 3

Case 4

Case 5

Case 6

Case 7

Age, sex

42, Male

32, Female

22, Male

20, Female

26, Male

23, Female

21, Female

Disease

CML

CML

CML

CML

T-ALL

B-ALL

CML

Stem cell source

PBSC

PBSC

BMT

PBSC

BM

BM

BM

Preparative regimen

Bu/CY

Bu/CY

Bu/CY

Bu/CY

Bu/CY

Bu/CY

Bu/CY

2

-

-

22

2

GVHD grade HC grade and time

III, day +69

II, day +32

III, day +27

III, day +60

IV, day +45

IV, day +60

Alternative therapies

Intravesical

-

-

-

-

-

IV, day +32

formalin, PGF2 Duration

3 weeks

21 days

5 weeks

21 days

3 months

?

HBO response

in 8 days

in 10 days

in 14 days

in 9 days

-

-

?

35 days

40 days

60 days

40 days

3 days

3 days

4 days

HBO complication

none

none

none

none

CMV reactivation??

none

none

Last visit

Exitus

Alive

Alive

Alive

Exitus

Exitus

Exitus

(Until asymptomatic) HBO duration

CML: Chronic Myelogenous Leukemia ALL: Acute Lymphoblastic Leukemia PBSC: Peripheral Blood Stem Cell BM: Bone Marrow

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the urothelial toxicity [1]. We use Mesna and hyperhydration protocol for the prevention of CY-induced HC in our Unit. In our study, since only four out of 49 HC cases (8.1%) developed in the first week of the transplantation, we consider this to be quite effective in preventing early-onset HC. Preparative regimens of these cases were also composed of busulfan, which may have been an additional risk factor for early HC in these patients [15]. We observed an incidence of HC of 30% in our study, which is comparable with the other reports (1,16).

We analyzed our patients to see if stem cell source had any effect on this complication, but were unable to determine any statistically significant difference with respect to incidence or severity of the HC (p=0.228 and p=0.445, respectively).

Hemorrhagic cystitis cases occurring weeks to months after HSCT are referred to as late-onset HC. Early HC is one of the recognized risk factors for late HC, but significant proportions of the late HC cases are reported to be associated with viruria and/or GVHD. The viruses most frequently implicated in HC are BK polyomavirus and adenovirus type 11 [16-18]. HC is generally self-limited and usually resolves in a few days to weeks without any sequelae. Occasionally, intermittent or persistent hematuria may be severe enough to require frequent transfusions. In more severe cases, cystoscopy should be performed to determine any problems with the vesical mucosa and to remove blood clots, followed by continuous bladder irrigation [19]. We diagnosed vesical sarcoma in one of our patients who required cystoscopy. Eight of the severe HC cases in our study failed to respond to standard management. Formalin and PGF2a were chosen for intravesical instillation in addition to bladder irrigation and platelet transfusions in two patients, respectively. Formalin was effective in controlling bleeding but PGF2 was not, which was likely due to insufficient doses because of the limited supply of this drug in our country [20]. Our first patient became asymptomatic in eight days with HBO therapy. We used this treatment modality in all seven cases with intractable HC. We used HBO therapy without any other alternative therapies in our other three responding patients, who became asymptomatic in 10, 14 and 9 days of HBO, respectively. We used HBO until microscopic hematuria disappeared in those patients. We followed our patients with weekly CMV antigenemia and determined CMV reactivation in our fifth patient after three days of HBO therapy. Although it might have been a coincidental finding in such a patient with severe GVHD, we thought HBO therapy might have facilitated the reactivation of GVHD and stopped the therapy despite persistent HC in that patient. We were unable to use HBO effectively in our last two patients because of severe CMV infection and graft failure symptoms and death was anticipated. We did not want to accept these cases as HBO- unresponsive because of the very short duration of the therapy.

Clinical and experimental studies suggest that HBO could be useful in preventing chemotherapy-induced HC [21-23].

HBO is considered as an adjunctive treatment to medical and surgical care. HBO induces the healing of tissue damage,

179

decreases edema and promotes capillary angiogenesis by increasing tissue oxygen levels. Plafki et al. [24] reviewed complications and side effects in 782 patients treated for various indications, with a total of 11,376 HBO therapy sessions, and summarized that the predominant complication is related with pressure equalization problems within the middle ear. We did not observe any complication directly related with HBO therapy. We did observe CMV reactivation coincidentally during HBO therapy, and even though we could not determine any deleterious effect such as viral reactivation, we stopped the therapy. There are some reports speculating that HBO therapy has an antiviral effect in hepatitis B and human immunodeficiency virus infection [25,26]. We conclude that moder ately severe HC can be treated conservatively in most patients. When hyperhydration, transfusion support and intravesical irrigation fail to treat HC, addition of HBO therapy may be beneficial, and HBO treatment should be considered without further delay.

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.

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Seber A, Shu XO, Defor T, Sencer S, Ramsay N. Risk factors for severe hemorrhagic cystitis following BMT. Bone Marrow Transplant 1999;23:35-40.

2. Sencer SF, Haake RJ, Weisdorf DJ. Hemorrhagic cystitis after bone marrow transplantation. Risk factors and complications.

Transplantation 1993;56:875-9.

3. Hows JM, Mehta A, Ward L, Woods K, Perez R, Gordon MY, Gordon-Smith EC. Comparison of mesna with forced diuresis to prevent cyclophosphamide induced haemorrhagic cystitis in marrow transplantation: a prospective randomised study. Br J Cancer 1984;50:753-6.

4. Kumar AP, Wrenn EL Jr, Jayalakshmamma B, Conrad L, Quinn P, Cox C. Silver nitrate irrigation to control bladder hemorrhage in children receiving cancer therapy. J Urol 1976;116:85-6.

5. Levine LA, Kranc DM. Evaluation of carboprost tromethamine in the treatment of cyclophosphamide-induced hemorrhagic cystitis.

Cancer 1990;66:242-5.

6. Mukamel E, Lupu A, deKernion JB. Alum irrigation for severe bladder hemorrhage. J Urol 1986;135:784-5.

7. Shrom SH, Donaldson MH, Duckett JW, Wein AJ. Formalin treatment for intractable hemorrhagic cystitis: a review of the literature with 16 additional cases. Cancer 1976;38:1785-9.

8. Bevers RF, Bakker DJ, Kurth KH. Hyperbaric oxygen treatment for haemorrhagic radiation cystitis. Lancet 1995;346:803-5.

9. Capelli-Schellpfeffer M, Gerber GS. The use of hyperbaric oxygen in urology. J Urol 1999;162:647-54.

10. Hughes AJ, Schwarer AP, Millar IL. Hyperbaric oxygen in the treatment of refractory haemorrhagic cystitis. Bone Marrow Transplant 1998;22:585-6.

11. Kalayoglu-Besisik S, Abdul-Rahman IS, Erer B, Yenerel MN, Oguz FS, Tunc M, Sargin D. Outcome after hyperbaric oxygen treatment for cyclophosphamide-induced refractory hemorrhagic cystitis. J.Urol 2003;170:922.

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12. Shameem IA, Shimabukuro T, Shirataki S, Yamamoto N, Maekawa T, Naito K. Hyperbaric oxygen therapy for control of intractable cyclophosphamide-induced hemorrhagic cystitis. Eur Urol 1992;22:263-4.

13. Weiss JP, Mattei DM, Neville EC, Hanno PM. Primary treatment of radiation-induced hemorrhagic cystitis with hyperbaric oxygen: 10-year experience. J Urol 1994;151:1514-7.

14. Grigg A. Hemorrhagic cystitis. In: Atkinson K, editor. Clinical Bone Marrow and Blood Stem Cell Transplantation. Cambridge: Cambridge University Press, 1993: 372-7.

15. Morgan M, Dodds A, Atkinson K, Szer J, Downs K, Biggs J. The toxicity of busulphan and cyclophosphamide as the preparative regimen for bone marrow transplantation. Br J Haematol 1991;77:529-34.

16. Bedi A, Miller CB, Hanson JL, Goodman S, Ambinder RF, Charache P, Arthur RR, Jones RJ. Association of BK virus with failure of prophylaxis against hemorrhagic cystitis following bone marrow transplantation. J Clin Oncol 1995;13:1103-9.

17. Childs R, Sanchez C, Engler H, Preuss J, Rosenfeld S, Dunbar C, van Rhee F, Plante M, Phang S, Barrett AJ. High incidence of adeno- and polyomavirus-induced hemorrhagic cystitis in bone marrow allotransplantation for hematological malignancy following T cell depletion and cyclosporine. Bone Marrow Transplant 1998;22:889-93.

18. Miyamura K, Takeyama K, Kojima S, Minami S, Matsuyama K, Morishima Y, Kodera Y. Hemorrhagic cystitis associated with urinary excretion of adenovirus type 11 following allogeneic bone marrow transplantation. Bone Marrow Transplant 1989;4:533-5.

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19. Kimura M, Tomita Y, Morishita H, Takahashi K. Presence of mucosal change in the urinary bladder in nonhematuric patients with long-term exposure and/or accumulating high-dose cyclophosphamide. Possible significance of follow-up cystoscopy on preventing development of cyclophosphamide-induced hemorrhagic cystitis. Urol Int 1998;61:8-11.

20. Shurafa M, Shumaker E, Cronin S. Prostaglandin F2-alpha bladder irrigation for control of intractable cyclophosphamideinduced hemorrhagic cystitis. J Urol 1987;137:1230-1.

21. Hughes MJ, Davis FM, Mark SD, Spearing RL. Hyperbaric oxygen for cyclophosphamide-induced cystitis. Br J Haematol 2002;119:575.

22. Oztas E, Korkmaz A, Oter S, Topal T. Hyperbaric oxygen treatment time for cyclophosphamide induced cystitis in rats.

Undersea Hyperb Med 2004;31:211-6.

23. Hader JE, Marzella L, Myers RA, Jacobs SC, Naslund MJ. Hyperbaric oxygen treatment for experimental cyclophosphamideinduced hemorrhagic cystitis. J Urol 1993;149:1617-21.

24. Plafki C, Peters P, Almeling M, Welslau W, Busch R. Complications and side effects of hyperbaric oxygen therapy. Aviat Space Environ Med 2000;71:119-24.

25. Gabrilovich DI, Musarov AL, Zmyzgova AV, Shalygina NB. The use of hyperbaric oxygenation in treating viral hepatitis B and the reaction of the blood leukocytes. Ter Arkh 1990;62:82-6.

26. Reillo MR, Altieri RJ. HIV antiviral effects of hyperbaric oxygen therapy. J Assoc Nurses AIDS Care 1996;7.

Research Article

181

The effects of low electromagnetic field and lead acetate combination on some hemato-biochemical and immunotoxicological parameters in mice Farelerdeki bazÕ hemato-biyokimyasal ve immunotoksikolojik parametrelerde düýük elektromanyetik alan ile kurýun asetat kombinasyonunun etkileri Mohamed Abd El-azim Hashem1, Nabela Imam El-sharkawy2 1Department 2Department

of Clinical Pathology, Faculty of Vet. Medicine, Zagazig University, El-Zeraa Street, Sharkia Province, Egypt of Forensic Medicine and Toxicology, Faculty of Vet. Medicine, Zagazig University, El-Zeraa Street, Sharkia Province, Egypt

Abstract Objective: The present study was carried out to investigate the potential effects of extremely low-frequency electromagnetic fields (ELF-EMF) and lead acetate on some hemato-biochemical, immune and pathologic variables in mice.

Material and Methods: A total of 90 female mice were equally divided into six groups. (Gp. 1) kept as control, (Gp. 2) exposed to EMF of 2 millitesla (mT) intensity and 50 Hz frequency (4h/day) for 30 days, (Gps. 3 and 4) were administered lead acetate orally at doses 1 and 5 mg/kg BW, respectively for 30 days. The last 2 groups (5, 6) were exposed to EMFlead combination for the same period.

Results: EMF exposure induced a significant increase in RBCs (p<0.001), WBCs (p<0.01) and platelets (p<0.001) counts, compared to control. However, anemia and leukopenia were recorded with oral administration of Pb acetate. The phagocytosis % and phagocytic index were significantly (p<0.05) increased in mice exposed to EMF for 30 days, but decreased (p<0.01) in the animals given the highest dose of lead. Comparing to unexposed mice, significant variation in biochemical parameters (glucose, enzymes, and protein profiles) were noticed. Combined lead and EMF treatments had antagonizing effect on some previous parameters, whereas mice given the highest dose of lead with EMF aggravated hemato-biochemical and pathological findings.

Conclusion: We concluded that combined exposure to ELF-EMF and Pb acetate produced significant changes in the hemato-biochemical and immune parameters which were both real and inconsistent. (Turk J Hematol 2009; 26: 181-9) Key words: Low electromagnetic field, lead acetate, hemato-biochemical parameters, phagocytic index Received: July 19, 2009

Accepted: September 1, 2009

Özet Amaç: Mevcut çalÕýma farelerdeki bazÕ hemato-biyokimyasal, immun ve patolojik deúiýkenler üzerinde aýÕrÕ düýük frekansta elektromanyetik alanlarÕn (ELF-EMF) ve kurýun asetatÕn potansiyel etkilerini araýtÕrmak amacÕyla gerçekleýtirilmiýtir.

Yöntem ve Gereçler: Toplam 90 diýi fare eýit sayÕda altÕ gruba ayrÕldÕ. 1. Grup kontrol olarak tutuldu, 2. Grup 30 gün boyunca 2 militesla (mT) yoúunlukta ve 50 Hz frekansta (4 saat/gün) EMF'ye maruz bÕrakÕldÕ, 3. ve 4. Gruplara 30 gün boyunca belirtilen sÕrayla oral olarak 1 ve 5 mg/kg BW dozlarda kurýun asetat verildi. Son 2 grup (5, 6) aynÕ süre boyunca EMF-kurýun kombinasyonuna maruz bÕrakÕldÕ.

Bulgular: EMF maruz kalÕm kontrol ile kÕyaslandÕúÕnda eritrosit’lerde (p<0.001), lökosit’lerde (p<0.01) ve trombositlerde (p<0.001) sayÕmlarÕnda önemli bir düýüý görüldü. Ancak Pb asetatÕn oral tatbiki ile anemi ve lökopeni kaydedildi. Fagositoz Address for Correspondence: Assoc.Prof. Mohamed Abd El-azim Hashem, 44511 Zagazig, Egypt Phone: 0020552273680 E-mail: mhashem_vet@yahoo.com

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yüzdesi ve fagositik indeksi 30 gün boyunca EMF’ye maruz bÕrakÕlan farelerde önemli ölçüde (p<0.05) yükseldi fakat yüksek dozlarda kurýun verilenlerde düýtü (p<0.01). Maruz bÕrakÕlmayan farelerle kÕyaslandÕúÕnda biyokimyasal parametrelerde (glukoz, enzimler ve protein profilleri) önemli deúiýiklik fark edildi. Kombine kurýun ve EMF tedavileri daha önceki bazÕ parametreler üzerinde antagonize edici etkiye sahipti, oysa EMF ile birlikte en yüksek dozda kurýun verilen fareler hematobiyokimyasal ve patolojik bulgularÕ ýiddetlendirdi.

Sonuç: ELF-EMF ve Pb asetatÕn birlikte maruziyeti hemato-biyokimyasal ve immun parametrelerde gerçek ama tutarsÕz aúÕr deúiýiklikler üretti. (Turk J Hematol 2009; 26: 181-9) Anahtar kelimeler: Düýük frekans elektromanyetik alan, kurýun asetat, hemato-biyokimyasal parametre, fagositik indeks Geliý tarihi: 19 Temmuz 2009

Kabul tarihi: 1 Eylül 2009

Introduction In recent years, several studies have suggested possible bio-effects of magnetic fields on body systems [1]. People are exposed to ELF-EMF daily at home or at work through power lines and the constant use of appliances in every day life such as refrigerators, washing machines and kettles. These household appliances alone may generate magnetic fields of up to 4 T [2]. EMF may interfere with memory performance as there is evidence suggesting impairing effects of stressinduced corticosterone release on object recognition in rats [3] and long-term significant occupational exposure to ELF-EMF may certainly increase the risk of both Alzheimer's disease and breast cancer [4]. The key events arising from exposure to EMF may include alterations in cell membrane activity and effects on various enzyme systems [5]. Previous data showed that EMFs are of minor importance in controlling heart rate, blood pressure, and biochemical parameters [6]. Exposure of mice to static magnetic field [SMF) increased the blood urea nitrogen, glucose and creatinine concentrations [7].

Lead (Pb) is a multiple-source pollutant, well known for its toxicity, of great risk both for the environment and human health. The main target organs of lead are the hematopoietic, nervous, and renal systems; there are also reports in support of its impairment effects on the hepatic, reproductive and immune systems [8, 9]. A significant decreased RBCs counts, hemoglobin levels and hematocrit values were reported in male and female mice given dietary lead [10]. Phagocytic cells, such as macrophages, may be used as a biomarker of immunotoxicity in wildlife studies [11]. There are only a few publications on medical examinations of workers exposed simultaneously to electromagnetic fields and chemicals [12].

Considering the lack of consensus on the biologic effects of static magnetic fields especially in combination with pollutants, this work aimed to investigate the impact of the combined exposure to EMF and Pb acetate on hematobiochemical, immunological and pathological findings in mice and to compare these with single treatment.

Material and methods Animals A total of 90 female healthy Swiss albino mice weighing 20-25 g BW (6-wk-old) were obtained from Laboratory Animal Housing, Faculty of Veterinary Medicine, Zagazig University,

Egypt. After 3 days of habituation to the laboratory environment, the animals were housed in metallic cages (50 cm length, 25 cm width and 30 cm height), with 15 mice in each cage. The mice were fed a standard pellet diet (El-Nasr Co., AbouZaabal, Cairo, Egypt), and water ad libitum. They were removed daily from their cages for cleaning the cages and renewing their food and water supply.

Exposure system

Electromagnetic field generator was designed and constructed in Biophysics Dept, Faculty of Science, Zagazig Univ., Egypt. The apparatus consists of an open box (width 100 x length 100 x height 50 cm) made of wood, painted mat gray inside.

Magnetic field chamber consisted of a parallel double walled cylindrical cage made from copper plate (2 mm thick) and was 114 cm internal diameter, 140 cm external diameter and 152 cm long (Fig. 1). The two cylinders were sealed at each end with copper to permit water flow between the two layers. A solenoid consisted of coils with 320 turns each from electrically insulated 2.2 mm copper wire were wound around the outer cylinder at equal distance. The four coils were connected in parallel to minimize the total impedance of the wire and allow a homogenous magnetic field within the chamber volume. The cylinder was grounded. A mesh from copper was used to cover both ends of the cylinder. The coils were connected to a Variac fed from the mains (220 V and 50 Hz). The magnetic field inside the chamber was measured at different locations using a hand-held Gauss/ Tesla Meter (Model 4048, F.W. Bell, Division of Bell Technologies, Orlando, FL). A probe T-4048.001 (USA) of ± 2% accuracy was used to calibrate the magnetic field. The field strength can be varied by means of Variac up to 2 mT inside the homogenous zone without an increase in the chamber temperature (± 0.5 0C).

The device was adjusted to induce extremely low frequency of 50 Hz alternating field with a high-intensity vertical magnetic field up to 2 mT (=20 Gauss). The mice cage put in the middle of the coils to get homogenous and magnetic field strength.

The cage in the EMF generator contained five mice for each exposure.

Treatments

The 90 mice were divided randomly into six groups including fifteen animals each: control group (Gp. 1) which received only water as a vehicle; EMF-exposed group (Gp. 2) which was exposed to EMF of 2 mT intensity and 50 Hz frequency, 4h/day, for 30 days; Pb acetate-treated groups

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thick paraffin sections were prepared and stained by hematoxylin and eosin (H&E.) according to [17].

Statistical analysis

The data were expressed as means ±standard errors (SE).

Differences between group means were estimated using a one-way analysis of variance (ANOVA) and the Duncan's Multiple Range Test was done for multiple comparisons using the SPSS 12.0 for Windows. Results were considered statistically significant at p<0.05.

Results Figure 1. Magnetic field exposure facility

(Gps. 3 and 4), administered Pb acetate orally at doses of 1 and 5 mg/kg BW, respectively for 30 days [13]; EMF + Pb acetate groups (Gps. 5 and 6) administered Pb acetate and exposed to EMF as Pb acetate and EMF groups. The animal experiments have been approved by the Committee of Animal Experimental Ethics of the Faculty of Veterinary Medicine, Zagazig University, Egypt.

Hematological and biochemical studies

Twenty-four hours following the last magnetic exposure, Blood samples were collected from the supra-orbital venous plexus of mice into two tubes. The first tube contained dipotassium salt of EDTA as anticoagulant for RBCs, hematocrit, hemoglobin, WBCs, neutrophils, eosinophils, lymphocytes, monocytes and platelets analysis, according to [14]. Blood sample in the other tube was left for a short time to allow clotting. Clear serum samples were obtained by centrifugation at 3000 r.p.m. for 20 min. and then kept at-20°C prior to biochemical analysis. Serum levels of glucose, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using the enzymatic methods according to kit manufacturer instructions. The serum creatinine and urea were measured colorimetrically, using commercial diagnostic Kits (Human-Germany).

Immunological studies

The estimation of the humoral immune response was based upon electrophoretic analysis of serum protein fractions by polyacrylamide-gel electrophoresis (Al-Ahram Lab., Tanta, Egypt). Serum total proteins (T.P) were determined by kits supplied from Bio-Analytic, Cairo, Egypt.

The phagocytic activity of polymorphnuclear neutrophil was carried out [15] for the evaluation of the cellular immunity.

Materials used for assessment of phagocytic activity were heparinized tubes for blood collection, Hank’s solution, phosphate buffer saline, Leishman’s stain [16], Candida albicans and foetal calf serum (supplied by Animal Health Institute, Dokki, Giza, Egypt). The total number of phagocytic cells, and the phagocytes which ingested yeast cells in individual phagocytes, were determined to calculate the percentage of phagocytosis, and phagocytic index.

Pathological studies

Specimens from the liver, kidneys and spleen were collected and fixed in 10% neutral buffered formalin solution. Five-micron

Blood hematology As shown in Table 1, EMF (2 mT intensity and 50 Hz frequency) exposure,4h/day, for 30 days (Gp. 2) caused a significant (p<0.001) increase of RBCs count, Hb concentration and Ht value compared to control mice (Gp.1). Mice administered Pb acetate at doses of 1 and 5 mg/kg BW alone (Gps. 3 and 4) or in combination with EMF (Gps. 5 and 6) showed a significant (p<0.01) decrease in the erythrocyte parameters producing macrocytic hypochromic anemia.

EMF-exposed mice revealed significant (p<0.01) leukocytosis with neutrophilia, lymphocytosis and monocytosis, in comparison with the control. Total and differential leukocyte counts did not significantly differ in (Gps. 3, 5 and 6) in comparison with the values of control mice, but significantly (p<0.01) decreased in groups 4. However, eosinophil count showed insignificant change in all groups. EMF exposure significantly (p<0.001) increased the platelets count, but Pb administered mice (Gp. 4) decreased it (p<0.05). It showed insignificant change in mice of gps. 3, 5 and 6.

Blood chemistry

As illustrated in Table 2, EMF and lead acetate-treated mice (Gps. 2 and 4) showed significant (p<0.01) higher levels of serum enzymes (ALT, AST), creatinine and urea than that of control. In contrast, significant (p<0.01) hyperglycemia (Gp. 2) and hypoglycemia (Gp. 4) were recorded, when compared with control mice. Insignificant change in these parameters was observed in (Gp. 3). Compared with the control mice, exposure to EMF- Pb acetate combinations (Gps. 5 and 6) resulted in the same findings for ALT, AST, creatinine and urea, while serum glucose showed insignificant change.

Immunological results

As shown in Table 3, EMF exposure of mice during 30 consecutive days significantly increased the serum total protein levels (p<0.05), gamma (p<0.01), and total globulins (p<0.01) compared to control group. The serum total proteins, albumin, and globulin fractions were significantly (p<0.01) decreased in mice given the highest dose of Pb acetate (5 mg/kg BW), but they showed no statistical change with the lowest one (1 mg/ kg BW). Combined EMF- Pb acetate treatments produced insignificant change.

As indicated in Table 4 and Figures 2-7, the percentage of phagocytosis and phagocytic index were significantly (p<0.001) increased in EMF exposed mice for 30 days (Gp. 2), while

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Table 1. Hematological parameters (Mean values ± SE) in different mice groups Groups Parameters

Gp. 1

Gp. 2

Gp. 3

Gp. 4

Gp. 5

Gp. 6

RBC (x106/ l)

7.22B

8.52A

6.12C

5.32D

7.05B

6.42C

±0.42

±0.16

±1.22

±0.66

±1.02

±0.86

Hb (g/dl)

11.50B

13.20A

9.45C

9.20C

10.95B

10.50B

±2.18

±1.26

±9.98

±4.23

±9.98

±6.05

Ht (%)

29.80B

38.00A

26.50C

25.00C

31.80B

28.55B

±2.54

±1.88

±3.40

±4.14

±1.55

±3.00

MCV (fl)

MCH (pg)

MCHC (%)

WBC (x103/ l)

Neutrophil (x103/ l)

Eosinophil (x103/ l)

Lymphocyte (x103/ l)

Monocyte (x103/ l)

Platelet (x103/ l)

41.3C

44.60

B

B

44.00

46.99

B

45.11

B

44.47

B

±1.08

±1.41

±0.90

±1.12

±1.50

±1.71

15.80B

15.50B

15.44B

17.29AB

15.53B

16.36B

± 0.90

±0.56

± 1.11

± 0.50

± 0.43

±0.56

35.20B

36.80B

34.43B

36.77B

38.40A

34.74

B

± 3.91

±2.44

± 4.50

± 8.91

± 1.43

±3.71

10.30B

14.61A

10.10B

8.32C

10.11B

10.08B

± 0.19

±0.87

± 1.19

± 1.11

± 1.05

±0.87

5.20B

7.42A

5.18B

4.28C

5.13B

5.00B

± 6.07

± 9.00

± 7.19

± 5.17

± 7.00

± 8.45

0.41A

0.42A

0.40A

0.41A

0.40A

0.41A

± 0.04

± 0.10

± 0.03

± 0.02

± 0.09

± 0.12

3.77B

5.39

A

3.8B

2.80C

3.70B

3.69B

± 3.87

± 3.70

± 1.84

± 1.11

± 4.05

± 5.92

0.93B

1.36

A

0.92B

0.60C

0.94B

0.92B

± 0.05

± 0.07

± 0.06

± 0.02

± 0.05

± 0.04

536.00B

707.20A

534.50B

350.40D

514.66B

498.90BC

±86.90

±55.17

±102.10

±92.10

±70.10

±100.17

Note: Values are expressed as means ±SE; n = 15 for each treatment group. ABCDE Within rows, means with different superscript letters differ significantly (P < 0.05)

Table 2. Serum biochemical changes (Mean values ± SE) in different mice groups Groups Parameters

Gp. 1

Glucose

102.00B

(mg/dl)

± 3.19

ALT (U/L)

18.35

E

±1.05 AST (U/L)

Creatinine (mg/dl)

23.83

E

(mg/dl)

Gp. 3 A

122.00 ±2.55 59.66

C

±2.11 60.01

C

B

97.00

± 2.19 19.30

E

±1.02 25.45

E

Gp. 4 75.00

C

± 2.33 44.62

D

±2.04 49.85

D

±3.14

±4.52

±2.09

±3.22

D

C

D

C

1.07

±0.08 Urea

Gp. 2

35.57

D

±1.93

1.99

±0.15 79.44

B

±4.14

1.05

±0.06 35.99

D

±1.66

1.97

±0.13 66.37

C

±3.12

Gp. 5 110.80AB ±3.11 68.33

C

±2.9 73.00

BC

±0.14 85.57

B

94.90

±1.28 B

84.35

±3.22 B

±4.13 2.11

Gp. 6

B

±4.11

A

85.83

±4.35 2.27

B

±0.12 105.34

A

±4.14

Note: Values are expressed as means±SE; n = 15 for each treatment group. ABCDE Within rows, means with different superscript letters differ significantly (P < 0.05)

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Table 3. Proteinogram (Mean values ± SE) in different mice groups Groups Parameters

Gp. 1

Total protein (g/dl)

6.10

B

Gp. 2 6.90

A

Gp. 3 6.32

B

Gp. 4 4.12

C

Gp. 5 5.99

B

±1.42

±1.50

±1.22

±1.03

±1.05

A

A

A

B

A

3.04

3.00

1.95

2.60

Albumin

3.10

(g/dl)

± 0.22

± 0.19

± 0.14

± 0.06

± 0.12

A

A

A

B

A

_1-Globulins (g/dl)

_2-Globulins (g/dl)

0.42

0.45

0.35

0.45

± 0.19

± 0.12

± 0.11

± 0.19

± 0.15

A

A

A

C

A

0.61

0.62

0.61

0.34

±0.11

±0.13

±0.05

±0.11

A

A

A

B

`-Globulins (g/dl)

1.14

a-Globulins (g/dl)

1.19

±0.50 B

±0.50 Total globulins (g/dl)

0.44

3.00

B

±1.00

1.09

±0.71 1.85

A

±0.65 3.80

A

±1.10

1.12

±0.80 1.21

B

±0.23 3.40

B

±1.03

0.85

±0.02 0.64

C

±0.45 2.47

C

±0.50

0.60

±0.13 0.99

AB

±0.06 1.23

B

±0.43 3.29

B

±1.14

Gp. 6 5.90

B

±1.12 2.60

A

± 0.10 0.44

A

± 0.07 0.55

AB

±0.09 1.06

A

±0.25 1.11

B

±0.36 3.30

B

±1.16

Note: Values are expressed as means±SE; n = 15 for each treatment group. ABC Within rows, means with different superscript letters differ significantly (P < 0.05)

Pathological findings

Figure 2. Phagocytosis of C. albicans by neutrophils of control mice (gp.1). One cell of C. albicans was engulfed by neutrophils (arrow), Leishman’s stain, X100.

Figures 3-4. Phagocytosis of C. albicans by neutrophils of EMF exposed mice (gp.2). One and two C. albicans engulfed by neutrophils were represented by arrow (Fig. 2) and arrow-head (Fig. 3) respectively, Leishman’s stain, X100.

Figures 5. Phagocytosis of C. albicans by neutrophils of mice administered lead acetate (gp.4). One C. albicans was engulfed by neutrophils (arrow), Leishman’s stain, X100.

Figures 6. Phagocytosis of C. albicans by neutrophils of mice administered lead acetate (gp.5) showing two cells of C. albicans were engulfed by neutrophils (arrow) and one cell attached to the surface of neutrophil (arrow-head), Leishman’s stain, X100.

Figures 7. Phagocytosis of C. albicans by neutrophils of mice administered lead acetate (gp.5) showing two cells of C. albicans were engulfed by neutrophils (A) and one cell attached to the surface of neutrophil (B) and/ or engulfed (arrow), Leishman’s stain, X100.

administration of Pb acetate exerted a significant (p<0.01) decrease (Gp. 4), in comparison with (Gp. 1). These tests were insignificantly changed in mice of groups 3 and 6, but increased significantly (p<0.05) in mice of (Gp. 5).

The liver, kidney and spleen sections from a control animal showing normal parenchyma (Figs. 8, 9 and 10) respectively.

The liver from exposed mice to EMF (Gp. 2) showed focal centrolobular necrosis of the hepatic cells surrounded by severe hydropic degeneration involving the majority of hepatic parenchyma (Fig. 11). The kidneys of EMF exposed mice showed congestion of renal blood vessels, contracted glomerular tufts of some glomeruli and focal leukocyte aggregation (Fig. 12). An area of coagulative necrosis infiltrated with few lymphocytes and plasma cells in the renal tubules was seen. Some splenic white pulps of spleen suffered from lymphoid depletion and the others became hyperplastic with proliferation of megakaryocytes, beside hemosiderosis in red and white pulps (Fig. 13). Mice given 1mg/kg BW of Pb acetate (Gp. 3) showed mild lesions in comparison with Pb acetate group given the high dose. The liver sections from mice given 5mg/kg BW of Pb acetate (Gp. 4) showed congestion of blood vessels, portal leukocyte infiltration and hydropic degeneration (Figs. 14), beside focal necrotic area invaded with lymphocytes in the hepatic parenchyma. Kidneys revealed large area of necrosis in the renal cortex and focal replacement of some renal tubules by lymphocytes and erythrocytes. Kidneys revealed focal replacement of renal parenchyma by lymphocytes (Fig. 15). Spleen showed the same lesion of (Gp. 2). In addition, few hemosiderin granules were scattered in the white and red pulps. The spleen showed severe lymphoid depletion, rudimentary white pulps, and hemosiderosis in red and white pulps, beside thickened splenic trabeculae (Fig. 16). Mice exposed to a combination of lead and EMF (Gps. 5 and 6)

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showed more severe lesions than previous groups. Liver (Gp. 6) showed portal and interstitial lymphocyte aggregations, hyperplastic Kuffer cells, hydropic degeneration, hyperchromatic nuclei and disorganized hepatic cords, (Fig. 17). Periglomerular lymphocytic infiltrations with individual coagulative necrosis were noticed in the renal tubules (Fig. 18).

Discussion For several decades, researchers have been concerning about the bioeffects of low-frequency, low-intensity EMFs, which are comparable to both residential and occupational exposure levels in many work fields [18]. The choice of 2 mT intensity/50 Hz magnetic fields which they are below ICNIRP standard [19] and since they are to a certain degree realistic in terms of human and animal exposure.

Our data demonstrated that 30 days (4h/day) of MF exposure was associated with an increase in the count of RBCs, platelets, Hb content, Ht values and leukocyte count with neutrophilia, lymphocytosis and monocytosis, compared to control. Previous study reported that the increase in Hb and RBCs may be explained by the hypoxia-like status induced by static MF resulting probably from the oxygen-binding impairment of Hb [6]. By contrast, hematocrit remained unchanged [20]. The differences of hematocrit values may be related to duration (number of h/day) of EMF- exposure. The same results were observed in rats after exposure to SMF of 128 mT for 1h/day during 30 consecutive days [21]. Contrarily, mice exposed to magnetic wave field of 60 Hz with a 0.11-mT intensity showed leukopenia and hemoglobin decrease [22].

They added that the bioeffects described could be correlated with spleen hyperfunction, which could have been produced by chronic exposure to EMF. Besides, rats exposed to low EMF reveal a decrease in platelets count [23]. Similarly, no EMF effects on the hematologic variables of rats exposed to 20 kHz MF at 6.25 microT, 8h/day, for 12 or 18 months [24].This discrepancy may be explained by the intensity and the duration of the exposure, as well as, type of experimental animal.

Lead is one of the most common toxic metals. Pb acetate administration alone or in combination with EMF produced macrocytic hypochromic anemia, associated with increased MCV and decreased MCHC. It was reported that anemia following Pb acetate poisoning is in part the result of various inhibitory effects of lead on heme biosynthesis [25]. Besides, excessive lead exposure inhibits heme synthesis, leading to anemia and erythrocytes degeneration or destruction [26].

Lead may inhibit the bodyâ&#x20AC;&#x2122;s ability to make hemoglobin by interfering with several enzymatic steps in the heme pathway, through inhibiting aminolevulinic acid dehydratase and ferrochelatase activity [27]. Our findings were in coincidence with other reporters [9,10,28]. Dose dependent changes in total and differential WBCs, as well as platelets counts were detected in Pb acetate alone or with EMF-treatment groups.

Leucopenia, neutropenia, lymphopenia, monocytopenia and thrombocytopenia were noticed in 5 mg/kg Pb acetate treated animals. On the other hand, injection of Pb acetate for 4 days was shown to stimulate a striking leukocytosis in female mice with an increase of monocytes and neutrophils [29]. In addition,

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insignificant change in hematological and enzymatic parameters were reported with Pb acetate given for 30 and 60 days [30].

Leukocytosis was previously reported in mice given Pb acetate orally at doses 1 and 10 mg/kg BW for one month [13].

Present data showed that exposure to EMF increased serum glucose level and transaminases (AST and ALT) activities. The increase in the glucose level agrees well with previous findings with EMF with different strengths [31].

Moreover, exposure to EMF had increased blood glucose and decreased insulin release, leading to a diabetic-like state in pregnant rats [6]. Formerly, hyperglycemia was explained by structural and functional changes in pancreas, in response to EMF stress [20]. Serum transaminases have been widely utilized in mammalian toxicology as biomarkers of specific organ dysfunction [6]. Magnetic field induced structural changes in hepatocytes, primarily in mitochondria [32]. In addition, a significant increase in ALT activity indicates citotoxic effect of non-ionizing radiation on hepatocytes inducing apoptosis and necrosis [33]. These results were in accordance with previous findings [7,21]. EMF- exposure increased the serum creatinine and urea concentrations. These results may be due to the renal dysfunction associated with congestion of renal blood vessels, contracted glomeral tufts of some glomeruli and focal leukocyte aggregation by pathologic examination.

Similarly, mice exposed to MFs (5 T) for 48 h increased significantly blood urea nitrogen, whereas creatinine levels remained unchanged [34]. Contrarily, subchronic exposure of rats to MF (128mT, 1h/day for 30 days) had no effect on serum creatinine and urea levels [21]. This discrepancy could be related to the difference of the intensity of the SMF and the exposure time (h/day) and duration.

Lead acetate administration (5, 10 mg lead acetate/kg, daily for 30 days) resulted in a dose- dependent increase in serum ALT, AST, creatinine, and urea; however, serum glucose was significantly decreased. Lead can cause adverse effects to hepatic cells owing to its storage in the liver after Pb acetate exposure [35]. Increases in serum enzyme activities are attributed to their release from the cells and this may be related to the tissue injury induced by lead. Moreover, an elevation of transaminases at 2-week intervals in male albino rats receiving Pb (500 mg/kg diet 3 times per week) for a period of 6 weeks was encountered [36]. Similar results were observed in albino rats orally administered 200 or 400 ppm Pb acetate [8], 1000 or 2000 ppm Pb acetate for 60 days [28] or in rabbits orally given 80 or 160 ppm Pb for 15 days [9]. The observed elevation in creatinine concentrations indicates impairment in kidney function [37]. Enhanced protein catabolism together with accelerated amino acid deamination for gluconeogenesis is probably an acceptable postulate to interpret the elevated levels of urea [28]. The latter authors added that the elevated serum urea levels may be due to the previously reported destruction of RBCs. Similarly, the increased concentrations of urea and creatinine in serum indicated a nephropathy possibly induced by lead [9,38].

Therefore, creatinine and urea could be considered as suitable prognostic indicators of renal dysfunction in case of lead exposure [39].

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Table 4. Percentage and index of phagocytosis (Mean values ± SE) in different mice groups Groups Parameters Phagocytic %

Gp. 1 43.10

C

±5.30 Phagocytic index

0.71

B

± 0.22

Gp. 2 56.90

A

±4.11 0.95

A

± 0.19

Gp. 3 43.31

C

±5.60 0.70

B

± 0.14

Note: Values are expressed as means±SE; n = 15 for each treatment group.

ABCDE

Figure 8-10. Liver, kidney and spleen sections from control mice (Gp. 1) showing normal structure respectively, H&E., x1200.

Figure 11. The liver section from mice of (Gp. 2) showed focal centrolobular necrosis of the hepatic cells surrounded by severe hydropic degeneration involving the majority of hepatic parenchyma and round cells infiltration, H&E., x1200.

Figure 12. The kidney section (Gp. 2) suffered from congestion of renal blood vessels, contracted glomerular tufts of some glomeruli and focal leukocyte aggregation, H&E., x300.

Figure 13. Spleen section (Gp. 2) showed lymphoid depletion in splenic white pulps and hemosiderosis in red and white pulps, H&E., x1200.

Figure 14. Liver section from mice given 5mg/kg BW (gp. 4) of lead acetate showed leukocyte infiltration in the portal area and hydropic degeneration, H&E., x1200.

Figure 15. Kidney section from mice given 10mg/kg BW of lead acetate (Gp. 5) revealed focal replacement of renal parenchyma by lymphocytes, H&E., x1200.

Figure 16. Spleen section from Gp. (5) showed severe lymphoid depletion, hemosiderosis in red and white pulps beside thickened splenic trabeculae, H&E., x1200.

Figure 17. Liver section of mice exposed to a combination of lead and EMF (Gp. 6) showed focal replacement of hepatic parenchyma by lymphocytes, hyperplastic Kuffer cells, hydropic degeneration, hyperchromatic nuclei and disorganized hepatic cords, H&E., x1200.

Figure 18. Kidney section from mice of Gp. (6) showed periglomerular lymphocytic infiltrations with individual coagulative necrosis, H&E., x1200.

Concerning the effect of EMF on immune parameters, present data showed that EMF exposure increased serum total protein levels, gamma, and total globulins, phagocytosis % and phagocytic index in mice. This hyperproteinemia may be attributed to the change in protein metabolism of stressed mice

Gp. 4 36.20 D ±3.82 0.65

C

± 0.17

Gp. 5 50.20

B

±6.12 0.85

A

± 0.12

Gp. 6 43.90

C

±4.72 0.75

B

± 0.13

Within rows, means with different superscript letters differ significantly (P < 0.05)

or the increase in the globulin component [20,40]. On the contrary, a significant decrease in the levels of total protein, `and a-globulins in steelworkers exposed to EMF (1.3mT intensity and 50 Hz frequency, mean 6.8h /day) for 5days was reported [7]. Significant decrease in blood levels of total protein, albumin and `-globulin were observed in rats exposed to EMF, 24 h/ day for 8 weeks [41]. This discrepancy could be attributed to the difference of the intensity of the EMF and the exposure scenario and duration. The increased phagocytosis % and phagocytic index in mice with EMF exposure indicated that the role of electromagnetic field is prevalent in the formation of effects of the intensity and completeness of phagocytosis [42]. There is no generally accepted mechanism to explain how extremely low frequency fields might initiate bioeffects, if any, on immune system [43]. In other way, some reports indicate that acute exposure to a 50 Hz magnetic field (10 microT, for 24 h) has no effect on hematological or immune parameters in healthy male volunteers [44]. The present results were supported by the pathologic lesions in the liver (Fig. 11) and kidney (Fig. 12).

Similarly, experimental exposition of mice by mobile telephones showed a slightly increased number of micronuclei and discrete perivenular fatty changes in liver [45]. Highly cytoplasmic vacuolation of liver and swelling of epithelial cells of kidney tubules with subsequently cell necrosis were shown in two groups of guinea pigs exposed to EMFs of 0.013 T or 0.207 T with 50Hz frequency, 2 or 4hours daily for 5 days [46].

Interestingly, decreased in serum proteins, albumin, and globulins may be attributed to hepatotoxicity or renal toxicity resulting from the highest dose of lead acetate. Contrarily, low dose of Pb acetate (1 mg/kg) produced insignificant change.

Experiments conducted in male rats exposed to 200 ppm dietary Pb acetate for 10 weeks showed normal serum total proteins and albumin [47]. However, blood lead > or = 25 microg/dL caused a significant decrease in immunoglobulins (IgG, IgM, IgA) with dysfunctions in different organ systems of the body, such as the immune system [48].

Observations of suppression of phagocytic activity neutrophil in relationship to Pb acetate exposure were in parallel with others [49]. The former authors mentioned that occupational exposure to lead interfered with humoral and especially cell-mediated immunity even at frequently occurring (<50 microg/dl) blood lead levels and caused a primary impairment of the chemotactic and phagocytic activities of neutrophil leucocytes. Similarly, a significant decrease in phagocytic activity and the number of peripheral leukocytes in

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Pb (35 mg/kg)- administered mice [50] was shown. Phagocytic and lytic functions of the polymorphonuclear cells collected from sublethal lead-injected toads and incubated with suspensions of Candida pseudotropicalis [51] were affected negatively.

The combined effects of EMF and of Pb acetate were found to potentiate the toxic effects. Similarly, the combined effects of EMF (300 T to 0.3T) and of chlorpyrifos at various concentrations (0.1,1, 50 and 100mg/kg) were found to potentiate the toxic properties of the pesticide [42].

The most common and constant findings was a portal leukocyte infiltration, hydropic degeneration and loss of normal architexture in the liver. Light microscopy of kidney revealed focal replacement of renal parenchyma by lymphocytes and coagulative necrosis. These findings were dose-dependent.

Similar histopathological lesions have been reported in experimental lead toxicity with different species [9,52-54].

In conclusion, several experiments are still necessary with the purpose of explaining which frequency, intensity, exposure time and other parameters involved with EMF, especially concurrent with environmental pollutants to protect ourselves from that harms.

Acknowledgments The author thanks Dr.Abd El-Moneim Aly, Prof. of Pathology, Faculty of Veterinary Medicine for their assistance in the histopathology, and Dr.Magda Hanafy, Prof. of physics, Faculty of Science, Zagazig University, Egypt for supplying electromagnetic field generator.

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.

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Research Article

190

Invasive fungal diseases in children with hematologic disorders Hematolojik hastalÕklÕ çocuklarda invazif fungal infeksiyonu

Birol Baytan1, Adalet Meral Güneý1, Solmaz Çelebi2, Ünsal Günay1 1Department 2Department

of Pediatric Hematology, Uludaú University, Medical Faculty, Bursa, Turkey of Pediatric Infectious Diseases, Uludaú University, Medical Faculty, Bursa, Turkey

Abstract Objective: Fungal infection is a significant problem, causing of infective deaths of leukemic patients. The situation in developing countries is not well documented. The purpose of this study was characterizing IFD by analyzing data retrospectively to determine the incidence, predisposing factors, diagnostic methods, efficacy of treatment, and the outcome in pediatric patients with hematological disorders.

Materials and Methods: There were 160 children with leukemia (22 AML, 129 ALL) and 9 with aplastic anemia (AA). The diagnostic criteria for IFD were defined according to the EORTC/MSG, 2008. IFD was classified as proven or probable.

Empiric antifungal treatment with L-AmB was commenced by day 5-7 of persistent fever. Patients with invasive aspergillosis (IA) who were refractory to primary treatment were commenced on voriconazole (VCZ). Salvage therapy as combination of VCZ and caspofungin was given to those with progressive infection.

Results: The incidence of IFD was found 23 (14.3%). 19 with leukemia (14 ALL, 5 AML) and 4 with aplastic anemia were diagnosed as IFD. IA was the dominant cause of infection (n=17) and the rest (n: 6) had candidiasis. Ten children had “proven” infection and 13 children were defined as “probable”. The most frequent site of infection was lungs. In our series, the most frequently used diagnostic methods were clinical findings (100%) and radiologic methods (84%). The success rate of treatment for candidiasis and IA were found 60%, 71% respectively. IFD related death rate was found 30%.

Conclusion: IFD is still a major morbidity and mortality reason in children with hematologic disorders. However, the availability of new antifungal treatments and diagnostic tests will improve the survival rates in these children.

(Turk J Hematol 2009; 26: 190-6) Key words: Hematologic disorders, invasive fungal infection, children Received: June 4, 2009

Accepted: October 13, 2009

Özet Amaç: Fungal infeksiyonlar lösemili hastalarda infeksiyonlardan ölümlerde önemli bir sorundur. Bu sorun geliýmekte olan ülkelerde iyi dökümente edilmemiýtir. Bu çalÕýmanÕn amacÕ, geriye dönük olarak çocuk hastalarda görülen hematolojik hastalÕklardaki invazif fungal hastalÕk (ûFH) insidansÕnÕ, predispozan faktörleri, tanÕ metodlarÕ, tedavi etkiliúi ve sonuçlarÕnÕ incelemektir.

Address for Correspondence: M.D. Birol Baytan, Uludag University, Children's Hematology Field 16059 Bursa, Turkey Phone: +90 224 295 04 31 E-mail: baytanbirol@yahoo.com

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Invasive fungal infections

Turk J Hematol 2009; 26: 190-6

191

Yöntem ve Gereçler: Merkezimizde Ocak 2003 ve AralÕk 2008 tarihleri arasÕnda tedavi edilen 160 lösemili (22 AML, 129 ALL) ve 9 aplastik anemili olgu çalÕýmaya alÕndÕ. EORTC-MSG 2008 kriterlerine göre olgulara olasÕ veya kesin ûFH tanÕsÕ kondu. Antibiyotik tedavisine raúmen 5 günden uzun süren ateý yüksekliúi saptanan olgulara ampirik antifungal tedavi olarak L-AmB baýlandÕ. ûnvasive aspergillozis (IA) saptanan olgularda tedavi voriconazole (VCZ) deúiýtirildi. Kurtarma tedavisinde VCZ ve kaspofungin kombinasyonu kullanÕldÕ.

Bulgular: ÇalÕýmamÕzda ûFH %14,3 (n: 23) bulundu. 19 lösemili(14 ALL, 5 AML) ve 4 aplastik anemili olguda IFH tanÕsÕ kondu. IA en sÕk rastlanan enfeksiyon oldu (n: 17), diúer 6 olguda kandidiazis saptandÕ. On olguya kesin ve 13 olguya kuvvetli olasÕ enfeksiyon tanÕsÕ kondu. En sÕk tutulan enfeksiyon bölgesi akciúerdi. ÇalÕýmamÕzda, en sÕk kullanÕlan tanÕ yöntemleri; klinik bulgular (%100) ve radyolojik metodlar (%84) olarak saptandÕ. Tedavide baýarÕ oranlarÕ kandidiasis ve IA’da %60 ve %71 bulundu. ûFH ile iliýkili ölüm oranÕ %30 saptandÕ.

Sonuç: Sonuç olarak, ûFH hala hematolojik hastalÕklÕ olgularda ciddi mortalite morbitide sebebidir. Ancak yeni antifungal ilaçlar ve tanÕ yöntemleri ile yaýam oranlarÕ artmaktadÕr. (Turk J Hematol 2009; 26: 190-6) Anahtar kelimeler: Hematolojik hastalÕklar, çocuk, invazif mantar infeksiyonu Geliý tarihi: 4 Haziran 2009

Kabul tarihi: 13 Ekim 2009

Introduction Invasive fungal diseases (IFD) are a serious cause of morbidity and mortality in immunocompromised children, especially those with hematological malignancies and bone marrow failure [1-3]. The incidence has dramatically increased in recent decades [4]. Various factors account for this increased frequency. Of these factors, dose-intensive regimens causing profound neutropenia and mucosal barrier damage, widespread use of broad spectrum antibiotics are important.

Moreover, the placements of indwelling central venous catheters, hematopoietic stem cell transplantation (HSCT) render these children more vulnerable to fungal infections [5]. Although, there has been a great improvement in the survival rates of children with leukemia and other hematological disorders, IFD still remains a life-threatening problem [6,7]. The pediatric data about IFD is scarce and the situation in developing countries is not well documented. The purpose of this study, therefore, was to investigate the incidence of IFD, risk factors, diagnostic methods, efficacy of treatment and the outcome in pediatric patients with hematological disorders treated in a single center.

Methods Patients and institution There were 160 children with leukemia (22 AML, 129 ALL) and 9 with aplastic anemia admitted to the pediatric hematology department between January 2003 and December 2008.

In this study, we evaluated only the patients diagnosed as proven and probable IFD. Each patient was evaluated and those with colonization were excluded. Children with ALL and AML were treated according to ALL- BFM 95 and AML-UKMRC 12 studies, respectively [8,9].

Patients during the phase of neutropenia were allocated in one ward in separate rooms with single bed without high efficiency particulate air filtration. They had restricted access to visitors and other particular behavioral measures: such as a ban on plants or flowers and hand washing.

Diagnostic Studies

Diagnostic works-ups for IFD included collection of blood, urine cultures at the onset of fever, serum galactomannan (GM) levels twice weekly (Platelia® Aspergillus; Bio-Rad Laboratories, France). A positive result was based on two consecutive

samples with a GM index of 0.5 or above [10]. High resolution computed tomography (HRCT) on the 4-7th day of fever was performed. Additional examinations (e.g., abdominal ultrasonography, sinus or cranial computerized tomography, tissue biopsy) were performed if indicated.

Definition of IFD

We identified patients who developed proven or probable IFD using standardized definitions set by the revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) Consensus Group in 2008 [11]. According to this group, proven IFD requires histopathology, cytopathology or direct microscopic examination showing hyphal invasion or yeast cells, or a positive culture from a radiological abnormal site that is normally sterile excluding bronchoalveolar lavage fluid, a cranial sinus cavity specimen, and urine. Probable IFD is defined by mycological evidence in a patient with a host factor and clinical features.

Treatment

Empiric antifungal treatment liposomal amphotericin B (L-AmpB) 3 mg/kg/day were commenced by day 5-7 of persistent fever that did not respond to broad-spectrum antibacterial therapy. Liposomal amphotericin B (5mg- 8mg/kg/day) was given to those with non-responded of 3 mg/kg/day.

Patients with invasive aspergillus (IA) who were refractory to primary treatment based upon clinical, radiological and serological progression were commenced on voriconazole (VCZ).

Salvage therapy as combination of VCZ and caspofungin for IA were given to those with suggestive progressive signs and/or symptoms and persistent neutropenic fever.

The criteria for responses to antifungal therapy; therapeutic success was classified as complete, partial, stable and progression of fungal disease, each defined according to EORTC/ MSG [12].

Results The Incidence and Patient’s demographics: IFD was identified in 23 (n:23/160; 14.3%) cases (F/M; 12/11). The incidence in leukemic children and AA was 12.5% [14 ALL (10%), 5 AML (22%)] and 44.4% (n:4), respectively.

The mean age was 7.8±5.3 years (range 2.4-17.2 years, median age 6,4 years). For 3 patients with AA, a concomitant

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Table 1. The Clinical Symptoms of Children Clinical symptoms

n: 23 (%)

Persistent fever

23 (100%)

Cough

10 (43%)

Dyspnea

4 (17%)

Pleural pain

3 (13%)

Hemoptysis

3 (13%)

Focal neurological signs

4 (17%)

Tachypnea

7 (30%)

Headache

4 (17%)

bacterial infection was diagnosed. Klebsiella pneumonia (n:2) and Stenotrophomonas maltophilia (n:1) were isolated from their blood cultures (patients 1, 14 and 17). IFD was more frequent (n: 5/22; 22%) in AML than (n: 14/129; 10%) in ALL. Of the 19 leukemic patients, 11 were in induction therapy, 3 had bone marrow relapse receiving induction therapy and the others were in complete remission at consolidation phase. All of the children with aplastic anemia (n:4) was diagnosed as severe AA with total leukocyte count below 500/mm3. None had fanconi. Of them, 3 received immunosuppressive (antithymocyte globulin, cyclosporine and corticosteroid) therapy. The other one was on the waiting list for HSCT with no therapy. IFD rate in these children was 44.4% (n:4/9), higher than the acute leukemia group. All patientsâ&#x20AC;&#x2122; clinical symptoms were given in Table 1.

Risk Factors

All children had a compromised immune function owing to underlying hematologic diseases. Nine-teen with leukemia were receiving intensive chemotherapy when IFD occurred. Of them, 14 children with ALL had taken steroids longer than 10 days. Five leukemic children were on relapse therapy. Of the AA patients, 3 were receiving immunosuppressive therapy. All children were suffering from long-term neutropenia (<500 mm3) longer than 10 days. These children were not on fungal prophylaxis. None of them was stem cell transplant recipient and had central venous catheter. All patients were on broad spectrum antibiotics when the diagnosis was made.

Clinical, radiological and microbiological factors

Host, clinical and microbiological factors were demonstrated in Table 2. All children had persistent fever longer than 5 to 7 days. Thorax HCT findings related to IFD were determined in 15 children. The radiographic findings on thorax HRCT related to IA were shown in Table 2.

Mycological studies

Of the 10 children with proven IFD, 6 had candidemia and 4 had IA. Candida albicans was the most frequently isolated yeast from blood (n:4). The others, Candida krusei and tropicalis were detected in blood culture (patient 1, 4).

Invasive procedures for defining fungal infection could be done only in 5 children. Cranial magnetic resonance imaging (MRI) was performed in 2 cases due to abnormal neurological findings. Both showed mass lesions, suggesting tonsillary herniation. Children were taken to surgery and the masses were

resected. In other two patients, sinus CT suggested IA, and aspergillus hypes were demonstrated taken by endonasal endoscopic biopsy. Aspergillus flavus was grown in cultures of these 4 children (patients 7,16,20,21). The fifth child had BAL due suspicion of IA in thorax HRCT. Aspergillus hypes were demonstrated in the BAL sample.

Treatment

Regarding antifungal treatment, all children empirically received first line therapy with L-AmpB. Eleven out of 23 (48%) patients were treated only with L-AmB. Dose ranged between 3 and 8 mg/kg/day. In 12 children, L-AmB had to be changed due to therapy failure. Of them, two children with candidiasis were successfully treated with caspofungin. Voriconazole was given to 10 children with IA due to progressive infection. Six of them were successfully treated VCZ monotherapy. Combined antifungal treatment as VCZ and caspofungin were required in the rest (n:5) in Table 3.

Outcome

Overall mortality in this cohort was 40% (n: 9/23). Of them, the cause of death was related to IFD in 7 (30%) children and resistant leukemia in 2 cases. The underlying disorders, treatments, site of IFD and definitions of IFD, antifungal therapy in those cured and lost ones are given in Tables 3 and 4, respectively. Two children following the treatment of cerebral IA had sequels as motor mental retardation (patient 7) and convulsion (patient 13).

Discussion In our series where 12.5% of the children treated within the last 5 years for hematologic malignancies had documented IFD. The largest epidemiologic study in pediatric patients with hematologic malignancies, reported the overall 11-year incidence of fungal infections as high as 9%-10% [13]. There are two main published studies from Turkey, regarding IFD in children with cancer. Kaya Z et al. [14] reported the incidence of IFD within a 10 year period as 13.6% in 154 leukemic children receiving fluconazole prophylaxis. In another report, a high rate of fungal colonization (69.2%) was found in children receiving chemotherapy for leukemia and lymphoma [15]. In the same study, proven IFD was found 5.8%, of which was less than observed by the other reports. The authors attributed this low incidence to sample selection and failure to isolate fungi. In the current study, IFD rate for aplastic anemia was 44.4% (n:4/9), higher than the acute leukemia group. We have a very limited data for this group to make a conclusion. However, in the aplastic anemia studies consisted of both adults and children; fungal infection rate was reported as 7% [16]. The major risk factor for the development of invasive mycoses in this group was prolonged and severe neutropenia [17]. For our patients, all of them were neutropenic for longer than 10 days.

Although Candida spp. has been the main cause of IFD, the recent epidemiological data indicated that an increasing number of infections in immunocompromised patients are being caused by Aspergillus spp. [18-20]. In our data, aspergillosis was also the major cause of IFD and the incidence was found 74%. Additional risk factor for the high incidence at our

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Table 2. The Patients Host, Clinical and Microbiological Factors Patients Suspected pathogen 1 Candidiasis 2 Candidiasis 3 Candidiasis 4 Candidiasis 5 Candidiasis 6 Candidiasis 7 IA4 8 IA 9 IA 10 IA 11 IA 12 IA 13 IA 14 15 16 17 18 19 20 21 22 23

IA IA IA IA IA IA IA IA IA IA

Host Factors Use of ATG1,cyc2,cs3 Neutropenia Neutropenia Neutropenia Neutropenia Prolonged use of Cs Prolonged use of Cs Prolonged use of Cs Neutropenia Neutropenia Prolonged use of Cs Prolonged use of Cs Neutropenia

Clinical Manifestations Bullâ&#x20AC;&#x2122;s eye lesion in spleen Bullâ&#x20AC;&#x2122;s eye lesion in spleen-liver

Focal lesion on Cranial MRI Thorax HRCT (nodules*) Thorax HRCT (air cresent) Thorax HRCT (nodul) Thorax HRCT (nodules, cavity) Thorax HRCT (nodules) Focal lesion on Cranial MRI, Thorax CT (nodules) Use of ATG,cyc,cs Thorax HRCT (halo signs) Spesific monoclonal antibodies Thorax HRCT (nodul) Neutropenia Sinus CT and black eschar Use of Cyc Thorax HRCT (halo signs) Neutropenia Thorax HRCT (air cresent) Neutropenia Sinus CT and black eschar Prolonged use of Cs Thorax HRCT (halo signs) Prolonged use of Cs Focal lesion on Cranial MRI Prolonged use of Cs Thorax HRCT (halo signs) Prolonged use of Cs Thorax HRCT (air cresent)

Mycological Evidence Blood Culture Blood Culture Blood Culture Blood Culture Blood Culture Blood Culture Cytology, culture BAL5 Cytology 6 GM +/Sputum cytology GM+ GM+ GM+ GM+

Definition Proven Proven Proven Proven Proven Proven Proven Probable Probable Probable Probable Probable Probable

GM+/Sputum cytology GM+ Cytology, culture GM+ GM+ GM+/ Cytology, culture Sputum /Cytology, culture Cytology, culture/GM+ GM+ GM+

Probable Probable Proven Probable Probable Proven Probable Proven Probable Probable

1ATG: Antithymocyt globulin, 2cyc: Cyclosporin, 3cs: Corticosteroid, 4IA: Invasive Aspergillosis, 5BAL: Bronchoalveolar lavage, 6GM: Galactomannan

*nodul: dense, well circumscribed lesions without a halo sign.

Table 3. The Features of Children Cured from Invazive Fungal Disease Patients Suspected Underlying pathogen Disease Site of IFD Definition 3 Candidiasis AML Blood Proven 4 Candidiasis ALL-RL Blood-Liver/Spleen Proven 6 Candidiasis ALL Blood Proven 7 IA ALL Brain Proven 9 IA AML Lung Probable 10 IA AML Lung Probable 12 IA ALL Lung Probable 13 IA ALL-RL Brain Probable 15 IA ALL Lung Probable 16 IA A.A Sinus Proven 18 IA ALL Lung Probable 20 IA ALL Lung Probable 21 IA ALL Brain Proven 22 IA ALL Lung Probable

Treatment L-amp(5), caspo4 L-amp(5) L-amp(5), caspo L-amp(3,5,8) L-amp(3,5) L-amp(5), VCZ L-amp(5) L-amp(5), VCZ L-amp(5), VCZ, caspo L-amp(5), VCZ L-amp(5), VCZ, caspo L-amp(5), VCZ L-amp(5), VCZ L-amp(5), VCZ

Outcome Cured from IFD Cured from IFD Cured from IFD Cured from IFD Cured from IFD Cured from IFD Cured from IFD Cured from IFD Cured from IFD Cured from IFD Cured from IFD Cured from IFD Cured from IFD Cured from IFD

Survival time (months) 17 19 21 34 22 17 33 20 9 11 13 15 22 34

1AA: Aplastic anemia, 2L-amp: liposomal amphotericine B, 3RL: Relapse leukemia, 4Caspo: caspofungin, 5VCZ: Voricanazole

center was ongoing hospital renovation in the last 5 years.

Several studies have suggested an association between IA and contamined ventilation systems, hospital construction or renovation [21-23]. In the current study, the respiratory tract was the most common site of the IA. Four children also had more than one site of infection where CNS was involved in three. The previous reports also suggested that respiratory tract followed

by CNS in IA were the most frequent IFD in the leukemia and transplant settings [2,3,24,25]. Therefore, children with pulmonary IA should have a careful neurological examination.

The diagnosis of IFD in immunocompromised patients is difficult. In the current study, the episodes according to EORTC/MSG criteria were defined as proven in 10 (43%) children and probable in 13 (57%) cases. Histopathologic and/or

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cytopathalogic evidence for proven infection is rather difficult, especially in children. This is in part because invasive diagnostic procedures are often contraindicated due to impaired clinical condition [26]. In ours study, we were able to do tissue biopsy only in 4 children. In recent years, early HRCT as a noninvasive method seems to be more valuable compared to chest X-ray in defining IFD [27]. The findings of thorax CT in IFD are given in details, mentioning that focal rather than diffuse infiltrates, macronodules with or without halo sign, wedgeshaped infiltrates, segmental and lobar consolidation can be found in pulmonary lesions. In addition radiologic data in pediatric population is limited and its presence is highly predictive, but not specific [28]. Although not totally specific, segmental, consolidation, multi-lobar consolidation, perihilar infiltrates and pleural based nodular masses can all be seen and raise suspicion about IFD in these immunosuppressive children [29,30]. In our setting, HRCT scans were obtained in the first week of febrile neutropenia in all children. Findings related to IFD were documented in 65% (n: 15/23) of these scans, although the concomitant chest X-rays had no signs related to infection.

Serum GM levels could lead to an early diagnosis and substantially improve the clinical outcome. The inclusion of GM in the EORTC/MSG criteria could at least partly resolve this problem [26]. We have determined GM positivity in 13 out of 17 patients with IA. In the previous reports, the sensitivity of the assay was found 64.5% for proven and 16.4% for IA [31-33].

However, the false positive rate was reported as 44% in children [34-36]. Therefore, the test should be used in conjunction with other diagnostic work-ups and serial sampling is required to maximize detection.

All children had clinical findings of infection (Table 1).

Despite the heterogeneity of the combinations of criteria used for diagnosis, we found that the most frequently used diagnostic methods were clinical findings (100%) and radiogical methods (84%).

The success rate of treatment for candidiasis and IA were found 60%, 71% respectively. Two children with hepatosplenic candidiasis, L-AmB were changed to caspofungin, due to stable response. Comparative trials with adults have demonstrated that caspofungin was effective as a first line drug for invasive candidiasis and as a second line agent for IA [37].

However, there is limited data on the use of caspofungin in pediatrics cases [37,38]. Groll et al. [39] have recently displayed favorable safety and tolerance in with caspofungin 64 immunocompromised children. In our study, three out of 6 children with candidiasis was lost. All of them had positive blood cultures; in patient 1 both C.crusei and K. pneumonia, in patient 2 and 5, C. albicans were grown. Patient 5 also had hepatosplenic candidiasis. Of these 3 deaths, only two (33%) (patients 1 and 5) were related to candidiasis. The underlying host factor for patient 1 was severe AA treated with ATG, cyclosporine and steroids, and for patient 2 and 5 was relapse/ resistant AML (Table 4).

In our study, the success rate of VCZ monotherapy in IA was found 75%. Patterson et al. [40] reported a good response in the primary treatment of IA with VCZ compared to amphotericin B (52.8% vs. 31.6%) and increased survival rates (70.8% vs. 57.9%). Therefore, VCZ was approved as a first line treatment in IA [41]. In our study, four children with IA required combination therapy with caspofungin due to stable response to VCZ. All of these children had impaired bone marrow function either due to relapse or bone marrow failure. Caspofungin has been shown effective as a salvage therapy for IA in adults [41]. Prospective controlled studies in analyzing the efficacy of combination therapy is a few in children. However, the recent reports on this issue addressed that combination therapy in children with hematological diseases increased the survival rates [38,41-43].

In current study, six out of 17 (35%) children with IA was lost. Among those 6 cases, underlying diseases were ALL in 4 and AA in 2. Only five deaths (30%) were related to IFD. The causes of death in these fatal cases were massive haemoptysis (patients 8, 11, 23), respiratory failure (patients 14, 17). Two of them had also positive blood cultures for K. pneumonia and S. maltophilia (patients 14, 17) in addition to fungal disease.

One death (patient 19) was related to relapse/resistant leukemia. Three out of 4 patients with leukemia were in remission but, all of them were on prolonged use of steroid and had neutropenia.

In our series, the total mortality of IFD was found 30% (n: 7/23) excluding patients with relapse leukemia. In a study

Table 4. The Features of Lost Children Patients Suspected pathogen 1 Candidiasis 2 Candidiasis 5 Candidiasis 8 IA 11 IA 14 IA

Underlying Treatment Disease phase ATG1,cyc2,cs3 A.A1 AML-RL Resistant leukemia AML-RL Consolidation ALL Induction ALL Induction A.A ATG,cyc,cs

Survival time Outcome (months) Died of IFD and sepsis 2,1 4 Died of RL3 Died of IFD 1,2 Died of IFD 1.8 Died of IFD 1.8 Died of IFD and 2 sepsis Probable L-amp(5), VCZ, Died of IFD and 1,4 caspo sepsis Proven L-amp(5), VCZ, caspo Died of RL 2 Probable L-amp(5), Died of IFD 1.8

Site of IFD Definition Blood Proven Blood-Liver/Spleen Proven Blood Proven Lung Probable Lung Probable Lung Probable

17

IA

A.A

Cyc

Lung

19 23

IA IA

ALL-RL ALL

Resistant leukemia Induction

Sinus Lung

Treatment L-amp(5)2 L-amp(5) L-amp(5) L-amp(5) L-amp(5) L-amp(5)

1AA: Aplastic anemia, 2L-amp: liposomal amphotericine B, 3RL: Relapse leukemia, 4Caspo: caspofungin, 5VCZ: Voricanazole

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from Japan, IFD related mortality in a total of 334 pediatric patients with hematologic malignancies, aplastic anemia and solid tumors was determined as 48.2% [44]. In the same study, when the children with hematologic malignancies and aplastic anemia were separately evaluated, IFD related death rate increased to 58.8% (n: 10/17). ALL-BFM study group including 2021 children, reported infection related death rate as 2.1% (n:43). The cause of death was IFD in 9 of these 43 fatal cases (21%) [45]. One of the major AML study group, MRC, has reported total death rate as 13.8% in 341 children. The main cause of death was infection (65.9%). Of them, fungal diseases were significant problem, causing 23% of all infective deaths [46]. Invasive fungal infections have been also identified as a growing threat in patients with AA and were reported as the major cause of death in this population [16, 17]. Considering all of these data, the incidence of fungal infections is considerably increasing in immunocompromised children causing high mortality and morbidity.

It is concluded that fungal infections are an important problem in children with hematologic malignancies and SAA. Both the underlying disease and prolonged neutropenia are important risk factors. It is rather difficult in children to prove the fungal disease. Therefore, any sign in children under risk of developing IFD should be carefully evaluated for an urgent treatment to decrease the morbidity and mortality. The availability of new antifungal treatments and early diagnostic tests will also help to improve the survival rates in this patient group.

Acknowledgement: The authors thank to following doctors for their contribution to the diagnostic work-ups; Prof.

Dr. Beyza Ener in the Department of Microbiology and Prof. Dr. Zeynep YazÕcÕ in the Department of Radiology. This study has the ethical committee approval numbered B.30.2.ULU.0.20.00.00.02.020/6979 at 08/06/2009.

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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.

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Research Article

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The levels of nitric oxide in megaloblastic anemia Megaloblastik anemide nitrik oksit düzeyleri

Mehmet Ali Erkurt1, ûsmet Aydoúdu2, Nihayet Bayraktar3, ûrfan Kuku1, Emin Kaya1 1Department

of Hematology, ûnönü University Faculty of Medicine, Malatya, Turkey of Hematology, Selçuk University Meram Medical Faculty, Konya, Turkey 3Department of Biochemistry, ûnönü University Faculty of Medicine, Malatya, Turkey 2Department

Abstract Objective: The purpose of this study was to investigate the relationship between nitric oxide degradation products (nitrate and nitrite) levels and megaloblastic anemia which is treated with cyalocobalamin.

Materials and Methods: A total of 30 patients with megaloblastic anemia (16 Male, 14 Female) were included in the study.

Cyanocobalamin was administered (1.000 g/day intramuscularly) until the reticulocyte crisis occurred to the normal range. The control group consisted of 30 healthy subjects (15 Male, 15 Female). Nitric oxide levels were measured before treatment and compared with the values obtained during peak reticulocyte count.

Results: Plasma direct nitrite, total nitrite and nitrate levels were 24,86±3,87, 60.56±7,01 and 36,02±5,24 in before treatment versus 15,48±3,05, 38,92±6,44 and 22,77±6,04 mol/dl in after treatment, respectively. Plasma direct nitrite, total nitrite and nitrate levels were significantly lower in after treatment compared with the before treatment (p<0.001).

Conclusion: Nitric oxide levels are seen to increase in megaloblastic anemia. This study suggested that abnormalities in the nitric oxide levels in megaloblastic anemia are restored by vitamin B12 replacement therapy.

(Turk J Hematol 2009; 26: 197-200) Key words: Megaloblastic anemia, vitamin B12, nitric oxide Received: January 6, 2009

Accepted: July 31, 2009

Özet Amaç: Bu çalÕýmada siyanokobalamin ile tedavi edilen megaloblastik anemi ile nitrik asit degranülasyon ürünleri olan nitrat ve nitrit arasÕndaki iliýki araýtÕrÕldÕ.

Yöntem ve Gereçler: ÇalÕýmaya 16’sÕ erkek, 14’ü kadÕn olan toplam 30 megaloblastik anemili hasta alÕndÕ. Hastalara retikü losit krizi görülene kadar 1.000 g/gün dozunda siyanokobalamin intramüsküler olarak uygulandÕ. Kontrol grubu tamamen saúlÕklÕ 15’i erkek, 15’i kadÕn toplam 30 kiýiden oluýturuldu. Nitrik oksit düzeyleri tedavi öncesinde ölçüldü ve retikülosit sayÕsÕnÕn en yüksek seviyesinde tekrar ölçülerek karýÕlaýtÕrÕldÕ.

Bulgular: Tedavi öncesindeki ortalama plazma direk nitrit seviyesi mol/dl olarak 24,86±3,87, total nitrit 60,56±7,01 ve nitrat 36,02±5,24 bulundu. Buna karýÕlÕk kontrol grubunda sÕrasÕyla 15,48±3,05, 38,92±4,42 ve 22,77±6,04 bulundu. Tedavi sonrasÕ

Address for Correspondence: M.D. Mehmet Ali Erkurt, Konya Education and Research Hospital, Division of Hematology, 4460 Konya, Turkey Phone: +90 332 233 42 45 E-mail: erkurtali@hotmail.com

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Megaloblastic anemia and nitric oxide

Turk J Hematol 2009; 26: 197-200

plazma direk nitrit, total nitrit ve nitrat seviyesinin tedavi öncesine göre istatistiksel olarak anlamlÕ derecede azaldÕúÕ tespit edildi (p<0.001).

Sonuç: Nitrik oksit düzeyleri megaloblastik anemide artmaktadÕr. Bu çalÕýma megaloblastik anemide nitrik oksit düzeylerindeki anormalliklerin B12 replasman tedavisi ile düzeldiúini ortaya koymaktadÕr. (Turk J Hematol 2009; 26: 197-200) Anahtar kelimeler: Megaloblastik anemi, vitamin B12, nitrik oksit Geliý tarihi: 6 Ocak 2009

Kabul tarihi: 31 Temmuz 2009

Introduction Megaloblastic anemia is characterized by megaloblastic erythropoiesis and is secondary to decreased activity of methionine synthase, one of two mammalian enzymes that requires vitamin B12 (cobalamin) as a cofactor. Methionine synthase catalyzes the transfer of the methyl group of 5-methyltetrahydrofolate to homocysteine via a methylcobalamin intermediate with cycling of cobalamin between the +1 valency state cobalamin and the +3 valency state cobalamin [1,2].

Methyltetrahydrofolate is the major intracellular storage form of folates, and its synthesis from 5,10-methylene tetrahydrofolate is essentially irreversible in vivo [2,3]. Thus, decreased methionine synthase activity leads to trapping of intracellular folates as 5-methyltetrahydrofolate, and the megaloblastic anemia of vitamin B12 deficiency is virtually indistinguishable from the megaloblastosis of folate deficiency [4]. Nitric oxide (NO) is produced by most cell types and regulates a diverse array of biological functions [5]. NO has been reported to inhibit methionine synthase activity in vitro [6-8], it might be expected to bind to the cobalt in cobalamin because (i) NO binds tightly to the iron in heme; (ii) ferrous heme and cbl (III) are isoelectronic; and (iii) in both heme and cobalamin, the metal ion is coordinated to four in-plane nitrogen atoms of a tetrapyrrole ring and has two out-of-plane ligands [2]. In Literatür published that NO inhibits methionine synthase activity in vivo and that NO produced by three different pharmacological agents or produced physiologically by rat C6 glioma cells inhibits carbon flow through the folate pathway [4].

In the light of above mentioned information, NO inhibits methionine synthase and direct cause of ineffective erythropoiesis. So, Nitric oxide leads megaloblastic anemia.

The onset of anemia due to B12 or folate deficiency begins the production of nitric oxide then a vicious cycle of anemia sets in due to inhibition of methionine synthase. There have been no studies in humans regarding the effects of nitric oxide on patients with megaloblastic anemia. Given the lack of studies regarding the relations between serum levels of nitrate and nitrite in patients with megaloblastic anemia, the aim of this study was to explore these relations and restoration effect of cyanocobalamin in adult with megaloblastic anemia.

Material and Methods This study was conducted in Turgut Özal Medical Center, Department of Hematology, between January 2005 and December 2006. Thirty patients (16 male and 14 female, age 17-75, average 55 years) with megaloblastic anemia were enrolled in the study. Patients with acute or chronic infections,

proven chronic inflammatory diseases, heart diseases and other anemia with patients were not included in the study.

Informed consent was obtained at the beginning of the study from all participants, both the megaloblastic anemia patients and the healthy control subjects. Diagnostic criteria of the patients are summarized in Table 1. All patients showed low serum levels of vitamin B12 (the average value and the normal range were 85 and 200-900 pg/ml, respectively). Diagnosis was based on the medical history, macroovalocytosis in peripheral blood, megaloblastic changes in bone marrow, low serum levels of vitamin B12, increased serum LDH and indirect bilirubin levels, and grade 4 atrophic gastritis in endoscopic biopsy. NO levels were measured before treatment and compared with the values obtained during peak reticulocyte count(average seventh day). Cyanocobalamin was administered (1.000 g/day intramuscularly) until the reticulocyte crisis occurred and serum vitamin B12 levels returned to the normal range. The control group consisted of 30 healthy subjects (15 M, 15 F, average age: 28 years), NO levels in the blood samples were measured from pro and post cyanocobalamin treatment and statistical significance was evaluated.

Assay for Nitric Oxide

Plasma nitrite/nitrate levels were measured with the Griess reaction using a spectrophotometer at 545 nm. Nitrite (0.1M sodium nitrite in water) has been mixed with sulfanilamide solution (1% sulfanilamide in 5% phosphoric acid) first, followed immediately by addition of NED solution (0.1% N-1napthylethylenediamine dihydrochloride in water). The absorbance has been measured within 30 minutes.

Statistical Analysis

Statistical analysis was done by SPSS (Statistical Program for Social Sciences, version 15.0). Significance of differences was evaluated with independent and paired Student’s t test; p < 0.05 was regarded as statistically significant.

Results The avarage hemoglobin level during diagnostic period 7,3 g/dl, leukocyte count 4,1/ 103/ l, platelet count 137,4 /103/ml, MCV 115,6 /fl, vitamin B12 83,8 pg/ml and folic acid 7,8 ng/ mldetected (Table 1).

Plasma direct nitrite, total nitrite and nitrate levels were 24,86±3,87, 60,56±7,01 and 36,02±5,24 in before treatment versus 15,48±3,05, 38,92±6,44 and 22,77±6,04 mol/dl in after treatment, respectively. Serum nitrite and nitrate levels were significantly higher in the before treatment than in the after treatment and control group (p< 0.001). Almost seven days later than cyanocobalamin treatment, the nitric oxide levels returned to normal and reached nearly the same levels as that of the control group (Table 2).

Erkurt et al.

Megaloblastic anemia and nitric oxide

Turk J Hematol 2009; 26: 197-200

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Table 1. Some blood parameters of patients in initial diagnosis Parameters

Min.

Max.

Mean

SD

Normal range

Hemoglobin, g/dl

3.4

10.7

7.3

1.9

11-16

Hematocrit, %

8.9

31.2

21.1

5.9

34-45

Mean corpuscular volume, fl

99

136

115.6

12.67

80-95

Platelet count, x103/ml

9

497

137.4

108.3

150-450

Leukocyte count, x103/ l

1.3

10.9

4.1

2.26

4-11

LDH, U/l

564

12773

4640

325.4

200-350

Potassium, mmol/l

3.4

4.7

4.4

0.32

3.5-5.5

123.6

563.8

342.1

91.6

154-422

Unconjugated bilirubin, mol/l

0.5

17.4

6.4

3.1

0-10

Conjugated bilirubin, mol/l

0.29

5.9

1.6

1.4

0-4

Vitamin B12, pg/ml

42

167

83.8

82.5

200-900

Folic acid, ng/ml

1.7

21.6

7.8

5.76

2.7-16.1

Uric acid, mol/l

Table 2. Nitric oxide levels of megaloblastic anemia patients and controls Parameters

Megaloblastic anemia patients (n=30)

Control group (n=30)

p value

Treatment Before

After

Direct nitrite ( mol/dl)

24.8 ± 3.8

15.4 ± 3

16.6 ± 4.5

p< 0.001

Total nitrite ( mol/dl)

60.5 ± 7

38.9 ± 4.4

35.4 ± 5.1

p<0.001

36.0 ± 5.2

22.7 ± 6

20.1 ± 3.2

p<0.001

Nitrate ( mol/dl) Statistically significant

Discussion NO plays a critical role in many different physiological processes including blood pressure regulation, platelet aggregation, neurotransmission, and macrophage cytotoxicity [5]. Many of NO’s effects (e.g. blood pressure regulation and platelet aggregation) are mediated via NO binding to the iron in the heme prosthetic group of guanylate cyclase, which markedly activates the enzyme and thereby increases the intracellular concentration of the second messenger cGMP [9,10]. NO has a remarkably high affinity for ferrous heme with a binding constant on the order of 1012 to 1014 M-1, and NO also binds to ferric heme. Iron and cobalt are transition metals adjacent in the periodic table, and the porphyrin ring of heme and the corrin ring of cobalamin are both substituted tetrapyrrole rings [11]. Thus, it is not surprising that NO binds to the cobalt in cobalamin. In the light of above studies, In megalobalstic anemia, serum levels of vitamin B12 decrease. The lack of vitamin B12 has been thought to be the main factor in this decrease, but another potential factor is nitric oxide, which has been shown to affect cobalamin metabolism in vitro and invivo.

Nitric oxide is an inhibitor of erythropoisis. Cytokine-induced NO is known to decrease human erythropoiesis, and NO is likely an important mediator of the anemia of chronic disease in humans. Also, NO inhibits methionine synthase and direct cause of ineffective erythropoiesis. Therefore, Nitric oxide leads megaloblastic anemia [12]. In megaloblastic anemia, reductions

in cobalamin synthesis are known to be associated with low levels of intracellular cobalamin, but nitrate and nitrite levels have not been previously implicated. In this study we found that patients with megaloblastic anemia, as defined by low levels of serum vitamin B12, had higher serum levels of nitrate and nitrite than did normal controls. NO levels are known to have increased in anemia. In published studies, NO levels in anemia with iron deficiency [13] and aplastic anemia [14] found increased. On the other hand, In another studies, NO bioavailability in thalassemia [15] and sickle cell anemia [16] reported decreased. In this study, NO levels were measured averagely one week later than the diagnosis, not a tangible improvement is anticipated within this duration. That the anemia increased the NO levels which hence deepened the megaloblastik anemia can be derived from this study. After treatment of cobalamin(average seventh day), the levels of nitric oxide returned normally. We observed that cyanocobalamin administration had restored the increased levels of nitric oxide which was the main abnormality. Our hypothesis is that when B12 vitamin is applied on the patients with megalobalstic anemia, there is a likelihood of decreased activity of inhibition caused by NO to methionine synthase which may contribute the NO level's decrease and return to normal at the end of the treatment. We, however, were unable to disclose this molecularly. In the future, to get a truer picture of the subject, more detailed molecular studies in higher numbers of patients are needed.

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Megaloblastic anemia and nitric oxide

In conclusion, we suggest that nitric oxide is associated with the serum level of vitamin B12 in patients with megaloblastic anemia. The replacement of vitamin B12 in patients with megaloblastic anemia restored, at least in the early phase of treatment, the significant increase in the levels of nitric oxide.

This study appears to be the first to investigate a relation between nitric oxide and vitamin B12 levels in a clinical setting.

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.

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Banerjee RV, Matthews RG. Cobalamin-dependent methionine synthase. FASEB J 1990;4:1450-9.

Ludwig ML, Matthews RG. Structure-based perspectives on B12-dependent enzymes. Annu Rev Biochem 1997;66:269-313.

Rosenblatt DS, Cooper BA, Lue-Shing S, Wong PW, Berlow S, Narisawa K, Baumgartner RJ. Folate distribution in cultured human cells. Studies on 5,10-CH2-H4PteGlu reductase deficiency. Clin Invest 1979;63:1019-25.

Danishpajooh IO, Gudi T, Chen Y, Kharitonov VG, Sharma VS, Boss GR. Nitric oxide inhibits methionine synthase activity in vivo and disrupts carbon flow through the folate pathway. J Biol Chem 2001;276:27296-303.

Lloyd-Jones DM, Bloch KD. The vascular biology of nitric oxide and its role in atherogenesis. Annu. Rev. Med 1996;47:365-75.

Brouwer M, Chamulitrat W, Ferruzzi G, Sauls DL, Weinberg JB. Nitric oxide interactions with cobalamins: Biochemical and functional consequences. Blood 1996;88:1857-64.

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Nicolaou A, Warefield CJ, Kenyon SH, Gibbons WA. The inactivation of methionine synthase in isolated rat hepatocytes by sodium nitroprusside. Eur J. Biochem 1997;244:876-82.

Nicolaou A, Kenyon SH, Gibbons JM, Ast T, Gibbons WA. In vitro inactivation of mammalian methionine synthase by nitric oxide.

Eur J Clin Invest 1996;26:167-70.

Stamler JS. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 1994;78:931-6.

Moro MA, Russell RJ, Cellek S, Lizasoain I, Su Y, Darley-Usmar VM, Radomski MW, Moncada. cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase. S Proc Natl Acad Sci U. S. A. 1996;93:1480-5.

Traylor TG, Sharma VS. Why NO?. Biochemistry 1992;31:2847-9.

Pradhan P. Malarial anaemia and nitric oxide induced megaloblastic anaemia: a review on the causes of malarial anaemia. J Vector Borne Dis 2009;46:100-8.

Odemis E, Koca C, Karadag A, Catal F, Aydin M, Turkay S, Yigitoglu R. Nitric oxide affects serum ferritin levels in children with iron deficiency. Pediatr Hematol Oncol 2007;24:189-94.

Chung IJ, Lee JJ, Nam CE, Kim HN, Kim YK, Park MR, Cho SH, Kim HJ. Increased inducible nitric oxide synthase expression and nitric oxide concentration in patients with aplastic anemia. Ann Hematol 2003;82:104-8.

Bayraktar N, Erkurt MA, Aydoúdu I, Baýaran Y. The levels of nitric oxide in beta-thalassemia minor. Turkish Journal Of Hematology 2008;25:187-9.

Eberhardt RT, McMahon L, Duffy SJ, Steinberg MH, Perrine SP, Loscalzo J, Coffman JD, Vita JA. Sickle cell anemia is associated with reduced nitric oxide bioactivity in peripheral conduit and resistance vessels. Am J Hematol 2003;74:104-11.

Case Report

201

Simultaneous occurrence of Kaposi’s sarcoma and nodular lymphocyte predominant subtype of Hodgkin’s lymphoma in the same lymph node AynÕ lenf nodülünde Kaposi sarkomu ve nodüler lenfosit predominant Hodgkin lenfoma birlikteliúi Duygu Kankaya1, Gülýah Kaygusuz1, IýÕnsu Kuzu1, Berna Savaý1, üule Mine Bakanay2, Muhit Özcan2 1Department 2Department

of Pathology, Ankara University, School of Medicine, Ankara, Turkey of Hematology, Ankara University, School of Medicine, Ankara, Turkey

Abstract Many cases have been established with coexisting Kaposi’s sarcoma (KS) and classical Hodgkin’s Lymphoma (C-HL) in the same lymph node. But composite presentation of KS and Nodular Lymphocyte Predominant subtype of Hodgkin’s lymphoma (NLPHL) in the same lymph node has not been described yet. KS is related to immunodeficiency most frequently due to human immunodeficiency virus (HIV) infection or immunosupression by other reasons. Our case presented here was not related to any immunodeficiency status. Besides of being the first case of composite KS and NLPHL in the same lymph node, it was also unusual with the indolent behaviour of the NLPHL without any therapy for 8 years follow up and primary lymph node presentation of KS without cutaneous involvement. (Turk J Hematol 2009; 26: 201-3) Key words: Hodgkin’s disease, Kaposi’s sarcoma, Lymph node Received: August 6, 2008

Accepted: January 7, 2009

Özet Kaposi Sarkomu (KS) ve Klasik Hodgkin LenfomanÕn (K-HL) aynÕ lenf nodülünde eý zamanlÕ infiltrasyon gösterdiúi pek çok vaka bildirilmektedir. Fakat aynÕ lenf nodülünde KS ve Nodüler Lenfosit Predominant Hodgkin Lenfoma (NLPHL) birlikteliúi henüz tanÕmlanmamÕýtÕr. KS sÕklÕkla “human immunodeficiency virus (HIV)” veya baýka nedenlerle ortaya çÕkan immunyetmezlik zemininde geliýim göstermektedir. Olgumu immunyetmezlik zemini bulunmamasÕ açÕsÕndan farklÕlÕk göstermekteydi. AyrÕca aynÕ lenf nodülünde KS ve NLPHL birlikteliúinin görüldüúü ilk olgu olmasÕnÕn yanÕsÕra, NLPHL’nÕn 8 yÕllÕk tedavisiz takip sürecine raúmen çok yavaý seyir göstermesi ve KS’nun cilt tutulumu olmaksÕzÕn primer olarak lenf nodülü tutulumu ýeklinde ortaya çÕkmasÕ açÕsÕndan önemliydi. (Turk J Hematol 2009; 26: 201-3) Anahtar kelimeler: Hodgkin hastalÕúÕ, Kaposi Sarkomu, Lenf Nodülü Geliý tarihi: 6 Aúustos 2008

Kabul tarihi: 7 Ocak 2009

Address for Correspondence: M.D. Duygu Kankaya, Ankara University, School of Medicine, Department of Pathology, Level 01, 06100, Sihhiye, Ankara, Turkey Phone: +90 312 310 30 10 / 303 E-mail: duygu.kankaya@gmail.com

202

Kankaya et al.

Kaposi sarcoma and Hodgkin's lymphoma in the same lymph node

Introduction Kaposi’s sarcoma (KS) is a neoplasm of endothelial cells, affecting mainly the skin but also involving regional lymph nodes and internal organs. It occurs in several clinical-epidemiologic forms, all associated with infection by the human herpesvirus-8 (HHV-8) [1]. An association between KS and lymphoreticular malignancies, especially B cell lymphomas, has been previously reported [2]. Coexistence of KS and Classical Hodgkin’s Lymphoma (C-HL), even in the same lymph node has also been established [3-6]. But, this is the first case of nodular lymphocyte predominant (NLPHL) subtype of HL associated with KS, without cutaneous or any other involvement. It is also important that both of them were diagnosed simultaneously and there was no predisposition of human immunodeficiency virus (HIV) infection or immunosupression due to any reason.

Material and Method Fifty seven years old male patient was admitted to the hospital in 1999, with complaints of gradually enlarged inguinal lymphadenopathy, reaching a diameter of 6.5 cm within five years. He had a history of lymph node excision from the same site in 1985, which had been reported as reactive lymphoid hyperplasia. The serology for HIV was negative, but HBsAg was positive. Excisional biopsy of the inguinal lymph node was performed.

The surgical specimens were fixed in unbuffered formalin and embedded in paraffin. 4-5 +m thick cut sections stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed on tissue sections by using Ventana Automated Immunostainer. The antibodies used included CD20 (Novacastra, 1 200), CD45 (Neomarkers, 1/500), EMA (Neomarkers, 1/1000), EBV (LMP1) (Novacastra, 1/100), CD34 (Neomarkers, 1/200), CD31 (Neomarkers, 1/50), Factor VIII (Neomarkers, 1/150), HHV-8 (Novacastra, 1/30), CD30 (Neomarkers, 1/50), CD15 (Neomarkers, 1/50) antibodies.

Appropriate positive tissue controls were used.

Written informed consent was obtained from the patient.

Results Histopathological examination showed effacement of the inguinal lymph node architecture by a nodular infiltrate of small lymphocytes, admixed with histiocytes and scattered atypical multilobated Hodgkin’s-like cells. Among these areas, nodules composed of fascicles of spindle shaped cells arranged between slit-like vascular spaces filled with red blood cells were detected. Immunophenotype of the Hodgkin’s-like cells were CD20, CD45 and EMA positive B lymphoid nature and negative for EBV(LMP1), CD30 and CD15. The spindle cells showed positive reaction with CD34, CD31, Factor VIII and HHV-8 (Figs. 1,2). The findings were consistent with coexistence of NLPHL and KS in the same lymph node. There was no cutaneous involvement of KS and no malignant infiltration was detected in the bone marrow biopsy specimen. The

Turk J Hematol 2009; 26: 201-3

patient was staged as early favorable stage I-A NLPHL. The clinical follow up with short intervals was performed instead of chemotherapy. Multiple axillary lymphadenopathies with the largest diameter of 3 cm were detected within three months time. Biopsy again revealed NLPHL. Follow-up of the patient continued without any significant progression during the next four years. In 2004, left inguinal lymph node enlargement was detected again and rebiopsied. The diagnosis was relapse of both tumors, NLPHL and KS. Following year, NLPHL relaps was diagnosed in axillary lymph node. The patient is being followed with left axillary lymphadenopathies which remain stable in size and without any progression of disease.

Discussion NLPHL is an infrequent (5%) form of HL and differs from C-HL both histologically and clinically. The presentation of the disease in our patient was consistent with the natural behavior of NLPHL which usually presents with early clinical stage and with cervical or inguinal involvement. The disease course is indolent and progresses slowly. NLPHL is reported to relapse frequently but the relapses are rarely fatal. Frequent local relapses occurred in our patient. However, the lymph nodes did not enlarge rapidly and usually were stable during the follow up. Establishing a standart treatment for NLPHL especially in the early favorable stages has been difficult. Involved field radiotherapy has been recommended as standart therapy for early favorable stage I-A disease. However, extended field radiotherapy, combined modality treatments and monoclonal antibodies have been tried [7].

Some pediatric groups reported that wait and watch strategy after initial lymph node surgery may be appropriate treatment for selected group of patients [8,9]. Our patient has survived for 8 years after the initial diagnosis without significant progression but frequent localized relapses.

KS is an indolent endothelial neoplasm for immunocompromised patients developed by the oncogenic effect of HHV-8 which is not restricted to endothelial cells. It also infects B lymphocytes and is associated with two B-cell lymphoproliferative diseases, primary effusion lymphoma [10] and multicentric Castleman’s disease [11]. The most common lymphoproliferative malignancies associated with KS were documented as lymphoid neoplasms of B-cell origin including NHL, CLL/SLL and MM [2]. But, a great many cases have also been established with coexisting KS and C-HL, some of whom were in association with HIV [3-6]. An analysis of 65 KS cases associated with lymphoreticular malignancies showed that 29% of them had C-HL as a second malignancy [2]. It has been known that 30-50% of HL’s were associated with Ebstein-Barr virus (EBV) [12] and %95 of KS’s were associated with HHV-8 [1].

EBV and HHV-8 are human a herpesviruses that establish persistent latent infection and prevent apoptosis of infected cells which result in malignant transformation in the presence of immunosupression. The concomitant occurrence of KS and C- HL in the same lymph node has led researchers to investigate a common ethiopathogenetic mechanism which could not be proved yet. Neither EBV nor HHV-8 were found in association with both of these malignancies by itself. HHV-8 was demonstrated in relation to KS in our case, but neither EBV nor HHV-8 were presented on the L&H cells of NLPHL. Role of

Kankaya et al.

Kaposi sarcoma and Hodgkin's lymphoma in the same lymph node

Turk J Hematol 2009; 26: 201-3

A

B

C

Figure 1. Neoplastic cells of KS and NLPHD (H&E x200). B,C: HHV8 positivity of

A of tumor cells (x1000)

EBV on C-HL pathogenesis has been well documented in the previous studies, but no such relation with any agent has been demonstrated for NLPHL [13]. Somatic hypermutations in several transcription factor genes has been speculated to play a role on the development of NLPHL and C-HL. Bcl-6 mutations have been documented most frequently in NLPHL cases [14,15] and it reveales the germinal center origin of neoplastic cells [13]. Longstanding antigenic stimulus causes B cells to be vulnerable to chromosomal aberrations by inducing B cell proliferation. In relation to the pathogenesis of composite presentation of KS and NLPHL, it can be speculated that the chronic antigenic stimulus of HHV-8 and immune response mechanisms may be playing a role in the development of NLPHL. Our case of composite neoplasia of KS and NLPHL has unique characteristics in several respects. It is the first case of NLPHL associated with KS. It is also important that both of the tumors were diagnosed simultaneously and there was no predisposition of HIV infection or immunosupression due to any reason. KS presented only in the lymph node without cutaneous or any other involvement The clinical follow up also revealed the indolent behaviour of both neoplasia without any therapy.

6.

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Huang YQ, Li JJ, Kaplan MH, Poiesz B, Katabira, Zhang WC, Feiner D, Friedman-Kien AE. Human herpesvirus-like nucleic acid in various forms of Kaposi’s sarcoma. Lancet 1995;345:759-61.

Ulbright TM, Santa Cruz DJ. Kaposi’s sarcoma:relationship with hematologic, lymphoid and thymic neoplasia. Cancer 1981;47:963-73.

Carbone A, Volpe R. Kaposi’s sarcoma in lymph nodes concurrent with Hodgkin’s disease. Am J Clin Pathol 1983;80:228-30.

Hayes MMM, Coghlan PJ, King H, Close P. Kaposi’s sarcoma, tuberculosis and Hodgkin’s lymphoma in a lymph node- possible acquired immunodeficiency syndrome. S Afr Med J 1984;66:226-9.

Ngan KW, Kuo TT. Simultaneous occurrence of Hodgkin's lymphoma and Kaposi's sarcoma within the same lymph nodes of a non-AIDS patient. Int J Surg Pathol. 2006;14:85-8.

C

Figure 2. Popcorn cells of NLPHD (H&E x1000). B, C: CD45 and CD20 positivity

KS’s cells ( x200, x400)

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.

B

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Mitsuyasu RT, Colman MF, Sun NCJ. Simultaneous occurrence of Hodgkin’s disease and Kaposi’s sarcoma in a patient with the acquired immune deficiency syndrome. Am J Med 1986;80:954-8.

Nogova L, Rudiger T, Engert A. Biology, clinical course and management of nodular lymphocyte-predominant hodgkin lymphoma. Hematology ASH Education Program. 2006;266-72.

Murphy SB, Morgan ER, Katzenstein HM, Kletzel M. Results of little or no treatment for lymphocyte-predominant Hodgkin disease in children and adolescents. J Pediatr Hematol Oncol.

2003;25:684-7.

Pellegrino B, Terrier-Lacombe MJ, Oberlin O, Leblanc T, Perel Y, Bertrand Y, Beard C, Edan C, Schmitt C, Plantaz D, Pacquement H, Vannier JP, Lambilliote C, Couillault G, Babin-Boilletot A, Thuret I, Demeocq F, Leverger G, Delsol G, Landman-Parker J;Study of the French Society of Pediatric Oncology. Study of the French Society of Pediatric Oncology. Lymphocyte-predominant Hodgkin's lymphoma in children:therapeutic abstention after initial lymph node resection--a Study of the French Society of Pediatric Oncology. J Clin Oncol. 2003;21:2948-52.

Cesarman E, Chang Y, Moore PS, Said JW, Knowles DM. Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N Engl J Med. 1995;332:1186-91.

Soulier J, Grollet L, Oksenhendler E, Cacoub P, Cazals-Hatem D, Babinet P, d'Agay MF, Clauvel JP, Raphael M, Degos L, Sigaux F. Kaposi's sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman's disease. Blood. 1995;86:1276-80.

Lyon:International Agency for Research on Cancer/World Health Organisation, IARC monographs on the evaluation of carcinogenic risks to humans. Epstein-Barr virus and Kaposi’s sarcoma herpesvirus/human herpesvirus 8, Vol. (1997) Re D, Küppers R, Diehl V. Molecular pathogenesis of Hodgkin's lymphoma. J. Clin. Oncol. 2005;23:6379-86.

Liso A, Capello D, Marafioti T, Tiacci E, Cerri M, Distler V, Paulli M, Carbone A, Delsol G, Campo E, Pileri S, Pasqualucci L, Gaidano G, Falini B. Aberrant somatic hypermutation in tumor cells of nodular-lymphocyte-predominant and classic Hodgkin lymphoma. Blood. 2006;108:1013-20.

Renné C, Martín-Subero JI, Hansmann ML, Siebert R Molecular cytogenetic analyses of immunoglobulin loci in nodular lymphocyte predominant Hodgkin's lymphoma reveal a recurrent IGHBCL6 juxtaposition. J Mol Diagn. 2005;7:352-6.

Case Report

204

Acute massive myelofibrosis with acute lymphoblastic leukemia Akut masif myelofibrozis ve akut lenfoblastik lösemi birlikteliúi Zekai AvcÕ1, BarÕý Malbora1, Meltem Gülýan1, Feride Iffet üahin2, Bülent Celasun3, NamÕk Özbek1 1Department

of Pediatrics, Baýkent University Faculty of Medicine, Ankara, Turkey of Medical Genetics, Baýkent University Faculty of Medicine, Ankara, Turkey 3Department of Pathology, Baýkent University Faculty of Medicine, Ankara, Turkey 2Department

Abstract Acute myelofibrosis is characterized by pancytopenia of sudden onset, megakaryocytic hyperplasia, extensive bone marrow fibrosis, and the absence of organomegaly. Acute myelofibrosis in patients with acute lymphoblastic leukemia is extremely rare. We report a 4ï-year-old boy who was diagnosed as having acute massive myelofibrosis and acute lymphoblastic leukemia. Performing bone marrow aspiration in this patient was difficult (a “dry tap”), and the diagnosis was established by means of a bone marrow biopsy and immunohistopathologic analysis. The prognostic significance of acute myelofibrosis in patients with acute lymphoblastic leukemia is not clear. (Turk J Hematol 2009; 26: 204-6) Key words: Acute myelofibrosis, acute lymphoblastic leukemia, dry tap Received: April 9, 2008

Accepted: December 24, 2008

Özet Akut myelofibrozis ani geliýen pansitopeni, kemik iliúinde megakaryositik hiperplazi, belirgin fibrozis ve organomegali olmamasÕ ile karakterize bir hastalÕktÕr. Akut myelofibrozis ile akut lenfoblastik lösemi birlikteliúi çok nadir görülmektedir.

Burada akut masif myelofibrozis ve akut lenfoblastik lösemi tanÕsÕ alan 4,5 yaýÕnda erkek hasta sunuldu. Kemik iliúi aspirasyonu yapÕlamayan (“kuru ilik”) hastanÕn tanÕsÕ, ancak kemik iliúi biyopsisi ve immünohistopatolojik inceleme ile konabildi.

Akut myelofibrozisin akut lenfoblastik lösemide prognostik önemi tam bilinmemektedir. (Turk J Hematol 2009; 26: 204-6) Anahtar kelimeler: Akut myelofibrozis, akut lenfoblastik lösemi, kuru ilik Geliý tarihi: 9 Nisan 2008

Kabul tarihi: 24 AralÕk 2008

Introduction Idiopathic primary myelofibrosis is a chronic disorder characterized by bone marrow fibrosis, extramedullary hematopoiesis, and a leukoerythroblastic blood profile, and it is usually classified as a myeloproliferative syndrome. A rapidly progressive form of myelofibrosis, called “malignant myelofibrosis”

or “acute myelofibrosis (AMF)”, is characterized by pancytopenia of sudden onset, megakaryocytic hyperplasia, excessive marrow fibrosis, and the absence of hepatosplenomegaly [1-3]. Primary myelofibrosis also occurs in those with acute leukemia (especially the myelocytic types); however, AMF that develops in individuals with acute lymphoblastic leukemia (ALL) is extremely rare. We report a 4ï-year-old boy diagnosed with AMF and ALL.

Address for Correspondence: M.D. Zekai AvcÕ, Baýkent Üniversitesi Hastanesi, Pediatri Polikliniúi, 6.cadde, 72/3, Bahçelievler, Ankara, Türkiye Phone: +90 312 212 68 68-1305 E-mail: zekaiavci@yahoo.com

AvcÕ et al.

Acute myelofibrosis with ALL

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Case Report A 4ï-year-old boy who presented with a one-month history of fever, fatigue, and abdominal pain was initially diagnosed as having typhoid fever and was treated with ceftriaxone for seven days. He was referred to our clinic because of coexisting anemia and leukopenia. This child was a monozygotic twin born to healthy nonconsanguineous parents, and his growth and development were within normal limits. His vital signs at admission were as follows: body temperature 38.4ºC, blood pressure 100/60 mmHg, and pulse rate 120 bpm. The initial physical examination did not reveal lymphadenopathy or hepatosplenomegaly. This patient demonstrated no phenotypic signs of Down syndrome or mosaicism. The results of a complete blood count revealed the following values: white blood cell count, 3.6 x 109/L (26% neutrophils, 7% monocytes, 64% lymphocytes, and 3% blasts); hemoglobin, 8.9 g/dl; platelet count, 315 x 109/L; and reticulocyte count, 0.93%. The results of serum biochemical analyses were all within the normal range, except for the serum lactate dehydrogenase level, which was 634 IU/L (normal range: 150-500 IU/L). The sedimentation rate was 120 mm/h, and the level of C-reactive protein was 168 mg/dl (normal range, 0-10 mg/dl). Direct Coombs’ test and an antinuclear antibody test were negative, and the levels of serum immunoglobulins G, M, and A as well as complements 3 and 4 were within the reference range. Chest radiograph and abdominal ultrasonographic findings were unremarkable. The results of viral serologic testing, including that for Epstein-Barr virus (EBV), cytomegalovirus (CMV), parvovirus B19, human immunodeficiency virus (HIV), and hepatitis A, B, and C, were negative, as were the results of the Widal test for Salmonella and the Wright agglutination test for Brucella. The bone marrow aspiration was a dry tap in each of three consecutive attempts. The bone marrow biopsies revealed a diffuse blastic infiltration (Figure 1). On immunohistopathologic examination, the blasts were positive for CD34, CD10, TdT, CD20, and CD79a. The results of staining for CD3, CD41, CD43, CD56, CD117, and myeloperoxidase were negative. The reticulin fibers were increased in number, especially at sites in which neoplastic infiltration was intense (Figure 2). Conventional cytogenetic and fluorescent in situ hybridization (FISH) analyses were performed from deparaffinized bone marrow biopsy. Unstimulated bone marrow lymphocyte

Figure 1. Bone marrow biopsy showing a diffuse infiltrate of lymphoblasts (hematoxylin-eosin stain, original magnification x 400)

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cultures were set up for incubation time of 48 and 72 hours, and metaphase spreads were examined after harvesting and GTG banding according to standard protocols. Three metaphases were found and revealed a 46, XY karyotype. FISH studies were also performed with probes specific for 5q31, 7q31, BCR-ABL fusion, and t(15;17) (Vysis, Downers Grove, IL, USA), and revealed normal signals except for an extra signal with the translocation probe t(8;21) (Vysis, Downers Grove, IL, USA) for chromosome 21q22 region in 22% of the cells. With the extra signal for 21q22, we decided to perform chromosome analysis from peripheral blood and skin biopsy samples of the patient, which revealed normal karyotypes.

The patient was diagnosed as having B-cell ALL with acute massive myelofibrosis. He is currently being treated according to the Berlin-Frankfurt-Munster (BFM) 95 treatment protocol for ALL. On day 15 of the chemotherapy, repeated bone marrow aspiration was a dry tap. A bone marrow biopsy showed necrosis between bone trabeculae and significant fibrosis, as well as an increased number of reticulin fibers and no hematopoietic cells. At day 33 of the chemotherapy regimen, a bone marrow biopsy revealed persisting necrosis with foci of immature bone formation and massive collagen fibrosis. No hematopoietic cells were noted. At day 70 of the chemotherapy and before continuation therapy, a bone marrow aspiration and biopsy showed normal hematopoietic cells and no fibrosis. At the time of this writing (20 months after diagnosis), the patient is still in clinical remission and is being treated with a continuation chemotherapy regimen.

Written informed consent was obtained from the patient and the family.

Discussion Acute myelofibrosis is known to progress to various types of acute myeloid leukemia and chronic myelogenous leukemia. Of particular interest is the rare termination of AMF as ALL, which has been reported in two pediatric patients. In one of those children, AMF terminated as ALL of T-cell origin, and in the other, AMF terminated as null-type ALL [4,5]. In addition to those two cases, Abla and Ye [1] recently reported a child with B-ALL and AMF. They did not know whether or not myelofibrosis was present previously and terminated to ALL in that patient, but his massive

Figure 2. Reticulin stain showing an increased number of fibers at the time of diagnosis (original magnification x 400)

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myelofibrosis was most likely due to secretion of cytokines from blastic cells. The same situation was probably present in our case.

To our knowledge, only four adult patients with AMF terminating as ALL have been described in the literature [2,3,6,7]. Three of seven reported cases including both children and adults in the literature died within two months of diagnosis. However, it is difficult to determine the prognostic significance of AMF in patients with ALL due to small number of cases reported to date.

Cytogenetic studies of patients with myelofibrosis have shown that the presence of 13q-, 20q-, +8, and abnormalities of chromosomes 1,7 and 9 constitute more than 80% of the chromosomal changes in those individuals and suggest that in many patients, gene loss and/or inactivation may be an important pathogenetic mechanism in the development of myelofibrosis [8].

Cytogenetic analysis of peripheral blood and skin fibroblast cultures in our patient revealed a 46,XY genotype. However, the results of a FISH analysis from bone marrow confirmed the presence of trisomy 21 in 22% of the cells in our patient. It is well known that AMF is associated with Down syndrome [9]. Our patient did not have any phenotypic or karyotypic feature of Down syndrome. Although we did not detect a karyotype abnormality in the peripheral blood and skin fibroblast culture, we concluded that there could be a cryptic chromosome abnormality limited to the leukemic cell population, and the extra copy of chromosome region 21q22 could be responsible for the myelofibrosis.

Finally, it should be remembered that dry taps are caused by a marked increase in reticulin fibrosis. In patients who experience that difficulty, bone marrow biopsy can be of great help in making an accurate diagnosis, as in our patient.

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.

Turk J Hematol 2009; 26: 204-6

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

Abla O, Ye CC. Acute lymphoblastic leukemia with massive myelofibrosis. J Pediatr Hematol Oncol 2006;28:633-4.

Amjad H, Gezer S, Inoue S, Bollinger RO, Kaplan J, Carson S, Bishop CR. Acute myelofibrosis terminating in an acute lymphoblastic leukemia: a case report. Cancer 1980;46: 615-8.

Dunphy CH, Kitchen S, Saravia O, Velasquez WS. Acute myelofibrosis terminating in acute lymphoblastic leukemia: case report and review of the literature. Am J Hematol 1996;51:85-9.

Chen JS, Lin DT, Chuu WM, Lin KH, Su IJ, Lin KS. Acute myelofibrosis terminating in acute lymphoblastic leukemia: report of one case. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi 1992;33:136-43.

Marino R, Altshuler G, Humphrey GB. Idiopathic myelofibrosis followed by acute lymphoblastic leukemia. Am J Dis Child 1979;133:1194-5.

Polliack A, Prokocimer M, Matzner Y. Lymphoblastic leukemic transformation (lymphoblastic crisis) in myelofibrosis and myeloid metaplasia. Am J Hematol 1980;9:211-20.

Amberger DM, Saleem A, Kemp BL, Truong LD. Acute myelofibrosis-a leukemia of pluripotent stem cell. A report of three cases and review of the literature. Ann Clin Lab Sci 1990;20:409-14.

Reilly JT. Cytogenetic and molecular genetic aspects of idiopathic myelofibrosis. Acta Haematol 2002;108:113-9.

Awasthi A, Das R, Varma N, Ahluwalia J, Gupta A, Marwaha RK, Garewal G. Hematological disorders in Down syndrome: ten-year experience at a tertiary care centre in North India. Pediatr Hematol Oncol 2005;22:507-12.

Case Report

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Intestinal mucus accumulation in a child with acute myeloblastic leukemia Akut myeloblastik lösemili bir çocukta intestinal mukus birikimi BarÕý Malbora1, Zekai AvcÕ1, Deniz Anuk ûnce2, Ünser ArÕkan3, NamÕk Özbek1 1Department

of Pediatric Hematology, Baýkent University, School of Medicine, Ankara, Turkey of Pediatrics, Baýkent University, School of Medicine, Ankara, Turkey 3Department of Pathology, Baýkent University, School of Medicine, Ankara, Turkey 2Department

Abstract Intestinal mucus accumulation is a very rare situation observed in some solid tumors, intestinal inflammation, mucosal hyperplasia, elevated intestinal pressure, and various other diseases. However, it has never been described in acute myeloblastic leukemia. The pathogenesis of intestinal mucus accumulation is still not clear. Here, we report a 14-year-old girl with acute myeloblastic leukemia and febrile neutropenia in addition to typhlitis. She was also immobilized due to joint contractures of the lower extremities and had intestinal mucus accumulation, which was, at first, misdiagnosed as intestinal parasitosis. We speculate that typhlitis, immobilization and decreased intestinal motility due to usage of antiemetic drugs might have been the potential etiologic factors in this case. However, its impact on prognosis of the primary disease is unknown. (Turk J Hematol 2009; 26: 207-9) Key words: Acute myeloblastic leukemia, intestinal mucus accumulation, parasite Received: November 25, 2008

Accepted: July 3, 2009

Özet ûntestinal mukus birikimi, solid tümörler, intestinal inflamasyon, mukozal hiperplazi, intestinal basÕnç artÕýÕ gibi çeýitli hastalÕklarda nadiren görülen bir durumdur. Ancak, bu durum akut miyeloblastik lösemili hastalarda bildirilmemiýtir. ûntestinal mukus birikiminin patogenezi ise hala tam olarak bilinememektedir. Burada, 14 yaýÕnda akut miyeloblastik lösemili, febril nötropenili ve tifilitli bir kÕz hastayÕ sunduk. HastamÕz, ayrÕca alt ekstremite eklem kontraktürü nedeniyle immobilize idi. ûntestinal mukus birikimi nedeniyle baýlangÕçta intestinal parazitoz tanÕsÕ almÕýtÕ. Biz burada, tiflit, immobilizasyon ve kullanÕlan antiemetikler nedeniyle barsak hareketlerinin azalmasÕnÕn potansiyel etyolojik faktörler olabileceúini düýündük.

Ancak, bu durumun primer hastalÕúÕn prognozu üzerine etkisi bilinmemektedir. (Turk J Hematol 2009; 26: 207-9) Anahtar kelimeler: Akut miyeloblastik lösemi, intestinal mukus birikimi, parazit Geliý tarihi: 25 KasÕm 2008

Kabul tarihi: 3 Temmuz 2009

Introduction Intestinal mucus accumulation is a rare clinical presentation manifested as mucocele in benign and malignant intestinal tumors and obstructions [1]. The entity has not been reported in patients with acute leukemia. Inflammation, mucosal

hyperplasia, elevated intestinal pressure similar to that seen in intestinal lesions, and mucus accumulation due to intestinal tumors play a role in the pathogenesis of intestinal mucus accumulation [2]. The morphology of worm-like mucus accumulations may be confused with parasites such as Ascaris lumbricoides, which can result in the unnecessary use

Address for Correspondence: M.D. Baris Malbora, Baýkent University Faculty of Medicine, Department of Pediatric Hematology, 6. Cadde, No: 72/3, Bahçelievler 06490 Ankara, Turkey Tel: +90 312 212 68 68 E-mail: barismalbora@gmail.com

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of antiparasitic drugs. In such cases, a pathology examination is required for definitive diagnosis. Here, we report an adolescent with acute myeloblastic leukemia and intestinal mucus accumulation.

Case Report A 14-year-old girl had been diagnosed with acute myeloblastic leukemia (FAB M2) and had been started on an acute myeloid leukemia Berlin-Frankfurt-MĂźnster 2004 (AML BFM-2004) chemotherapy protocol at another center. After the induction therapy, febrile neutropenia and invasive pulmonary aspergillosis developed. She was given wide-spectrum antibiotics (meropenem, teicoplanin) and antifungal (voriconazole) treatment, and was also given total parenteral nutrition. During this time, she had lower right abdominal pain and constipation. Afterwards, a 35-cm long parasite was observed in her stool, and she was therefore treated with levamisole hydrochloride. She was referred to our hospital following the development of stupor during the consolidation phase of the chemotherapy protocol.

The physical examination revealed joint contractures due to immobilization. The findings of cranial magnetic resonance imaging were typical of a posterior reversible leukoencephalopathy syndrome. Her stupor resolved in one week, after which the remainder of her treatment protocol was started. After the consolidation phase, the patient developed febrile neutropenia and recurrent invasive pulmonary aspergillosis. On the 8th day of febrile neutropenia, she experienced vomiting, bloody stool, and again, lower right abdominal pain. Results of another physical examination were negative for abdominal masses, perianal lesions, and rectal masses. Thickening of the cecum wall (4.8 mm) was observed on abdominal ultrasonography. A diagnosis of neutropenic enterocolitis (typhlitis) was made.

She was treated with wide-spectrum antibiotics and antifungal combination therapy that included imipenem, amikacin, metronidazole, liposomal amphotericin-B, and caspofungin. She was also given granulocyte colony-stimulating

Figure 1. Macroscopic view of mucus accumulation

Turk J Hematol 2009; 26: 207-9

factor and granulocyte suspension infusions. Enteral nutrition was stopped and total parenteral nutrition was started. On the 10th day of neutropenia, a round, worm-like, yellowish substance approximately 35 cm long was observed in her watery stool (Figure 1). During this time, serum electrolyte levels were normal. Microbiologic examination of the stool showed no parasites or other pathogens. On pathologic examination, the sample was defined as acellular and basophilic mucus (Figure 2). We concluded that the material was due to mucus accumulation. Intestinal mucus accumulation did not recur. Treatment of the patient ended successfully. The patient has been followed without chemotherapy for 7 months and has had no problems.

Informed consent was obtained from the patient and the family.

Discussion Intestinal mucus accumulation is rare; it presents mostly as mucocele and is usually located in the appendix [1]. Clinical manifestations of appendiceal mucocele include lower right abdominal pain, nausea, vomiting, weight loss, changes in bowel habits, gastrointestinal bleeding, and a palpable abdominal mass [1,3,4]. Two major pathological mechanisms have been postulated for the formation of intestinal mucocele.

One is elevated intestinal pressure as a sequela of luminal obstruction caused by inflammation, mucosal hyperplasia, or intestinal lesions such as fecaliths, endometriosis, diverticula, and polyps. The other is mucus accumulation due to intestinal tumors [2]. In diagnosing mucocele, imaging techniques including ultrasonography, computed tomography, and intestinal endoscopy may be beneficial [5,6]; however, certain diagnosis is always made by pathology.

The pathologic diagnosis in our patient was intestinal mucus accumulation but not mucocele, because no cells were seen in the specimen. To the best of our knowledge, there are no reports in the literature regarding intestinal mucus accumulation in patients with acute leukemia. In our patient, we concluded that the mucus accumulation had been caused

Figure 2. Basophilic mucus without cellular component (hematoxylin-eosin Ă&#x2014;10 original magnification)

Turk J Hematol 2009; 26: 207-9

by neutropenic enterocolitis. The massive mucus production could have been caused secondarily by inflammation due to an invasive microorganism in the cecum.

Our patient had lower right abdominal pain, nausea, vomiting, weight loss, constipation, and gastrointestinal bleeding, as also seen in mucocele. The evaluation of the clinical findings plus the results of the abdominal ultrasonography showing intestinal wall thickening led to the diagnosis of neutropenic enterocolitis. However, we were unable to distinguish whether the mucus accumulation had any effects on the clinical manifestations or determine its impact on the prognosis of the primary disease. The intestinal mucus accumulation could have been the consequence of neutropenic enterocolitis or changes in the intestinal flora arising from the various wide-spectrum antibiotics administration or of the reduction in intestinal motility due to antiemetic use and immobilization. While we were unable to reach a diagnosis based on the clinical, microbiological, and radiologic methods, the definitive diagnosis was made with pathological examination.

In such cases, the clinician should be able to distinguish this situation from parasitosis to avoid unnecessary use of antiparasitic treatment. Pathologic examination is an efficient tool for the differential diagnosis.

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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. Landen S, Bertrand C, Maddern GJ, Herman D, Pourbaix A, de Neve A, Schmitz A. Appendiceal mucoceles and pseudomyxoma peritonei. Surg Gynecol Obstet 1992;175:401-4.

2. Roberge RJ, Park AJ. Mucocele of the appendix: an important clinical rarity. J Emerg Med 2006;30:303-6.

3. Sasaki K, Ishida H, Komatsuda T, Suzuki T, Konno K, Ohtaka M, Sato M, Ishida J, Sakai T, Watanabe S. Appendiceal mucocele: sonographic findings. Abdom Imaging 2003;28:15-8.

4. Stocchi L, Wolff BG, Larson DR, Harrington JR. Surgical treatment of appendiceal mucocele. Arch Surg 2003;138:585-9; discussion 589-90.

5. Kim SH, Lim HK, Lee WJ, Lim JH, Byun JY. Mucocele of the appendix: ultrasonographic and CT findings. Abdom Imaging 1998;23:292-6.

6. Isaacs KL, Warshauer DM. Mucocele of the appendix: computed tomographic, endoscopic, and pathologic correlation. Am J Gastroenterol 1992;87:787-9.

Images in Hematology

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Recurrent bruising of lower eyelids: a presenting symptom of neuroblastoma in a child Alt gözkapaklarÕnda tekrarlayan morluklar yakÕnmasÕyla baývuran nöroblastomlu bir çocuk olgu Mustafa BüyükavcÕ, Zühal Keskin YÕldÕrÕm Atatürk University, Faculty of Medicine, Division of Pediatric Oncology, Erzurum, Turkey

A 21-month-old girl referred to our hospital for investigation of bleeding diathesis. She had four months history of intermittent bruising on lower eyelids. Physical examination was unremarkable except the 0.5x1 cm of ecchymosis on the right lower eyelid (Figure 1). The laboratory investigation revealed a hemoglobin level of 11.8 gr/dL, white blood cell count of 8900/mm3 and platelet count of 248000/mm3. The coagulation workup including PT, aPTT and bleeding time was within normal range.

Serum lactate dehydrogenase and ferritin were measured as 2248 U/L (normal range 211-411) and 1444 ng/ml (normal range 7-140), respectively. Urinary vanillylmandelic acid level was 23 mg/g creatinine (normal range <18.8). Abdominal USG showed a left supra-renal mass (55x74x68 mm in diameter) with calcifications. Orbital MRI was normal. Bilateral bone marrow aspiration smear showed severe infiltration with neuroblasts (Figure 2). The examination of an open-biopsy material of the abdominal mass revealed neuroblastoma.

It is well known that periorbital ecchymosis is easily recognized and associated with basal skull fractures. In cases without a history of trauma it may be a sign of systemic abnormalities such as neuroblastoma [1]. Particularly in mild cases, the correct diagnosis of this condition is sometimes delayed because of workup for other conditions such as child abuse and hematological coagulopathies. In this case, recurrent bruising of lower eyelids had been previously thought the result from disorders of coagulation.

The characteristic ‘’raccoon eyes’’ appearance associated with neuroblastoma is probably related to obstruction of the palpebral vessels by tumor tissue in and around the orbits [2].

Interestingly, there was no typical raccoon eyes appearance and metastatic involvement of the periorbital tissue on MRI in our case.

Written consent for publication of the photo from the parents was obtained.

Figure 1. The ecchymosis on the right lower eyelid of the patient at admission

Figure 2. The mononuclear cells (neuroblasts) in bone marrow aspiration smear

References 1. Bay A, Faik Oner A. Raccoon eyes. Indian Pediatr. 2005;42:949.

2. Timmerman R. Images in clinical medicine. Raccoon eyes and neuroblastoma. N Engl J Med.2003;349:e4.

Address for Correspondence: Assoc. Prof. Mustafa BüyükavcÕ, AraýtÕrma Hastanesi Çocuk Servisi 25240 Erzurum, Turkey Phone: +90 442 231 68 91 E-mail: buyukavci@hotmail.com

Letter to the Editor

211

Treatment of primary myelofibrosis Primer miyelofibroz tedavisi üinasi Özsoylu Fatih University Medical School Hospital, Ankara, Turkey

To the Editor, I have read with interest the presentation of three adult patients with primary myelofibrosis by Kar et al. in the recent issue of the journal [1]. The authors, without mentioning dose schedule, stated that parenteral and oral methylprednisolone was used in their patients.

Although our cases of primary myelofibrosis (PM) were not included in Kar et al.’s review, we were the first to successfully treat those patients, beginning in 1980, with megadose methylprednisolone (MDMP) [2-5]. Five children and four adult patients (a 60- year-old woman was not reported) with PM were all treated with MDMP (daily, 30 mg/kg for 3 days, followed by 20 mg/kg for 4 days, and then by 10, 5, 2 mg/kg doses, each dose given 1 week, continued with 1 mg/kg dose until hemoglobin (Hb) level reached 12 g/dl). Each dose was given within 5 to 10 minutes, intravenously earlier and orally recently. Each dose was given before 9 a.m., preferentially between 5 and 6 a.m.

It must be emphasized that MDMP treatment should not be compared with conventional prednisone administration (2 mg/kg, usually divided into 2 or 4 doses). Despite elevation in Hb level in the authors’ patients, correction of the myelofibrotic process was not investigated by the authors. In 6 of our 9 patients, normalization of bone marrow was shown by bone marrow biopsy. During the 6-year follow-up period, no recurrences were observed in our patients.

Increased reticulin score of the bone marrow was mentioned by the authors. I would like to call attention to the fact that the presence of increased collagen and osteoblasts in the bone marrow as well as increased number of white cells and megakaryocyte precursors in the peripheral blood were more informative for the diagnosis [6.7].

References 1. Primary autoimmune myelofibrosis: a report of three cases and review of the literature. Turk J. Hematol.

2009;26:146-50.

2. Ozsoylu S, Ruacan S. High-dose intravenous corticosteroid treatment in childhood idiopathic myelofibrosis., Acta Haematol. 1986;75:49-51.

3. Ozsoylu S. High-dose intravenous methylprednisolone for idiopathic myelofibrosis, Lancet. 1988 Apr 2;1(8588):766.

4. Ozsoylu S. High-dose intravenous methylprednisolone in hematologic disorders., Hematology Res 1990;4:197-207.

5. Ozsoylu S, Ruacan S. Karaaslan Y. Dürdar S, Megadose intravenous methylprednisolone in adult myelofibrosis.

Hematology Res 1991;5:121-8.

6. Han ZC, Briere I, Nedellec G, Abgrall JF, Sensebe L, Parent D, Guern G. Characteristics of circulating megalocyte progenitors (GFF-MK) in patients with primary myelofibrosis.

Eur J Haematol 1988;40:130-5.

7. Pereira A, Cervantes F, Brugues R, Rozman C. Bone marrow histopathology in primary myelofibrosis: clinical and haematologic correlations and prognostic evaluation.

Eur J Haematol. 1990 Feb;44:95-9.

Author Reply Dear Editor, We would thank Prof. Özsoylu for critically evaluating our paper and giving some valuable comments.The review of cases in our paper specifically refers to the distinct entity of primary autoimmune myelofibrosis reported in the literature.

In this study we have not included cases of idiopathic myelofibrosis and hence references quoted by Prof. Özsoylu have not been mentioned.

The patients 1 and 2 received iv methyl prednisolone 1g IV OD x 3 days followed by wysolone 50 mg PO. At 5 months and one year follow up, there was improvement in cytopenias and spleen size was regressed. The patient 3 received oral steroids (dose 1mg/kg/day). At one year of follow up the total leucocyte counts and hemoglobin were normal and paravertebral mass had regressed. However, spleen was

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palpable 2 cm. below costal margin. Unfortunately, all three patients had a short period of follow up and thus a repeat trephine biopsy could not be performed to assess the marrow fibrosis.

As regards to increased collagen fibrosis and osteosclerosis, that would indicate progression of the same disease process to more advanced stages. None of our cases showed osteosclerosis and there was absence of significant leukoerythroblastosis as opposed to cases idiopathic myelofibrosis.

Sincerely,

Turk J Hematol 2009; 26: 211-2

Seema Tyagi MD. Associate Professor Department of Hematology All India Institute of Medical Sciences, New Delhi - 110029, India

Letter to the Editor

213

Megadose methylprednisolone for granulocytic sarcoma Granülositik sarkomda yüksek doz metilprednizolon üinasi Özsoylu, Fatih University Medical School Hospital, Ankara, Turkey

To the Editor, Kaya et al.’s [1] case report entitled “Granulocytic sarcoma after stem-cell transplantation in a child with biphenotypic leukemia” gives me an opportunity to remind physicians that short-course megadose methylprednisolone (MDMP; daily, 30 mg/kg for 3 days then 20 mg/kg for 4 days) has been effectively used in the treatment of granulocytic sarcoma (GS) [2-4]. With this treatment, extramedullary relapse rarely occurs, even if the patient develops marrow relapse [3].

On this occasion, I would like to emphasize that, to my knowledge, neutropenic sepsis has not been reported with this treatment, and it is cheaper than the conventional treatment. In addition, it can be easily applied at home on outpatient basis, which makes the treatment much cheaper.

References 1. Kaya Z, Koçak U, Albayrak M, Gürsel T, Akyürek N, Oktar SÖ. Granulocytic sarcoma after stem cell transplantation in a child with biphenotypic leukemia. Turk J Hematol 2009;26:151-3.

2. Hiçsönmez G, Özsoylu ü, Tuncer M, Erer B. High dose intravenous methylprednisolone in the treatment of acute non-lymphoblastic leukemia with ocular involvement. Turk J Pediatr.1988;30:181-3.

3. Hiçsönmez G, Kale G, Erdem G, Bilgic S, Hazar V. Dramatic effects of high-dose methylprednisolone on orbital granulocytic sarcoma. Pediatr Hematol Oncol.

1996;13:187-90.

4. Özsoylu ü. Megadose methylprednisolone for granulocytic sarcoma. Acta Haematol 2001;105:118

Address for Correspondence: üinasi Özsoylu, M.D., Fatih University Medical School Hospital, Alpaslan Türkeý Cad. No. 57, Emek 06510 Ankara, Turkey Phone: +90 538 968 72 01 E-mail: sinasiozsoylu@hotmail.com

Letter to the Editor

214

Premarital screening in Muüla region of Turkey Muúla bölgesi'nde evlilik öncesi tarama

Sibel Özdemir1, ûsmail HakkÕ Timur1, ûskender Gencer2, Nejat Akar3 1Muúla

State Hospital, Biochemistry, Muúla, Turkey Local Health Department, Muúla, Turkey 3Pediatric Molecular Genetics Department of Ankara University, Ankara, Turkey 2Muúla

Received: August 20, 2009 Accepted: September 01, 2009

To the Editor, We read with interest Dr. Sarper et al.’s [1] paper discussing the premarital screening of hemoglobinopathies in the ûzmit region. They also reviewed the previously reported Turkish studies. An editorial on the same subject by Dr. Gürgey also appeared in the same issue [2]. Although they reviewed all the published studies, the data from the Muúla region was missing since it has not been reported previously.

Our group started hemoglobinopathy screening in the Muúla region in the early 1990’s. The city lies in the Mediterranean area of Turkey. Our first screening revealed 8.7% heterozygosity of beta thalassemia, and Hb S, Hb O Arab and Hb D Los Angeles were also observed with frequencies of 0.41, 0.27 and 0.14%, respectively. These values indicated the establishment of a premarital screening program in this region of Turkey [3].

The first official premarital screening was started in January 1997. Besides the obligatory screening for the couples, primary school students were also screened.

A total of 220,689 individuals had been screened by the end of 2008. This is almost 28% of the Muúla’s

population. Of these, 90,530 individuals were screened premaritally. The frequencies of beta thalassemia and abnormal hemoglobins were found to be 3% and 0.6%, respectively, in this group.

During these screenings, 131 couples at risk were detected and families who were at risk for children with hemoglobinopathy were directed to prenatal diagnosis.

Of these, 75 mothers were subjected to prenatal diagnosis and 21 were found to have beta thalassemia major; the pregnancy was terminated in all families.

Since the beginning of the official premarital screening in Muúla, there has been no new beta thalassemia major birth. However, over this period, there have been some new cases; these cases are from couples who married outside the Muúla region or who were married before the official start of the premarital screening.

Several additional hemoglobin variants, including Hb J Iran and Hb Hamadan, were also detected in several families, all of whom needed genetic counseling [4-6].

Sarper et al.’s review and our unpublished data reveal that a great success has been achieved towards the prevention of hemoglobinopathies in Turkey.

References 1. Sarper N, üenkal V, Güray F, üahin Ö, Bayram J. Premarital hemoglobinopathy screening in Kocaeli, Turkey: a crowded

Address for Correspondence: Prof. Nejat Akar, Koru Mah 606. Sok. Uyum Sitesi 19 Çayyolu, Ankara, Turkey Phone: +90 312 241 39 80 E-mail: akar@medicine.ankara.edu.tr

Özdemir et al.

Premarital screening in Muúla

Turk J Hematol 2009; 26: 214-5

industrial center on the north coast of Marmara Sea. Turk J Hematol

215

Author Reply

2009;26:62-6.

2. Gürgey A. From the editor. Turk J Hematol 2009;26:46.

3. Arcasoy A, Turan F, Yeýil N, KemahlÕ S, Uysal Z, Canatan D, Akar N. Muúla ili ve çevresinde thalassemia ve anormal hemoglobin sÕklÕúÕnÕn taranmasÕ. Pediatride Yöneliýler 1994;1:78-80.

4. Yenice S, KemahlÕ S, Bilenoúlu O, Gül Ö, Akar E, Basak AN, Akar N. Two rare hemoglobin variants in the Turkish population (Hb G-Coushatta (B22 (B4) GluÕAla) and Hb J-Iran (B77 (EF1) HisÕAsp).

Turk J Hematol 2000;171:27-8.

5. Akar E, Ozdemir S, Hakki Timur I, Akar N. First observation of

I thank to Özdemir et al. for their attention to our manuscript.

Their letter reveals that 3% of the population in Muúla is thalassemia carier. It seems that premarital screening and prenatal diagnosis are quite successful in preventing births with thalassemia major in this city.

I guess that official premarital screening studies in Turkey are generally unpublished. My aim to prepare this manuscript was to contribute to the epidemiological studies in Turkey. I wish the present study will motivate colleagues to publish their data on hemoglobinopathy screening studies.

homozygous hemoglobin Hamadan (B 56 (D7) Gly-Arg) and beta thalassemia (-29 G>A)- hemoglobin Hamadan combination in a Turkish family. Am J Hematol 2003;74:280-2.

6. Akar N, Akar E, Özdemir S. A further case of Hb J-Iran [beta77(EF1) His>Asp] in Muúla, Turkey. Turk J Hematol 2007;24:37-8.

Nazan Sarper Kocaeli University, Medical Faculty, Pediatric Hematology Department of Child Health and Diseases 41380 Umuttepe, Kocaeli, Turkey

Letter to the Editor

216

Chemotherapeutic trial for acute leukemia in Iraq Irak'ta akut lรถsemi iรงin kemoterapรถtik deneme

Abbas Hashim Abdulsalam Hematology Unit, Lab Department, Al-yarmouk Teaching Hospital, Baghdad, Iraq

Received: July 28, 2009

Accepted: September 1, 2009

Letter to the Editor In Iraq, the diagnosis of acute leukemia is essentially based on clinical presentation and the basic hematological investigations including complete blood count and blood smear morphology, after which a bone marrow aspirate and sometimes a trephine biopsy will follow. These measures usually permit the diagnosis of most varieties of acute leukemias with a very acceptable level of reliability. However, there is still a small percentage of cases that can never be diagnosed based only on morphological features.

Moreover, here in Iraq, there is absence of genetic analysis, apart from limited molecular by genetics polymerase chain reaction (PCR) only for CML BCR-ABL1 oncogene. There is also an absence of flow cytometry and immunophenotyping panels, apart from individual, never in complete panels and inconsistently available/few CD markers study using immunohistochemistry.

Therefore, a chemotherapeutic trial for those who can not afford to seek a more precise diagnosis with genetic study and lineage specification outside this country is a realistic option, as the response to treatment could be a very useful confirmation of the provisional diagnosis. The two examples already faced are AML-M3v diagnosed provisionally only by morphology but with a dramatic response to all-trans-retinoic acid (ATRA) trial, confirming the diagnosis [1] and a few cases of morphologically undifferentiated acute leukemia in which the induction therapy for ALL is tried first. If the patient responds, then a diagnosis of ALL can be deduced; if not, the regimen should be shifted to chemotherapy of AML.

References 1.

Hashim A, Sabeeh N. Acute promyelocytic leukaemia. Slide atlas, BloodMed, British Society of Haematology; 2009, Blackwell Publishing.

Address for Correspondence: M.D. Abbas Hashim Abdulsalam, Hematology Unit, Lab Department, Al-yarmouk Teaching Hospital, Baghdad, Iraq Phone: 964 7904 188690 E-mail: dr.abbas77@yahoo.com

26th Volume Index 26. Cilt Dizini SUBJECT INDEX - KONU DûZûNû March 2009 - December 2009 Mart 2009 - AralÕk 2009 Acute Leukemias

Acute Leukemias, Thyroid Carcinoma

Acute lymphoblastic leukemia / avasküler osteonekroz, 34

Acute myeloid leukemia / akut miyeloid lösemi, 97

Acute myeloblastic leukemia / Akut miyeloblastik lösemi, 207

Radioactive iodine / Radyoaktif iyot, 97

Acute promyelocytic leukemia / Akut promiyelositik lösemi, 47

Thyroid carcinoma / tiroid karsinomu, 97

All-trans-retinoic acid / all-trans retinoik asit, 47 Arsenic / arsenik, 47

Anemias (Acquired)

Avascular osteonecrosis / Akut lenfoblastik lösemi, 34

Gingival and lip hypertrophy induced by cyclosporin A treatment in

Child / çocukluk çaúÕ, 34

an aplastic anemia patient / Aplastik anemili bir hastada siklosporin A

Histone deacetylase / histon deasetilaz, 47

tedavisi ile indüklenen gingiva ve dudak hipertrofisi, 104

ûntestinal mucus accumulation / intestinal mukus birikimi, 207 Metabolism / metabolizma, 47

Anemias (Acquired), Coombs-positive autoimmune hemolytic

Parasite / parazit, 207

anemia, bone marrow aplasia

Resistance / direnç, 47 Therapy / tedavi, 47 Acute erythroid leukemia (AML-M6) - Is it rare? / Akut eritroid lösemi (AML-M6) - Nadir mi görülür?, 38 Megadose methylprednisolone for granulocytic sarcoma / Granülositik sarkomda yüksek doz metilprednizolon, 213 Chemotherapeutic trial for acute leukemia in Iraq / Irak'ta akut lösemi için kemoterapötik deneme, 216 Acute Leukemias, Fanconi anemia Acute myeloid leukemia / akut miyeloid lösemi, 118 Congenital abnormalities / konjenital anormallikler, 118 Diepoxybutane / diepoksibütan, 118 Fanconi / Fanconi, 118 Acute Leukemias, Myelodysplastic Disease Myeloid sarcoma / Myeloid sarkom, 90 Unclassified myelodysplastic/myeloproliferative disease / sÕnÕflandÕrÕlamayan myeloproliferatif/myelodisplastik hastalÕk, 90 Urinary bladder / mesane, 90

Severe bone marrow aplasia and Coombs-positive autoimmune hemolytic anemia in microfilariasis - coincidental or causal? / Mikrofilariaziste aúÕr kemik iliúi aplazisi ve Coombs-pozitif otoimmün hemolitik anemi- sebep, 157 Bleeding Disorders (Hereditary), Trombocytopenia Familial thrombocytopenia associated with ovarian agenesis, umbilical hernia, bicuspid aortic valve, patent ductus arteriosus and epilepsia / Ovaryen agenez, umblikal herni, biküspis aortik kapak, patent duktus arteriyozus ve epilepsi ile birliktelik gösteren ailesel trombositopeni, 42 Chronic Lymphoproliferative Disorders Aortic aneurysm / aort anevrizmasÕ, 31 Fibrillin / Fibrillin, 31 Marfan syndrome / Marfan sendromu, 31 Non-Hodgkin's Lymphoma / Hodgkin dÕýÕ lenfoma, 31 TGF-beta / TGF-beta, 31 Extramedullary hematopoiesis in the axillary lymph node in a patient with an accelerated phase of chronic myeloid leukemia / Akselere faz kronik myeloid lösemili hastada aksiller lenf nodülünde ekstrameduller

Acute Leukemias, Myelofibrosis

hematopoezis, 40

Acute lymphoblastic leukemia / akut lenfoblastik lösemi, 204 Acute myelofibrosis / Akut myelofibrozis, 204

Chronic Myeloproliferative Disorders

Dry tap / kuru ilik, 204

Treatment of primary myelofibrosis / Primer miyelofibroz tedavisi, 211

Gene therapy

Infection, Crimean-Congo

Usage of U7 snRNA in gene therapy of hemoglobin S disorder - is

Crimean-Congo hemorrhagic fever / KÕrÕm-Kongo hemarojik ateýi, 161

it feasible? / Hemoglobin S hastalÕúÕnÕn gen tedavisinde U7 snRNA

Hematological manifestation / hematolojik belirtiler, 161

kullanÕmÕ uygun mu?, 159

Hemophagocytosis / hemafagositoz, 161

Hemoglobinopathies

Infection, Immune disorder

Genetic origin / gensel köken, 17 Hb Beograd / Hb Beograd, 17 Hb D-Los Angeles / Hb D-Los Angeles, 17 Laboratory diagnosis / laboratuvar tanÕsÕ, 17 Premarital diagnosis / premarital tanÕ, 17 Premarital screening / evlilik öncesi tarama, 62 Sickle cell anemia trait / orak hücreli anemi taýÕyÕcÕlÕúÕ, 62 B-thalassemia trait / B,-talasemi taýÕyÕcÕlÕúÕ, 62 Compound heterozygosity for Hb D-Punjab / `-thalassemia and blood donation: case report / Hb D-Punjab / `-talasemi ve kan baúÕýÕ için bileýik heterozigozite: olgu raporu, 100 Premarital screening in Muúla region of Turkey / Muúla bölgesi'nde evlilik öncesi tarama, 214

Fas-Fas ligand / fas-fas ligand, 12 Natural killer cell / NK hücre, 12 Perforin expression / perforin, 12 Varicella / Varicella, 12 Depletion of innate immunity or bone marrow suppresion by viral infection ? / Viral enfeksiyonla kemik iliúinin baskÕlanmasÕ mÕ, yoksa doúal baúÕýÕklÕúÕn tükenmesi mi?, 158 Infection, Stem Cell Transplantation Catheter-related bacteremia / Kateter ile iliýkili bakteriyemi, 67 Diagnosis / tanÕ, 67 Hematopoietic stem cell transplantation / hematopoietik kök hücre nakli, 67 Infectious complication / infeksiyöz komplikasyon, 67

Hemoglobinopathies, Haplotype Beta globin haplotypes / beta globin haplotipi, 129

Iron Overload

Beta thalassemia / Beta talasemi, 129

Atomic absorption / atomik absorbsiyon, 114

Mutation / mutasyon, 129

Chelator / ýelatör, 114 Ciprofloxacin / siprofloksasin, 114

Hemoglobinopathies, Iron Deficiency

Iron overload / demir yüklenmesi, 114

B-Thalassemia trait / B-talasemi taýÕyÕcÕlÕúÕ, 138

Thalassemia / Talasemi, 114

Discriminative indices / ayÕrÕcÕ indeksler, 138 ûndices / ûndeksler, 138

Kala-azar, Leishmaniasis

Iron deficiency anemia / demir eksikliúi anemisi, 138

Leishmaniasis in Yemeni children / Yemenli çocuklarda Leishmaniasis, 102

Red blood cell / kÕrmÕzÕ kan hücresi, 138 Hemophagocytic Lymphohistiocytosis, cyclosporin side effect Corticosteroids / kortikosteroid, 154 Cyclosporin A / Siklosporin A, 154 Hemophagocytic lymphohistiocytosis / hemofagositik lenfohistiyositoz, 154 Hypertrichosis / hipertrikoz, 154 Hypercoagulabiliy Heparin-induced thrombocytopenia / Heparine baúlÕ trombositopeni, 171 Low molecular weight heparin / düýük molekül aúÕrlÕklÕ heparin, 171 medical patients / medikal hastalar, 171

Kala-azar, Parasites Clinical / Klinik, 25 Hematological / Hematolojik, 25 Leishmania / Leishmaniasis, 25 Kaposi’s Sarcoma, Hodgkin’s Lymphoma Hodgkin’s disease / Hodgkin Lenfoma, 201 Kaposi’s sarcoma / Kaposi Sarkomu, 201 Lymph node / Lenf Nodülü, 201

Turkish cohort / Türk hasta grubu, 171

Laboratory Hematology

Unfractionated heparin / fraksiyone olmayan heparin, 171

Acute leukemia / akut lösemi, 72 Blood smear / kan yaymasÕ, 21

Infection

Erythropoietin / Eritropoetin, 72

Children / çocuk, 190

Estimation of platelet count / Trombosit sayÕsÕnÕn tahmin edilmesi, 21

Hematologic disorders / Hematolojik hastalÕklar, 190

Fanconi anemia / Fanconi anemi, 72

Invasive fungal infection / invazif mantar infeksiyonu, 190

Gold nanoparticle / AltÕn nanopartikülü, 29

Hemato-biochemical parameters / hemato-biyokimyasal parametre, 181

Megaloblastic anemia, Laboratory Hematology

Hypercholesterolemia / hiperkolesterolemi, 77

Megaloblastic anemia / Megaloblastik anemi, 197

Hyperglycemia / Hiperglisemi, 77

Nitric oxide / nitrik oksit, 197

Iron deficiency anemia / demir eksikliúi anemisi, 72

Vitamin B12 / vitamin B12, 197

Lead acetate / kurýun asetat, 181 Low electromagnetic field / Düýük frekans elektromanyetik alan, 181

Molecular Hematology (Benign Diseases)

Lymphocyte / lenfosit, 29

Congenital neutropenia / Konjenital nötropeni, 1

Mice / fare, 181 Morphology / morfoloji 77 Phagocytic index / fagositik indeks, 181 Red cell: platelet ratio / KÕrmÕzÕ kan hücresi:trombosit oranÕ, 21 Thallasemia / talasemi, 72 Laboratory Hematology, Hemoglobinopathy Premarital and antenatal screening of Hb-Los Angeles / Hb-Los Angeles’in evlenme ve doúum öncesi tayini, 103

Molecular basis / moleküler temeller, 1 Myelopoiesis / miyelopoezis, 1 RNA processing / RNA ilerlemesi, 1 Myelodysplastic Disease CMML / KMML, 93 CMPD / KMPH, 93 Fibrosis / Fibrozis,93 Leukemia / lösemi, 93 Myelodysplastic syndrome / miyelodisplastik sendrom, 93

Laboratory Hematology, Myelofibrosis

Neuroblastoma

Autoantibodies / otoantikorlar, 146

Recurrent bruising of lower eyelids: a presenting symptom of

Autoimmune / otoimmün, 146

neuroblastoma in a child / Alt gözkapaklarÕnda tekrarlayan morluklar

Leukoerythroblastosis / lökoeritroblastozis, 146

yakÕnmasÕyla baývuran nöroblastomlu bir çocuk olgu, 210

Myelofibrosis / Miyelofibrozis, 146 Splenomegaly / splenomegali, 146

Stem Cell Transplantation Hemorrhagic cystitis / hemorajik sistit, 176

Laboratory Hematology, Thrombocytopenia

Hyperbaric oxygen / Hiperbarik oksijen, 176

Gestational thrombocytopenia / gestasyonel trombositopeni, 123

Stem cell transplantation / kök hücre nakli, 176

HELLP / HELLP, 123 Preeclampsia / preeklampsi, 123

Stem Cell Transplantation, Acute Leukemia

Pregnancy / gebelik, 123

Isolated granulocytic sarcoma / ûzole granulositik sarkom, 151

Thrombocytopenia / Trombositopeni, 123

Relapse / relaps, 151

Liver Diseases, Bleeding disorders Actin polymerization / aktin polimerizasyonu, 82 Adenine nucleotides / adenin nükleotidler, 82 Cirrhosis / Siroz, 82 Cytosolic calcium / sitozolik kalsiyum, 82 Platelets Secretion / trombosit salÕnÕmÕ, 82 Serotonin / serotonin, 82 Malignant Diseases Cancer / kanser, 106 Chemotherapy / kemoterapi, 106

Stem cell transplantation / kök hücre nakli, 151 Thalassemia Cap+1(A-C) mutation / Cap+1(A-C) mutasyon, 167 Polymerase chain reaction (PCR) / polimeraz zincir reaksiyonu (PCR), 167 Silent beta thalassaemia / sessiz beta talasemi, 167 Insulin-like growth factor-1 and zinc in children with Beta thalassemia minor / Beta talasemi minörlü çocuklarda insülin benzeri büyüme faktörü-1 ve çinko, 44 Brilliant cresyl blue staining for screening hemoglobin H disease: Reticulocyte smear / Hemoglobin H hastalÕúÕnda görüntüleme için brilliant cresyl blue boyamasÕ: Retikülosit yaymasÕ, 45

Cryopreservation / kriyoprezervasyon, 106

Thrombosis

Fertility preservation / DoúurganlÕúÕn korunmasÕ, 106

Factor V / Faktör V, 9

Radiotherapy / radyoterapi, 106

Turkish / Türk, 9

26th Volume Index 26. Cilt Dizini AUTHOR INDEX - YAZAR DûZûNû March 2009 - December 2009 Mart 2009 - AralÕk 2009 Khosrou Abdi, 114 Abbas Hashim Abdulsalam, 216 Muhammad Usman Abdul Karim, 167 Ece Akar, 17 Nejat Akar, 9, 17, 214 Sinem Akgül, 154 Eda AkpÕnar, 40 Nalan Akyürek, 34, 151 Meryem Albayrak, 34, 151 Michael Alemayechou, 100 Ayfer Alikaýifoúlu, 154 Manel Almela, 67 Çiúdem Altay, 72 Jain Ankit, 97 Sam Annie-Jeyachristy, 82 Deniz Anuk ûnce, 207 Abdessamad Arabi, 21 Ayten Arcasoy, 118 Unser ArÕkan, 207 Sarangapani Arulprakash, 82 Ayfer Atalay, 17, 129 Erol Ömer Atalay, 17, 129 Suresh Atilli, 97 Zekai AvcÕ, 204, 207 ûsmet Aydoúdu, 197 Emel Babacan, 118 Sadia Usman Babar, 167 Anzel BahadÕr, 17, 129 üule Mine Bakanay, 201 Yasemin IýÕl BalcÕ, 154 Neslihan BaýçÕl Tütüncü, 42 Ullas Batra, 97 Nihayet Bayraktar, 197 Jülide Bayram, 62 Birol Baytan, 190 Amine Bekadja Mohamed, 21 IýÕk Bökesoy, 118 Mohamed Brahimi, 21 Mustafa BüyükavcÕ, 210 Enric Carreras, 67 Bülent Celasun, 204 Premalata CS, 97 Ayhan O. Çavdar, 44, 118 Solmaz Çelebi, 190

Mualla Çetin, 72 Ümit Çobanoúlu, 90 Prasenjit Das, 93 Reena Das, 93 Lokanatha Dasappa, 97 Muzaffer Demir, 171 Enver Duran, 171 Feride Duru, 161 Shyamali Dutta, 146 Mustafa Edis, 171 Mohamed Abd El-azim Hashem, 181 Belkheir Smain Elkahili, 21 Mitra Elmi, 114 Nabela Ibraheem El-sharkawy, 181 Badra Enta-Soltan, 21 Nursat Erdemli, 12 Mehmet Ali Erkurt, 197 Mehmet Ertem, 118 Tunç FÕýgÕn, 161 Shantveer G. Uppin, 38 Gurjeevan Garewal, 93 Arumugam Geetha, 82 ûskender Gencer, 214 Kumar Ghosh Sandip, 31 Subrata Ghosh, 31 Sujoy Ghosh, 31 Ghada A. Gobah, 25 Babu Govind, 97 Sevgi Gözdaýoúlu, 44, 118 Meltem Gülýan, 204 Fatma Gümrük, 45, 72 Ünsal Günay, 190 Adalet Meral Güneý, 190 Fatih Güray, 62 Aytemiz Gürgey, 12, 72, 154 Türkiz Gürsel, 34, 151 Nuray Gürses, 176 Alptekin Gürsoy, 42 Nilgün Güvener Demiraý, 42 Gamal Abdul Hamid, 25 Deepali Jain, 93 Subburayan Jeevan Kumar, 82 Gayathri K., 38 Sevgi Kalayoúlu-BeýÕýÕk, 176

Duygu Kankaya, 201 Rakhee Kar, 146 Ateý Kara, 12 Theano Karafoulidou, 100 Emin Kaya, 197 Zühre Kaya, 34, 151 Gülýah Kaygusuz, 40, 201 Bijan Keikhaei, 138 Zühal Keskin YÕldÕrÕm, 210 Georgia Kiriakopoulou, 100 Üker Koçak, 34, 151 Hasan Koyuncu, 17 Aylin Köseler, 17 ûrfan Kuku, 197 ûbrahim Kulaç, 45 IýÕnsu Kuzu, 40, 201 Saini KV, 97 Sajeevan KV, 97 Turan Kürüm, 171 BarÕý Malbora, 204, 207 Massoud Mahmoudian, 114 Josep Mensa, 67 Srinivas Upendra Mogalluru, 157 Moinuddin Moinuddin, 167 Sevdagül Mungan, 90 Babu Narayanan, 77 Hamza Okur, 12 Soufi Osmani, 21 Muhit Özcan, 201 NamÕk Özbek, 1, 204, 207 Sibel Özdemir, 214 Suna Özhan Oktar, 151 Sinan Özkavukcu, 106 Bülent Özpolat, 47 üinasi Özsoylu, 102, 103, 158, 211, 213 Onur Öztürk, 129 Rasin Özyavuz, 90 Rashmi Patnayak, 38 Tara Roshni Paul, 38 Oktay Perdeci, 176 Parthena Perperidou, 100 Bapsy PP, 97 Permeshwar R, 97 Fakher Rahim, 138

Parvaneh Rahimi-Moghaddam, 114 Senthil Rajappa, 38 Raghunadha Rao Digumarti, 38 Rojrit Rojanathanes, 29 Montserrat Rovira, 67 Nawsherwan Sadiq Muhammed, 123 Muhammed Salih Jaff, 123 Deniz SargÕn, 176 Nazan Sarper, 62 Berna Savaý, 201 Gülten Seçmeer, 12 Amornpun Sereemaspun, 29 Mehdi Shafiee-Ardestani, 114 Pouls Shamoon Rawand, 123 Tejinder Singh, 97 Clio Sinopoulou, 100 Mehmet Sonmez, 90 Bircan Sönmez, 90 Murat Sönmezer, 106 Asuman Sunguroúlu, 118 Rajagopal Surendran, 82 Feride Iffet üahin, 204 Özcan üahin, 62 Vijdan üenkal, 62 Alpaslan üenköylü, 34 Zohra Taghezout, 21 Illias Tazi, 104 Emre Tekgündüz, 171 Stamatia Theodoridou, 100 ûsmail HakkÕ Timur, 214 Ayýe Nur Torun, 42 Murat Tunç, 176 Seema Tyagi, 146 AkÕn Uysal, 40 Zümrüt Uysal, 118 üule Ünal, 12, 45, 72, 154 Maria Velasco, 67 Viroj Wiwanitkit, 29, 159 Mustafa Nuri Yenerel, 176 Ömer YiúitbaýÕ, 171 Mahmut Yüksel, 171 Memnune Yüksel, 118

Advisory Board of This Issue (December 2009) Ali Uúur Ural Ali Ünal Alpay Azap Aydan ûkincioúullarÕ Ayýe Erden Ayýegül Ünüvar Burhan Ferhanoúlu Burhan Turgut Bülent Antmen Celalettin Üstün Cengiz Beyan Dilber Talia ûleri Duygu Uçkan Emel Özyürek Erol Erduran Evren Özdemir

Fatma Gümrük Feride ûffet üahin Fevzi Altuntaý Geng Duyan Güçhan Alanoúlu Güray Saydam Hakan Göker Hakan Özdoúu Hale Ören Hamdi Akan IýÕnsu Kuzu ûsmet Aydoúdu Kenan Keven Lebriz Yüksel Soycan Mehmet Ertem Meliha Nalaci

Mine Hekimgil Mualla Çetin Mustafa Nuri Yenerel Mutlu Arat NamÕk Özbek Nazan Sarper Nurdan TaçyÕldÕz Ömer Devecioúlu Prasama Pradhan Ramazan Çöl Semra Paydaý Teoman Soysal Teresa Coleman Tiraje Çelkan Zahit Bolaman

Announcements 28-30 January 2010 T-cell Lymphoma Forum Maui - USA

22-25 May 2010 The 56th Annual ISTH Conference Cairo, Egypt

30 January 2010 7th Annual Mayo Clinic Hematology Review Minneapolis - USA

4-8 June 2010 2010 ASCO Annual Meeting Chicago, ABD

12-13 February 2010 Turkish School of Hematology 7 Researcher Education Program Ankara Turkey

10-13 June 2010 15th Congress of the EHA Barcelona, Spain

24-28 February 2010 The 2010 BMT Tandem Meetings Orlando, Florida, USA 26-28 February 2010 6th European Congress on Hematologic Malignancies: from Clinical Science to Clinical Practice Cannes - France 4-6 March 2010 6. Turkish National Bone Marrow Transplantation and Stem Cell Therapies Congress Antalya Turkey 21-24 March 2010 The 36th Annual Meeting of the European Group for Blood and Marrow Transplantation (EBMT)Hematology Medical Congress Viena, Austria 16-18 April 2010 Turkish School of Hematology 8 Hematopathology and Consultation Hematology Samsun Turkey 6-9 May 2010 Turkish Society of Hematology Post-Graduate Hematology Education Congress Update on Hematological Oncology Cyprus

10-14 June 2010 World Congress of the World Federation of Hemophilia Buenos Aires, Argentina 25-27 June 2010 ESH-EHA Type II Tutorial on Myeloid Malignancies Istanbul Turkey 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, ABD