2010-4

Volume 27 Issue 4 December 2010 40 TL

ISSN 1300-7777

TURKISH JOURNAL OF HEMATOLOGY • VOL.: 27 ISSUE: 4 DECEMBER 2010

Review Article

Parvovirus-associated hematologic disorders

Sevgi Yetgin et al.; Ankara, Turkey

Research Articles

LTC-IC of CD34-selected cord blood

Gülderen Yankkaya Demirel et al.; stanbul, Turkey, USA

Heparin and human B-lymphoblast cell cycle

Aye Aksoy et al.; Trabzon, Turkey

Dysautonomia and cytokines in cobalamine deficiency

Özcan Çeneli et al.; Ankara, Turkey

Primary HLH in Turkey

Tunç Fgn et al.; Samsun, Kayseri, stanbul, Konya, Ankara, zmir, Trabzon, Van, Turkey

Importance of modified mixed lymphocyte culture test

Hülya Saylan en et al.; stanbul, Turkey

Changes in CBC after radioactive iodine ablation therapy

Bircan Sönmez et al.; Trabzon, Turkey

Blood donation characteristics among university students

Bülent Eser et al.; Kayseri, Turkey

Angiopoietin-2 and soluble Tie-2 in AML

Mohamed A. Attia et al.; Tanta, Egypt

Cover Picture:

Dr. engün Ulutin

The Lake, 2010

4

3 rd International Congress

on

Leukemia

Lymphoma

Myeloma

11-14 May 2011

Istanbul, TURKEY

www.icllm2011.org

Turkish Society of Hematology

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Editor-in-Chief

Aytemiz Gürgey

Associate Editors

Mutlu Arat

Muzaffer Demir

Reyhan Diz Küçükkaya

Mehmet Ertem

Hale Ören

Mehmet Ali Özcan

Ayegül Ünüvar

Celalettin Üstün

Nee Yaral

Akif Selim Yavuz

Past Editors

Erich Frank

Orhan Ulutin

Hamdi Akan

Senior Advisory Board

Orhan Ulutin

Yücel Tangün

Osman lhan

Language Editor

Corinne Can

Statistic Editor

Mutlu Hayran

Editorial Secretary

pek Durusu

Bengü Timoçin

Cover Picture:

engün Ulutin was born in 1935, Mersin

Turkey. She was retied from Internal

Clinic of Cerrahpasa Medical Faculty of

Istanbul University,Cerrahpasa Hospital,

Istanbul, Turkey. The Lake (Oil painting)

2010 was exhibited at 36. Turkish National

Hematology Meeting.

International Review Board

Nejat Akar (Turkey)

Görgün Akpek (USA)

Serhan Alkan (USA)

Çidem Altay (Turkey)

Koen van Besien (USA)

Ayhan Çavdar (Turkey)

M.Sraç Dilber (Sweden)

Ahmet Doan (USA)

Peter Dreger (Germany)

Thierry Facon (France)

Jawed Fareed (USA)

Gösta Gahrton (Sweden)

Dieter Hoelzer (Germany)

Marilyn Manco-Johnson (USA)

Andreas Josting (Germany)

Emin Kansu (Turkey)

Winfried Kern (Germany)

Nigel Key (USA)

Korgün Koral (USA)

Contact Information

Editorial Correspondence should be addressed to

Dr. Aytemiz Gürgey

Editor-in-Chief

Address: Hacettepe University Faculty of Medicine

Pediatric Hematology Department

06100 Shhiye, Ankara/Turkey

Phone: +90 312 305 41 17

Fax: +90 312 305 41 17

E-mail: agurgey@hacettepe.edu.tr

Turkish Society of Hematology

Muhit Özcan, President

Mutlu Arat, General Secretary

Hale Ören, Vice President

Muzaffer Demir, Research Secretary

Teoman Soysal, Treasurer

Fahir Özkalemka, Member

Mehmet Sönmez, Member

Türk Hematoloji Dernei, 07.10.2008 tarihli ve

6 no’lu karar ile Turkish Journal of Hematology’nin

Türk Hematoloji Dernei kdisadi letmesi

tarafndan yaynlanmasna karar vermitir.

Abdullah Kutlar (USA)

Luca Malcovati (Italy)

Robert Marcus (United Kingdom)

Jean Pierre Marie (France)

Ghulam Mufti (UK)

Gerassimos A. Pangalis (Greece)

Santiago Pavlovsky (Argentina)

Antonio Piga (Italy)

Ananda Prasad (USA)

Jacob M. Rowe (Israel)

Jens-Ulrich Rüffer (Germany)

Norbert Schmit (Germany)

Orhan Sezer (Germany)

Anna Sureda (Spain)

Ayalew Tefferi (USA)

Nüket Tüzüner (Turkey)

Catherine Verfaillie (USA)

Srdan Verstovsek (USA)

Claudio Viscoli (Italy)

All other inquiries should be adressed to

TURKISH JOURNAL OF HEMATOLOGY

Address: lkbahar Mahallesi, Turan Güne Bulvar

613. Sk. No:8 06550 Çankaya, Ankara/Turkey

Phone: +90 312 490 98 97

Fax: +90 312 490 98 68

E-mail: info@tjh.com.tr

ISSN: 1300-7777

Web page

www.tjh.com.tr

Sahibi

Türk Hematoloji Dernei adna

Muhit Özcan

Sorumlu Yaz leri Müdürü

Aytemiz Gürgey

Yayn ve Yönetim Yeri

Türk Hematoloji Dernei

Türk Oca Cad. 17/6

Caalolu-Eminönü-stanbul

Üç ayda bir yaynlanan yerel bilimsel dergidir.

Publisher: AVES Yaynclk

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Place of printing: Görsel Dizayn Ofset Matbaaclk Tic. Ltd. ti. - +90 212 671 91 00

Date of printing: December 2010 Broadcast as: Local periodical

A-I

AIMS AND SCOPE

The Turkish Journal of Hematology is the regular publishing organ of the Turkish Society of Hematology. This periodical journal covers subjects

on hematology. The journal is an independent, peer-reviewed international periodical, published quarterly (March, June, September and December)

in English language. The Turkish Journal of Hematology is a nonprofit scientific peer reviewed journal.

Editorial Board of Turkish Journal of Hematology works under the principles of The World Association of Medical Editors (WAME), the International

Council of Medical Journal Editors (ICMJE), and Committee on Publication Ethics (COPE).

The aim of the Turkish Journal Hematology is to publish original research papers of highest scientific and clinical value on hematology. Additionally,

educational material, reviews on basic developments, editorial short notes, case reports, original views and letters from specialists on hematology,

and hematology medicine covering their experience and comments as well as social subjects are published.

General Practitioners interested in hematology, and internal medicine specialists, are also our target audience, and we will arrange the Turkish Journal

of Hematology according to their needs. The Turkish Journal of Hematology is indexed in

- Science Citation Index Expanded

- Embase

- Scopus

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- EBSCO

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Subscription Information

The Turkish Journal of Hematology is sent free of charge to hematologists and academicians in our country as well as to other specialists interested

in hematology. All published volumes in full text can be reached free of charge through the web site www.tjh.com.tr

Adress: Ilkbahar mah. Turan Güne Bulvar 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


Permissions

Requests for permission to reproduce published material should be sent to the editorial office.

Editor: Prof.Dr. Aytemiz Gürgey

Adress: Ilkbahar mah. Turan Güne Bulvar 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


Instructions for Authors

Instructions for authors are published in the journal and on the web page www.tjh.com.tr

Material Disclaimer

The author(s) is (are) responsible from the articles published in the Turkish Journal of Hematology. The editor, editorial board and publisher do not

accept any responsibility for the articles.

The journal is printed on acid-free paper.

A-II

INSTRUCTION TO AUTHORS

The Turkish Journal of Hematology accepts

invited review articles, research articles, brief

reports, case reports, letters to the editor, and

images in Hematology on subjects within

the scope of hematology, on the condition

that they have not been previously published

elsewhere. All papers are subject to editorial

revision for purpose of conformity to the style

adopted by the Journal. Evaluation is a double

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Original research articles

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Maximum length for a Regular Article

is 4,000 words of text. Abstracts must

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objective, material and methods, results,

conclusion. Submissions are limited to a

total of 7 figures/tables. References should

be limited to 50. The sections of a Regular

Article should include Abstract, Introduction,

Material and Methods, Results, Discussion,

References, Figure Legends. Editorial Board

of Turkish Journal of Hematology works

under the principles of The World Association

of Medical Editors (WAME), the International

Council of Medical Journal Editors (ICMJE),

and Committee on Publication Ethics (COPE).

Brief Reports

Short manuscripts definitively documenting

either experimental results or informative

clinical observations will be considered as

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figure legends, and references; abstracts must

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Review Articles

Review articles should not exceed 4,000

words in length, must include an abstract of

300 words or fewer, and may not have more

than 100 references.

Letters to the Editor

Letters can include no more than 400 words of

text, 5-10 references, and 1 figure or table. No abstract

is required, but please include a brief title.

Images in Hematology

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

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Preparation of Manuscript

Each of the following sections of the

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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

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The signed statement on absence of conflict of

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When reporting experiments on human subjects

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Declaration of 1975, revised 2002 available

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“Guide for the care and use of laboratory

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A-III

Abstract and key words: The second page

should include an Abstract which does not

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methods, main findings, specific data, statistical

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Provide 3 to 10 key words below the abstract

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Do not repeat in the text all the data in the tables

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Discussion: Emphasize the new and important

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References: Identify references in text,

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in Index Medicus; consult List of Journals

Indexed in Index Medicus. Include among

the references any papers accepted but not

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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;336:684-7.

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;2:742-4.

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: 780-94.

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;84:2821-3.

Tables

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Place explanatory statistical measures of

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Measurements should be reported in the metric

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New England Journal of Medicine Books

1992, when necessary.

Abbreviations and Symbols

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A-IV

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PROCESS

Manuscripts can be submitted online at

www.journalagent.com/tjh/

The online system consists of four main

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Table 1.

Supported formats

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Windows or Macintosh)

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Submission online

To submit a manuscript, you must first

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This is followed by a keyword input page,

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Use this page to also send your images. Send

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Manuscripts that have passed an initial

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Following initial peer-review, articles judged

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Otherwise Turkish Journal of Hematology

will reject manuscripts which do not received

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A Copyright transfer and conflict of interest

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com.tr

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English-language editing

All manuscripts are professionally edited by

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Online Early

Turkish Journal of Hematology published

abstracts of accepted articles online in advance

of their publication in a printed issue.

A-V

CONTENTS

Review Article

224 Parvovirus-B19 and hematologic disorders

Sevgi Yetgin, Selin Aytaç Elmas, Ankara, Turkey

Research Articles

234 Long-term culture-initiating cells (LTC-IC) produced from CD34+ cord blood cells with limiting dilution method

Gülderen Yankkaya Demirel, Tülin Budak-Alpdoan, Sema Akta, Mahmut Bayk, stanbul, Turkey, New Jersey, USA

242 The effect of heparin on the cell cycle in human B-lymphoblasts: an in vitro study

Aye Aksoy, Erol Erduran, Yavuz Tekeliolu, Mehmet Mutlu, Yusuf Gedik, Trabzon, Turkey

250 Autonomic nervous system dysfunction and serum levels of neurotoxic and neurotrophic cytokines in patients with

cobalamin deciency

Özcan Çeneli, ahika Zeynep Ak, Betül Çevik Küçük, Aye Bora Tokçaer, Reha Kuruolu, Münci Yac, Ankara, Turkey

257 Clinical and laboratory data of primary hemophagocytic lymphohistiocytosis: A retrospective review of the Turkish

Histiocyte Study Group

Tunç Fgn, Türkan Patrolu, Akif Özdemir, Tiraje Celkan, Ümran Çalkan, Mehmet Ertem, Nee Yaral, Erol Erduran,

Canan Vergin, Cengiz Canpolat, Feride Duru, Ali Bay, Namk Özbek, Deniz Ylmaz Karapnar, Samsun, Kayseri, stanbul,

Konya, Ankara, zmir, Trabzon, Van, Turkey

263 The predictive value of stimulation index calculated by modied mixed lymphocyte culture in the detection of

GVHD following hematopoietic stem cell transplantation

Hülya Saylan en, Tülay Klçaslan Ayna, Hayriye entürk Çiftçi, Sevgi Kalayolu Bek, Emel Aye Önal, Arzu Akçay,

Hülya Bilgen, Mehmet Gürtekin, Deniz Sargn, Mahmut Çarin, stanbul, Turkey

269 The changes in complete blood count in thyroid cancer patients treated with radioactive iodine ablation therapy

Bircan Sönmez, smail Doan, Canan Yavruolu, Gamze Can, Mehmet Sönmez, Trabzon, Turkey

275 Are university students a favorable target group for blood donation campaigns?

Bülent Eser, Fatih Kurnaz, Leylagül Kaynar, Mehmet Yay, Serdar vgn, Ali Ünal, Mustafa Çetin, Kayseri, Turkey

282 Prognostic value of soluble angiopoietin-2 and soluble Tie-2 in Egyptian patients with acute myeloid leukemia

Mohamed A. Attia, Sahar M. Hazzaa, Salwa A. Essa, Mahmoud F. Seleim, Tanta, Egypt

Case Reports

289 Acquired pure megakaryocytic aplasia successfully treated with cyclosporine

Halima El Omri, Firyal Ibrahim, Ruba Yasin Taha, Riham Hassan Negm, Aisha Al Khinji, Mohammed Yassin, Ibrahim Al Hijji,

Hanadi El Ayoubi, Hussein Baden, Doha, Qatar

A-VI

294 Syngeneic peripheral blood stem cell transplantation with immunosuppression for hepatitis-associated severe

aplastic anemia

Aleksandar Savic, Bela Balint, Ivana Urosevic, Nebojsa Rajic, Milena Todorovic, Ivanka Percic, Stevan Popovic, Novi Sad,

Belgrade, Serbia

299 A case of acute lymphoblastic leukemia with additional chromosomes X and 5 associated with a Philadelphia chromosome

in the bone marrow

Burak Durmaz, Asude Alpman Durmaz, Emin Karaca, Güray Saydam, Özgür Çoulu, Ferda Özknay, zmir, Turkey

303 Niemann - Pick disease associated with hemophagocytic syndrome

Serap Karaman, Na ye Urganc, Günsel Kutluk, Feyzullah Çetinkaya, stanbul, Turkey

308 Autoimmune hemolytic anemia and giant cell hepatitis: Report of three infants

ule Ünal, Bar Kukonmaz, Necati Balamtekin, Gökhan Baysoy, Selin Aytaç Elmas, Diclehan Gülsev, Gulsev Kale, Aysel Yüce,

Figen Gürakan, Fatma Gümrük, Mualla Çetin, Ankara, Turkey

Letters to the Editor

314 Mega-dose methylprednisolone (MDMP) for chronic idiopathic myelobrosis

inasi Özsoylu, Ankara, Turkey

316 Pancreatitis in a child with acute lymphoblastic leukemia after Erwinia asparaginase: Evaluation of ultrasonography

and computerized tomography as diagnostic tools

Fatih Mehmet Azk, Gonca Klç, Ankara, Turkey

318 Thrombosis and risk factors

Nejat Akar, Ankara, Turkey

320 Laboratory diagnosis of acute leukemia in Iraq, the available options

Abbas Hashim Abdulsalam, Baghdad, Iraq

322 The incidence of alpha-thalassemia in Setif, Algeria

Ayenur Öztürk, Bakhouche Houcher, Nejat Akar, Ankara, Turkey, Setif, Algeria

Images in Hematology

324 FDG-PET in mantle cell lymphoma involving skin

Mehmet Sönmez, Ümit Çobanolu, Sava Karyaar, Bircan Sönmez, Trabzon, Turkey

326 Spinal cord involvement of multiple myeloma detected by F-18 FDG PET/CT Scan

lknur Ak Sivrikoz, Havva Üsküdar Teke, Zafer Gülba, Eskiehir, Turkey

A-VII

224 Review

Parvovirus-B19 and hematologic disorders

Parvovirus B-19 ve hematolojik hastalklar

Sevgi Yetgin, Selin Aytaç Elmas

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

Abstract

Parvovirus-B19 (PV-B19) is a member of Parvoviridae, which is one of the smallest DNA viruses.

PV-B19-associated diseases usually serve as a good representation of the balance of virus, host response

and the immune system. The diseases manifested with PV-B19 are erythema infectiosum, which is common

in children, hydrops fetalis, transient pure red cell aplasia in patients with chronic hemolytic anemia,

arthralgia - mostly observed in women, and chronic pure red cell aplasia in immunocompromised

individuals. Cytopenia (bicytopenia, monocytopenia or pancytopenia) may also accompany the diseases

mentioned above. On the other hand, there are many diseases, including neurologic, vasculitic, hepatic,

rheumatoid, nephritic, autoimmune, myocardial, and others in which the mechanisms of the diseases are

not clear, which may be associated with PV-B19. The virus may manifest with unexpected and unexplained

clinical pictures and lead to misdiagnosis. Therefore, hematologic disorders in any unestablished

clinical diagnosis should be investigated for PV-B19 infection. However, serologic examination for

PV-B19 diagnosis is not sufficient in immunocompromised status. The virus can be determined with

polymerase chain reaction (PCR) in the serum or tissue samples. Supportive therapy, blood transfusion

and immunoglobulin are the conventional therapeutic interventions for PV-B19 today. Vaccination studies

are under examination. (Turk J Hematol 2010; 27: 224-33)

Key words: Children, hematologic findings, parvovirus, treatment

Received: July 28, 2010 Accepted: November 22, 2010

Özet

Parvovirus-B19 (PV-B19) küçük bir DNA virusu olup Parvoviridia ailesinin bir üyesidir. PV-B19 ile ilgili

hastalklar virus, konakç cevab ve immun sistem etkilemesini temsil eden iyi birer örnektir. PV-B19

ilikili hastalklar, çocukluk çanda eritema infeksiozum, hidrops fetalis ve kronik hemolitik anemilerde

geçici eritroid aplazi, kadnlarda artralji, immun yetmezlikli olgularda ise kronik eritroid aplazidir.

Sitopeni (bisitopeni, monositopeni yada pansitopeni) yukardaki hastalklara elik edebilir. Öte yandan

PV-B19 ile ilikili olabilecek nörolojik, vaskülitik, hepatik, romatoid, nefritik, otoimmun, miyokard hastalklar

ve mekanizmas tam olarak açklanamam birçok hastalk da vardr. Virus beklenmeyen ve

açklanamayan klinik tablolara yol açabilecei için yanl tan konulabilmektedir. Bu nedenledir ki henüz

klinik tan konulmam, hematolojik bulgular olan hastalarda PV-B19 infeksiyonu dikkate gelmelidir.

Ancak immun yetmezlik durumunda serolojik testler tan için yeterli olmayabilir. Virus serumda yada

doku örneinde PCR ile tespit edilebilir. Destek tedavisi, kan transfüzyonu yada immunglobulin günümüzde

kullanlan tedaviler olup a çalmalar aratrma aamasndadr. (Turk J Hematol 2010; 27: 224-33)

Anahtar kelimeler: Çocuk, hematolojik bulgular, parvovirus, tedavi

Geli tarihi: 28 Temmuz 2010 Kabul tarihi: 22 Kasm 2010

Address for Correspondence: Prof. Dr. Sevgi Yetgin, Department of Pediatric Hematology, Hacettepe University, Ankara, Turkey

Phone: +90 312 305 11 72 E-mail: yetgins@hacettepe.edu.tr

doi:10.5152/tjh.2010.43

Yetgin et al.

Turk J Hematol 2010; 27: 224-33 Parvovirus-associated hematologic disorders 225

Introduction

Parvovirus-B19 (PV-B19) was discovered in the

mid 1970s by Yvonne Cossart in London [1]. The

name of this virus is derived from occupying well

B19 in a large series of petri dishes numbered

accordingly. Since the discovery of PV-B19, many

reports have been published showing an association

between the virus and many other clinical diseases

[2-10]. In the early 1980s, it was demonstrated

as an agent of human disease erythema infectiosum

(fifth disease) in children, transient aplastic

anemia and hydrops fetalis [3-7]. Gradually, reports

showed that this virus had a pathogenic role in a

large perspective in addition to sometimes being

asymptomatic [8-39]. The literature of PV-B19 infection

with a growing number of publications has

reported its association with erythema infectiosum

in children, transient aplastic crisis in persons with

high red cell turnover, chronic pure red cell (PRC)

aplasia in primary or secondary immunocompromised

patients, hydrops fetalis with infection during

pregnancy, and arthropathy, mostly in females.

Today, according to the results obtained from

PV-B19 studies, in the case of unexpected or unexplained

anemia or hematologic disorders, PV-B19

should be investigated. In addition, there are also

many diseases with an unusual clinical presentation

in which PV-B19 is identified as the pathogen.

In this review, principally PV-B19 and hematologic

disorders will be discussed on the basis of the

literature and our experience. Special characteristics

of the virus are presented beforehand.

PV-B19 characteristics

Human PV-B19 is from the group Parvoviridae,

and is one of the smallest DNA viruses. The

Parvoviridae family includes many animal viruses

that are pathogenic for animals. Parvoviruses form

small capsids about 25 nm in diameter and contain

a genome consistent with single-stranded DNA [38-

40]. The viral genome encodes three proteins of

known function [38-41]. The gene product, the nonstructural

protein NS1, has been shown to be

involved with DNA, helps with replicase function

and is cytotoxic for host cells. It also initiates apoptosis

with cytokine stimulation. The other two structural

proteins, viral protein1 (VP1) and VP2, take

part in viral capsid proteins [38,40]. PV-B19 exerts

its pathogenicity by the cellular receptor that is present

on erythroid precursor cells and erythrocytes as

the blood group P antigen [42]. Since this receptor

protein is necessary for viral infection, a person

without P antigen on red cells is immune against

PV-B19 infection. Various other cells presenting P

antigen are platelets and cells on the liver, kidney,

heart, lung, endothelium, and on the synovium [43].

PV-B19 infection is seen in all ages worldwide.

This infection is common in childhood, and approximately

80% of the population is immune to this

virus after 50 years [44]. The infection rate of PV-B19

is determined by assessment of antiparvovirus

immunoglobulin (Ig)G antibody in serum samples.

The virus is spread by respiratory droplets and has

also been transmitted by blood products because of

its resistance to heat inactivation and solvent detergents

[45,46]. PV-B19 infection is usually asymptomatic.

The most common clinical presentations

of infection are fifth disease or erythema infectiosum,

arthropathy and hydrops fetalis, which are

found among immunocompetent hosts [2,3,38].

The infection may cause clinically significant

arthropathy in adults, especially middle-aged

women. Arthropathy includes not only arthralgia

but also inflammatory arthritis that may occur with

sequelae in some cases. It may mimic rheumatoid

arthritis with clinical presentation and positive rheumatoid

factor [34-38,47]. Hematologic diseases

such as transient PRC aplastic crisis, hydrops fetalis

and single or multiple transient parvovirus-related

cytopenias (neutropenia, autoimmune thrombocytopenic

purpura, pancytopenia) are also seen in

immunocompetent patients [4,5,7-10,39]. Hepatic,

neurologic, vasculitic, rheumatologic, and nephritic

syndromes, and myocardial disorders associated

with PV-B19 are a heterogeneous group of diseases

in which the etiological role of the virus is not clear

[24-33,36,37,47-55]. Immunocompromised hosts

such as cancer patients, transplant patients and/or

patients receiving immunosuppressive drugs and

those with acquired immune deficiencies are at risk

of PV-B19 infection and PRC aplasia resulting in

chronic anemia [11-23]. Although erythroid lineage

is mostly affected, thrombocytopenia, neutropenia

and pancytopenia have also been reported with

PV-B19 infection in this group of patients.

Chronic anemia is the result of a selective

decrease in red cell precursors in the bone marrow

that presents as reticulocytopenia in the peripheral

226

Yetgin et al.

Parvovirus-associated hematologic disorders Turk J Hematol 2010; 27: 224-33

blood [56-58]. All immunocompromised patients

with anemia or any other abnormal hematologic

findings, even if they have no specific symptoms of

viral infection, should be suspected of PV-B19.

Antibody-based diagnostic tests cannot help in the

diagnosis because of immunosuppressive status

that leads to lower or no Ig production. Virus

genome by polymerase chain reaction (PCR) or

other assays should be preferred according to

patient characteristics [2].

Clinical associations of PV-B19 infection, such as

skin eruption or gloves and socks syndrome and

chronic fatigue syndrome, are seen rarely [24,59-61].

Intrauterine PV-B19 infection is also very rarely

associated with developmental abnormalities [62].

Since the diseases associated with PV-B19 may be

related with any system of the body, PV-B19 infection

should be kept in mind in cases of an unsolved

clinical picture even when there is no suggestion of

an infection.

PV-B19 and immune response

In PV-B19 infection, fever and influenza–like

symptoms occur early during viremia. This feature is

followed by cutaneous eruption and arthropathy, and

this phase corresponds to the appearance of antibody

in the blood. While the titers of the virus fall in

the blood, IgM antibody appears at the same time

and continues for 2-3 months in the serum. IgG antibodies

are observed after 2-3 weeks of infection and

continue life-long [2,4,61]. The virus can persist in the

body for a long time, months and even years [11].

In fact, PV-B19 is eliminated from the patients within

a few weeks; however, in 20% of immunocompetent

patients and in patients with a defective Ig

production against the virus, PV-B19 infection is

persistent [11,30,58,61,62]. These patients are a

source for PV-B19 infection if they are a donor.

Proteins on the surface of the virus capsids, VP1

and VP2, are recognized by the host immune system

and are antigenic determinants. These many

linear epitopes due to the VP1 region of the capsid

are combined by neutralizing antibodies. VP1 is

principally required for an effective immune

response and also clearance of the virus. In the

early phase of convalescence, the patient’s serum

reacts to VP2, but it is not effective in the late phase

[47,63,64]. Commercial Igs are obtained from the

plasma of normal persons with strong anti–VP1

activity.

Hematologic disorders: major hematologic

disorders of PV-B19 with underlying disease and

conditions

Parvovirus-B19 is the causative agent of various

forms of hematologic diseases, among which transient

aplastic crisis in patients with underlying hemolytic

anemia, chronic hypoplastic anemia-PRC aplasia

in immunocompromised patients and hydrops

fetalis in pregnant women are the most common.

Besides these hematological disorders, PV-B19 precedes

or is associated with other hematological diseases

or present hematologic features [39].

Transient aplastic crisis: In transient PRC aplasia,

PV-B19 has a direct effect on the hematopoiesis,

mainly on erythroid progenitor and erythrocytic cell

line, and thus anemia is principally the most frequent

hematologic presentation. In addition to the

increased red cell destruction, patients also produce

red blood cells and thus PV-B19 infection may result

in transient suppression of erythropoiesis [5-10].

Some patients with clinically inapparent and uncomplicated

PV-B19 infection can present with a compensated

phase of anemia with a smaller decrease

or stable level of hemoglobin. Reticulocytopenia is

clear because of cessation of the erythropoiesis in

that compensated anemia. Anemia occurs during

viremia since PV-B19 infects erythroid progenitor

cells, resulting in temporary cessation of red cell

production, and lasts about 1-2 weeks. Although

specifically erythroid lineage is affected, myeloid

lineage, which may also include megakaryocytic

cells, may also be suppressed [65-67]. The PV-B19

infection in immunocompetent persons classically

presents with an isolated red cell aplasia [5].

However, in immunocompromised patients, the

virus can also affect all hematopoietic lineages and

lead to pancytopenia [15,58].

Viral infections cause transient bone marrow

aplasia or selective erythroid aplasia. Anemia on

this basis is rare in healthy subjects without underlying

disease because of the long life span of the red

blood cell. In contrast, patients with hemolytic anemia

can experience a rapid fall in hemoglobin level.

Although transient aplastic crisis is self-limited, red

cell transfusion as a supportive treatment should be

provided in case of acute anemia.

Parvovirus-associated transient hypoplasia of

multiple peripheral blood cell lines has also been

reported in children with chronic hemolytic anemia

and even in healthy individuals [4,9,39,65-67]. In

Yetgin et al.


sickle cell anemia, erythrocyte membrane defects,

red cell enzyme defects, thalassemia, and acquired

hemolytic anemia result in stressed erythrocyte production

as in hemorrhage, iron deficiency anemia

and bone marrow transplantation, in which there

may be a red cell hemolytic process, and the preceding

PV infection can present with transient

aplastic crisis [38,39,47]. In a study of a group of

sickle cell patients with aplastic crisis, it was shown

that nearly all patients had PV-B19 infection close to

their crisis [7,8].

In patients with hemolytic anemia, bone marrow

examination is not required to establish the diagnosis

of aplastic crisis. However, bone marrow aspiration

is necessary for a patient who was previously

well or has an atypical clinical presentation. The

most common finding is normocellular bone marrow

with a decrease in erythroblasts [38,39]. It is

known that the virus may cause severe pancytopenia

and aplastic anemia [68]. A girl from our clinic

with PV-B19 infection presenting with a severe

aplastic anemia without underlying disease was

reported. This patient underwent bone marrow

transplantation from her HLA-identical sibling,

resulting in complete recovery [69]. Hanada et al.

[65] experimentally showed that PV-B19 significantly

inhibited erythroid (CFU-E), myeloid (CFUmyeloid)

and megakaryocytic (CFU-Mgk) growth in

a patient with hereditary spherocytosis. PV-B19-

infected patients seem to infer hematological findings

as related with their own body physiology and

response to virus toxicity.

Persistent PV-B19 infection: chronic pure red cell

aplasia

This clinical aspect of chronic PRC aplasia principally

occurs in the context of primary or secondary

immune deficiency in patients who cannot produce

neutralizing antibody against PV-B19 [12-

23,58,70,71]. Since antigen-antibody complexes are

not produced in persistent PV-B19, which results in

chronic PRC aplasia, fifth disease does not develop.

Acquired PRC aplasia was described in a patient

with Nezelof syndrome who was infected with

PV-B19 infection for the first time [11]. One report

suggested that transplacental transmission of

PV-B19 caused a severe anemia in three infants

[56]. A separate report described three infants with

Diamond-Blackfan anemia in whom PV-B19 might

have had an unclear role in that hypogammaglobulinemia

[57]. Acquired immune deficiency secondary

to human immunodeficiency virus (HIV) infection

[14], immunosuppression occurring in malignant

disease or during chemotherapy, and transplantation

or autoimmune diseases can lead to

chronic anemia due to persistent PV-B19 infection

[12-23]. Persistent PV infection in children with leukemia

may be associated with anemia or cytopenia

and cause prolonged interruption of chemotherapy

[70]. Patients who were under chemotherapy for

malignant disease such as leukemia may also present

PRC aplasia even in remission. The PRC aplasia

due to persistent PV infection emerges in the disease

course of that patient. However, the two features

occur simultaneously only rarely. In the case

of PRC aplasia, it is important to investigate and

determine PV-B19 in order to prevent any confusion

surrounding disease recurrence and interruption of

chemotherapy due to suspicion of a toxic effect on

the bone marrow. Moreover, PV-B19 should be

investigated at the time of the diagnosis of malignant

disease [72].

Today, stem cell transplantation of bone marrow

and organ transplantation are established therapy

methods for many diseases representing both

malignant and non-malignant groups. PV-B19 can

produce a picture known as PRC aplasia in transplant

patients. PV has been identified in patients

with engraftment failure due to PRC aplasia [73].

Since the deep immune suppression period continues

one year after transplantation, the virus can be

increased and more effective in that time [15-23].

Hydrops fetalis

In utero, PV-B19 infection may cause nonimmune

hydrops fetalis especially during the second

trimester of pregnancy [6,38,47,57]. It is known

that the fetal red cell source is the liver in the early

period of intrauterine life. PV-B19 infection in the

liver leads to anemia and also virus, which can

directly affect the myocardium and may cause cardiac

failure that results in hydrops fetalis [38]. About

10-20% of non-immune hydrops fetalis is related

with PV infection [38]. Prospective studies reported

a 30% risk of transplacental infection of PV, and it

was reported that half of the pregnant women in the

German population are susceptible to PV infection

[38,47]. On the other hand, many newborns are

born from an infected mother without any symptoms.

However, death rate among fetuses with

hydrops is about 9% [38,47]. Furthermore, in a

mother with PV infection in early pregnancy, the

228

Yetgin et al.


spontaneous abortion rate is reported to be 5%.

PV-B19 may also cause premature birth with hematological

disorders. Severe anemia in newborns

diagnosed as PRC aplasia (Diamond-Blackfan anemia)

by bone marrow examination can be the

result of transplacental transmission of PV-B19

infection [57]. If a pregnant woman’s immune status

shows a positive PV-B19 infection via positive

IgM antibodies, this mother should be examined by

ultrasonography weekly or bi-weekly for at least

10-12 weeks. Amniotic fluid and umbilical cord

blood are used to detect the virus and IgM antibodies

to PV-B19. Nevertheless, PV-B19 IgM antibody of

the mother may be negative at the time of diagnosis

of hydrops fetalis. During the follow-up of this fetus,

intrauterine transfusion therapy may be necessary.

PV-B19 infection with other hematological

disorders

In addition to the well-known common hematological

presentation, PV can present more than the

major hematologic manifestations. In this group,

the clinical presentation of PV infection can be

missed. Based on peculiarities of the host and properties

of the pathogen, clinical and hematological

symptoms may result in unexpected findings that

are not suggestive of PV infection at first evaluation.

Since PV-B19 is a hematopoietic, principally an

erythrogenic virus, anemia is the most frequent

hematologic finding of PV infection. Pancytopenia

with mild or masked findings may occur in patients

with underlying hemolytic anemia or immunocompromised

conditions [8,9,15,39,58]. Autoimmune

thrombocytopenia and/or autoimmune isolated

neutropenia has been reported in immunocompetent

individuals to be caused by or associated with

PV-B19 [74,75]. Cartron et al. [74] showed that in 5

of 11 chronic autoimmune neutropenic children,

antineutrophil antibody was positive and was an

evidence of primary PV infection. Neutropenia and

PV association was determined by bone marrow

culture studies in another report [4].

Immune thrombocytopenic purpura (ITP) may

follow a viral infection or immunization and is caused

by an abnormal response of the immune system

[76,77]. Antiplatelet antibody production is the main

mechanism of ITP. In PV-B19 infection, immune

response to the virus is characterized by the appearance

of the specific autoantibodies. Host response

contributes to the progress of the disease. Persistent

infection can be seen in patients who have a low

level of neutralizing antibodies. ITP association with

PV-B19 infection was reported by Murray et al. in

1994 [75]. In a study in our clinic of 19 children with

ITP (8 acute, 11 chronic), the rate of 47% PV-B19 DNA

also indicated a strong association of PV-B19 infection

with ITP [78]. This relation was also supported

by high seropositivity of anti-PV-B19 IgM and IgG. A

literature search revealed case reports of chronic ITP

and neutropenia and a case report of sarcoidosis

complicated by chronic red blood cell aplasia and

severe thrombocytopenia with PV-B19 infection [79-

81]. In virus-associated ITPs, antiplatelet antibodies

or virus antibody-immune complex has been associated

with platelet destruction [75]. In acute ITP cases

in whom PV-B19 was detected, a direct antibodymediated

destruction is a likely explanation. The

chronic ITP cases with PV-B19 could be associated

with autoantibodies or viral persistence. The pathogenic

mechanism of thrombocytopenia associated

with PV-B19 has been proposed as autoimmune

activity and submicroscopic megakaryocytopathic

effect leading to less platelet production [78,81,82]. It

was also suggested that even stem cells are affected

by PV-B19. The three lineage dysplasias in ITP that

have been reported from our clinic may be evidence

suggestive of the virus effect on stem cells [83].

Acute PV-B19 infection associated with myelodysplastic

syndrome (MDS) has been reported

rarely in immunocompetent children [84]. Chronic

hemolytic anemia or subclinical immune deficiency

by contributory predisposing factors with PV-B19

infection might provide the appropriate condition

for the outcome of dysplastic presentation. Acute

PV-B19 infection mimicking juvenile myelomonocytic

leukemia (JMML), classified under MDS, has

also been reported [85]. In this case, which was

reported from our clinic, clinical and hematological

findings strongly suggested JMML, and immunologic

examinations were normal. On the basis of this

case and a report of PV with myelodysplasia [86],

it might be postulated that PV-B19 not only affected

the proliferative capacity of hematopoiesis but

also the differentiation process, which are the principal

findings of MDS, a group of clonal stem cell

disorders.

Leukoerythroblastosis characterized by the presence

of leukocytosis and erythroid and myeloid blast

cells in the peripheral blood in a premature baby was

explained to be related to intrauterine PV-B19 infection

[87]. Dysplasia restricted to only one lineage

such as dyserythropoiesis was reported [88].

Yetgin et al.


There are case reports of PV-B19 infection presenting

hemophagocytic lymphohistiocytosis [89,90].

Parvovirus infection with an expanded clinical

manifestation may precede or be associated with

leukemia. This infection preceding leukemia has

been suggested in several reports [72,91,92]. It has

been hypothesized that at the time of diagnosis of

acute leukemia, the virus may not be present in the

serum but may be present at the cryptic site such as

the cerebrospinal fluid [91,92]. We experienced two

cases of PV-B19 infection and bone marrow infiltration

with pre-B cell lymphoblasts in which the diagnosis

was established based on morphological and

flow cytometric examinations. One of these patients

progressed to acute lymphoblastic leukemia (ALL),

while the other showed total resolution of the blastic

morphology and phenotype in the short followup

period [72]. A report pointed out the expression

of PV-B19 receptor on leukemic cells with a weak

association [93]. Temporary effects of the virus on

the hematopoietic system may lead to a limited

capacity of clonal proliferation, as in our patient

with a resolution of ALL. In the case of compromised

status of the host, the effect of the virus on

hematopoiesis prompts the clonal proliferation,

which allows this speculation in our other case with

persistent blastic presentation.

Diagnosis

Constitutional symptoms of PV infection, such as

fever and erythroid progenitor cell depletion in the

bone marrow, are accompanied by viremia during

the first week of infection. PV causes fifth disease in

children and polyarthropathy in adults. Transient

aplastic crisis occurs in the case of underlying

chronic hemolytic anemia conditions, and chronic

PRC anemia is presented by persistent infection in

immunocompromised individuals. In pregnant

women, IgM to PV-B19 is a predictive finding for

infection.

Reticulocytopenia occurs at the height of viremia

and is followed by anemia according to the host

physiology. Bone marrow examination shows normocellularity

with findings of profound depletion of

erythroid cells, abnormal very large pronormoblasts

and normoblasts exhibiting intranuclear eosinophilic

inclusion bodies (Lampion or Lantern cells).

Specific antibody IgM begins to appear when the

titer of virus falls in the blood and continues for 3-6

months; IgG antibodies can appear in the third

week of inoculation of the virus and persist throughout

life. Immunologic assays remain the most sensitive

method to detect PV-B19 infection in almost all

cases of fifth disease [94,95]. Because of interindividual

variation of antibody production according to

physiologic conditions, in persistent infection, DNA

assay should be performed for diagnosis of PV-B19.

In immunocompromised patients, antibody production

is minimal or absent. Viral DNA testing is crucial

for the diagnosis of PV-B19 infection. PCR can

detect PV genome. DNA amplification by PCR assay

may be false-positive because of contamination.

Real-time PCR is used for determining virus load on

tissues, and immunohistochemistry (IH) and in situ

hybridizations (ISHs) allow the topographical identification

of virus-specific target cells in the histological

sections. PCR and nested–PCR have been introduced

in pathology to detect the PV-B19 genome

[95,96].

Treatment

Clinical approaches to PV-B19 infection should

be planned based on the infection symptoms and

severity. Infection is self-limited in healthy children

and in adults. Isolation is not proposed except for

those admitted to the hospital [45]. In patients with

chronic hemolytic anemia, blood transfusion may

be needed especially in patients with clear spleen

sequestration. Blood transfusion is also required in

case of intrauterine infection and hydrops fetalis.

Specific therapy is adapted according to infection

related to the body system and severity of symptoms.

Ig therapy has been used in immunocompromised

patients and/or autoimmune hematological

or non-hematological presentations.

Persistent infection in PV is a treatable cause of

anemia in immunocompromised patients. Nearly

all HIV-infected patients with PV-B19 PRC aplasia

responded to therapy with intravenous immunoglobulin

(IVIG). This regimen is also curative in

patients with congenital immune deficiency

[96,97]. Immunocompromised hosts are particularly

at risk of PV-B19 infection, including patients

with congenital or acquired immune deficiency,

acquired immunodeficiency syndrome (AIDS) and

cancer, and in transplant patients on immunosuppressive

treatment. Ig therapy is effective in ITP

patients with a repeated given regimen [98].

Studies regarding vaccination against PV-B19 infection

are under way [64,65].

In conclusion, since PV-B19 expresses expanded

clinical and laboratory findings, it should be evalu-

230

Yetgin et al.


ated and kept in mind in patients showing rapid cell

destruction, in primary and secondary immune

compromised patients, and in pregnant women

with any hematological manifestations during their

pregnancy. Different clinical findings in any body

system with unexplained clinical pictures should

also be investigated with satisfactory methods for

PV-B19. Investigation of this virus in malignant diseases

before starting chemotherapy is an important

approach. It is possible that there are more manifestations

due to PV-B19 that will be uncovered in the

future.

Conflict of interest

No author of this paper has a conflict of interest,

including specific financial interests, relationships,

and/or affiliations relevant to the subject matter or

materials included in this manuscript.

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234 Research Article

Long-term culture-initiating cells (LTC-IC)

produced from CD34+ cord blood cells with

limiting dilution method

Kstl dilüsyon yöntemi ile CD34+ kordon kan hücrelerinden uzundönemli

kültür-balatan hücreler (UDK-BH) üretimi

Gülderen Yankkaya Demirel 1 , Tülin Budak-Alpdoan 2 , Sema Akta 1 , Mahmut Bayk 3

1Yeditepe University, Stem Cell Laboratory of Hematology Department, Istanbul, Turkey

2University of Medicine and Dentistry of New Jersey, Department of Medicine, New Jersey, USA

3Marmara University, Department of Internal Medicine, Hematology-Immunology Section, Istanbul, Turkey

Abstract

Objective: Even though much progress has been made in defining primitive hematologic cell phenotypes by

using flow cytometry and clonogenic methods, the direct method for study of marrow repopulating cells still

remains to be elusive. Long Term Culture-Initiating Cells (LTC-IC) are known as the most primitive human

hematopoietic cells detectable by in vitro functional assays.

Materials and Methods: In this study, LTC-IC with limiting dilution assay was used to evaluate repopulating

potential of cord blood stem cells.

Results: CD34 selections from cord blood were completed succesfully with magnetic beads (73,64%±9,12).

The average incidence of week 5 LTC-IC was 1: 1966 CD34+ cells (range 1261–2906).

Conclusion: We found that number of LTC-IC obtained from CD34+ cord blood cells were relatively low in

numbers when compared to previously reported bone marrow CD34+ cells. This may be due to the lack of

some transcription and growth factors along with some cytokines and chemokines released by accessory cells

which are necessary for proliferation of cord blood progenitor/stem cells and it presents an area of interest

for further studies. (Turk J Hematol 2010; 27: 234-41)

Key words: Stem cell, cord blood, LTC-IC, limiting dilution, CFU assay, cytometry

Received: May 24, 2010 Accepted: August 19, 2010

Özet

Amaç: Akm hücre ölçer ve klonojenik yöntemler kullanm ile öncül hematolojik hücre fenotiplerinin tanmlanmasnda

çok aama kaydedilmi olmasna ramen, kemik iliini yenileyen hücrelerin dorudan bir yöntemle

tanmlanmas henüz mümkün olmamaktadr. Uzun Dönemli Kültür-Balatan Hücreler (UDK-BH) in

vitro fonksiyonel yöntemlerle saptanabilen en öncül insan hematopoietik hücreleridir.

Yöntem ve Gereçler: Bu aratrmada, kordon kan kök hücrelerinin kemik iliini yenileyen hücre yeteneini

ölçümlemek üzere kstl dilüsyon ile UDK-BH yöntemi uygulanmtr.

Bulgular: Kordon kanndan CD34+ hücrelerin seleksiyonu manyetik boncuklarla baar ile gerçekletirilmitir

(%73,64±9,12). UDK-BH’lerin 5. haftada ortalama görülme skl 1: 1966 CD34+ hücredir (aralk:

1261-2906).

Address for Correspondence: M.D. Gülderen Yankkaya Demirel, Devlet Yolu, Ankara Cad. 102 -104, Kozyata 34752 Istanbul, Turkey

Phone: +90 216 578 40 85 E-mail: gulderen.ydemirel@yeditepe.edu.tr

doi:10.5152/tjh.2010.44

Demirel et al.

Turk J Hematol 2010; 27: 234-41 LTC-IC of CD34-selected cord blood 235

Sonuç: CD34+ kordon kan hücrelerinden elde edilen UDK-BH says daha önce yaynlarda bildirilen kemik

ilii hücreleri deerlerinden daha düük düzeyde saptanmtr. Bu bulgunun proliferasyon için gerekli olan

sinyal transkripsiyon molekülleri, büyüme faktörleri, kemokin ve sitokinlerin aksesuar hücreler tarafndan

salnarak ortamda olmamasna bal olduu düünülmütür. Bu alanda gelecekte daha çok aratrma yaplmas

bilgilerimizin artmasn salayacaktr. (Turk J Hematol 2010; 27: 234-41)

Anahtar kelimeler: Kök hücre, kordon kan, LTC-IC, kstl dilüsyon, CFU kültürü, akan hücre ölçer

Geli tarihi: 24 Mays 2010 Kabul tarihi: 19 Austos 2010

Introduction

Although much progress has been made in

defining primitive hematologic cell phenotypes

using flow cytometry and clonogenic methods, a

direct method for study of marrow repopulating

cells remains elusive. The long-term culture-initiating

cells (LTC-IC) are the most primitive human

hematopoietic cells detectable by in vitro functional

assays. The validity of the LTC-IC assay is dependent

on the existence of a linear relationship between

the endpoint measured (clonogenic cell output as

assessed after 5 weeks of culture) and the number

of LTC-IC in the original test suspension down to

limiting numbers of LTC-IC. To evaluate cord blood

stem cells for their repopulating potential, we used

limiting dilution assay (LDA) for LTC-IC. We believe

that identification of defined conditions for extensive

and rapid amplification of these cells would

help in analyzing the benefit of these cells for therapeutic

applications in the future [1-3].

LDAs are designed to define an unknown frequency

of effector cells in a population. As described

by Frisan et al. [4], “LDA are dose-response assays

that allow detection of an all-or-none (positive or

negative) immunoresponse in each individual culture

within replicates that vary in the number of

responder cells tested. The frequency of positive

cultures is not informative because it is never clear

whether one or more precursors in the culture well

are giving the positive response. The negative

response instead demonstrates that there are no

precursors of a given specificity. Therefore, the

evaluation of the cell frequency in the original population

is possible by determining the number of

cultures that are negative in the experiment”. The

LDA method for LTC-IC was the method of choice in

our study to achieve more efficient results.

The type of the feeder layer, cell concentration,

presence of accessory cells, change of culture

media, and additional growth factors in the media

are important for the proliferation of LTC-IC in culture

media [3]. While many different cell lines have

been published previously as feeder layer [4], it has

been reported that the murine fibroblast M210-B4

cell line can support the maintenance of LTC-IC

from human bone marrow as effectively as standard

human marrow adherent cells [5].

Materials and Methods

Sample Collection

Seventeen cord blood samples were collected

from healthy mothers delivering in Zeynep Kamil

Obstetrics Hospital, stanbul, after their permission

was obtained. An ethical committee approval

(021098/90) was provided from Marmara University

Medical Faculty Ethical Committee. Collections

were made when placenta was still in utero. No

samples were collected from mothers with obstetric

complications or from those with a history of

hepatitis C infection or inherited disease. Gender of

the baby was not considered in the collection and

evaluation. After the cord was clamped from two

distal sites, collection was performed with a 50 ml

syringe containing phosphate-buffered solution-ethylenediaminetetraacetic

acid (PBS-EDTA), and

40-100 ml blood was collected from each cord.

Samples less than 40 ml were not processed.

Samples were transported at 18-25°C in specific

transport boxes.

Blood Count from the Collected Cord Blood

Samples

All cord blood samples were counted with automated

cell counter (Coulter AcT cell counter) within

2 hours (h) of sample collection. Cell counts

were also performed after erythrocyte lysis and

CD34+cell selection. Cell viability was measured

with trypan blue dye after each processing. Trypan

blue stock solution was diluted in a 3:4 ratio with

isotonic saline. Under the microscope, those cells

staining blue were counted as dead cells.

236

Demirel et al.

LTC-IC of CD34-selected cord blood Turk J Hematol 2010; 27: 234-41

Immunophenotyping of Cord Blood Cells

For immunophenotyping, cord blood samples

were labelled with CD45, CD34, HLA-DR, CD38,

CD71, CD90, and CD135 after blood count. Cell concentrations

were adjusted to <5x10 6 /ml. All of the

antibodies and absolute count beads (FlowCount

Fluorospheres) were obtained from Beckman

Coulter, Turkey. Since all of the antibodies, except

CD135, were in conjugated form, direct staining

method was used. CD135 tube was labelled with

goat anti mouse-FITC as secondary antibody.

DHR123 was obtained from Molecular Probes,

Eugene, OR, USA. Tubes were prepared with the

following combinations after careful titrations of

antibodies: IgG1-FITC/IgG1-PE (isotypic control),

CD45-FITC/CD34-PE, Rhodamine (Rhd)123/CD34-

PE, CD34-FITC (for absolute counts), CD34-FITC/

HLA-DR-PE, CD34-FITC/CD38-PE, CD71-FITC/CD34-

PE, CD90-FITC/CD34-PE, and CD135-FITC/CD34-PE.

Flow count fluorospheres were used for absolute

CD34 counts (concentration 1043 beads/l). After

labelling with antibodies, tubes were incubated at

room temperature (18-25°C), in the dark for 15 minutes

(min); then, a lysing solution (ammonium

chloride, pH 7.2-7.4) was added. After 10 min of

incubation, cells were washed twice, and cell fixation

was done with 1% PFA (paraformaldehyde,

Sigma) in a 1:1 concentration.

For absolute counts, 100 l of flow count fluorospheres

were vortexed and added to CD34 absolute

count tubes, and absolute counts were calculated

using the following formula:

Absolute Count = Total counted cell number x Concentration of flow count

Total counted fluorospheres

fluorospheres

Immunophenotyping analyses were performed

with EPICS XL-MCL flow cytometry system

(Beckman Coulter, USA). Before analysis, Flow

Check beads were used for checking the laser

alignment, and Flow Set beads were used for fluorescence

calibration and to minimize the day-to-day

variations.

For each tube, cytometric measurements were

made using SS/FS (Side Scatter-Granularity/Forward

Scatter-Size), FL1 (green fluorescence) and FL2

(orange fluorescence) parameters. For CD34 counts,

50,000 nucleated cells were counted in each tube.

All of the list mode data were recorded and later

analyzed with System 2.0 software (Beckman

Coulter, Turkey).

Selection of CD34+ Cord Blood Cells with

Magnetic Beads

All of the cell selection and follow-up procedures

were performed in a clean environment inside the

laminar air flow hood.

Magnetic separation of the cell suspensions: Magnetic

separation beads were obtained from Miltenyi,

Germany. To prevent non-specific or Fc receptor

(FcR) binding, 100 l of FcR blocking reagent was

added onto cells. For each 10 8 cells, 100 l of CD34

MultiSort MicroBeads were added, mixed well and

incubated at +4°C for 30 min. The Miltenyi LS columns

used for magnetic separation were able to

select a total of 2 x 10 9 cells. LS columns, which were

kept at -20°C the night before the cell processing,

were taken out of the -20°C 5 min before the selection

time and attached to MidiMACS magnet area,

and columns were washed three times with ice cold

PBS containing EDTA and 2% bovine serum albumin

(PBS-EDTA-BSA buffer) solution. Due to the hydrophilic

structure of the column, buffer solution passed

through the columns easily and quickly. Cells were

passed through the column after this priming.

To eliminate the residual non-CD34 cells, columns

were washed with buffer solution twice.

Then, the column was taken out of the magnet area

and 5 ml of buffer solution was added to the column,

which was placed over a 15 ml conical tube of

appropriate size. By applying sudden force to the

column piston, the CD34+ cells that were attached

to magnetic beads were pushed into the collection

tube. The force applied to the column pistol was

critical for the CD34+ cell yield. A small portion of

cells were taken for immunophenotypic analysis.

For disattachment of MACS MultiSort beads, Multi-

Sort Release Reagent (20 l/ml) was used, and cells

were incubated at +4°C for 10 min. After cells were

centrifuged at 400xg, pellet was resuspended to 10 7

cells/50 l. 30 l MACS MultiSort Stop Reagent was

added for each 10 7 cells. Cells were diluted in RPMI-

1640 before cell culture.

CD34 Immunophenotyping of CD34-Selected

Samples by Flow Cytometry

Isotypic control (IgG1-FITC/IgG1-PE), CD34-FITC,

CD34-PE and CD34-+Rhd123 tubes were labelled

using the direct method. After incubation at room

temperature for 15 min, cells were washed with

Demirel et al.


PBS-EDTA buffer solution twice. Cells were then

analyzed by flow cytometry (EPICS XL-MCL, Beckman

Coulter, Ltd), and the results were evaluated

with System 2.0 software.

Cell Culture and Assays for CD34+ Cells

LTC-IC (Long-Term Culture-Initiating Cells)

Even though there have been many developments

for determination of the precursor and progenitor

cells of hematopoietic cells, there is still no

specific analytical means of determining the bone

marrow repopulating cells. Cells obtained through

LTC-IC are the most primitive cells that can be analyzed

by in vitro functional assays. The validity of the

LTC-IC assays is dependent on the existence of a

linear relationship between the endpoint measured

(clonogenic cell output as assessed after 5 weeks of

culture) and the number of LTC-IC in the original

test suspension down to limiting numbers of LTC-IC

[6,7]. We used the limiting dilution method for LTC-

IC in order to evaluate the bone marrow renewal

capacity of the cord blood CD34+ cells (Figure 1).

As feeder layer, a murine bone marrow stroma,

M2-10B4 (ATCC, CRL 1972), known to release the

necessary cytokines and growth factors, has been

used [8,9]. M2-10B4 cells were fed with RPMI 1640

(Sigma, USA) cell culture medium with 10% fetal

bovine serum (FBS) in cell culture flasks. After they

became confluent in approximately one week, cells

were irradiated with 15 Gy in 137 Cs, trypsinized,

washed and diluted in IMDM (Iscove’s Modified

Dulbecco’s Medium) containing 10% FBS (Sigma),

10% horse serum (Sigma), and 10 -6 M hydrocortisone

(Sigma). After cell count and adjustment of cell concentration,

cells were distributed to 96- well plates,

15,000 - 22,000 cells/well. These feeder layer plates

were kept ready with change of medium twice a

week and were used in two weeks. CD34+ cord

blood cells, selected with MACS magnetic beads,

were cultured on these M2-10B4 feeder layers with

six different dilutions [1, 10, 100, 300, 1000, 5000) in

16 wells for each concentration. Cultures were incubated

in a humidified atmosphere containing 5%

CO2 in air at 37°C. Culture plates were fed with half

medium changes for five weeks. CAFCs (Cobblestone

Area Forming Cells) were defined as cell groups of at

least 15 cells under phase contrast inverted microscope

with typical appearance at weeks 5-8. LTC-IC

absolute counts with LDA were evaluated as

described previously [10-12].

MethoCult GF+ Semi-Solid Culture System

MethoCult obtained from Stem Cell Technologies,

Canada was used for colony-forming unit (CFU)

assays. This culture media consists of methyl cellulose,

FBS, BSA, 2-mercaptoethanol, L-glutamine,

recombinant human (rh) stem cell factor, rh granulocyte-macrophage

colony-stimulating factor

(GM-CSF), rh interleukin (IL)-3, rhIL-6, rhG-CSF, and

rhErythropoietin. Cells were seeded in 300

CD34 + cell/ml concentration. Colonies were first

observed between days 12-15 under inverted microscope

and counted on day 14 when the number of

colonies was at the maximum. Two independent

scientists counted the colonies on the same day

and mean values were reported. Groups containing

more than 40 cells were considered as colonies.

CFU-GM, BFU-E (Burst Forming Unit-Erythroid) and

CFU-GEMM (granulocyte, erythroid, monocyte,

macrophage) colonies were differentiated according

to the following criteria: CFU-GM colonies were

observed as transparent, flat and non hemoglobinconsisting

colonies, and as very dense colonies;

BFU-E were discriminated easily as dense, with

color changing from orange to red due to their

hemoglobin content; CFU-GEMM could be determined

easily, since they contained both erythroid

and myeloid cells with both transparent and orangered

hemoglobin-containing cells [12].

Results

Immunophenotypic characteristics of the cord

blood samples are summarized in Table 1. The

CD45 percentage of the cord blood mononuclear

cells was lower compared to bone marrow or

peripheral blood due to the presence of nucleated

erythrocytes in the analysis area. CD71 was used to

determine the percentage of nucleated erythrocytes

and found to be 10.2±1.78%.

The mononuclear cell content and CD34 values

before and after selection are summarized in Table 2.

Mean CD34 percentage before positive selection

with MACS beads was 1.2±0.26%, and there was a

purity of 73.64±9.12% after selection.

CAFCs were obtained after 5 weeks of culture.

After CAFC was observed, cells from the CAFCpositive

wells were transferred to MethoCult GF+

semi-solid cultures to observe the colonies on

days 14-15.

238

Demirel et al.


CD34+ Cell Analysis by FC

Cord Blood Collection

Blood Count

CD34+ cell selection by beads

Thawing M210-B4 cells

Culture of M210-B4 cells

Confluence of cells

Irradiation of M210-B4 cells

Twice/week medium exchange

Inoculation of CD34+ cells with LDA method on

pre-prepared irradiated murine cell line M210-B4

Observing CAFC by 5 th week

Half medium change/twice a week

Transfer to semi-solid medium, MethoCult

Colony count on day 14

Calculation of results

Figure 1. A summary of the LTC-IC by LDA method. FC: Flow cytometry, LDA: Limiting dilution assay, CAFC: Cobblestone area forming cells

Table 1. Immunophenotypic characteristics of cord blood cells before

selection

Immunophenotypic Markers

Positivity (%) +/- SD1

CD34 (Stemness marker) 1.2±0.26

CD45 (Pan leukocyte marker) 84.8±2.0

CD38 (Activation marker, positive on SCs) 57.0±7.45

CD71 (Activation marker, positive on erythroid cells) 10.2±1.78

CD90 (Stem cell marker) 0.4±0.39

HLA-DR (MHC Class II marker) 11.3±1.31

CD135 (FLT3-Stem cell marker) 11.9±3.11

CD45 percentage is less than 90% due to presence of nucleated erythrocytes in

cord blood

Table 2. MNC and CD34 levels before and after CD34+ cell selection

with MACS beads

Before Selection After Selection

Percentage of CD34 1.2±0.26 73.64±9.12

Mononuclear Cell Count 1.8±0.65 0.19±0.12

(x 10 6 /ml)

Only 60% of the MethoCult GF+ cultures obtained

from CAFC of LTC-IC gave rise to HPP-Q (High

Proliferative Potential-Quiescent) colonies, while all

cultures had BFU-E, GM and GEMM colonies by day

14, as shown in Figure 2. LTC-IC numbers calculated

as LTC-IC/CD34+ cells are presented in Table 3.

Discussion

Although the number of articles on cord blood

LTC-IC are somewhat limited, our LTC-IC/CD34+

cell numbers were lower compared to some of the

published studies [13,14], but results of several

studies were consistent with ours [15]. There are

several controversial issues, which will be discussed

in the remainder of this article.

LTC-IC assay with limiting dilution is relatively

easier than the other LTC-IC assays, since the preprepared

murine bone marrow stromal cell line

plates can be used in five weeks, and counting

CFUs is easier than counting CAFCs. This method is

Demirel et al.


used as an “ex vivo expansion” assay for CD34 +

cells. When only the CAFCs were scored, a higher

frequency of LTC-IC was estimated in cultures utilizing

the M2-10B4 cell line [16].

Table 3. LTC-IC counts from CD34+ selected cord blood cells (range:

1261-2906, mean: 1966±808)

Samples

LTC-IC/CD34+ cells

1 2705

2 2906

3 2580

4 1649

5 1781

6 1280

7 1649

8 2499

9 1350

10 1261

Mean Value 1966±808

The role of LTC-IC in long-term engraftment is

unknown; however, they are the most primitive progenitors

that can be detected in an in vitro assay.

Among the cultured LTC-IC, some cells remain quiescent

and others are thought to be triggered into

proliferation. Clinical studies have been performed

with ex vivo expanded cells from autologous stem

cell harvests; however, it is unknown if the longterm

engraftment was achieved from ex vivo

expanded cells including LTC-IC or from surviving

stem cells in patients.

A recent study reported that type of stromal

feeder layer used in LTC-IC LDA affects the determination

of LTC-IC frequencies in uncultured cells and

also has a significant effect on cultures. In their

study, Nadali et al. [16] noted that proliferative

capacity of CB LTC-IC can be strongly influenced by

culture conditions and that the frequency of LTC-IC

estimated using these cell lines as stromal support

is not identical. They emphasized that long-term

Figure 2. CAFC and CFUs obtained from CD34+ cord blood cells are shown. CAFC were measured at week 5 and then cells were transferred

to MethoCult, a semicellulose commercial media used for CFU assays. CFUs were counted on day 12 (week 7). A. Cobblestone Area

Forming Cells (CAFCs) after week 5. B. CFU-GEMM: Colony Forming Unit-GranulocyteErythroidMonocyteMacrophage. C. BFU-E: Burst Forming

Unit – Erythroid. D. Giemsa-stained BFU-E. E. A HPP-Q (High Proliferative Potential-Quiscent) colony. F. A BFU-E and CFU-GM are seen in

a nearby area of the petri dish in MethoCult media

240

Demirel et al.


culture on stromal support other than normal bone

marrow can strongly influence the in vitro behavior

of LTC-IC and may lead to errors in estimating the

frequency of LTC-IC. Even though it is easier to grow

and maintain cell lines such as M2-10B4, one can

not be sure if they are releasing all of the necessary

cytokines, chemokines and growth factors.

Due to use of various cell lines or bone marrow

stromal cells, there have been diverse outcomes

from different laboratories worldwide [17-19]. It has

been published that CD34+ purification is one of

the prerequisites for successful expansion. However,

it is also the reason for the lower number of

colonies counted in this assay, since the cells such

as monocytes and lymphocytes, which release different

growth factors, cytokines and chemokines,

are not present to help the stem cell proliferation.

The temperature of the LTC-IC was kept at 35°C

in our study. Some of the earlier studies have used

37°C for the first three to four days and then

switched to 33°C, but most of these studies were for

murine LTC-IC [17]. Podesta et al. [19] published

that temperature has an effect on colony growth of

human progenitor cells, and LTC-IC performed at

37°C gave rise to more colonies than those at 33°C.

One of the reasons for the lower numbers of colonies

in our study could be the temperature.

Clinical trials of ex vivo expanded cord blood are

under way in the United States and Europe, using

CD34+ selected cells expanded in Teflon culture

bags and non-selected cord blood mononuclear

cells expanded in bioreactors. These experiments

are testing different combinations of cytokines to

determine the optimal dose of cytokines to lead to

improved stem cell engraftment [20].

STAT-5 is a signal tranducer and activator of transcription,

involved in self renewal, proliferation and

apoptosis of hematopoietic cells. Schepers et al.

[21] performed STAT5 RNAi in sorted cord blood

cells by lentiviral transduction, and investigated the

effects of STAT5 downmodulation on normal stem/

progenitor cell compartment. They showed that

LDAs with cord blood had a 3.9-fold reduction in

progenitor numbers. Their data indicated that STAT5

expression is required for the maintenance and

expansion of primitive hematopoietic stem and progenitor

cells, both in normal and leukemic hematopoiesis

[21]. STAT5 and expression of other transcription

factors may also have played a role in the

lower number of colony counts in our study.

Even though there has been some progress in

the standardization of the LTC-IC culture methods, a

more standardized approach will help us to achieve

better results for ex vivo expansion of hematopoietic

cells. We believe that identification of defined

conditions for extensive and rapid amplification of

LTC-IC would help to analyze the benefit of these

cells for therapeutic applications in the future. We

now know that standardization of the method with

human originated feeder layer cells and use of animal-free

medium are necessary to use these cells in

therapeutic applications. By implementing the LDA

method for LTC-IC in our laboratory, we have taken

a step forward for the ex vivo expansion of stem

cells and will proceed with further research in the

future with more studies on cell-to-cell contact and

cytokine/chemokine effect on LTC-IC.

We also suggest that more information about the

in vivo scenario can be gained with further insight

into the use of human cord blood stromal cells or

HUVEC (human umblical vein endothelial cells) as

feeder layers and with exploration of in vitro behavior

of LTC-IC for CFC production.

Acknowledgments

The authors thank Zeynep Kamil Obstetrics Hospital

Delivery Room personnel, especially Dr. Vedat

Dayolu and Dr. Ate Karateke, for their help in cord

blood collection; Dr. Pakize I. Tarzi Laboratuvarlar for

permission to use their flow cytometry system; and

Marmara University Blood Bank personnel for their

help and kindness during the work in the stem cell

laboratory. Special thanks to Asl Önder Gül for the

technical assistance with flow cytometry and to Prof.

Dr. Sami Kart, Yeditepe University Medical Faculty,

for careful reading of the manuscript.






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The effect of heparin on the cell cycle in human

B-lymphoblasts: An in vitro study

Heparinin insan B-lenfositlerinin hücre döngüsüne etkisi: Bir in vitro

çalma

Aye Aksoy 1 , Erol Erduran 2 , Yavuz Tekeliolu 3 , Mehmet Mutlu 4 , Yusuf Gedik 5

1Department of Pediatrics, Division of Child Neurology, Karadeniz Technical University, Trabzon, Turkey

2Department of Hematology, Karadeniz Technical University, Trabzon, Turkey

3Department of Histology and Embryology, Karadeniz Technical University, Trabzon, Turkey

4Department of Pediatrics, Karadeniz Technical University, Trabzon, Turkey

5Department of Cardiology, Karadeniz Technical University, Trabzon, Turkey

Abstract

Objective: Heparin has been shown to be a strong inhibitor of the proliferation of several cell types. In

this in vitro study, we investigated whether different heparin concentrations can affect the cell cycle of

lymphoblasts in newly diagnosed acute lymphoblastic leukemia (ALL) patients.

Materials and Methods: Lymphoblasts were incubated in different heparin concentrations (0, 10, 20 U/

ml), and the percentages of lymphoblasts in each phase of the cell cycle were simultaneously measured

by flow cytometry at 0, 1, and 2 hours (h).

Results: The percentages of lymphoblasts at the G2/M and S phases were significantly increased in 20

U/ml heparin concentration at 1 h compared to 0 U/ml (without heparin) concentration. We demonstrated

that heparin increases the percentages of lymphoblasts in the S and G2/M phases in a concentration-

and time-dependent manner.

Conclusion: It was shown that heparin expands the proliferation of lymphoblasts by increasing the

transition to G2/M and S phases and the S-phase fraction ratio. Heparin thus appears promising for

its contribution to new treatment fields such as by providing a synergistic effect with chemotherapeutic

drugs. (Turk J Hematol 2010; 27: 242-9)

Key words: Cell cycle, heparin, leukemia

Received: December 11, 2009 Accepted: April 30, 2010

Özet

Amaç: Heparin, çeitli hücre tiplerinde proliferasyonun güçlü bir inhibitörü olduu gösterilmitir. Bu

in vitro çalmada; akut lenfoblastik lösemi (ALL) tans alan hastalarda farkl heparin

konsantrasyonlarnn lenfoblast hücre döngüsündeki fazlara etkisi aratrld.

Yöntem ve Gereçler: Lenfoblastlar 0 (heparinsiz), 10 ve 20 U/mL heparin konsantrasyonlarna maruz

braklarak 0, 1 ve 2 saatlerde lenfoblastlarn hücre döngüsü oranlar FCM ile ölçüldü.

Address for Correspondence: M.D. Aye Aksoy, Department of Pediatrics, Division of Child Neurology, Karadeniz Technical University,

Trabzon, Turkey Phone: +90 533 235 39 87 E-mail: aysechild@gmail.com

doi:10.5152/tjh.2010.45

Aksoy et al.

Turk J Hematol 2010; 27: 242-9 Heparin and human B-lymphoblast cell cycle 243

Bulgular: Lenfoblastlar, 0 U/mL heparinsiz konsantrasyonuna göre; 20 U/mL heparin konsantrasyonunun

1. saatinde en fazla olmak üzere G2/M ve S döngüsü anlaml derecede artmtr. Heparinin

doza ve zamana bal olarak lenfoblastlarn G2/M ve S fazlarnda arta neden olduunu gösterdik.

Sonuç: Heparin, hücre döngüsünde G2/M ve S fazna geçii artrarak lenfoblastlarda proliferasyonu

artrd gösterildi. Böylece heparinin, kemoterapi ilaçlar ile sinerjistik etki salayarak yeni tedavi

alanlarna olas katklar umut verici olarak görünüyor. Bu ön çalmamzn sonuçlar, heparinin lenfoblastlardaki

proliferatif etkisini aratrmak amac ile daha ayrntl ve kapsaml çalma yaplmas

gerektii gösterir. (Turk J Hematol 2010; 27: 242-9)

Anahtar kelimeler: Hücre siklusu, heparin, lösemi

Geli tarihi: 11 Aralk 2009 Kabul tarihi: 30 Nisan 2010

Introduction

Heparin, which is generally used as an anticoagulant

but has been shown to have additional biological

activities, was determined in several clinical trials to

have an effect on malignancies. The activities of

heparin include anticoagulant, tissue factor pathways

inhibitor release, heparinase inhibition, selectin-mediated

interaction inhibition, modulation of the

activity of some proteases and extracellular matrix

components, and growth factor binding [1-6].

Leukemia cells were found sensitive to chemotherapeutic

agents that either interfere with the cell

cycle or cause apoptosis [7-9]. Some anti-cancer

reagents cause cell death through interfering with

the processes of the cell cycle, while others cause

cell death by apoptosis, which plays an important

role in the balance between cell replication and cell

death [7,10]. Almost all conventional anticancer

drugs are primarily effective against rapidly dividing

cells [11]. Compared with proliferating lymphocytes,

a 500-fold higher concentration of chemotherapeutic

drug is required to kill resting cells [12].

In initial acute lymphoblastic leukemia (ALL), a

higher fraction of proliferating cells seems to result

in a higher efficacy of the chemotherapeutic drug

targeting the cell division cycle. Interestingly, at first

presentation of childhood ALL, the prognostic value

of the proliferative capability of lymphoblasts

remains controversial, and there is no clear evidence

of an association with prognosis [13-15].

However, in in vitro experiments, an increased proliferation

rate was associated with good response to

treatment [15].

The S-phase fraction (SPF) measures the percentage

of a given cycling subpopulation between

G0/G1 and G2/M phases, and it reflects cell proliferation

[16]. The SPF has been studied in different

malignancies including ALL for the purpose of classification

or prognosis. It has been proven to be a

useful prognostic marker in types of solid cancers

and leukemia [14,17-19]. However, contradictory

results have been reported concerning the prognostic

value of the SPF of leukemia cells [13-15].

To date, no study dealing with the proliferative

activity of heparin in human lymphoblasts has been

undertaken. In this in vitro study, we investigated

whether heparin can affect proliferation of lymphoblasts

in newly diagnosed ALL patients. We also

attempted to establish by flow cytometry (FCM)

which phase in the cell cycle of the lymphoblasts

can be considered heparin-sensitive. This was

determined by analyzing the correlation between

the SPFs and the effect of heparin on the lymphoblast

cell cycle.


Twelve children (8 girls, 4 boys; age: 2-15 years)

with newly diagnosed ALL (all had B-cell leukemia)

were included in the study with the written consent

of their parents. The clinical features of the 12

patients are shown in Table 1. Diagnosis of the

patients was done according to the findings of complete

blood counts, peripheral and bone marrow

aspiration (BMA) smears, histochemical staining of

BMA smears, and FCM analysis (Coulter Epics Elite

ESP Flow Cytometry) of BMA materials. ALL was

diagnosed by examining a lymphoblast population

of more than 25% in the BMA smears. No patients

had Philadelphia chromosome.

Cell typing

CD3 PE (Coulter PN IM1282), CD7 FITC (Coulter

PN IM0585), CD10 FITC (Coulter PN IM0471), CD13

FITC (Coulter PN IM0778), CD14 FITC (Coulter PN

IM0650), CD19 FITC (Coulter PN IM1284), CD20 FITC

(Coulter PN IM1455), CD33 FITC (Coulter PN

IM1179), CD45 FITC (Coulter PN IM0782) and MPO

FITC (Coulter PN IM1874) monoclonal antibodies

244

Aksoy et al.

Heparin and human B-lymphoblast cell cycle Turk J Hematol 2010; 27: 242-9

Table 1. Clinical features of children with acute lymphoblastic leukemia

Patient no Sex Age (yr) Stage of disease WBC % Blast Immunophenotype

(x 10 3 /l) (BM)

1 F 4 Diagnosis 1.2 98 CD 10 , CD 19 , CD 20

2 F 3 Diagnosis 120.0 88 CD 10 , CD 19 , CD 20

3 F 3 Diagnosis 25.2 96 CD 10 , CD 19

4 M 5 Diagnosis 2.8 95 CD 10 , CD 19

5 F 2 Diagnosis 7.8 88 CD 10 , CD 19

6 M 7 Diagnosis 44.7 89 CD 10

7 F 4 Diagnosis 30.3 95 CD 10 , CD 20


9 F 15 Diagnosis 127.0 90 CD 19 , CD 20

10 F 2 Diagnosis 13.1 90 CD 10


12 F 5 Diagnosis 9.7 98 CD 19 , CD 20

WBC: Leukocyte counts at diagnosis; BM: Bone marrow

were used for the diagnosis of ALL. All patients had

monoclonal antibody positivity for B-cell leukemia.

Separation of Blast Cells

Bone marrow aspiration (BMA) materials were

drawn into a tube with ethylene diamine tetraacetate

(EDTA). The same quantities of phosphatebuffered

saline (PBS) and the BMA sample were

added into the tube. The sample was stirred, and

waited for 30 minutes (min) at room temperature.

Buffy coat obtained from the upper surface of the

specimen was added onto Ficoll-Hypaque 1077

(lymphocyte separation medium Gibco BRL 13010-

12, Grand Island, NY) and centrifuged at 700 g for 30

min. Mononuclear cells containing 90% lymphoblasts

were obtained from the upper surface of the

specimen and washed twice with PBS to exclude

debris. The blast cells were suspended at a concentration

of 3-5 x10 5 cells/ml in RPMI with L-glutamine

without sodium bicarbonate medium (Sigma

R-6504, Miami, FL). 100 ml of each material was

studied for immunotyping by FCM. The remaining

amount of each sample was kept at -80°C approximately

five-seven months until the study was performed.

Viability of lymphoblasts was again determined

after having the samples using acridine

orange. Lymphoblasts with viability higher than 70%

were used in the study.

Pure heparin was used in the study (Sigma

Biochemicals and Reagents-2001 Catalog, Sigma H

3149). The purity and activity of heparin were Grade

I, 140 USP unit/mg. The heparin did not include

the additional stabilizing agents. The blast cell suspensions

were thawed at room temperature. No

heparin (0 U/ml) or different heparin concentrations

(10, 20 U/ml) were added on the blast cells

(1 ml). Each tube included different heparin concentrations

and was divided into three different

tubes. All the samples were processed with a

Coulter DNA-prep reagent kit (CN: 640445) at 0, 1,

and 2 hours (h). The DNA-prep reagent kit contained

DNA prep-LPR solution (<0.1% potassium

cyanide, <0.1 sodium nitride, nonionic detergents,

saline, and stabilizers) and the DNA-prep stain

(50 g/ml propidium iodide [PI], <0.5% NaN 3 ,

saline, and stabilizers). The blast membranes were

pored by the DNA-prep LPR solution, and RNAs and

DNAs of the blasts were stained with PI. Stained

RNAs were removed from the medium by RNAse;

therefore, DNA content was marked by PI. The aliquots

were taken following 0, 1, and 2 h, and FCM

analyses were carried out for cell cycle of the blast

cells in a Coulter Epics Elite Flow Cytometer

(Multicycle DNA, Phoenix Flow Systems, San Diego,

CA). The FCM analyses were immediately done

within 3-5 seconds after adding no heparin or different

heparin concentrations (0, 10 and 20 U/ml) on

to the blasts.

Flow cytometric (FCM) analyses could not be

performed in 10 and 20 U/ml heparin concentrations

at 3h because the samples were seen to have

transformed into a gelatinous substance. A gelatinous

substance similarly developed following the

addition of higher heparin concentrations (30 and

50 U/ml) into the lymphoblast samples.

Aksoy et al.


The percentages of the cells in the G0/G1, G2/M

and S phases were determined from an analysis

using the PEAK computer program, generously provided

by Dr. Phillip Dean [20]. Since only samples

that contained more than 80% leukemic cells were

included, the lymphoblast proliferative activity was

expressed in terms of the calculated percentage of

cells in the S phase of their cycle.

The fraction of cells in the G0/G1, S and G2/M

phases of the cell cycle is mathematically determined

from the DNA distribution. The SPF is the

fraction of the total cell population in the S phase of

the cell cycle [16].

S

SPF=

G 0 /G 1 +S+G 2 /M

X 100

Statistical Analysis

Data obtained from FCM and fluorometrik measurements

were analyzed by the SPSS version 10.0

statistical package program. Variance analysis was

used in the repeated measurements and the comparison

of groups. The normal distribution of the

data was assessed by Kolmogorov-Smirnov test.

Paired t test (post hoc) was used to determine the

statistically significant differences between measurements

using different heparin concentrations.

Results were calculated as arithmetic mean ±standard

deviation (x±SD).

Results

The cell cycle analyses were performed without

heparin and with heparin at varying concentrations

(0, 10 and 20 U/ml) at 0, 1, and 2h after adding

heparin on to the lymphoblasts. However, the samples

with heparin were seen to transform into a

gelatinous substance after 2 h; hence, FCM analysis

could not be performed.

The mean percentage of blast cells in the G0/G1

phase in different heparin concentrations at 0 (without

heparin), 1, and 2 h are shown in Table 2. In 0 U/

ml (without heparin) concentration, the mean percentage

of blast cells in the G0/G1 phase at 2 h was

significantly lower than those at 0 h and 1 h

(p<0.001). The highest percentage of blast cells in

the G0/G1 phase was established in 0 U/ml (without

heparin) at 0 h. The lowest percentage of blast cells

in the G0/G1 phase was determined in 20 U/ml

heparin level at 1 h. The mean percentage of blast

cells in the G0/G1 phase in 20 U/ml heparin concentration

at 1 h was significantly lower than in 0 U/ml

(without heparin) and 10 U/ml heparin concentrations

at 1 h (p<0.001), and in 20 U/ml heparin concentration

at 0 h (p<0.001). There were significant

differences between the percentage of the lymphoblasts

in 20 U/ml and 10 U/ml heparin concentrations

and 0 U/ml (without heparin) at 2 h (p<0.001).

The mean percentage of blast cells in the G2/M

phase in the different heparin concentrations at 0, 1,

and 2 h are shown in Table 3. There were significant

differences in the mean percentage of blast cells in

the G2/M phase in 0 U/ml (without heparin) concentration

at 0, 1, and 2 h (p<0.001). The lowest percentage

of blast cells in the G2/M phase was determined

in 0 U/ml without heparin concentration at 2 h. The

highest percentage of blast cells in the G2/M phase

was determined in 20 U/ml heparin level at 1 h. The

mean percentage of blast cells in the G2/M phase in

20 U/ml heparin concentration at 1 h was significantly

higher than those in 0 U/ml and 10 U/ml heparin

concentrations at 1 h (p<0.001), and in 20 U/ml

heparin concentration at 0 h (p<0.003). The mean

percentage of blast cells in 20 U/ml heparin level at 1

Table 2. Percentages of lymphoblasts in the G0/G1 phase in 0 U/ml

(without heparin), 10 and 20 U/ml heparin concentrations at 0, 1,

and 2 hours (%, mean±SD, min.-max.)

Heparin

Time (hour)

0 1 2

0 U/ml 97.80±0.51 a 97.72±0.47 d 97.62±0.48 g

97.0 - 98.5 97.0 - 98.3 96.9 - 98.2

10 U/ml 85.38±4.3 b 85.54±2.06 e 90.77±0.81 h

78.9 - 92.0 82.4 - 89.0 90.0 - 92.0

20 U/ml 88.38±3.12 c 76.91±4.58 f 78.34±2.12 i

83.3 - 92.0 69.6 - 84.5 74.9 - 81.0

a-b, a-c, c-f, c-i, d-e, d-f, e-h, e-f, g-h, g-i, h-i: p<0.001, b-h: p<0.001

Table 3. Percentages of lymphoblasts in the G2/M phase in 0 U/ml


and 2 hours (%, mean±SD, min.-max.)

Heparin

Time (hour)

0 1 2

0 U/ml 1.16±0.42 a 1.18±0.42 d 1.08±0.43 g

0.5 - 1.9 0.5 - 1.9 0.4 - 1.8

10 U/ml 7.24±1.9 b 4.50±1.74 e 3.00±0.76 h

4.1 - 10.1 1.8 - 7.4 1.3 - 4.0

20 U/ml 6.00±1.95 c 10.21±3.49 f 9.37±2.65 i

3.0 - 9.0 5.4 - 18.4 4.9 - 13.0

a-b, a-c, b-e, b-h, d-f, e-f, g-i, h-i: p< 0.001, c-f: p<0.003, d-e: p<0.003,

c-i, e-h: p<0.006, g-h: p<0.0

246

Aksoy et al.


h was significantly higher than those in 0 U/ml (without

heparin) and 10 U/ml heparin levels (p<0.001).

The mean percentage of blast cells in the S

phase in different heparin concentrations at 0, 1,

and 2 h are shown in Table 4. The mean percentage

of blast cells in the S phase in 0 U/ml (without heparin)

concentration was the same at 0, 1, and 2 h.

The lowest percentage of blast cells in the S phase

was determined in 0 U/ml (without heparin) concentration.

The highest percentage of blast cells in

the S phase was determined in 20 U/ml heparin

concentration at 1 h. The mean percentage of the

blast cells in the S phase in 20 U/ml heparin concentration

at 1 h was significantly higher than in 0 U/ml

(without heparin) and 10 U/ml heparin concentrations

(p<0.001, p<0.006, respectively) and in 20 U/

ml heparin concentration at 0 h (p<0.001). There

were significant differences between the percentages

of the blast cells in 20 U/ml and 10 U/ml, and

20 U/ml and 0 U/ml heparin levels in S phase at 1 h

(p<0.001), and the highest percentage of the lymphoblasts

in S phase at 2 h was detected in 20 U/ml

heparin level.

In the 0 U/ml (without heparin) concentration at

0 h, 97.80% of the lymphoblasts were in the G0/G1

phase, and this value dropped to 76.91% in 20 U/ml

heparin concentration at 1 h (p<0.001). Similarly,

at 0 h, 1.16% of the lymphoblasts were in G2/M

phase, and this value increased to 10.21% in 20 U/

ml heparin concentration at 1 h (p<0.001). Also at

0 h, 1.03% of the lymphoblasts were in S phase,

and this value increased to 13.63% in 20 U/ml

heparin concentration at 1 h (p<0.001). G0/G1

phase cell population decreased while G2/M and S

phase cells increased in 10 U/ml and 20 U/ml concentrations

at 1 and 2 h compared to cell populations

at 0 h (p<0.001).

Table 4. Percentages of lymphoblasts in the S phase in 0 U/ml


and 2 hours (%, mean±SD)

Heparin

Time (hour)

0 1 2

0 U/ml 1.03±0.60 a 1.03±0.60 d 1.03±0.60 g

0.2 - 2.0 0.2 - 2.0 0.2 - 2.0

10 U/ml 7.43±3.61 b 10.12±2.71 e 6.22±0.95 h

3.0 - 14.4 7.0 - 15.3 5.0 - 7.9

20 U/ml 5.45±2.31 c 13.63±3.47 f 12.11±2.73 i

2.2 - 7.5 10.0 - 21.4 9.9 - 17.4

a-b, a-c, c-f, c-i, d-e, e-h, d-f, g-h, g-i, h-i: p<0.001, e-f: p<0.006

The SPF was determined in the 0 U/ml (without

heparin), 10 U/ml and 20 U/ml heparin concentrations

at 0, 1 and 2 h, with values ranging from

1-13.6% (data not shown). While the SPF values in

the 0 U/ml (without heparin) at 0 h, 1 h and 2 h were

1%, the highest SPF value was 13.6% in the 20 U/ml

heparin concentration at 1 h. We found a statistically

significant difference in the mean SPF ratio in

the 20 U/ml heparin concentration at 1 h compared

to the other groups (p<0.05).

Prominent alterations in the cell cycle distribution

were observed in 20 U/ml heparin concentration

at 1 h. There was a movement of lymphoblast

cells from G0/G1 to G2/M and S phases. The percentage

of lymphoblasts in the G0/G1 phase was

decreased while the percentage of lymphoblasts in

the G2/M and S phases was increased in 20 U/ml

heparin level at 1 h. This study showed that the

effect of heparin in the cell cycle of lymphoblasts

was associated with a decrease in the percentage

of G0/G1 phase cells and an increase in the percentage

of G2/M and S phase cells in a dose- and time–

dependent manner. In other words, we found that

heparin increases both the percentage of lymphoblast

cells in G2/M and S phases and the SPF ratio in

20 U/ml heparin level at 1 h. The FCM histograms of

lymphoblasts depicted characteristic DNA distributions

in 0 U/ml (without heparin) and 20 U/ml heparin

concentrations at 1 h in a patient (no: 6) with

ALL [Figure 1(A) and (B)]. Comparisons of the percentages

of the lymphoblastic cell populations in 0

U/ml (without heparin) and 20 U/ml heparin levels

in the G0/G1, S and G2/M cell cycle phases at 1 h are

shown in Figure 1(A) and 1(B), respectively.

Discussion

In the present study, we have demonstrated that

heparin increases the percentages of lymphoblasts

in the S and G2/M phases and the SPF ratio in a concentration-

and time-dependent manner. This indicates

that heparin may induce the proliferation of

lymphoblasts by increasing the cell cycle percentages

in the G2/M and S phases and the SPF ratio. To

the best of our knowledge, no study has reported on

the influence of heparin on the cell cycle of lymphoblasts

and the correlation with the SPF.

Although previous works demonstrated that heparin

could inhibit proliferation of hepatoma cells,

vascular smooth muscle cells, renal mesangial

cells, and cervical epithelial cells, we have shown

that heparin induced lymphoblast proliferation by

Aksoy et al.


1000

HI009514.HST HISTO PMT4 CELL CYCLE

SO

DATA

1000

PE009632.HST ped

SL CL SO

CELL CYCLE

DATA

Cell Number

800

600

400

200

Mean G1=54.8

CV G1 =4.9

%G1 =98.3

Mean G2 108.5

CV G2 =3.2

%G2 =1.2

% S = 0.6

G2/G1 = 1.980

Cell Number

800

600

400

200

Mean G1=54.4

CV G1 =3.4

%G1 =71.9

Mean G2 113.1

CV G2 =2.7

%G2 =18.2

% S = 9.9

G2/G1 = 2.077

A

0

0 32 64 96 128 160 192 224 256

Chi Sp.=1.3

DNA Content

B

Chi Sp. = .9

0

% Agg. = .1

0 32 64 36 128 160 192 224 256

DNA Content

Chi Sp. = 3.0

Figure 1(A) and (B). Comparison of the percentage of the lymphoblastic cell populations in 0 U/ml (without heparin) (A) and 20 U/ml (B)

heparin levels in the G0/G1, S and G2/M cell cycle phases at 1 hour (respectively, left and right) (No: 6)

increasing the cell cycle percentages in the S and

G2/M phases [1,21-24]. The antiproliferative activity

of heparin has been linked to the blockage of the G1

phase of the cell cycle, and also reduces the number

of cells entering the cycle from G1 [1,3,22,25,26].

Even though it is known from earlier studies that

heparin has an antiproliferative effect on non-cancerous

cells, it is quite interesting that it was shown

in this study to increase proliferation in lymphoblasts.

The mechanism responsible for these effects

of heparin must be investigated in further studies.

On the other hand, heparin causes apoptosis in

human peripheral blood neutrophils, lymphoblasts

and mononuclear cells, and indicates its apoptotic

effect on lymphoblasts via extrinsic or intrinsic pathways

[27-29].

We demonstrated in this study that the greatest

proliferation effect of heparin and the highest SPF

ratio were determined in 20 U/ml concentration at 1

h. Recent studies from Erduran et al. [30] determined

that the greatest apoptotic effect of heparin

on the lymphoblasts was detected in 20 U/ml concentration

at 1 h. The results of some recent studies

were found to be concordant [28-30]. Apoptosis

and proliferation are important regulators of normal

development and homeostasis in the bone marrow.

According to the knowledge gained in previous

experimental studies, a greater regenerative capacity

of hematopoiesis may be reflected by an

increased rate of apoptosis and/or proliferation and

therefore is associated with a more favorable outcome

[31]. According to these results, we suggest

that heparin may have a dual effect on lymphoblasts

by stimulating apoptosis in a portion of them,

while increasing proliferation in others by stimulating

S and G2/M phases in the cell cycle. Whether or

not this dual effect contributes to prognosis will

require further investigation. We did not aim to

show which molecular pathway is involved in the

proliferative effect of heparin on the lymphoblast

cell cycle. In view of the preliminary nature of our

study, our purpose was only to establish whether or

not heparin had any effect on the cell cycle of lymphoblasts.

We believe that the relationship among

heparin, cell cycle phases and apoptosis needs to

be studied at the molecular level.

Chemotherapy agents may work in only one

phase of the cycle (termed “cell-cycle specific”)

(e.g. azathioprine, cytosine arabinoside (Ara-C),

hydroxyurea, and vincristine), or in all phases (‘cellcycle

nonspecific”). Many chemotherapeutic drugs

are effective on the cells in the S phase of the cell

cycle [8-11,32]. Bone marrow samples generally

had a higher SPF ratio than blood samples in children

with leukemia [33]. The relationship of leukemic

blast proliferative activity to prognosis is controversial.

Duque et al. [14] reported that the SPF was

not prognostic for treatment response or response

duration in acute leukemia in general. On the other

hand, several studies reported that bone marrow

SPFs of >6% were strongly predictive of outcome in

childhood ALL [33,34]. In contrast, Braess et al. [13]

found that a high proliferative activity was associated

with a higher complete remission rate in 187

patients with acute myeloblastic leukemia. On the

other hand, it has been demonstrated that stimula-

248

Aksoy et al.


tion of cell proliferation of leukemia cells by pretreatment

with growth factors (granulocyte colonystimulating

factor, granulocyte-macrophage colonystimulating

factor, interleukin-3) in combination

with cycle-specific cytotoxic drugs as chemosensitizing

agents could enhance in vitro sensitivity to

some chemotherapeutic agents for killing of leukemic

stem cells [35-38]. It was reported that an

increased in vitro sensitivity to several chemotherapeutic

drugs, such as vincristine and L-asparaginase,

was related to higher SPF of lymphoblasts isolated

from initial childhood ALL patients. Especially the

SPF of pretreatment childhood ALL samples

appeared to correlate with the in vitro sensitivity to

several chemotherapeutic drugs [15]. Since

increased proliferative activity may increase the

sensitivity to chemotherapeutic drugs, pretreatment

with growth factors prior to treatment with these

proliferation-dependent drugs may increase the

response rate in childhood acute leukemia. The

increased proliferative effect of heparin on lymphoblasts

will be important for its contribution to new

treatment fields. Our results presented here indicate

that heparin increases the proliferation of lymphoblasts

in G2/M and S phases and the SPF.

Heparin might increase the effect of chemotherapeutic

drugs on lymphoblasts that are in the G2/M

and S phases. Alternatively, heparin combined with

chemotherapeutic agents might be a feasible

approach to increase the effectiveness of the chemotherapy

with relative specificity for the lymphoblasts,

but these results should be tested in vivo. We

lack data for comparison of the effect of heparin on

various types and stages of leukemia.

Thromboembolism is a common finding in

patients with malignancy and a well-recognized

serious complication during chemotherapy, such as

with Ara-C. Anticoagulation with heparin has also

been suggested in children undergoing some kinds

of ALL therapy [39]. Our results might suggest that

pretreatment with heparin as a chemosensitizing

approach could be useful for patients with ALL who

are prone to thrombosis. The proliferative and

apoptotic effects of heparin on lymphoblasts might

present new opportunities in the treatment of childhood

ALL. This study provides pilot data for a future

randomized trial of the use of heparin during ALL

therapy for the prevention of some chemotherapeutic

agent-associated thrombotic events.

In conclusion, in addition to its anticoagulant

effect, heparin might be useful in children with ALL

because it induces the transition of the lymphoblasts

from the G0/G1 phase to G2/M and S phases.

In addition, heparin might increase the effect of

chemotherapeutic drugs on lymphoblasts that are

in the G2/M and S phases. There are no reports

related to the enhancement of chemotherapeutic

drug sensitivity by heparin in the treatment of ALL or

any type of leukemia. The findings of this preliminary

study indicate that further and more comprehensive

research on the effects of heparin on the

lymphoblast cell cycle is needed to explore the

therapeutic potential of heparin in patients with ALL

or any kind of leukemia.






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Autonomic nervous system dysfunction and

serum levels of neurotoxic and neurotrophic

cytokines in patients with cobalamin deficiency

Kobalamin eksiklii olan hastalarda otonom sinir sistemi bozukluu

ve nörotoksik, nörotropik sitokinlerin serum düzeyleri

Özcan Çeneli 1 , ahika Zeynep Ak 1 , Betül Çevik Küçük 2 , Aye Bora Tokçaer 2 ,

Reha Kuruolu 2 , Münci Yac 1

1Department of Internal Medicine, Division of Hematology, Gazi University Faculty of Medicine, Ankara, Turkey

2Department of Neurology, Gazi University Faculty of Medicine, Ankara, Turkey

Abstract

Objective: The imbalance between neurotoxic cytokine tumor necrosis factor- (TNF-) and neurotrophic

cytokines epidermal growth factor (EGF) and interleukin-6 (IL-6) plays a role in the pathogenesis

of cobalamin (Cbl) deficiency-induced neuropathy. The aim of this study was to evaluate autonomic

nervous system dysfunction and to look for any relationship between autonomic nervous system

disturbances and serum cytokine levels (TNF-, EGF, IL-6) in patients with Cbl deficiency.

Materials and Methods: Serum levels of TNF-, EGF and IL-6 were studied in patients with Cbl deficiency

(n=41) and a healthy control group (n=17) and after 3 months in patients who underwent Cbl

replacement therapy (n=22). All patients with Cbl deficiency underwent electrophysiological studies

(EPS) for the diagnosis of neuropathy. Statistical analysis was performed using SPSS for Windows

11.5 software.

Results: With EPS, 29 of 41 Cbl-deficient patients (70.73%) demonstrated neurological dysfunction

[3 (7.32%), 19 (46.34%) and 7 (17.07%) patients with sensorimotor peripheral neuropathy, parasympathetic,

and sympathetic autonomic dysfunction, respectively]. Although there was no significant

difference in serum levels of EGF and IL-6 between patients with versus without autonomic dysfunction,

levels were significantly lower in Cbl- deficient patients than healthy controls.

Conclusion: Presence of autonomic dysfunction seems to be a frequent neurological finding in patients

with Cbl deficiency. However, we could not find any relationship between serum cytokine levels and

autonomic dysfunction by EPS. (Turk J Hematol 2010; 27: 250-6)

Key words: Cobalamin deficiency, autonomic dysfunction, dysautonomia, autonomic neuropathy,

serum cytokine levels

Received: March 10, 2010 Accepted: April 30, 2010

Address for Correspondence: M.D. Özcan Çeneli, 18. Sokak 55/10 Emek 06500 Ankara, Turkey

Phone: +90 532 362 95 50 E-mail: cenelio@yahoo.com

doi:10.5152/tjh.2010.46

Çeneli et al.

Turk J Hematol 2010; 27: 250-6 Dysautonomia and cytokines in cobalamine deficiency 251

Özet

Amaç: Nörotoksik sitokin tumor necrosis factor- (TNF) ile nörotrop sitokinler epidermal growth

factor (EGF) ve interleukin-6 (IL-6) arasndaki dengesizlik kobalamin eksikliine bal nöropati patojenezinde

rol oynamaktadr. Bu çalmann amac, kobalamin eksiklii olan hastalarda otonom sinir

sistemi bozukluunu deerlendirmek ve otonom sinir sistemi bozukluklar ile serum sitokin düzeyleri

(TNF, EGF, IL-6) arasndaki ilikiyi aratrmaktr.

Yöntem ve Gereçler: Kobalamin eksiklii olan hastalar (n=41), salkl kontrol grubu (n=17) ve

kobalamin yerine koyma tedavisinden 3 ay sonra hastalarda (n=22) serum TNF, EGF, IL-6 düzeyleri

çalld. Nöropati tans için, Cbl eksiklii olan tüm hastalarda elektrofizyolojik çalma (EPS) yapld.

statistik analizde “SPSS for Windows 11.5” yazlm kullanld.

Bulgular: EPS ile 41 kobalamin eksiklii olan hastadan 29’unda nörolojik bozukluk gösterildi [ 3

(7.32%), 19 (46.34%) ve 7 (17.07%) hastada srasyla, sensorimotor periferal nöropati, parasempatik

ve sempatik otonom bozukluk]. Otonom bozukluk olan ve olmayan hastalar arasnda serum EGF ve

IL-6 düzeylerinde anlaml fark yoksa da, bu sitokin düzeyleri kobalamin eksiklii olan hastalarda,

salkl kontrolerden anlaml derecede düüktü.

Sonuç: Kobalamin eksiklii olan hastalarda otonom bozukluk, sk nörolojik bulgu olarak görünmektedir.

Bununla birlikte, elektrofizyolojik çalmayla saptanan otonom bozukluklar ve serum sitokin

düzeyleri arasnda iliki bulamadk. (Turk J Hematol 2010; 27: 250-6)

Anahtar kelimeler: Kobalamin eksiklii, otonom bozukluk, disotonomi, otonom nöropati, serum

sitokin düzeyi

Geli tarihi: 10 Mart 2010 Kabul tarihi: 30 Nisan 2010

Introduction

The clinical presentation of patients with vitamin

B 12 (cobalamin-Cbl) deficiency varies in a spectrum

ranging from hematological disorders to neuropsychiatric

diseases. As the level of Cbl deficiency does

not correlate with the severity of neurological disorders

and the lesions may become irreversible if not

treated promptly, neurological abnormalities are a

matter of clinical concern in patients with Cbl deficiency.

Cbl deficiency may affect both the peripheral

and central nervous system, with diminished

vibratory sensation as the most common abnormality

[1,2]. In rare cases, autonomic nervous system

dysfunction presented as orthostatic hypotension,

impotence, constipation, and urinary retention have

been attributed to Cbl deficiency [1,3,4]. Although

the association between Cbl deficiency and neurological

abnormalities is well known, the precise

mechanism by which neurological impairment

occurs is not yet obvious [5]. Neurological abnormalities

may occur not only in patients with classic

Cbl deficiency but also in subtle or atypical deficiencies

in which anemia is absent [6]. In previous

reports, the pathogenesis of neurological abnormalities

due to Cbl deficiency has been attributed to

the accumulation of methylmalonic acid (MMA)

and homocysteine (Hcys). However, in totally gastrectomized

Cbl-deficient rat models, Scalabrino et

al. [7-9] showed that the severity of the neuropathological

damage in the spinal cord white matter did

not correlate with the progressive accumulation of

MMA and Hcys, and it was suggested that some

cytokines and/or growth factors other than MMA

and Hcys might play a role in the pathogenesis of

neurological damage in Cbl deficiency. Experimental

studies over the last few years have demonstrated

that Cbl deficiency increases the local overexpression

of neurotoxic cytokine tumor necrosis factor-

(TNF-) and decreases the synthesis of the neurotrophic

epidermal growth factor (EGF) and interleukin-6

(IL-6) in the cerebrospinal fluid (CSF) of rats

[10-12]. On the basis of these experimental observations,

we measured the serum levels of TNF-,

EGF and IL-6 in adult patients with Cbl deficiency

and investigated the effect of these factors on neurological

abnormalities, especially on autonomic

dysfunction as assessed by electrophysiological

studies (EPS). We also investigated the effect of

replacement therapy with Cbl in some of these

patients.


Subjects

The study population consisted of newly diagnosed

and untreated patients with Cbl deficiency

(n=41), healthy volunteers as a control group

(n=17) and patients after three months of treatment

for Cbl deficiency (n=22).

252

Çeneli et al.

Dysautonomia and cytokines in cobalamine deficiency Turk J Hematol 2010; 27: 250-6

Patient group

The diagnosis of Cbl deficiency was based on the

finding of low serum Cbl with normal serum folate

levels and high plasma levels of Hcys. Cut-off value

of serum Cbl was accepted as 160 pmol/L.

The patients with Cbl deficiency aged 16-80 years

who had none of the exclusion criteria were enrolled

into the study. Patients with a history of diabetes

mellitus, uremia, chronic hepatitis, concomitant

malignancies, alcohol abuse, drug use, folate deficiency,

or other possible causes of polyneuropathy

were excluded. None of the patients had apparent

infection or any generalized/localized infection sign

such as fever, leukocytosis or erythema, and none

of them had malabsorption, were following a vegetarian

diet, or had previous gastric surgery.

All the patients underwent careful medical history

and physical and neurological examination. As

a part of the medical history, all patients were queried

regarding the presence of autonomic dysfunction

symptoms such as orthostatic hypotension,

impotence, constipation, diarrhea, and incontinence.

Routine blood chemistry tests, complete

blood cell count and measurements of Hcys levels,

serum Cbl levels and folate concentrations were

performed. Serum MMA levels could not be measured

due to technical limitations in our hospital.

Serum levels of TNF-, EGF and IL-6 were studied in

patients with Cbl deficiency, the healthy control

group and after 3 months of Cbl replacement. All

patients with Cbl deficiency underwent EPS for the

diagnosis of neuropathy. Anti-parietal cell antibody

(APA) test, gastroduodenoscopy and endoscopic

biopsies were also performed in patients with Cbl

deficiency.

Control group

Healthy volunteers, who served as normal controls,

were defined following a detailed clinical history

and physical examination, with normal baseline

blood tests including full blood count, fasting

glucose, renal function, hepatic function, electrolytes,

and serum Cbl. They had no acute or chronic

disease such as diabetes mellitus, infections, rheumatologic

disorders, immune dysfunctions, or any

neurological disorders.

Cobalamin replacement therapy group

After tests had been completed, intramuscular

Cbl injections were started for patients with Cbl

deficiency. Cobalamin was given at a dose of 1000

g per day with intramuscular injection (week 1),

1000 g twice weekly (week 2), 1000 g/week for

the third and fourth weeks, and then 1000 g/

month. Three months after the initiation of therapy,

available patients were reevaluated for the presence

of neurological findings and serum cytokine

levels.

All participants gave informed consent prior to

participation in the study, and the study was

approved by the local Ethics Committee.

Methods

Peripheral venous blood samples of 2 ml and 8

ml were drawn after an overnight fast without stasis

from an antecubital vein with a 21-gauge needle

into ethylene diamine tetra-acetic acid (EDTA)-

containing tubes and empty tubes, respectively.

Serum samples were separated within 1-3 hours

and stored at -80°C until assayed. Serum Cbl, folate

and plasma Hcys concentrations were measured

using commercial kits.

Serum Cbl assay was performed by chemiluminescence

method using commercial kits on the

ADVIA Centaur chemistry analyzer (Siemens

Healthcare Diagnostics, Deerfield, IL, USA).

Serum TNF-, EGF and IL-6 assays were measured

by means of an enzyme immunoassay kit

(Biosource human TNF- ultrasensitive ELISA,

human EGF ELISA and human IL-6 ultrasensitive

ELISA kit, Biosource International Inc., Camarillo,

CA, USA).

Electrophysiological study (EPS)

Electrophysiological studies (EPSs) carried out in

all the patients with Cbl deficiency included motor

and sensory nerve conduction studies, RR interval

variation (RRIV) and sympathetic skin response

(SSR).

Patients underwent motor and sensory nerve

conduction studies of the right median, peroneal

and sural nerves. Motor studies included wristelbow

and knee-ankle segments of the median and

peroneal nerves, respectively. Sensory nerve conduction

was performed orthodromically on the digit

II-wrist segment of the median nerve. The sural

nerve was stimulated at midcalf, and recordings

were made antidromically just lateral and posterior

to the lateral malleolus. Stimuli were delivered

Çeneli et al.


supramaximally by surface stimulating electrodes.

Whenever nerve conduction abnormalities were

found in the studied nerves, other motor and sensory

nerves in the upper or lower extremities were

also studied to rule out mono- or polyneuropathies.

Recordings were performed by surface electrodes.

RRIV was studied by a Dantec Cantata electromyograph

as described previously [13]. Recordings

were made by using silver-silver chloride electrodes

with a 7x4 mm recording area placed on the dorsum

of each hand. Bandpass filters were set at 20-100 Hz

and the sensitivity was 200-500 V per division.

Oscilloscope was triggered at a sweep of 200 ms per

division, which permitted display of 2 QRS complexes

on the screen. Latency variations of the second

complex were measured in each sweep. Five groups

of 20 tracings were recorded at rest and two during

deep breathing. The range of RR intervals (a) and the

mean RR interval (b) were measured, and RRIV was

calculated according to the following formula: a/

bx100. The averages of five recordings at rest and

two recordings during deep breathing were termed

R% and D%, respectively. D%-R% and D%/R% were

also calculated and compared to normative data of

Shahani et al. [13]. Values lower than the 95% confidence

limits in any of the four calculated variables

were considered abnormal [parasympathetic (cardiovagal)

dysfunction].

SSR was recorded from both hands and feet

using the same equipment and electrodes described

in RRIV testing. The cathode was attached either to

palms or soles, referenced as the dorsum of the

hand or foot. Bandpass filters were set at 0.5-2000

Hz. Sensitivity was 500 V per division, with a sweep

of 500 ms per division for upper extremity and 1 s

per division for lower extremity recording. Responses

were elicited by delivering electrical stimuli to the

contralateral median or tibial nerves. At least four

tracings were obtained from each site. Abnormality,

sympathetic (sudomotor) dysfunction, was defined

as absence of any response in these recordings.

Statistical analysis

Statistical analysis was performed using SPSS for

Windows 11.5 software. Student’s paired-t test and

Wilcoxon ranks test were used for within-group

comparison. Mann-Whitney U test was used for

comparisons between groups. The relationships

between the variables were assessed using either

Spearman’s or Pearson correlation tests. Data for

TNF- and IL-6 levels are expressed as

median±standard deviation (SD). Data for EGF levels

are expressed as mean±SD. Differences were

considered significant if p<0.05.

Results

A total of 41 consecutive patients with newly

diagnosed and untreated Cbl deficiency (17 male,

24 female, age range: 19-75 years, median: 43 years)

were included. Twenty-two of the 41 patients were

re-evaluated after three months of parenteral Cbl

replacement treatment. The control group consisted

of 17 healthy volunteers (11 female, 6 male, age

range: 16-70 years, median: 42 years).

Initial presenting symptoms and signs of the

patients were: fatigue in 16/41 (39%), neurological

symptoms including paresthesias, dizziness, ataxia,

memory loss, and syncope in 31/41 (75.6%), anemia

and/or macrocytosis in 19/41 (46.34%), and gastrointestinal

symptoms in 3/41 (7.3%). Tingling (pins

and needles) and/or numbness in hands and/or

feet, evaluated as paresthesias, were present in

12/41 patients (29.26%). Neurological examination

showed positive Romberg’s sign in 15/41 patients

(36.58%), extensive vibratory sense loss in 13/41

patients (31.70%), and decreased deep tendon

reflex in 5/41 patients (12.19%).

Upper gastrointestinal system endoscopic examination

was performed in 30/41 patients. 16/30

patients (53.3%) had Helicobacter pylori infection.

Nine of 30 patients (30%) had atrophic gastritis. In 5

of 9 patients with atrophic gastritis (55.5%), APAs

were positive.

Eleven (26.80%) patients had macrocytic anemia

and in 4 of these patients, leukopenia and/or thrombocytopenia

was also present. In 9 of 41 patients

(21.95%), additional iron deficiency was present.

Hematological variables in patients with Cbl deficiency

were as follows: hemoglobin 11,33±2,48 g/

dl, mean corpuscular volume (MCV) 91,85±17,19 fl,

leukocyte 5.85±1.65 x10 9 /L, and platelet count

240.20±93.84 x10 9 /L Median value of serum Cbl levels

was 114,83±39,82 pmol/L. Median serum levels

of TNF- and IL-6 were 0,17±0,27 pg/ml (range:

0,13-1,9) and 0,09±0,21 pg/ml (range: 0,08 - 1,47),

respectively. Mean serum level of EGF was 1,15±0,73

pg/ml (range: 0,06 - 2,68). Hematological variables,

serum Cbl levels, and serum IL-6, TNF- and EGF

levels in Cbl-deficient patients and in healthy controls

are summarized in Table 1.

254

Çeneli et al.


Serum EGF and IL-6 levels were significantly

lower in patients with Cbl deficiency than healthy

controls [1.15±0.73 vs 1.96±0.93 (p=0.001) and

0.09±0.21 vs 0.13±0.84 (p=0.006), respectively].

Serum TNF- levels were not significantly different

between Cbl-deficient patients and healthy controls

[median 0.17±0.27 (range: 0.13 - 1.9) vs 0.18±0.97

(range: 0.15 - 3.12) pg/ml, p>0.05]. Serum TNF-

and IL-6 levels were not significantly different

between Cbl-deficient patients and after Cbl

replacement treatment [median 0.17±0.27 (range:

0.13 - 1.9) vs 0.19±0.17 (range: 0.07 - 1.47) pg/ml,

and 0.09±0.21 (range 0.07 - 1.47) vs 0.09±0.08

(range 0.04 - 0.45) pg/ml, respectively, p>0.05].

However, serum EGF levels significantly increased

after Cbl replacement treatment (1.13±0.73 vs

1.90±0.74, p=0.005). Serum levels of TNF-, IL-6

and EGF did not correlate with any of the considered

hematological variables in patients with Cbl

deficiency or in controls. Serum Cbl, IL-6, TNF-,

and EGF levels were not significantly different in

Cbl-deficient patients with autonomic neuropathy

than in patients without neuropathy (p>0.05).

Serum levels of IL-6, EGF and TNF- were not significantly

different between patients whose autonomic

neuropathy findings responded versus did

not respond to Cbl replacement treatment (p>0.05).

According to the EPS, 12/41 patients (29.27%)

had normal EPS findings, while 29/41 patients

(70.73%) demonstrated neurological abnormalities.

Sensorimotor peripheral neuropathy and autonomic

dysfunction according to EPS were present in

3/41 patients (7.32%) and 26/41 patients (63.41%),

respectively. In detail, autonomic dysfunction by

EPS was parasympathetic (cardiovagal) dysfunction

in 19 patients (46.34%) and sympathetic (sudomotor)

dysfunction in 7 (17.07%) (Figure 1). Twentytwo

of 41 patients were re-evaluated with hematological

variables, neurological examination and

serum cytokine levels after three months of parenteral

Cbl replacement treatment. After Cbl replacement,

a follow-up EPS could be evaluated in 13

patients with neuropathy, and improvement was

achieved in only 5 patients (38.46%).

Discussion

In our study, we demonstrated that serum EGF

and IL-6 levels were significantly decreased in

Distribution of neurop athies in patients with cobalamine

deficiency by electrophysiological studies (n=41)

no

neuropathy

n=12

29.27%

Sensorimotor peripheral neuropathy

n=3

7.32% Symphathetic (sudomotor)

autonomic dysfunction

n=7

17.07%

n=19

46.34%

Parasymphatethic

(Cardiovagal autonomic

dysfunction)

Figure 1. Distribution of neuropathies according to electrophysiological

studies in cobalamin-deficient patients

Table 1. Hematological variables, serum cobalamin and cytokines levels of study groups

Patients with Healthy controls p Value

Cbl deficiency

(n=17)

(n=41)

Hematological variables (mean values)

WBC (x10 9 /L) 5.85±1.65 6.99±1.70 p>0.05

Hemoglobin (g/dl) 11.33±2.48 13.19±1.62 p<0.05

Mean corpuscular volume (fl) 91.85±17.19 84.79±9.37 p>0.05

Platelets (x10 9 /L) 240.20±93.84 274.90±39.15 p>0.05

Serum cobalamine level (pmol/L) mean 114.83±39.82 217.00±48.18 p<0.001

Serum IL-6 level (pg/ml) median 0.09±0.21 0.13±0.84 p=0.006

Serum EGF level (pg/ml) mean 1.15±0.73 1.96±0.93 p=0.001

Serum TNF level (pg/ml) median 0.17±0.27 0.18±0.97 p>0.05

Çeneli et al.


patients with Cbl deficiency. However, serum TNF-

levels were not significantly different between Cbldeficient

patients and healthy controls. After Cbl

replacement, only serum EGF levels increased significantly.

Neurological abnormalities were demonstrated

in EPS in 29 of 41 patients (70.73%) and

among them, autonomic dysfunction was present

in 26 of 41 Cbl-deficient patients (63.41%). There

was no association between serum levels of EGF,

TNF-, IL-6, and autonomic dysfunction as assessed

by EPS (p>0.05). In our study group, serum Cbl

levels were 120 pg/ml in only 22 of 41 patients, so

not all our patients had severe Cbl deficiency. This

situation might be considered a limitation of our

study.

Mean corpuscular volume (MCV) of our patient

group with Cbl deficiency was 91.85±17.19 fl

(mean). Generally, severe macrocytosis is expected

in Cbl deficiency anemia. Nevertheless, our

patient population consisted of not only megaloblastic

anemia patients due to Cbl deficiency but

also patients with Cbl deficiency without anemia

who had neurological symptoms and signs.

Furthermore, some of the patients had additional

iron deficiency. Relatively moderate macrocytosis

in our patients with Cbl deficiency can be explained

by these factors.

It has been demonstrated that the Cbl-deficient

central neuropathy of totally gastrectomized (TGX)

rats is not caused by the withdrawal of the vitamin

itself, but reflects a simultaneous increase in the

production of the neurotoxic cytokine TNF- and a

decrease in the synthesis of the neurotrophic

growth factor EGF and IL-6 [10-12,14]. In two studies,

cytokine levels were detected in humans with

Cbl deficiency [15,16]. In the first report by Peracchi

et al. [15], it was demonstrated that overproduction

of serum TNF- and underproduction of

serum EGF were present in humans with newly

diagnosed severe Cbl deficiency, as in rats. Later,

Scalabrino et al. [16] reported higher TNF- levels

and lower EGF levels in the CSF of Cbl-deficient

patients with neurological manifestations of subacute

combined degeneration (SCD). We could

not demonstrate an increase in serum TNF- levels

in the Cbl-deficient patients or a relationship

between serum IL-6, TNF- and EGF levels and

autonomic dysfunction. Severity of Cbl deficiency

and the presence of clinical SCD in the study of

Peracchi et al. [15] may be the major factor

explaining the discrepancy between the two studies.

The duration of Cbl deficiency may also be a

leading factor for the changes in serum neurotrophic

and neurotoxic cytokine levels.

Although the most common clinical manifestations

of Cbl deficiency are SCD of the spinal cord

and peripheral neuropathy, optic atrophy, dementia,

and autonomic nervous system dysfunction

have been reported rarely [1-4]. In the previous

studies, parameters of heart rate variability in

patients with Cbl deficiency were found to be significantly

lower as compared to healthy controls

[17,18]. Orthostatic hypotension as a function of

autonomic neuropathy was systematically assessed

in patients with Cbl deficiency by Beitzke et al. [3].

In their study, a significant fall in systolic blood

pressure directly after head-up tilt, stroke index,

and cardiac index, and a lack of increase of total

peripheral resistance index for the duration of tilt in

patients with Cbl deficiency as compared to healthy

controls were reported. In patients with Cbl deficiency,

gastric emptying by scintigraphy has also

been studied as a function of the autonomic nervous

system. Mean gastric emptying t1/2 in patients

with Cbl deficiency was reported to be prolonged.

Although mean gastric emptying t1/2 after Cbl

replacement therapy was somewhat shorter, a statistically

significant difference persisted after Cbl

replacement [4].

In conclusion, our present study showed that

neurotrophic cytokines EGF and IL-6 serum levels

decreased while neurotoxic cytokine TNF- serum

level did not change in patients with Cbl deficiency

as compared with healthy controls. Nevertheless,

we could not find a relation between autonomic

neuropathy and changes in serum neurotrophic

and neurotoxic cytokines levels in Cbl-deficient

patients. Presence of asymptomatic autonomic

neuropathy seems to be a frequent neurological

finding in patients with Cbl deficiency. To determine

the clinical relevance of neurotoxic and neurotrophic

cytokine levels, further studies are needed.






256

Çeneli et al.


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hemophagocytic lymphohistiocytosis: A retrospective

review of the Turkish Histiocyte Study Group

Primer hemofagositik lenfohistiositozisli hastalarn klinik ve labarotuvar

verileri; Türk Histiosit Çalma Grubunun retrospektif derlemesi

Tunç Fgn 1 , Türkan Patrolu 2 , Akif Özdemir 2 , Tiraje Celkan 3 , Ümran Çalkan 4 ,

Mehmet Ertem 5 , Nee Yaral 6 , Erol Erduran 7 , Canan Vergin 8 , Cengiz Canpolat 9 ,

Feride Duru 1 , Ali Bay 10 , Namk Özbek 11 , Deniz Ylmaz Karapnar 12

1Department of Pediatric Hematology, Ondokuz Mays University Faculty of Medicine, Samsun, Turkey

2Department of Pediatric Hematology and Oncology, Erciyes University Faculty of Medicine, Kayseri, Turkey

3Department of Pediatric Hematology, stanbul University Cerrahpaa Faculty of Medicine, stanbul, Turkey

4Department of Pediatric Hematology, Selçuk University Faculty of Medicine, Konya, Turkey

5Department of Pediatric Hematology, Ankara University Faculty of Medicine, Ankara, Turkey

6Dr. Sami Ulus Children’s Hospital, Ankara, Turkey

7Department of Pediatric Hematology, Karadeniz Technical University Faculty of Medicine, Trabzon, Turkey

8Dr. Behçet Uz Children’s Hospital, zmir, Turkey

9Department of Pediatric Hematology, Marmara University Faculty of Medicine, stanbul, Turkey

10Department of Pediatric Hematology, Yüzüncü Yl University Faculty of Medicine, Van, Turkey

11Department of Pediatric Hematology, Bakent University Faculty of Medicine, Ankara, Turkey

12Department of Pediatric Hematology, Ege University Faculty of Medicine, zmir, Turkey

Abstract

Objective: This study analyzes the clinical and laboratory findings of children with primary hemophagocytic

lymphohistiocytosis (HLH) followed in various referral centers of Turkey.

Materials and Methods: A simple three-page questionnaire prepared by the Turkish Histiocyte Study Group

was used for documentation of patient data.

Results: Age at diagnosis varied from 0.6 to 78 months (median±SD, 16.5±26.1). Sex distribution was

almost equal (F/M=10/12). The frequencies of parental consanguinity and sibling death in the family history

were 100% and 81.1%, respectively. The most common clinical findings were hepatomegaly (100%)

and fever (95%). The most common laboratory findings were anemia (100%), hyperferritinemia (100%) and

thrombocytopenia (90.9%). Triglyceride and total bilirubin levels in the deceased versus surviving group

appear to be high (triglyceride: 394±183 mg/dl, 289±7 mg/dl; total bilirubin: 2.7±6.9 mg/dl, 0.5±1.2 mg/

dl, respectively).

Address for Correspondence: M.D. Tunç Fgn, Ondokuz Mayis University, Faculty of Medicine Department of Pediatric Hematology 55139 Samsun, Turkey

Phone: +90 362 312 19 19 / 3658 E-mail: fisgint@yahoo.com

doi:10.5152/tjh.2010.47

258

Fgn et al.

Primary HLH in Turkey Turk J Hematol 2010; 27: 157-62

Conclusion: We concluded that fever, hepatosplenomegaly, anemia, thrombocytopenia, and hyperferritinemia

are the most common clinical and laboratory findings in primary HLH. Increased triglyceride and

total bilirubin level at the time of diagnosis might be an indicator of poor prognosis in HLH.

(Turk J Hematol 2010; 27: 157-62)

Key words: Primary hemophagocytic lymphohistiocytosis, clinical and laboratory findings

Received: June 7, 2009 Accepted: May 11, 2010

Özet

Amaç: Primer hemofagositik lenfohistiositoz tans ile Türkiye’deki farkl merkezlerde takip edilen

hastalarn klinik ve laboratuar deerlerini deerlendirmektir.

Yöntem ve Gereçler: Hasta verilerinin deerlendirilmesi için Türk Histiosit Çalma Grubu tarafndan

düzenlenmi 3 sayfalk soru formu kullanld.

Bulgular: Olgularn tan srasndaki yalar 0.6 ile 78 ay arasnda idi (median±SD, 16.5±26.1). Cinsiyet

dalm eite yaknd (K/E=10/12). Anne-baba akrabal ve karde ölüm öyküsü oranlar sras ile %100

ve %81.1 idi. Ensk görülen klinik bulgular hepatomegali (%100), ate (%95), labaratuvar bulgular ise

anemi (%100), hiperferritinemi (%100) ve trombositopeniydi (%90.9). Ölen olgularn trigliserid ve bilirubin

düzeyleri yüksek gibi gözükmekteydi (trigliserid; 394±183 mg/dL, 289±7 mg/dL, total bilirubin;

2.7±6.9 mg/dL, 0.5±1.2 mg/dL)

Sonuç: Ate, hepatosplenomegali, anemi, hiperferritinemi ve trombositopeni primer HLH'li hastalarda en

sk görülen klinik ve labaratuvar bulgulardr. Tan srasndaki artm trigliserid ve bilirubin düzeyleri kötü

prognostik belirteç olabilir. (Turk J Hematol 2010; 27: 157-62)

Anahtar kelimeler: Primer hemofagositik lenfohistiositozis, klinik ve laboratuvar veriler

Geli tarihi: 7 Haziran 2009 Kabul tarihi: 11 Mays 2010

Introduction

Hemophagocytic lymphohistiocytosis (HLH) is a

hyperinflammation disorder characterized by fever,

splenomegaly, bicytopenia, hypertriglyceridemia

and/or hypofibrinogenemia, hyperferritinemia, elevated

soluble CD25 levels, decreased or absent

natural killer cell activity, and hemophagocytosis in

the bone marrow, spleen, lymph nodes or other tissues

[1]. Primary or familial hemophagocytic lymphohistiocytosis

(FHLH) is an autosomal recessive

disorder of immune dysfunction caused by mutations

in Perforin, Munc13-4, Syntaxin 11 and STBX2

genes [2-5]. Secondary HLH develops in some disorders

such as infections, toxins, malignancies, and

autoimmune or immune deficiency disorders [6].

Males and females are affected at equal frequencies.

Onset of FHLH occurs generally in infancy, but

rarely, patients can be symptomatic at later ages,

even in adulthood [7]. The main pathogenetic

mechanism is defective natural killer cell function

and uncontrolled T cell activation leading to

increased levels of cytokines such as interferon-

(IFN-), tumor necrosis factor- (TNF-), interleukin-1

(IL-1), IL-6, IL-10, and soluble IL-2 receptor [8].

Profound hypercytokinemia activates macrophages,

and hemophagocytosis ensues. Infiltration of bone

marrow, liver, spleen, and the central nervous system

(CNS) with macrophages and T lymphocytes

results in multiorgan dysfunction with high mortality.

Prognosis is poor for FHLH patients treated with

chemotherapy alone, with an estimated five-year

survival (SE) below 20% [9].

Allogenic hematopoietic stem cell transplantation

(HSCT) is the only accepted curative therapy [1,6].

In this study, we report the clinical and laboratory

data of 22 children with HLH who were followed in

different pediatric referral centers in Turkey over the

past 10 years, together with a comprehensive review

of this rare disorder.


This study retrospectively analyzed the data of 22

children with HLH who were diagnosed and followed-up

in 13 pediatric referral centers in Turkey.

A simple three-page questionnaire was sent to each

center by the Turkish Histiocyte Study Group to standardize

data collection. This study has local ethical

committee approval.

All children were diagnosed as HLH between

January 1995 to June 2005 according to Diagnostic

Guidelines for HLH 1994 and 2004. Patients with

parental consanguinity and history of sibling death

were accepted as having FHLH. Parental consanguinity

was present in all of the patients and history

Fgn et al.

Turk J Hematol 2010; 27: 157-62 Primary HLH in Turkey 259

of sibling death in 18 of them. All patients fulfilled at

least five cardinal criteria of HLH at the time of diagnosis,

including fever, hepatosplenomegaly, bicytopenia

and/or pancytopenia, hypertriglyceridemia

and/or hypofibrinogenemia, hyperferritinemia, and

hemophagocytosis in the bone marrow, liver or

lymph nodes [10]. They had no evidence of malignancy,

infection, metabolic disease, or any other

disorder that might have been the cause of presenting

symptoms and hemophagocytosis. Patients

were evaluated regarding age, clinical findings,

laboratory data, treatment, and prognosis, using

descriptive statistics. A molecular genetic study

could be performed in only 4 patients.

Results

The age of the patients ranged from 0.6 to 78

months at the time of diagnosis. Sex distribution

was almost equal (F/M=10/12). Initial clinical findings

are shown in Tables 1 and 2. Rates of parental

consanguinity and sibling death were 100% (22/22)

and 81.1% (18/22), respectively. The patients were

diagnosed by bone marrow aspiration in 19 (86.4%)

and biopsy from the liver in 2 (9%), spleen in 1

(4.5%) and lymph nodes in 1 (4.5%).

Hepatomegaly was present in all the patients,

and 95.5% (21/22) had fever and splenomegaly. The

most common laboratory findings were anemia

(100%), hyperferritinemia (100%) and thrombocytopenia

90.9% (20/22). Other findings are shown in

Tables 1 and 2.

Cerebromeningeal symptoms including seizures

or cerebral nerve palsies occurred in 27.3% (6/22) of

the patients in our study group. Fourteen (63.3%)

children received supportive treatment (fresh frozen

plasma and erythrocyte concentrates) and corticosteroid,

intravenous immunoglobulin (IVIG),

and vincristine therapy. HLH-94 and HLH-2004 treatment

protocols were given in 4 (18.8%) and 3

(13.6%) children, respectively. One child had undergone

bone marrow transplantation, and she died

with chronic graft-versus-host disease and pulmonary

hypertension. Eighteen (86.3%) patients died

due to progressive disease, infection and bleeding.

Three children were alive at the time of preparation

of the article.

Discussion

Familial hemophagocytic lymphohistiocytosis

(FHLH) is a rare autosomal recessive disease characterized

by fever, hepatosplenomegaly, cytopenia,

Table 1. Clinical findings of patients with hemophagocytic lymphohistiocytosis

at diagnosis

n=22 Number %

Gender

Girl 10 45.5

Boy 12 54.5

Age (months) 16.5 0.6 - 78

(median) (range)

Positive History

Consanguinity 22 100

Fever 21 95.5

Sibling death 18 81.8

Skin eruptions 5 22.7

Clinical Findings

Hepatomegaly 22 100

Splenomegaly 21 95.5

Fever 21 95.5

Lymphadenomegaly* 7 31.8

Cerebromeningeal symptoms 6 27.3

Bleeding** 4 18.2

Skin eruptions 4 18.2

*Cervical, occipital, inguinal, **Gingival, epistaxis

Table 2. Laboratory findings of patients with hemophagocytic lymphohistiocytosis

at diagnosis

Number of patients (n)

n=22

Laboratory Findings mean±SD range

Hb (g/dl) 6.85±1.6 3.6 8 9.8

Platelet (x10 9 /L) 42±157 2 - 752

ANC (/mm 3 ) 895± 5632 50 - 24000

AST (U/L) 132±302 21 - 1176

ALT (U/L) 102± 1171 22 - 5446

Triglyceride (mg/dl) 374±179 284 - 999

LDH (U/L) 930±968 106 - 3547

Ferritin (mg/dl) 1434±1485 576 - 5654

Serum sodium (mEq/L) 133±5.7 124 - 148

T. Bilirubin (mg/dl) 2±6.5 0.4 - 22

PT (sec) 14±18 2 - 90

APTT (sec) 39±32 27 - 180

Fibrinogen (g/L) 1.28± 1.38 0 - 4.2

Hb: Hemoglobin: ANC: Absolute neutrophil count; AST: Aspartate aminotransferase;

ALT: Alanine aminotransferase; LDH: Lactic acid dehydrogenase; T. bilirubin:

Total bilirubin; PT: Prothrombin time; APTT: Activated partial thromboplastin time

260

Fgn et al.


and hemophagocytosis in the bone marrow or

other tissues [1]. It is known to occur frequently in

children under 2 years of age [6,10]. Due to the very

high rate of consanguineous marriages (21%) in

Turkey, the expected incidence of HLH should be

high as well. Many reports on FHLH in Turkish

patients justify this conclusion [11-13]. Although

there are no nationwide epidemiologic data, in one

study, the frequency of the HLH among hospitalized

patients was found to be 7.5/10000, and the rate of

consanguineous marriage was reported as 68% in

HLH patients [14]. This was 100% in our study

group, and 81.8% had family history of a sibling

death.

In this report, we analyzed the data of 22 patients

with HLH who were diagnosed and treated in different

pediatric referral centers between January 1995

and 2005. Along with the high frequency of consanguineous

marriages in the study group, each patient

fulfilled at least five cardinal criteria of HLH. Although

molecular genetic study is now available in a center

in Turkey (Hacettepe University, Ankara), it could

only be performed in a very limited number of

patients. Despite this limitation, we considered that

it would be informative to document the available

clinical and laboratory data of these children with

HLH from Turkey, where it is encountered relatively

more frequently than in other parts of the world.

The clinical findings of HLH are generally nonspecific,

and the most common symptoms are

known to be fever and hepatosplenomegaly. Many

patients with HLH present with mild to moderate

signs of infection, which delays the diagnosis.

Consistent with previous studies, the most common

initial findings in our patients were hepatomegaly,

splenomegaly and fever. There was only 1

patient who was afebrile at the time of diagnosis.

Although fever is a very common finding in HLH, it

may not occur in a small number of patients for

unknown reasons. Some patients might develop

fever during follow-up, as in our patient. Clinicians

should be aware of this possibility in the diagnosis

of HLH. The most common laboratory findings

were anemia, hyperferritinemia and thrombocytopenia,

as expected. This may be due to undetectable

infections, or the patients may have acquired

HLH. Although platelet and absolute neutrophil

counts were normal in 2 patients in the initial period,

these patients had cytopenia in more than 2 of 3

lineages and fulfilled the criteria of HLH. As is

known, the presence of infection does not discriminate

FHLH from the acquired form. FHLH episodes

are triggered by infectious agents [6].

Hemophagocytosis in the bone marrow or reticuloendothelial

system including liver, spleen and

lymph nodes was reported to be detected in onehalf

or two-thirds of the patients with HLH, so

hemophagocytosis may not always be observed in

the bone marrow aspiration, especially at the disease

onset. When the diagnosis of HLH is strongly

suspected, clinicians should make a thorough

microscopic examination of the bone marrow aspiration

smear or serial repeated aspirations should

be done. In our series, we observed hemophagocytosis

in all of the patients in the microscopic examination,

and it seems to be the highest ratio of determining

hemophagocytosis in HLH when compared

with previous reports in the literature.

Although elevated lactate dehydrogenase (LDH)

is emphasized as a supporting criterion by some

authors, it is not accepted as a diagnostic laboratory

criterion for diagnosis of HLH [10]. In our group, 19

of 22 (86.7%) patients had elevated LDH level. It is

notable that in our study group, the frequency of

elevated LDH was remarkably high.

On the other hand, cerebromeningeal symptoms

including seizures or cerebral nerve palsies occurred

in 27.3% (6/22) of the patients in our study group.

This figure is slightly less than those reported in previous

studies [13-16].

There is limited data regarding the association

between the prognosis of disease and triglyceride

metabolism. Monitoring the triglyceride level is a

recently defined follow-up parameter in the evaluation

of treatment response in secondary HLH as

reported by Okamoto et al. [17]. Sarper et al. [18]

reported low triglyceride levels in 2 patients diagnosed

with HLH.

In addition to perforin, the Munc13-4, syntaxin 11,

and recently Munc18-2 mutations are described

[2-5]. A molecular genetic study was performed in

only 4 patients, and syntaxin mutation was detected

in 1 of them [19].

We cannot discuss event-free survival because

of data from heterogeneous hospital records and

the differences between the therapy regimens of

the centers. Seven of the 22 children received specific

therapy for HLH and only 1 of them survived,

Fgn et al.

Turk J Hematol 2010; 27: 157-62 Primary HLH in Turkey 261

while 2 out of 14 patients who received Vepesid

(etoposide), IVIG and steroids survived. All surviving

patients had a history of parental consanguinity

and 2 of them had sibling death.

Etoposide, dexamethasone, cyclosporine A, and

intrathecal methotrexate have been the mainstay of

all HLH treatment protocols because of their different

properties. One of the basic findings of HLH at

the cellular level is decreased apoptosis, and etoposide

has a more significant proapoptotic effect. Use

of these medications at different dosages and durations

in 2 of our surviving patients might explain the

favorable outcome. The genetic analysis of these

patients will further clarify our results.

The prognosis of patients with HLH who require

admission to any center has been regarded as

extremely poor. SCT is the only curative treatment

option for patients with HLH [1,6]. There are two

major problems before bone marrow transplantation:

finding a matched sibling donor and keeping

the patients alive until the transplantation [1].

Without a bone marrow transplant, the child is

always at risk of a severe or fatal activation and will

have difficulty surviving beyond their first birthday.

Unfortunately, only 1 patient underwent peripheral

SCT from a fully matched mother in the study period,

and she died because of chronic graft-versushost

disease and pulmonary hypertension.

In conclusion, fever, hepatosplenomegaly, anemia,

thrombocytopenia, and hyperferritinemia are

the most common clinical and laboratory findings

in primary HLH. Parental consanguinity and presence

of affected siblings seem to be an important

clue for diagnosis. Increased triglyceride and total

bilirubin levels at the time of diagnosis might be

poor prognostic indicators in HLH, but this requires

further investigation.






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Research Article

263

The predictive value of stimulation index

calculated by modified mixed lymphocyte

culture in the detection of GVHD following

hematopoietic stem cell transplantation

Hematopoetik kök hücre naklini takiben gelien GVHD’nin

saptanmasnda modifiye mikst lenfosit kültür testinde hesaplanan

stimülasyon indeksinin belirleyici deeri

Hülya Saylan en 1 , Tülay Klçaslan Ayna 2 , Hayriye entürk Çiftçi 2 ,

Sevgi Kalayolu Bek 3 , Emel Aye Önal 4 , Arzu Akçay 1 , Hülya Bilgen 5 ,

Mehmet Gürtekin 2 , Deniz Sargn 3 , Mahmut Çarin 2

1Department of Hematology, Bakrköy Maternity and Children’s Diseases Hospital, stanbul, Turkey

2Department of Medical Biology, Istanbul University, Istanbul Medical Faculty, stanbul, Turkey

3Department of Internal Medicine, Division of Hematology, Istanbul University, Istanbul Medical Faculty, stanbul, Turkey

4Department of Public Health, Istanbul University, Istanbul Medical Faculty, stanbul, Turkey

5Blood Bank, Istanbul University, Cerrahpaa Medical Faculty, stanbul, Turkey

Abstract

Objective: Mixed lymphocyte culture (MLC) is one of the routine tests performed prior to hematopoietic

stem cell transplantation (HSCT) as a predictive assay for assessing the quality of donor matching and graftversus-host

disease (GVHD). The stimulation index is one of the formulas of the MLC test, and it is used

for evaluation of matching between donor and recipient. Modified MLC (mMLC) test is produced by adding

various cytokines to the MLC test, and increased sensitivity has been reported with this modification.

Materials and Methods: The importance of the stimulation index values in MLC and mMLC tests was

evaluated in 59 patients who received HSCs from human leukocyte antigen-identical sibling donors. In the

mMLC test, cytokines were added as interleukin (IL)-2, IL-2 + IL-4 and IL-2 + interferon (IFN)-gamma

+ tumor necrosis factor (TNF)-alpha. Stimulation index values in mMLC test were compared with stimulation

index values in MLC test.

Results: Twenty-three (39%) patients developed GVHD. When evaluated in terms of stimulation index >1

patients, in MLC, 55% of the patients developed GVHD (p=0.229), whereas these values were 75% in the

IL-2 added mMLC test (p=0.035), 100% in the IL-2 + IL-4 added mMLC test (p=0.076) and 85.7% in

the IL-2 + IFN-gamma + TNF-alpha added mMLC test (p=0.015).

Conclusion: mMLC increased the sensitivity of the test. The relation between the positive results and evidence

of GVHD after transplantation was found significant. (Turk J Hematol 2010; 27: 263-8)

Key words: Stimulation index, mixed lymphocyte culture, hematopoietic stem cell transplantation, cytokines

Received: July 5, 2009 Accepted: June 21, 2010

Address for Correspondence: M.D. Hülya Saylan en, emsettin Günaltay Caddesi. Sultan Sokak. Çada Apartman. No: 9/22 Erenköy stanbul, Türkiye

Phone: +90 532 296 66 86 E-mail: hulyasn@gmail.com

doi:10.5152/tjh.2010.48

264

en et al.

Importance of modified mixed lymphocyte culture test Turk J Hematol 2010; 27: 263-8

Özet

Amaç: Mikst lenfosit kültür testi hematopoetic kök hücre naklinden önce, donör uyumunu ve graft versus

host hastaln önceden belirlemek amac ile yaplan bir testtir. Stimülasyon indeksi mikst lenfosit kültür

testinde kullanlan formüllerden biridir ve alc ile verici arasndaki uyumu belirlemek için yaplr. Modifiye

mikst lenfosit kültür testi ise mikst lenfosit kültür testine çeitli sitokinlerin ilave edilmesiyle yaplr ve

yaplan bu deiiklikle duyarllkta art olduu saptanmtr.

Yöntem ve Gereçler: HLA uyumlu donörden hematopoetik kök hücre nakli yaplacak 59 hastada Mikst

Lenfosit Kültür ve Modifiye Mikst Lenfosit Kültürdeki stimülasyon indeksi deerlerine baklmtr.

Modifiye mikst lenfosit kültür testinde sitokinler interlökin-2, interlökin-2 + interlökin-4 ve interlökin-2 +

interferon-gama + tümör nekrozis faktör-alfa eklinde eklenmi ve modifiye mikst lenfosit kültüründeki

stimülasyon indeksi deerleri, mikst lenfosit kültüründeki stimülasyon indeksi deerleriyle karlatrlmtr.

Bulgular: Yirmi üç hastada (%39) graft versus host hastal olumutur. Mikst lenfosit kültürde stimülasyon

indeksi >1olan hastalara bakldnda hastalarn %55'inde graft versus host hastal görülmütür

(p:0,229). Bunun yan sra interlökin-2 ilavesi ile yaplan modifiye mikst lenfosit kültür testinde stimülasyon

indeksi >1 olan hastalarn %75'inde (p=0,035); interlökin-2 + interlökin-4 ilavesi ile yaplan modifiye mixt

lenfosit kültür testinde stimülasyon indeksi >1 olan hastalarn %100’ünde (p=0,076) ve interlökin-2 +

interferon-gama + tümör nekrozis faktör-alfa ilavesi ile yaplan modifiye mixt lenfosit kültür testinde stimülasyon

indeksi >1 olan hastalarn %85,7’sinde (p=0,015) graft versus host hastal görülmütür.

Sonuç: Modifiye mikst lenfosit kültür, testin duyarlln arttrmtr ve pozitif sonuçlar ile transplantasyondan

sonra graft versus host hastalnn gelimesi arasndaki iliki anlaml bulunmutur.

(Turk J Hematol 2010; 27: 263-8)

Anahtar kelimeler: Stimülasyon indeksi, mikst lenfosit kültür, hematopoetik kök hücre nakli, sitokinler

Geli tarihi: 5 Temmuz 2009 Kabul tarihi: 21 Haziran 2010

Introduction

Receiving a hematopoietic stem cell transplantation

(HSCT) from a matched donor is a lifesaving

treatment modality in some diseases. The degree of

human leukocyte antigen (HLA) donor/recipient

match has a significant association with graft-versus-host

disease (GVHD). To date, donor selection

has been based on match for the antigens encoded

by the HLA class I (A, B ,C) and class II (DR) loci.

Unrecognized or undefined mismatch for class II

genes between the donor and recipient can exist

despite a match for HLA-A, B, C and DR and could

contribute to the high risk of GVHD after transplantation.

Mixed lymphocyte culture (MLC) test is a

method used in determination of class II antigens

that evaluates cell proliferation based on compatibility

of HLA antigens between the recipient and

donor. MLC is a cellular test that exhibits the appropriateness

of HLA antigens of the donor/recipient.

One of the formulas used for this evaluation is the

stimulation index (SI) [1,2]. However, the MLC test

fails to detect the minor histocompatibility antigens

contributing to GVHD and tissue rejection and also

the response stimulated by HLA-DP. On the other

hand, it is reported that GVHD and tissue rejection

could be predicted by cytokine-modified mixed

lymphocyte culture (mMLC) test [3-6]. Cytokines

play a critical role after allogeneic recognition in the

MLC. The MLC is not only a clinical method in HSCT

but also an important model to show T cell activation

and cytokine interaction following alloantigen

recognition. The T helper 1 cytokine interferongamma

(IFN-) is known to induce cytotoxic T lymphocytes

(CTL) by enhancing the expression of

both HLA class I and class II molecules. Synergistic

effects of interleukin-2 (IL-2) as well as tumor

necrosis factor-alpha (TNF-) on the production of

IFN- are supposed, whereas IFN- itself also shows

a stimulatory influence on the expression of the IL-2

receptor in T lymphocytes [7].

Interleukin-4 (IL-4) is the developmental factor for

B-lymphocytes and among the molecules that establishes

association with HLA class II products [5,6].

TNF- activates leukocytes, particularly neutrophils

[8]. In both murine and human MLC reactions, TNF-

enhances the proliferative response [9].

In this study, cytokine-spiked mMLC test in addition

to the MLC test were administered to patientdonor

pairs. In mMLC, cytokines were added to the

test individually or in combination. The probability

of GVHD was thus determined by stimulating the

antigens that were not exhibited in MLC, with an in

vivo test. The association of SI results obtained from

the MLC and mMLC tests with GVHD development

was investigated. The relation between mMLC

results and grade or organ involvement of GVHD

was not considered.

en et al.

Turk J Hematol 2010; 27: 263-8 Importance of modified mixed lymphocyte culture test 265


MLC and mMLC tests were performed in a total

of 59 full match-related, recipient and donor pairs at

Istanbul University Medical Faculty, Medical Biology

Department. These cases were received from eight

different centers. Written informed consents were

obtained from all patients or their parents. This

study was approved by local ethics committee.

Allogeneic HSCT was performed from full matchrelated

donors, and bone marrow was used as HSC

sources for each patient.

The conditioning regimen was changed according

to the underlying disease and was mainly with

busulfan and cyclophosphamide (BU/CY). In patients

with multiple myeloma, the regimen consisted of

high-dose melphalan. In patients with hemoglobinopathy

and aplastic anemia, antithymocyte globulin

(ATG) was incorporated into the regimen.

GVHD prophylaxis consisted of cyclosporin A

(CsA)+short course methotrexate (MTX) in all cases.

The diagnostic tests for GVHD usually depended

on the symptoms, but could include: gastrointestinal

endoscopy, with or without a biopsy, liver function

tests (aspartate aminotransferase [AST], alkaline

phosphatase [ALP] and bilirubin levels are

increased), liver biopsy (if the patient only has liver

symptoms), lung X-rays, and skin biopsy [10,11].

The MLC test was administered in one-way and

two-stage manner (recipient and donor) in the culture

laboratory of the department. In this study, we

evaluated donor-directed. The peripheral blood

mononuclear cells of the recipient and donor pairs

were obtained via Ficoll Hypaque gradient centrifuge

method. The lymphocytes obtained were

washed three times with RPMI 1640 (with glutamine,

Hepes, Sigma-50 u/ml penicillin and 50 g/ml

streptomycin) consequently followed by counting

the cells in a medium containing RPMI 1640 and

human serum (9:1). Donor lymphocytes were used

at a final concentration of 1×10 6 cell/ml. Recipient

lymphocytes were used at a final concentration of

2×10 6 cell/ml. The recipient cells were irradiated at

5000 rad (cGy). Donor cells (100 l) and irradiated

recipient cells (50 l) were added to the culture

plates. All experiments were performed in triplicate.

Results were normalized and presented as means.

Cytokines were added in the same way in addition

to MLC test, by producing cell batch (IL-2: 2.4 U/l,

IL-4: 14.5 U/l, IFN-: 200 U/l, TNF-: 100 U/l)

(Table 1).

3H Thymidine was placed in each well after the

cells were incubated in 96-well plates for 96 hours

(h) at 37°C and 5% CO 2 . All the cells were collected

after 16-18 h. Cultures were harvested onto glass

fiber filters. Glass fiber filters are dried at room temperature.

The numeric information is defined in

counter (Packard Tricarb 1000 TR) by placing filter

papers in scintillation solution (toluol + 2,5 diphenyl

oxazole). Blank values are deducted from the measurements.

Sample filters were counted using

counter. SI formula was used for evaluation of the

results. On the MLC test, a SI value 1 indicates that

the donor is not reactive to the recipient.

SI=DR*/ DD

Statistical analyses were performed using SPSS

10.0 statistical software program. The relationship

between development of GVHD and MLC/mMLC

was analyzed with Fisher’s exact and Independent

Samples T Test.

Results

The mean age of the 59 patients who underwent

MLC test and HSCT was 19.91±1.96 (1-51)

and the mean age of the donors was 20.61±1.91

(1-51). Forty-one percent of the patients were

female (n=24) and 59% were male (n=35). The

median age was 21.50±2.88 (1-45) for the female

patients and 20.51±2.53 (1-51] for the males.

Twelve female recipients received stem cells from

a female donor and 14 from a male donor, whereas

17 of the male recipients received stem cells

from a female donor and 16 from a male donor.

Eighteen patients were previously diagnosed with

chronic myeloid leukemia (CML) and the rest of

the group were as follows: 15 acute myeloid leukemia

(AML), 9 aplastic anemia, 7 acute lymphoid

Table 1. Localization of the cells on the culture plate on the MLC and mMLC test

MLC MLC mMLC mMLC

Donor-Donor Donor-Recipient* DD + IL-2 DR* + IL-2

(DD) (DR) DD + IL-2 + IL-4 DR* + IL-2 + IL-4

DD + IL-2 + IFN- + TNF-

DR* + IL-2 + IFN- + TNF-

* irradiated cells

266

en et al.


leukemia (ALL), 3 thalassemia major, 3 multiple

myeloma (MM), 2 non-Hodgkin lymphoma (NHL),

1 myelodysplastic syndrome (MDS), and 1 sickle

cell anemia (Table 2).

Cytokine-spiked mMLC test was administered to

recipient and donor pairs with an adequate number

of cells.

GVHD developed in 23 (39%) of the 59 patients

who underwent HSCT. Acute and chronic GVHD

were not classified or analyzed separately. The

evaluation of the MLC test for SI in 59 patients

Table 2. Patient characteristics

Age

n %

15 (Pediatric Group)

Male 16 27

Female 11 19

15-51 (Adult Group)

Male 19 32

Female 13 22

Gender

Male 35 59

Female 24 41

Malignant Diseases

CML 18 31

AML 15 25

Aplastic Anemia 9 15

ALL 7 12

Thalassemia Major 3 5

Multiple Myeloma 3 5

NHL 2 3

MDS 1 2

Sickle Cell Anemia 1 2

revealed SI values 1 in 50 patients, while 9 patients

had SI values >1. In patients with SI values 1, after

transplantation, GVHD was detected in 18 patients

and not detected in 32 patients. In the group with SI

>1, 4 had no complications while 5 had GVHD. The

statistical evaluation of the association between the

SI values and GVHD development revealed no significance

(p=0.229) (Table 3).

mMLC test was performed by adding IL-2 in 53 of

these patients. While 45 patients revealed SI values

1, 8 patients had SI values >1. While no GVHD was

observed in 30 of the patients with SI 1, GVHD

occurred in 15 patients. Six of the 8 patients with SI

>1 developed GVHD while 2 patients did not. The

statistical evaluation of the relation between SI values

and GVHD development revealed a statistically

significant result (p=0.035) (Table 3).

mMLC test was performed by adding IL-2+IL-4

in 24 of these patients. While 22 patients revealed SI

values 1, 2 patients had SI values >1. While no

post-transplant GVHD occurred in 17 of those

patients with a SI 1, 5 patients developed this disease.

GVHD could be determined in 2 patients with

SI >1. In the group spiked with IL-2+IL-4 cytokines,

the statistical evaluation of the relation between SI

values and GVHD development revealed a non-significant

result (p=0.076) (Table 3).

mMLC test was performed by adding IL-2+IFN-

+TNF- in 51 of the patient-donor pairs. While 44

patients revealed SI values 1, 7 patients had SI values

>1. In 29 of those patients with SI 1, no posttransplant

GVHD occurred, while 15 patients experienced

GVHD. While it was not possible to determine

GVHD in 1 patient with SI >1, the disease had developed

in the remaining 6 of those 7 patients. There

was a statistically significant relation between SI values

and GVHD development (p=0.015) (Table 3).

Table 3. GVHD status and SI values calculated by MLC, MLC+IL-2, MLC+IL-2+ IL-4, and MLC+IL-2+IFN-+ TNF-

MLC MLC + IL-2 MLC + IL-2 + IL-4 MLC+IL-2+IFN-+

TNF-

SI 1 SI >1 Total SI 1 SI >1 Total SI 1 SI >1 Total SI 1 SI >1 Total

GVHD (-) n 32 4 36 30 2 32 17 0 17 29 1 30

% 64.0 44.4 (61.0) 66.7 25 (60.4) 77.3 0 (70.8) 65.9 14.3 (58.8)

GVHD (+) n 18 5 23 15 6 21 5 2 7 15 6 21

% 36.0 55.6 (39.0) 33.3 75 (39.6) 22.7 100 (29.2) 34.1 85.7 (41.2)

Total n 50 9 59 45 8 53 22 2 24 44 7 51

% 100 100 (100) 100 100 (100) 100 100 (100) 100 100 (100)

Fisher’s exact test p=0.229 p=0.035 p=0.076 p=0.015

en et al.

Turk J Hematol 2010; 27: 263-8 Importance of modified mixed lymphocyte culture test 267

When the mean SI values obtained on the MLC

and mMLC tests were evaluated with respect to

GVHD development, the mean SI value of the 36 of

the 59 patients undergoing MLC test who did not

develop GVHD was 0.63±0.14, while the mean SI

value in the 23 patients who developed GVHD was

0.71±0.10; the difference between the two groups

was not significant (p=0.956) (Table 4).

The mean SI value of the 32/53 patients undergoing

IL-2-spiked mMLC test who did not develop

GVHD was 0.52±0.07 while this value was 1.01±0.25

in the 21/53 patients who did develop GVHD; the

difference was statistically significant (p=0.008)

(Table 4).


IL-2+IL-4-spiked mMLC test who did not develop

GVHD was 0.59±0.05, while this value was

2.44±1.87 in the 7/24 patients who did develop

GVHD; the difference was statistically significant

(p=0.001) (Table 4).


IL-2 + IFN- + TNF--spiked mMLC test who did

not develop GVHD was 0.41±0.05, while this value

was 3.99±2.51 in the 21/51 patients who did develop

GVHD; the difference was statistically significant

(p=0.005) (Table 4).

Discussion

Despite the technological and scientific advances

in pre-transplant tissue typing tests and the fact

that HSCT is performed between HLA-identical

pairs, GVHD still represents the most significant

Table 4. The results for mean MLC, MLC+IL-2, MLC+IL-2+IL-4

and MLC+IL-2+IFN-+TNF- SI values and GVHD

GVHD (-) GVHD (+) p

MLC n 36 23

% 61 39 0.956

SI 0.63 ±0.14 0.71±0.10

MLC + IL-2 n 32 21

% 60.4 39.6 0.008

SI 0.52 ±0.07 1.01±0.25

MLC + IL-2 + IL-4 n 17 7

% 70.8 29.2 0.001

SI 0.59±0.05 2.44±1.87

MLC + IL-2+ n 30 21

IFN- + TNF- % 58.8 41.2 0.005

SI 0.41±0.05 3.99±2.51

complication occurring after transplantation [12].

The possible reasons for this are thought to be polymorphic

HLA determinants and/or minor histocompatibility

systems not yet detected [6,13].

In the present study, the results from the MLC

and cytokine-spiked mMLC tests were compared

with regards to GVHD development in HSCT recipients.

The association between GVHD development

and SI values, as 1 or >1, was investigated.

In mMLC, cell surface antigens were increased

by providing treatment of stimulator cells with cytokines

(IL-2, IL-4, TNF- , IFN-) that are known to

increase the expression of HLA and non-HLA antigens.

By adding exogenous cytokines to MLC cultures,

amplification of weak proliferative responses

was achieved. Using these amplifications, positive

MLC reactions were frequently achieved amongst

HLA-identical siblings [6,7,14].

In a study by Bishara et al. [9], pre-determination

of GVHD and tissue rejection was targeted by modifying

the one-way MLC test. Three separate modifications

were used in this study. In the first modification,

IL-1 , IL-2 and IL-4 were added separately and

in combination to the MLC test. Addition of IL-2 and

IL-4 increased the MLC response in all unpaired

controls and in certain HLA-identical pairs. The second

modification was made by pre-treatment of the

stimulator cells with IFN-, TNF- and IL-4. This

modification resulted in positive response in all the

cases. The third modification was performed by readdition

of cytokines to the MLC test where stimulator

cells were pre-treated with added cytokines. As

a result of this combined application, a high rate of

positivity was also detected among the HLAidentical

pairs. It was concluded that this result

could be used in determining undetectable minor

antigenic differences. The Bishara study reported

that the mMLC study could be beneficial in choosing

the most compatible donor in the presence of

multiple HLA-identical donors.

In the study by Visentainer et al. [15], exogenous

cytokines were added to the one-way MLC test. The

investigators, thus intending to increase the sensitivity

of the MLC test, stimulated the stimulator cells by

IL-4 or IFN- and added IL-2 or IL-4 to the responding

cells at the start of the culture. By addition of different

doses of cytokine in the autologous cultures and

compatible recipient-donor cultures, advantageous

results were obtained as compared to the MLC test.

Their study revealed that pre-treatment of stimulator

cells with IL-4 or IFN- did not increase allogeneic

response in the MLC test; however, IL-2 and IL-4

268

en et al.


addition at the start of the culture increased all the

responses including the autologous response.

Another trial by Visentainer et al. [15] investigated

the association between chronic GVHD occurring

after SCT and the one-way MLC test. They found in

that study that the MLC test was not adequate for predetermining

acute GVHD; however, it could be the

predictor of chronic GVHD when a result above

Relative Response Index (RRI)=4.5% was achieved.

As could be understood from the study conducted,

the objective of modification of the MLC test is to

predict post-transplant GVHD and tissue rejection. In

our study, in addition to the MLC test, three different

series were formed with addition of different cytokines.

In the first series, IL-2 was added separately to

the MLC test. In the second series, IL-2+IL-4 were

added. In the third series, IL-2+IFN-+TNF- were

added. In the mMLC tests, an increase was observed

in the SI values. GVHD developed in 39% of the

patients undergoing HSCT. GVHD was detected in

55.6% of the group with SI >1 in the MLC test, while

these figures were 75.6%, 100% and 85.7%, respectively,

in the series spiked with IL-2, IL-2+IL-4 and

IL-2+IFN-+TNF- [15].

When the mean SI values obtained on the MLC

and mMLC tests were evaluated with respect to

GVHD development, in the cytokine-spike groups,

the statistical evaluation of the relation between

mean SI values and GVHD development revealed

significant results.

In conclusion, the mMLC test sensitized by addition

of IL-2 and IL-2+IFN-+TNF- cytokines is

important in determining post-transplant GVHD. We

believe that studying the mMLC test could be beneficial

in choosing the most compatible donor

when multiple HLA-identical donors are present.






References

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2. Tiercy JM. Molecular basis of HLA polymorphism:

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6. Tanaka J, Imamura M, Kasai M, Kobayashi S, Hashino

S, Kobayashi H, Sakurada K, Miyazaki T. Cytokine gene

expression in the mixed lymphocyte culture in allogenic

bone marrow transplants as a predictive method

for transplantation-related complications. Br J Haematol

1994;87:415-8.

7. Danzer SG, aCampo C, Rink L. Interferon-gamma plays

a key role in the human mixed lymphocyte culture.

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8. Matthews SJ, Sullivan JS. A role for tumour necrosis

factor-alpha in the human mixed lymphocyte culture

reaction. Immunol Cell Biol 1992;70:107-10.

9. Bishara A, Brautbar C, Nagler A, Slavin S, Leshem B,

Cohen I, Kedar E. Prediction by a modified mixed leukocyte

reaction assay of graft-versus-host disease and

graft rejection after allogeneic bone marrow transplantation.

Transplantation 1994;57:1474-9.

10. Przepiorka D, Weisdorf D, Martin P, Klingemann HG,

Beatty P, Hows J, Thomas ED. Consensus conference

on acute GVHD grading. Bone Marrow Transplant

1995;15:825-8.

11. Rowlings PA, Przepiorka D, Klein JP, Gale RP, Passweg

JR, Henslee-Downey PJ, et al. IBMTR Severity Index for

grading acute GVHD: retrospective comparison with

Glucksberg grade. Br J Haematol 1997;97:855-64.

12. Storb R, Thomas ED. Allogeneic bone-marrow transplantation.

Immunol Rev 1983;71:77-102.

13. Pawelec G, Müller C, Ehninger G. Predictive strength of

mixed lymphocyte cultures for acute graft-versus-host

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14. Pawelec G, Baumgartner P, Rehbein A, Reusch U,

Schaudt K. Cytokine regulation of the balance between

alloindifferent and allospecific suppressor induction in

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1990;49:615-9.

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SC, Vigorito AC, Aranha FJ, Eid KA, Oliveira GB, Miranda

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Biol Res 2002;35:567-72.

Research Article

269

The changes in complete blood count in thyroid

cancer patients treated with radioactive iodine

ablation therapy

Radyoaktif iyot tedavisi alan tiroid kanserli hastalarda tam kan saym

deiiklikleri

Bircan Sönmez 1 , smail Doan 1 , Canan Yavruolu 1 , Gamze Can 2 , Mehmet Sönmez 3

1Department of Nuclear Medicine, Karadeniz Technical University School of Medicine, Trabzon, Turkey

2Department of Public Health, Karadeniz Technical University School of Medicine, Trabzon, Turkey

3Department of Hematology, Karadeniz Technical University School of Medicine, Trabzon, Turkey

Abstract

Objective: The aim of this study was to evaluate the effect of radioactive iodine (RAI) ablation therapy on

the complete blood count (CBC) in thyroid cancer patients.

Materials and Methods: One hundred sixty four patients undergoing RAI ablation therapy after total thyroidectomy

were included. CBC results were available from the patients’ medical records at the time of

ablation and at the 1 st , 6 th , and 12 th months after RAI therapy.

Results: Hemoglobin (Hb), white blood cell (WBC) and platelet (Plt) values were significantly lower than

baseline at 1 month after treatment (p<0.0001). Hb and WBC values were increased at the 6 th month and

at the 1 st year. Plt values increased at the 6 th month but had decreased again at the 1 st year. The values were

usually in normal ranges except in the patients with low pretreatment Hb and WBC values.

Conclusion: RAI ablation therapy in thyroid cancer patients is a safe treatment modality without any serious

or persistent hematological side effects. (Turk J Hematol 2010; 27: 269-74)

Key words: Radioactive iodine therapy, complete blood count, thyroid cancer

Received: June 1, 2010 Accepted: July 26, 2010

Özet

Amaç: Bu çalmann amac total tiroidektomi sonras artakalan tiroid dokusu için radyoaktif iyot (RAI)

tedavisi alan tiroid kanserli hastalarda tam kan saymndaki deiimleri deerlendimek.

Yöntem ve Gereçler: Total tiroidektomi sonras RAI tedavisi alm 164 hasta deerlendirildi. Hastalarn

tedaviden önce ve tedavi sonras 1. ay, 6. ay ve 12. ay hemoglobin (Hb), lökosit (WBC) ve trombosit (Plt)

düzeyleri deerlendirildi.

Bulgular: RAI tedavisinden 1 ay sonra Hb, WBC ve Plt deerlerinde anlaml bir azalma izlendi. Plt says

6. ayda düzelirken 1. ylda tekrar azald. Buna karlk Hb ve WBC deerlerinde ilk ayda gözlenen azalma

Address for Correspondence: M.D. Bircan Sönmez, Department of Nuclear Medicine, Karadeniz Technical University School of Medicine,

Trabzon, Turkey Phone: +90 462 377 57 42 E-mail: sonmezbircan@yahoo.com

doi:10.5152/tjh.2010.49

270

Sönmez et al.

Changes in CBC after radioactive iodine ablation therapy Turk J Hematol 2010; 27: 269-74

6. ay ve 1. ylda izlenmedi. Ancak izlenen bu deiiklikler tedavi öncesi deerleri düük hastalar hariç

normal snrlar içindeydi.

Sonuç: Tiroid kanserli hastalarda RAI tedavisi ciddi bir hematolojik yan etkiye yol açmakszn güvenilir bir

tedavi yöntemidir. (Turk J Hematol 2010; 27: 269-74)

Anahtar kelimeler: Radioaktif iyod tedavi, tam kan saym, tiroid kanseri

Geli tarihi: 1 Haziran 2010 Kabul tarihi: 26 Temmuz 2010

Introduction

Treatment of differentiated thyroid cancer (DTC)

with radioactive iodine (RAI) is a standard procedure

for the ablation of remnant thyroid tissue following

surgery and for the treatment of iodine-avid

metastases [1]. Usually, high doses of RAI (100-150

mCi) are used for total ablation of thyroid remnants.

RAI treatments may be repeated especially in metastatic

patients, and this delivers high cumulative

doses to non-thyroid organs. The side effects of RAI

treatment may occur in many areas and organ systems.

The most common side effect is a decreased

saliva production, but severe long-term side effects

are rare. Other organ-specific side effects are found

in the lacrimal glands, bone marrow, lungs, and

reproductive organs. Incidence of secondary malignancies

and leukemia might increase with higher

RAI doses [2,3].

Bone marrow suppression can arise after RAI

treatment [4]. There are many factors that affect the

frequency and severity of bone marrow suppression.

These include the prescribed and total cumulative

RAI doses, frequency of treatments and clearance

rate of RAI therapy, and additively, the patient’s

bone marrow reserve and degree of bone marrow

metastases [2]. Although bone marrow suppression

with repeated high-dose RAI treatments is reported

[5,6], changes in the peripheral complete blood

count (CBC) after an initial RAI ablation treatment is

not well defined. Therefore, we retrospectively

evaluated the hemoglobin (Hb), platelet (Plt) and

white blood cell (WBC) counts before ablation and

at the 1 st month, 6 th month and 1 st year following

initial RAI ablation treatment.


One hundred and sixty-four patients undergoing

RAI remnant ablation therapy after total thyroidectomy

were retrospectively evaluated. Gender, age

and demographic characteristics were recorded.

The patient’s CBC results were available in the

medical records at the time of ablation and at the 1 st

and 6 th months and 1 st year after RAI therapy.

Patients taking any medications known to affect the

CBC; having any hematological conditions; and/or

receiving a second dose of RAI during the first-year

follow-up or external beam radiotherapy or chemotherapy

before RAI therapy or within 1 year after

ablation were excluded from the study. None of the

patients had bone marrow metastases, but one

patient had pulmonary metastases and seven

patients had lymph node metastases. Levothyroxine

(L-T4) and triiodothyronine (L-T3) treatments were

discontinued after 4-6 weeks and 2 weeks, respectively,

and a low-iodine diet for 2 weeks was recommended

before RAI treatment. None of the patients

was prepared for RAI ablation with recombinant

human thyroid stimulating hormone (rhTSH). After

total thyroidectomy, RAI remnant ablation treatment

with standard-high RAI doses [3.7-7.4 GBq (100-200

mCi)] was performed in 6-8 weeks. RAI therapy

was administered as follows: 122 patients (74.4%),

37 patients (22.6%), and 5 patients (3%) received

100 mCi, 125-150 mCi, and 175-200 mCi, respectively.

L-T4 treatment was restarted after 48 hours.

Diagnostic imaging with low-dose RAI (2-5 mCi)

was performed within 6-12 months. However, no

second RAI treatment or radiotherapy was applied

during the one-year follow-up. All blood tests were

analyzed with Full Automated CBC analyzer

(Beckman Coulter LH-750). Hb reference values

were 13-17 g/dl and 11.5-16 g/dl for males and

females, respectively. WBC and Plt count reference

values were 4.8-10.8 x10 3 /UL and 130-400 x10 3 /UL,

respectively. Written informed consent was obtained

from the patients.

Statistical Analysis

Data are presented as the mean±standard deviation

(SD); medians with ranges are given when

Sönmez et al.

Turk J Hematol 2010; 27: 269-74 Changes in CBC after radioactive iodine ablation therapy 271

appropriate. Continuous variables were compared

using repeated measures variance analysis for changing

over time. Bonferroni test was used for post-hoc

analysis. CBC values between RAI groups were compared

by Student’s t test. p<0.05 was considered

statistically significant. All statistical analyses were

performed using SPSS version 13.01 for Windows.

Results

The mean age of patients (24 males, 68 females)

was 47±13 years (range: 22-81 years). The baseline

characteristics of the patients are presented in Table

1. Median TSH level was 82 mU/L (range: 32-100

mU/L) because of cessation of L-T4 or L-T3 treatments.

Hb (Males/Females), WBC, and Plt values

were significantly lower than baseline at 1 month

after treatment (p<0.0001). Hb and WBC values

had increased at the 6 th month and 1 st year. The

decreases in the Hb levels were not different

between the male and female patients. Similarly, it

was observed that neutrophil and lymphocyte

counts decreased equally. Plt values increased at

the 6 th month, but decreased again at the 1 st year

(Table 2, Figure 1a, b). There was no requirement of

blood transfusion or febrile neutropenia occurrence

in any of the patients. Although all hematological

parameters decreased at the 1 st month and for Plt

also at the 1 st year, the values were usually in normal

ranges except for those patients with low pretreatment

Hb and WBC values. Before treatment,

Hb values were low in 21 patients (12.8%, 8 males,

13 females) while WBC values were low in 7

patients (4.3%); no patients had iron, vitamin B12 or

folate deficiency. The change in Hb and WBC values

is probably related to the disease status or the

medications used before (Table 3). No significant

difference in the decreasing rate was found between

patients with low pretreatment and normal pretreatment

Hb and WBC counts. Similarly, there was

no significant difference when comparing patients

receiving different doses of RAI therapy. No complications

were observed.

Discussion

Bone marrow suppression is a serious and, if not

diagnosed, potentially life- threatening complica-

Table 1. Baseline characteristics of patients

Characteristics

Age 47±13

Histology

PTC 152 (92.7%)

FTC 11 (6.7%)

HCC 1 (0.6%)

TNM Stage

I 130 (79.3%)

II 20 (12.2%)

III 13 (7.9%)

IV 1 (0.6%)

Administered Activity (MBq/mCi)

Mean±SD 4144±851 (112.74±23)

Median 3700 (100)

Range 3700-7400 (100-200)

Post-Therapy Scan

Thyroid remnant 156 (95.1%)

Lymph node metastases 7 (4.3%)

Pulmonary metastases 1 (0.6%)

Bone metastases 0 (0%)

PTC: Papillary thyroid cancer; FTC: Follicular thyroid carcinoma; HCC: Thyroid carcinoma,

Hurthle cell; TNM: TNM staging system (tumor, node, metastasis)

Table 2. CBC changes with RAI therapy

Pre-treatment 1 st Month 6 th Month 1 st Year

Hb (g/dl)/M

Mean±SD 13.7±1.9 a,b 13.3±1.8 c,d 13.5±1.9 13.8±1.9

Median 14 13.6 13.8 14

Range 9.1-16.8 8.8-15.4 7.9-16 9.1-16.2

Hb (g/dl)/F

Mean±SD 13.4±1.2 a,b 6.7±1.9 a 12.6 ±1.1 c,d 6.0±1.8 e

Median 13.2 6.4 12.8 5.8

Range 9.4-16.4 2.5-16.7 8.6-15.2 1.7-12.9

WBC (10 3 /L)

Mean±SD 12.9±1.1 6.5±2.6 13.2±1 6.5±1.7

Median 13 6.1 13 6.3

Range 9.6-15.4 3.0-28.6 10.5-15.9 3.3-15.7

N/L 3.9±1.5/2.1±0.6 3.7±1.5/1.6±0.5 4±2.4/1.8±0.5 4±1.5/1.9±0.5

Plt (10 3 /L)

Mean±SD 289±71 a,f,g 259±57 265±62 h 258±65

Median 285 258 265 255

Range 152-538 142-423 134-422 135-498

a: pre-treatment-1st month p<0.0001; b: pre-treatment-6th month p<0.0001;

c: 1 st month-6 th month p<0.0001; d: 1st month-1st year p<0.0001; e: 1st month-

1st year p=0.001; f: pre-treatment-6 th month p<0.0001; g: pre-treatment-1 st year

p<0.0001; h: 6 th month-1 st year p=0.009.

Hb/M: Hemoglobin/males; Hb/F: Hemoglobin/females; WBC: White blood cell;

Plt: Platelets; N/L: Neutrophils/lymphocytes

272

Sönmez et al.


16

14

12

10

8

6

4

2

0

pre 1 st 6 th 12 th

Month

Hb (g/dl)/M

Hb (g/dl)/F

WBC (x1000/ L)

Plt(x10 3 / L)

290

280

270

260

250

240

pre 1 st 6 th 12 th

Month

Figure 1a. Hb and WBC changes with RAI therapy

Table 3. CBC changes in the patients with low Hb and WBC values

Pre-treatment 1 st Month 6 th Month 1 st Year

Hb (g/dl)/M

Mean±SD 11.5± 1.3 11.3±1.4 11.3±1.9 12.1±1.3

Median 11.7 11.3 11.4 12.2

Range 9.1-12.9 8.8-13 7.9-14.1 10.4-14.1

Hb (g/dl)/F

Mean±SD 10.7±0.7 10.6±1.1 11.2±1.1 11.8±1

Median 11 10.9 11.1 11.8

Range 9.4-11.5 8.6-11.9 9.6-13.4 10.5-14.4

WBC(10 3 /L)

Mean±SD 3.4±0.5 3.6±0.9 4.4±0.5 4.2±0.7

Median 3.4 3.9 4.4 4.2

Range 2.5-4 1.7-4.4 3.5-5.1 3.3-5.3

Hb/M: Hemoglobin/males; Hb/F: Hemoglobin/females; WBC: White blood cell

tion of RAI treatment. Transient leukopenia, anemia

and thrombocytopenia may be observed after RAI

administration, but severe cytopenia is usually seen

with high doses of RAI (>600 mCi) [7]. The World

Health Organization classification is used for bone

marrow suppression after RAI treatment. While

mild and reversible blood count alternations are

observed in grade I-II, persistent severe cytopenia

and aplasia or acute myeloid leukemia are detected

in grade III and grade IV, respectively. Although RAI

therapy induces chromosome damage in the lymphocytes,

the effect of RAI depends on lymphocyte

phenotype and RAI activity. Natural killer cells are

most sensitive, followed by B lymphocytes and

T-helper lymphocytes. However, these do not result

clinically in an immunosuppression [8,9]. Severe

and permanent bone marrow suppression was

reported by Benua et al. [10] in 8 of 59 patients

treated with RAI. In the dosimetric evaluations, six of

these eight patients received in excess of 3Gy (300

Figure 1b. Plt changes with RAI therapy

rads) to the blood. When using 2Gy (200 rads) to the

blood as the upper limit, Von Nostrand et al. [11]

found that mild transient decreases in blood cell

counts were seen in 90% of the patients. We usually

applied 100 mCi RAI and the dose usually did not

exceed 2Gy.

Hypothyroid patients have an increased serum

creatine and decreased glomerular filtration rate.

Decreased renal clearance results in increased RAI

retention. Therefore, the bone marrow-absorbed

dose after treatment with RAI would be expected

to be lower for patients given rhTSH, which is protective

for hypothyroidism and additionally may

reduce the half-life of RAI, than for patients subjected

to L-T4 withdrawal [12-14]. Molinaro et al.

[15] reported recently that RAI ablation treatment is

associated with a decline in WBC and Plt that persists

for at least one year after ablation without differences

between the rhTSH and the L-T4 withdrawal

groups. On the other hand, Rosario et al.

[16] demonstrated that the decrease in WBC and

platelets in the first three months was significantly

lower in the rhTSH group than in the L-T4 withdrawal

group. This suggests that the transient

effects on the bone marrow may be more of a

dose-related phenomenon, while the late persistent

effects are more influenced by individual susceptibility.

In this study, we documented that Hb,

WBC, and Plt counts in the first month were

decreased, but did not persist for a long time. The

patients with low pre-treatment Hb and WBC values

were not more susceptible to the suppressive

effect of the treatment than the patients having normal

values. Although statistically significant, the

decreases in Hb, WBC and Plt were small and without

evidence of clinical importance. Interestingly,

Sönmez et al.

Turk J Hematol 2010; 27: 269-74 Changes in CBC after radioactive iodine ablation therapy 273

Plt counts were slightly decreased at the 1 st year,

similar to the observation of Molinaro et al. [15],

but different from their report, we think that the

decreased Plt count was probably associated with

the low-dose RAI (2-5 mCi) applied for diagnostic

imaging at 6-12 months. Although CBC values were

normal after RAI treatment, the statistical results

showed that RAI doses were important for bone

marrow suppression. Therefore, physicians should

be careful regarding additive or overdoses.

Sublethal radiation doses damage bone marrow

cells and may lead to leukemia. Acute and chronic

myeloid leukemia were reported with RAI treatment,

especially in those with bone metastasis.

Incidence of leukemia was increased in the patients

who received more than 800 mCi, were >45 years

and were treated within short intervals. Only very

rarely is acute leukemia found in patients receiving

a small RAI dose of <300 mCi [17-19]. Similarly, we

did not observe the development of an acute or

chronic myeloid leukemia due to RAI treatment

within one year. However, the long-term outcome

of RAI therapy may be different from these results.

In conclusion, we suggest that RAI therapy can

be associated with slight and reversible changes in

Hb, WBC and Plt counts; however, it is a safe treatment

modality for ablation without any serious or

persistent hematological side effects.






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Research Article

275

Are university students a favorable target group

for blood donation campaigns?

Üniversite örencileri kan ba kampanyalar için uygun bir

hedef kitle midir?

Bülent Eser 1 , Fatih Kurnaz 1 , Leylagül Kaynar 1 , Mehmet Yay 2 , Serdar vgn 1 , Ali Ünal 1 ,

Mustafa Çetin 1

1Department of Hematology, Erciyes University Medical School, Internal Medicine, Kayseri, Turkey

2Blood Bank, Erciyes University Medical School, Kayseri, Turkey

Abstract

Objective: The aim of this study was to investigate the willingness of university students regarding

blood donation and to compare results among residents living in the Kayseri city center.

Materials and Methods: Admission for blood donation after donor acquisition campaigns and the rates

of repeated donation over a one-year period were compared between the two groups.

Results: Between November 2006 and August 2008, a total of 29614 people were included in the

study. After educational campaigns, the rate of admission for blood donation was 66% among university

students, while it was only 29% among the city residents. Although the deferral rate and adverse

events during donation were found to be higher in the student group, they had a higher repeated donation

rate and higher return rate after a short message system.

Conclusion: University students appear to be good candidates for long-term regular blood donation.

Use of a short message system to issue reminders about blood donation may be a reasonable method

to replenish the blood supply. (Turk J Hematol 2010; 27: 275-81)

Key words: Blood donation, university, student

Received: June 24, 2010 Accepted: August 10, 2010

Özet

Amaç: Bu çalmann amac üniversite örencilerinin kan ba konusundaki istekliliklerini aratrmak

ve bu grubu Kayseri ehir merkezinde yaayan insanlarla karlatrmakt.

Yöntem ve Gereçler: ki grup donor kazanm kampanyalar sonras kan ba için bavurma ve bir yl

içinde tekrar kan ba yapma oranlar yönünden karlatrld.

Bulgular: Kasm 2006 ile Austos 2008 arasnda toplam 29614 kii çalmaya dahil edildi. Eitimsel

kampanyalar sonrasnda kan ba için bavurma oran üniversite örencilerinde %66 iken bu oran

üniversite d grupta sadece %29 idi. Reddedilme ve kan verme ilemi srasnda istenmeyen etki

Address for Correspondence: M.D. Bülent Eser, Department of Hematology, Erciyes University Medical School, Internal Medicine, 38280, Kayseri, Turkey

Phone: +90 535 202 99 74 E-mail: beser@erciyes.edu.tr

doi:10.5152/tjh.2010.42

276

Eser et al.

Blood donation characteristics among university students Turk J Hematol 2010; 27: 275-81

gözlenme oran örenci grubunda daha fazla gözlenmesine ramen bu grupta toplam tekrar donor

olma ve ksa mesaj sistemiyle hatrlatma sonras yeniden donor olma oran daha yüksekti.

Sonuç: Üniversite örencileri uzun sureli düzenli kan baçs olmak için iyi bir aday grubudur. Kan

bann hatrlatlmas için ksa mesaj sisteminin kullanlmas kan stoklarnn yerine konmas için

aklc bir yöntem olabilir. (Turk J Hematol 2010; 27: 275-81)

Anahtar kelimeler: Kan ba, üniversite, örenci

Geli tarihi: 24 Haziran 2010 Kabul tarihi: 10 Austos 2010

Introduction

The recruitment and retention of sufficient numbers

of regular, volunteer blood donors are important

issues for maintaining an adequate and safe

blood supply. The World Health Organization (WHO)

and the Council of Europe recommend that blood

and blood components should be collected only

from voluntary donors in order to ensure the safety

of blood products [1,2]. According to this recommendation,

a person donates of his/her own free

will and receives no payment; thus, the donation

should be voluntary and non-remunerated. It is of

the utmost importance to prevent the transfusiontransmissible

infectious diseases. Paid donations

are prohibited by law in Turkey. Blood product

requirements are generally provided from voluntary

blood donors and partially from patients’ relatives in

the country. Acquisition of regular volunteer blood

donors remains an important issue for maintaining

the blood supply.

Volunteer blood donation recruitment and retaining

strategies include all the activities that increase

the number of volunteer donors. Television, posters,

bulletins, newspapers, and the internet are some of

the methods that promote blood donation and

acquaint individuals with the process [3]. It seems

the best means of providing face to face information

to blood donor candidates about blood donation

and to inform them regarding the importance

of the safety of blood and blood products for

patients in order to increase donor retention [4]. To

remind regular donors and request their donations

are also important steps toward replacing depleted

blood supplies. There are some methods that have

been used for this purpose; however, the short message

system (SMS) request via mobile phones has

not been widely used in our country.

It is not enough to just inform candidates about

the benefits and necessity of blood donation; they

must also be convinced to put aside any misconceptions

they may have on the subject [5,6].

Educational and social status and prior misconceptions

are important factors in blood donation. Young

people may be good candidates for becoming regular

blood donors. Furthermore, red blood cells

obtained from those younger in age have a longer

survival potential than the cells obtained from older

individuals because of less deformability of the red

blood cells [7]. As university students are well educated

and young, we investigated their willingness

regarding blood donation and compared results

with the willingness among other residents in the

city center.


Approximately 25,000 units of whole blood are

collected annually in Erciyes University Blood Bank.

The data between November 2006 and August 2008

were evaluated. Up to November 2006, it was not a

routine practice for regular volunteer donors to

donate blood because limited regular blood donors

were available in the city. In general, blood products

were obtained from occasional replacement donors

(from the close friends and family of the patients).

In order to increase the number of voluntary blood

donations, an acquisition program was started in

November 2006. Two experienced blood bank

employees were trained for two weeks concerning

donor motivation. Then, they were charged with

donor motivation members to reach the volunteers

effectively and to create a high awareness about the

importance of blood donation. Six persons, including

three nurses and one doctor, were also assigned

as a mobile blood collection team. People were

informed with announcements and publications via

television, the internet, brochures, and posters. The

information contained a brief education about the

necessity of blood donation for the patients and the

Eser et al.

Turk J Hematol 2010; 27: 275-81 Blood donation characteristics among university students 277

safety of the blood donation process. Each group

consisted of 40-50 persons and information was

given in approximately 20 minutes. Any suspicions

or questions from the audience were addressed

and a face to face interview was conducted if

required. After being given information about blood

donation, candidates were kindly asked whether or

not they wanted to be a blood donor. A questionnaire

was then distributed for all candidates older

than 18 years who were admitted for donation. All

candidates underwent a medical examination and

laboratory tests including pulse rate, arterial blood

pressure, fever and hemoglobin (Hb) level. If the

physical examination results and Hb level were

within normal ranges, a physician interviewed the

candidates to investigate the risk of syphilis and

blood transmissible viral infections (hepatitis B, C

and human immunodeficiency virus [HIV]). The

deferral criteria of donor candidates were evaluated

according to the directives of Turkey’s Ministry of

Health. Candidates were compared regarding reasons

for deferral after pre-donation screening interviews.

The appropriate candidates were asked to

donate blood.

Adverse events during blood donation were also

recorded. Samples for syphilis and viral markers

(HBsAg, Anti-HCV, and Anti-HIV) were obtained

from the blood bags. The syphilis test was conducted

with VDRL and others with the ELISA method.

Tests were studied with the micro ELISA method in

Etimax 3000 device; a second generation bio-assay

(Diasorin) was used for HBsAg, a third generation

bio-assay (Diasorin) for anti-HCV, and a fourth generation

bio-assay (Diasorin) for HIV. Viral parameters

(HBsAg, anti-HCV, anti-HIV), reasons for donor

deferral and adverse events were compared

between the two groups. The donors’ personal data

were recorded on the computer, and they were followed

up for further donations. Mobile phone numbers

were also requested in order to recall them via

the SMS. They were invited for blood donation by

SMS when stores of rare blood groups were depleted

or in the case of emergent blood product

requirements. In the content of the SMS, the candidate

was asked to donate for emergency patients by

admitting to our blood bank or to call us regarding a

donation. Donations could be taken in the donor’s

own locale, if they so desired. SMS requests were

sent only once for each donor, and all volunteer

donors were thus asked once a year for blood donation

sequentially. At the time of donation, the candidates

were queried regarding whether the admission

was voluntary or in reply to a SMS request.

The data were evaluated and analyzed by

Pearson’s chi-square statistical method. A p value

less than 0.005 was considered statistically significant.

Analyses were performed with SPSS, release

16.0 (SPSS, Inc., Chicago, IL).

Standard written informed content forms were

obtained from all donors for blood donation, according

to the directives of Turkish Ministry of Health.

Results

From November 2006 to August 2008, 29614

people were informed about the safety of the donation

procedure and the importance of voluntary

contribution of blood products for patients. A total

of 8730 students were registered on the university

campus, and 5832 (66%) of them applied for blood

donation. Blood was drawn from 4424 (75%) of

those who were eligible for donation. Median age of

the students was 20 years (range: 18-22 years). Of

the donors, 1198 (27%) were female and 3226 (73%)

were male. Outside the university, 20884 people

residing in the city center were informed, 6111

(29%) of those applied as volunteer donor, and 5341

(87%) of them were eligible for blood donation. The

group consisted of 267 (5%) females and 5074

(95%) males, and the median age of the group was

31 years (range: 18-60 years). Most of the donors

(19840 of 20884) from outside the university had

high school or lower educational levels (95%). The

rate of application for donation after the brief education

was significantly higher among the student

group than the other donors (p<0.001).

A total of 2178 persons were deferred; from the

whole group, the top deferral reason was low Hb

level in 892 (40%). Types of deferrals and their distribution

are outlined in Table 1. There were significant

differences between the two groups with

respect to the deferral reasons of low body mass

index (BMI), fear and age (p<0.001).

Infectious screening test results were as follows:

HBsAg was detected in 46 of the students (1.03%)

while anti-HCV was detected in only 1 (0.02%).

278

Eser et al.


Table 1. Causes of donor deferral

Causes of donor deferral U group OU group Statistics

n (%) n (%)

1 Hb levels outside normal limits 654 (15.8) 238 (4.5) p<0.001

2 Hypertension or hypotension 33 (0.8) 48 (0.9) p>0.05

3 Outside age limits (>65, <18 years old) 119 (2.7) 48 (0.9) p<0.001

4 BMI <18 kg/m 2 208 (4.7) 72 (1.3) p<0.001

5 High risk for hepatitis or HIV infection 35 (0.8) 59 (1.1) p>0.05

6 Fear (hospital, nurse, needle, hematophobia) 88 (2.0) 40 (0.7) p<0.001

7 Acute infections 28 (0.6) 31 (0.6) p>0.05

8 Chronic diseases or drug use 170 (3.8) 172 (3.2) p>0.05

9 Blood donation within last 2 months 26 (0.6) 35 (0.6) p>0.05

10 Other 47 (1.1) 27 (0.5) p<0.05

Total 1408 (24.1) 770 (12.6) p<0.001

U: University; OU: Outside university; BMI: Body mass index

HBsAg was detected in 92 (1.7%) of the donors from

outside the university and anti-HCV was detected in

1 (0.018%). The rate of HBsAg positivity was significantly

higher (p=0.005) in the group from outside

the university. There was no statistically significant

difference in the rate of anti-HCV positivity between

the university group and those from outside the university.

HIV and syphilis test results were negative in

both groups.

During the study period, volunteers were followed

concerning repeated donation for a oneyear

after their donation. A total of 1403 volunteer

donors (32%) from the university group applied

more than once for blood donation (1088 once

more, 315 more than twice). In this group of volunteers,

533 were responders to SMS and 870 applied

of their own accord (without any call or SMS). On

the other hand, 582 donors (11%) in the group outside

the university applied more than once (419

once more, 163 more than twice). While 434 of

them applied to the blood bank after SMS messages,

148 applied of their own accord. The rate of

candidates who donated blood more than once

was significantly higher in the university donor

population when compared with the other group

(p<0.001). The donation rate after SMS requests

was also significantly higher in the university group

(12% vs. 8%; p<0.001).

There were 106 recorded adverse events. The

most commonly observed adverse reaction related

to the donation procedure was vasovagal symptoms

(n=97), including sweating, pallor, nausea

and dizziness. Others were hematomas near the

venipuncture site (n=5) and signs of hypocalcemia

(n=4). There was no serious adverse reaction.

Adverse events were significantly more frequently

observed (p<0.001) in the university group (n=77)

than in the group from outside the university (n=29)

(1.7% and 0.5%, respectively) (Figure 1a, 1b).

Discussion

In recent years, there has been an increase in

blood consumption. Improvements in the areas of

surgery, stem cell transplantation and cancer chemotherapy

are some of the factors responsible for

this increment in blood demand [8-10]. Stringent

eligibility criteria for donors increase safety standards

in blood transfusion, but this approach may

decrease the number of voluntary donors due to

donor deferrals [8,9,11]. Young adults, in general,

have good health and may have a long donor

career. An increase in the number of younger

blood donors gives us an opportunity to improve

donor recruitment and its maintenance [4,12]. In

Turkey, the number of young people under 28.5

years represents approximately 50% of the population,

so it is very important to make efforts in

recruiting and retaining this source of young donors

in the country [13].

Lack of request was the most frequently reported

reason for not donating blood among young

donors [12,14]. Effective communication is one way

of raising awareness among eligible donors while

Eser et al.


adverse events (university students)

adverse events (city residents)

vasovagal symptoms: 71

hematomas: 3

hypocalcebia: 3

vasovagal symptoms: 26

hematomas: 2

hypocalcebia: 1

Figure 1a. Distribution of adverse events during donation in university

students

Figure 1b. Distribution of adverse events during donation in city

residents

also increasing the frequency of donations. Personal

communication, advertisement using classical

communication instruments (i.e. newspaper, TV),

the internet, brochures, and posters have all been

used in the recruitment of new donors. In the present

study, it was observed that after receiving brief

information about the importance of volunteer

blood donation, willingness rates among individuals

were significantly higher in the university population

than among those from outside the university

(66% and 29%, respectively). Some studies have

suggested that a higher educational level is associated

with a higher return rate [5], and our study

supports these findings.

Phone calls, SMS, letters, and e-mails can be

used both to remind donors to give blood and to

retain regular donors [4,14]. In one study, a survey

of 3,167 blood donors revealed that only 15.7% of

those who received automated telephone recalls

returned for blood donation, whereas 35% of those

who received a telephone call from a donor recruiter

returned to donate blood [2007, unpublished].

This shows that direct communication is a more

effective way to retain donors [4]. In the present

study, volunteer donors were reminded by SMS to

replenish the increased blood need, especially

when stores of rarely found blood groups were

depleted. In this way, a total of 9625 volunteer

donors were requested and 967 (10%) of them

returned for blood donation. The return rate was

higher in the university student population. The university

students might be more sensitive about the

importance of blood donation. Difficulty in getting

permission from employers, transportation difficulties,

and physical and economic loss may be some

of the reasons for low blood donation, particularly

in developing countries. To establish a wider donation

web, the formation of a donor access team to

take blood from donors in their own area should

lead to an increase in donation rates.

There are some eligibility criteria in order to

maintain blood safety and to protect blood donors

and recipients. In the present study, 24.1% of the

student donors and 12.6% of the other donors were

deferred. The most common deferral reason was a

low Hb level, and the others were chronic diseases

or drug use, a low BMI, being outside the age limits,

and fear (e.g. of hospitals, nurses, needles, and

hematophobia), respectively. Low Hb levels and a

low BMI were significantly more frequent among

donors from the university population than from

outside the university. These results may be due to

the higher rate of female donors in the university

population (27%) than in the population from outside

the university (5%). In the literature, it was

shown that approximately 10-15% of potential

donors were deferred [15-17]. Our relatively higher

deferral rate may be explained by our strict deferral

criteria in order to ensure safe donation and to provide

safer blood products.

Safe blood donors are the cornerstone of a safe

and adequate supply of blood and blood products.

There was no positive result for HIV or syphilis in

our volunteer donor group. HBsAg was detected in

46 of the students (1.03%) and anti-HCV was detected

in only 1 (0.02%), while the rates were 1.7% and

0.018%, respectively, for HBsAg and anti-HCV in the

volunteer blood donor population outside the uni-

280

Eser et al.


versity. HBsAg incidence was significantly lower in

the university population. Experiences show that

the safest blood donors are voluntary, non-remunerated

blood donors. Paid donors are statistically

more likely to carry some infection. Their blood is

more likely to be of a lower standard, as they tend

to donate more frequently. Fortunately, paid donations

have been prohibited by law in Turkey.

Voluntary blood donation from a low-risk population

requires identifying such a population and

motivating them to donate blood regularly. A younger

population is considered more impressionable

and has low risks [18]. This may be due to low

exposure risk to blood transmissible infections

because of youth. In the present study, university

student donors were found to be safer than other

donors in terms of HBsAg positivity. Donor programs

and researches should be focused primarily on

retaining regular blood donors since they have a

lower incidence of transfusion-transmissible infectious

diseases [4,19].

Although complications and adverse reactions

during the blood donation process are rare, it is

thought that they play a role in subsequent willingness

to donate blood [20,21]. In the present study,

the most common adverse events were fatigue and

vasovagal symptoms. Total adverse events were

significantly higher in the university population than

in the population outside the university, and this

may have been due to the higher number of female

donors and the young age. It was shown previously

that adverse events were seen frequently in the

young donor population [22]. In one study, it was

detected that the most common systemic adverse

events were fatigue (7.8%), vasovagal symptoms

(5.3%), nausea and vomiting (1.1%), and those

adverse events were frequent in donors younger

than 30 years old [23].

In conclusion, efforts to increase the number of

volunteer donors to ensure an adequate and safe

blood supply are of great importance. Since it is

easy to convince university students and they potentially

have a long donor career, they are good candidates

for becoming regular volunteer blood donors.

A continuous educational program about blood

donation and the correction of misconceptions

about blood donation will increase donation rates.

All technological utilities should be used effectively

to reach more donors and to increase the success

of the donor acquisition programs. Well-documented

records facilitate easy access to donors in case of

increased blood demand. Finally, easy access to

donation centers will motivate donors. Mobile blood

donation teams for easier access to donors and to

facilitate their making blood donations in their own

locale may increase donation rates.


The authors declare that no conflicting or competing

interests of any nature exist between the

authors of this work and their academic activity.

References

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quality assurance of blood components:

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2003.

2. Dhingra N. Blood safety in developing world and WHO

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3. Moog R, Fourne K. Recruitment of prospective donors:

what do they expect from a homepage of a blood

transfusion service? Transfus Med 2007;17:279-84.

4. Maghsudlu M, Nasizadeh S, Abolghasemi H, Ahmadyar

S. Blood donation and donor recruitment in Iran from

1998 through 2007: ten years’ experience. Transfusion

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5. Ownby HE, Kong F, Watanabe K, Tu Y, Nass CC.

Analysis of donor return behavior. Transfusion

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Papaioannou D. Blood donor behaviour in Greece: implications

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McGurk S. Collection and transfusion of blood and

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and transfusion in the United States in 2001.


10. Lemmens KP, Abraham C, Hoekstra T, Ruiter RA, De

Kort WL, Brug J, Schaalma HP. Why don’t young people

volunteer to give blood? An investigation of the

correlates of donation intentions among young nondonors.


11. Council of Europe: Guide to the preparation, use and

quality assurance of blood components. 13th ed.

Strassbourg: Council of Europe Publishing, 2007.

12. Misje AH, Bosnes V, Heier HE. Recruiting and retaining

young people as voluntary blood donors. Vox Sanguinis

2008;94:119-24.

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13. < www.tuik.gov.tr >; 2008.

14. Moog R. Retention of prospective donors: a survey

about services at a blood donation centre. Transfus

Apher Sci 2009;40:149-52.

15. Custer B, Johnson ES, Sullivan SD, Hazlet TK, Ramsey

SD, Hirschler NV, Murphy EL, Busch MP. Quantifying

losses to the donated blood supply due to donor deferral

and miscollection. Transfusion 2004;44:1417-26.

16. Newman B. Blood donor suitability and allogeneic whole

blood donation. Transfus Med Rev 2001;15:234-44.

17. Arslan O. Whole blood donor deferral rate and characteristics

of the Turkish population. Transfus Med

2007;17:379-83.

18. Mansour AH. Blood donor recruitment. ISBT Science

Series 2009;4:11-3.

19. Schreiber GB, Sanchez AM, Glynn SA, Wright DJ;

Retrovirus Epidemiology Donor Study. Increasing blood

availability by changing donation patterns. Transfusion

2003;43:591-7.

20. Newman BH, Newman DT, Ahmad R, Roth AJ. The

effect of whole-blood donor adverse events on blood

donor return rates. Transfusion 2006;46:1374-9.

21. Stewart KR, France CR, Rader AW, Stewart JC.

Phlebotomist interpersonal skill predicts a reduction in

reactions among volunteer blood donors. Transfusion.

2006;46:1394-401.

22. Zervou EK, Ziciadis K, Karabini F, Xanthi E,

Chrisostomou E, Tzolou A. Vasovagal reactions in

blood donors during or immediately after blood donation

Transfus Med 2005;15:389-94.

23. Newman BH, Pichette S, Pichette D, Dzaka E. Adverse

effects in blood donors after whole-blood donation: a

study of 1000 blood donors interviewed 3 weeks after

whole-blood donation. Transfusion 2003;43:598-603.


Prognostic value of soluble angiopoietin-2 and

soluble Tie-2 in Egyptian patients with acute

myeloid leukemia

Akut miyeloid lösemili Msr’l hastalarda çözünür anjiyopoitein -2 ve

çözünür Tie-2’nin prognostik deeri

Mohamed A. Attia 1 , Sahar M. Hazzaa 1 , Salwa A. Essa 1 , Mahmoud F. Seleim 2

1Department of Clinical Pathology, Tanta University Faculty of Medicine, Tanta, Egypt

2Department of Internal Medicine, Tanta University Faculty of Medicine, Tanta, Egypt

Abstract

Objective: Angiogenesis plays a critical role in the development and growth of solid tumors and hematologic

malignancies. The system involving angiopoietin-2 [Ang-2] and its receptor Tie-2 appears to play an

important role in tumor angiogenesis and in the biology of hematological and non-hematological malignancies.

We evaluated the levels of soluble (s)Ang-2 and sTie-2 in acute myeloid leukemia (AML) patients and

investigated the impact of their circulating levels on the overall survival in those patients.

Materials and Methods: Ang-2 and Tie-2 were measured in plasma samples from AML patients and controls

using enzyme-linked immunosorbent assay (ELISA).

Results: The levels of sAng-2 and sTie-2 were significantly higher in AML patients (2382.1±1586.1 pg/ml

and 6.74±3.47 ng/ml, respectively) than in controls (649.5±402.6 pg/ml and 2.63±0.57 ng/ml, respectively;

p<0.01). AML patients with high levels of sAng-2 and sTie-2 (2500 pg/ml and 8 ng/ml, respectively)

had significantly shorter overall survival than those patients with low levels (<2500 pg/ml and <8

ng/ml, respectively).

Conclusion: The results of our study demonstrated the prognostic significance of circulating sAng-2 and

sTie-2 in AML patients. Modulation of the angiopoietin / Tie-2 axis may be a promising approach to

improve the outcome in those patients. (Turk J Hematol 2010; 27: 282-8)

Key words: Ang-2, Tie-2, AML, ELISA

Received: May 16, 2009 Accepted: February 22, 2010

Özet

Amaç: Anjiyojenez, kat tümörler ve hematolojik hastalklarn geliimi ve büyümesinde kritik bir rol

oynamaktadr. Anjiyopoietin-2 [Ang-2] ve onun reseptörü Tie-2’yi kapsayan sistemin, tümör anjiyojenezinde

ve hematolojik ve hematoloji d hastalklarn biyolojisinde önemli bir rol oynad görülmektedir.

AML hastalarnda çözünebilen sAng-2 ve sTie-2 düzeyleri deerlendirilmi olup, bu hastalarda dolamdaki

düzeylerinin toplam sakalm üzerindeki etkileri aratrlmtr.

Address for Correspondence: Asst. Prof. Mohamed A. Attia, Clinical Pathology Department, Faculty of Medicine, Tanta University, 31511

Tanta, Egypt Phone: 0020403348954 E-mail: attia17@yahoo.co.uk

doi:10.5152/tjh.2010.50

Attia et al.

Turk J Hematol 2010; 27: 282-8 Angiopoietin-2 and soluble Tie-2 in AML 283

Yöntem ve Gereçler: AMLli hastalar ve kontrollerden alnan plazma örneklerinde, Enzim bal immünassay

(ELISA) ile Ang-2 ve Tie -2 ölçülmütür.

Bulgular: AML hastalarnda, sAng-2 ve sTie-2 deerleri [srasyla 2382.1±1586.1 pg/ml ve 6.74±3.47ng/

ml], kontrollere [srasyla 649.5±402.6 pg/ml ve 2.63±0.57 ng/ml] kyasla anlaml derecede daha yüksekti

(p<0.01). Toplam sakalm deeri,yüksek sAng-2 ve sTie-2 [srasyla 2500pg/ml ve 8ng/ml]

deerli AML hastalarnda düük düzeylere [<srasyla 2500pg/ml ve <8ng/ml] kyasla anlaml derecede

daha ksayd.

Sonuç: Çalmada elde edilen sonuçlarda, AML hastalarnn dolamdaki Ang-2 ve sTie-2 düzeylerinin

prognostik anlamll gösterilmitir. Anjiyopoietin / Tie2 aksisinin modülasyonu, söz konusu hastalarda

sonucu gelitirmeye yönelik ümit verici bir yaklam olabilir. (Turk J Hematol 2010; 27: 282-8)

Anahtar kelimeler: Ang-2, Tie-2, AML, ELISA

Geli tarihi: 16 Mays 2009 Kabul tarihi: 22 ubat 2010

Introduction

Angiogenesis, the formation of new blood vessels,

plays a critical role in the development and

growth of solid tumors and hematologic malignancies

[1,2]. Angiogenic activity has been demonstrated

to be significantly increased in acute myeloid

leukemia (AML) bone marrow as compared to normal

BM [3,4]. Padro et al. [5] stated that leukemic

blasts release several angiogenic molecules that

increase vessel density in neoplastic marrow.

The interplay of BM endothelial cells and growth

factors derived from leukemic blasts contributes to

the pathogenesis of hematologic malignancies. It

has become clear that angiogenic factors produced

by leukemic blasts may also act in an autocrine or

intracrine fashion, thereby stimulating cell proliferation

and survival through a mechanism independent

from angiogenesis [6].

Among many angiogenic mediators, the members

of the vascular endothelial growth factor

(VEGF), basic fibroblast growth factor (bFGF) and

angiopoietin (Ang) family have been established as

the major regulators of tumor-associated angiogenesis

[7]. Cellular VEGF has been found to be upregulated

in AML blasts [5,8], and to be negatively correlated

with survival [9].

The angiopoietins constitute a novel family of

angiogenic mediators, which have been shown to

be important regulators of neovascularization, vascular

stability and maturation [10]. Ang-1 and its

naturally occurring antagonist Ang-2 act via the

Tie-2 receptor tyrosine kinase, which is broadly

expressed in the endothelium of the adult vasculature

and in a subset of hematopoietic stem cells.

Although Ang-1 and Ang-2 have very similar protein

structure, they elicit opposing responses when

binding to endothelial Tie-2 [11]. Binding of Ang-1

causes autophosphorylation of Tie-2 and ensures the

integrity of blood vessels by strengthening interaction

between endothelial cells and peri-endothelial support

cells. In contrast, Ang-2 specifically disrupts Ang-

1-mediated receptor activation, resulting in vessel

destabilization, thereby facilitating the angiogenic

response to mitogenic factors such as VEGF or leading

to vessel regression in its absence [12].

With its role in both angiogenesis and vascular

maintenance, Tie-2 seems to have a dual function

defined by the quantitative balance between Ang-1

and Ang-2 activity. While Ang-1 is constitutively

expressed throughout adult tissues, providing a stabilizing

signal, normal postnatal Ang-2 expression is

only observed at the sites of active vascular remodeling

[13]. Thus, angiogenesis is controlled by a

dynamic balance between vessel regression and

growth that is mediated by the VEGF and the Ang/

Tie-2 system.

The role of this complex system has been extensively

examined in the neovascularization of a wide

variety of tumors, and many reports have documented

a correlation between Ang expression and

clinical features or prognosis [14]. Loges et al. [15]

reported that BM neoangiogenesis plays an important

pathogenic and possibly prognostic role in

AML. Therefore, this study aimed to evaluate the

circulating levels of soluble (s)Ang-2 and sTie-2 in

Egyptian patients with AML and to investigate their

impact on the overall survival in those patients. We

preferred to measure the circulating levels of these

parameters because they are easily accessible and

applicable for routine use.


The present study was carried out on 60 newly

diagnosed AML patients (Table 1) and 30 sex- and

284

Attia et al.

Angiopoietin-2 and soluble Tie-2 in AML Turk J Hematol 2010; 27: 282-8

Table 1. Patient characteristics

Parameters

AML patients

No 60

Age/year 15-63

Male 35

Female 25

FAB subtypes

M0 5

M1 15

M2 10

M4 16

M5a 7

M5b 5

M6 2

WBCs x 10 3 /cmm 16.0 (9.5-89.5)

Peripheral blood blast cells % 10

Bone marrow blast cells % 50

Follow-up

24 months

Main causes of death

Hemorrhagic complications

and infections

age-matched healthy controls in accordance with

local institutional ethical protocols. All the patients

were admitted to the Hematology/Oncology unit of

the Internal Medicine Department of Tanta University

Hospital. The diagnosis was made according to the

clinical picture and morphological and cytochemical

criteria of the French-American-British (FAB)

study group and immunophenotypic studies.

Patients were treated with a combination of Ara-C 1

g/m 2 /12h (day 1 to day 3) or mitoxantrone 12 mg/m 2

(days 3, 4 & 5) [16].

Patients were considered to be in complete

remission (CR) when BM aspirates showed trilineage

regeneration with less than 5% blasts by morphological

and immunocytochemical analysis in

the presence of a normal blood count that persisted

for at least one month. All other patients were considered

to be non-responsive. Three milliliters of

blood were withdrawn from each subject into

Vacutainer tubes containing EDTA as anticoagulant.

The blood was centrifuged for 15 minutes, and then

plasma was collected and stored at -20°C until

analysis. Blood samples were obtained from patients

at diagnosis before the initiation of induction chemotherapy.

Plasma levels of Ang-2 and Tie-2 were assayed by

enzyme-linked immunosorbent assay (ELISA) using

commercially available kits (Quantikine, R&D systems)

according to the manufacturer›s instructions.

Statistical analysis

The collected data were organized, tabulated

and statistically analyzed using SPSS software statistical

computer package version 12. For quantitative

variables, the median, range, mean and standard

deviation were calculated. The difference between

two medians was statistically analyzed using Mann-

Whitney test. For comparison between more than

two medians, the F value of analysis of variance

(ANOVA) was calculated and Scheffe test was performed

to compare between two medians if the F

value was significant. Pearson’s correlation coefficient

(r) was calculated to test the association

between two variables. Univariate and multivariate

Cox regression analysis were performed to evaluate

the predictive effects of each angiogenic factor.

Values of p<0.05 were considered statistically significant

[17].

Results

Plasma levels of Ang-2 and Tie-2 in AML patients

and controls

Medians, means and ranges of plasma levels of

Ang-2 and Tie-2 in AML patients and healthy controls

are presented in Table 2. There was significant

increase in the circulating plasma levels of both

Ang-2 and Tie-2 in AML patients when compared to

the control group (p=0.001 for both parameters)

(Table 2).

Correlation between plasma levels of Ang-2 and

Tie-2 and clinicopathological features of AML

patients

In AML patients, there were significant positive

correlations between plasma Ang-2 level and total

leukocytic counts (TLC), lactate dehydrogenase

(LDH) levels and percentage of blast cells in the BM

(r=0.48, 0.54 and 0.48, respectively). On the other

hand, there was a significant strong negative correlation

between Ang-2 plasma levels and survival in

AML patients (r=-0.84) (Table 3). Tie-2 plasma levels

were positively correlated with TLC and LDH

levels and the percentage of blasts in the BM (r=0.5,

0.52 and 0.52, respectively). Also, a significant negative

correlation was found between Tie-2 levels and

survival in AML patients (r=-0.8) (Table 3).

Attia et al.


Association between plasma levels of Ang-2, Tie-2

and overall survival

To assess the effect of circulating Ang-2 and sTie-

2 levels on the AML overall survival, univariate

analysis was done. We had sub-classified the studied

patients into risky and non-risky subgroups

regarding their age (risk with age 20 or 60 years),

TLC (risk 50,000) and their response to induction

therapy (risk in the absence of CR). LDH level was

considered as a risk factor when it was >400 U/L

and non-risky when it was 400 U/L.

The angiogenic factors Ang-2 (2500 vs <2500)

and Tie-2 (8.0 vs <8) were significantly associated

with effect on AML survival (Figures 1 and 2, respectively).

These cut-off values were statistically calculated

using a Receiver Operating Characteristic

(ROC) curve. The relative risk (RR) of death of Ang-2

was significant higher when baseline was 2500 pg/

ml (RR 6.3, 95% confidence interval (CI) 0.071-0.610,

p=0.050). In addition, the RR of death of Tie-2 was

significant higher when baseline was 8 pg/ml (RR

5.65, 95% CI 0.061-0.710, p=0.005) (Table 4).

Furthermore, a multivariate Cox regression analysis

incorporating all variables that were found to

have a significant effect on univariate analysis was

performed (Table 5). Response to treatment was

independently and significantly affected by Tie-2

and Ang-2 (p<0.001 and p<0.01, respectively),

while WBC was affected by Ang-2 (p<0.01) and

LDH was affected by Tie-2 (p=0.034).

Discussion

The system involving Ang-2 and its receptor Tie

-2 appears to play an important role not only in

tumor angiogenesis, but also in the biology of

hematological and non-hematological malignancies.

The elevated vessel density in neoplastic BM is

the result of the action of several angiogenic molecules

released from leukemic blasts. The expanded

endothelial microenvironment is able to support

leukemic cell survival and growth by secretion of

hematopoietic growth factors [18]. Moreover, angiogenic

mediators produced by AML cells also act

through external and internal autocrine loops,

thereby directly promoting cell survival, proliferation

and disease progression independently from

the mechanisms of angiogenesis [19].

In the present study, the plasma levels of sAng-2

and sTie-2 were evaluated in patients with newly

diagnosed AML. Levels of circulating Ang-2 and

Table 2. Plasma Ang-2 and Tie–2 levels in the studied groups

Control group AML group F P

Number 30 60

Ang-2 (pg/ml):

Range 189-1215 345-4210

Mean±SD 649.53± 402.67 2382.10±1586.12 9.361 0.001*

Median 550 3000

Tie-2 (ng/ml):

Range 1.9-3.5 1.4-10.5

Mean±SD 2.63±0.57 6.74±3.47 10.592 0.001*

Median 2.6 8.8

*Significant

Table 3. Relationship between plasma Ang-2 and Tie-2 levels

and measured variables in AML patients

Variables Ang-2 in AML Group Tie-2 in AML Group

(n=60)

(n=60)

r p r p

Age in years 0.249 0.185 0.276 0.140

TLC 0.477 0.008* 0.501 0.005*

LDH 0.543 0.002* 0.528 0.003*

Blast cells % 0.483 0.007* 0.520 0.003*

Survival -0.843 0.001* -0.806 0.001*

*Significant

Table 4. Univariate analysis of overall survival in AML patients

Risk factors No RR 95% CI p-value

Age:

20 & 60 40 1.1 0.346-2.231 0.817

21-59 8 0.9

TLC:

50.000 40 0.2 0.033-0.291 <0.001

<50.000 8 3.9

LDH:

> 400 28 1.2 0.051-0.430 0.05*

400 20 5.1

Response to treatment:

No remission 36 4.7 0.038-0.420 0.001*

Complete remission 12 0.3

Tie-2:

8 28 5.6 0.061-0.710 0.005*

<8 20 6.3

Ang-2:

2500 32 6.3 0.0710-0.610 0.05*

<2500 16 1.6

*Significant

286

Attia et al.


Table 5. Multivariate analysis for risk factors associated with

Tie-2 and Ang-2

Risk factors Tie-2 Ang-2

Wald p Wald p

TLC 0.656 0.418 6.683 0.010*

LDH 4.471 0.034* 3.285 0.070

Response to treatment 31.137 0.001* 25.138 0.010*

*Significant

Cum Survival

1.0

0.8

0.6

0.4

0.2

0.0

6 12 18 24 30

Survival in months

ang22

<=2500

>2500

<=2500-

censored

>=2500-

censored

Figure 1. Kaplan-Meier curve for overall survival in AML patients in

relation to plasma Ang-2 levels. The overall survival is significantly

shorter in AML patients with plasma Ang-2 2500 pg/ml than in

those with levels <2500 pg/ml

Cum Survival

1.0

0.8

0.6

0.4

0.2

0.0

5.00 10.00 15.00 20.00 25.00

Survival in months

TE22

<=8

>8

<=8-censored

>8-censored

Figure 2. Kaplan-Meier curve for overall survival in AML patients in

relation to plasma Tie-2 levels. The overall survival is significantly

shorter in AML patients with plasma Tie-2 8 ng/ml than in those

with levels <8 ng/ml

Tie-2 were found to be significantly higher in patients

group when compared to the control group. The

plasma levels were found to be strongly associated

with TLC, LDH levels and the extent of BM infiltrations.

The observed differences could be explained

in part by the source of circulating Ang-2, which

originates not only from leukemic blasts but also

from other cell types such as endothelial cells. In

fact, it is entirely possible that a predominant proportion

of circulating Ang-2 could be secreted by

activated BM endothelial cells, given the increased

angiogenic activity in the leukemic BM. Furthermore,

it is conceivable that Ang-2 could also be released

by the endothelium of the normal vasculature upon

dysregulated stimulation by leukemic blasts [19].

Our findings are in line with a recent observation of

elevated Ang-2 and Tie-2 levels in patients with

chronic myeloid leukemia (CML) and multiple

myeloma [14]. It was found that blood levels of

sAng-2 and sTie-2 were strongly associated with the

extent of BM infiltration, TLC and LDH, providing

indirect evidence that the leukemic blasts themselves

at least significantly contributed to the plasma

levels of the tested angiogenic factors [19].

Furthermore, a strong correlation could be demonstrated

between plasma levels of Ang-2 and Tie-2

and the overall survival in AML patients after chemotherapy.

The overall survival rate of AML patients

was significantly lower in the group having higher

Ang-2 and Tie-2 levels (2500 pg/ml and 8 ng/ml,

respectively).

Schliemann et al. [19] found that pre-therapeutic

levels of circulating Ang-2 and Tie-2 were significantly

higher in AML patients as compared to normal

controls, and elevated levels were also observed

in patients with CML and multiple myeloma.

Furthermore, they could demonstrate a strong correlation

between systemic levels of Ang-2 and overall

survival in intensively treated AML patients.

Patients with high plasma levels displayed significantly

worse overall survival than those with low

levels. The RR of death was more than four times

higher for patients with high Ang-2 levels. The threeyear

survival rate for AML patients with high levels

of Ang-2 was only 14.7% compared to 64.7% for

those with low Ang-2 levels. Furthermore, Lee et al.

[20] reported that AML patients with lower Ang-2

and Tie-2 levels displayed a survival advantage.

The mechanisms responsible for the difference in

prognosis between AML patients with high and low

Ang-2 expression remain unclear. The observed differences

in survival might result from modulation of

BM neovascularization. Angiogenesis is an inevitable

step in the development and progression of malignancy.

In the absence of vascularization, tumors

cannot grow to more than 1-2 mm in diameter, probably

because this size is the maximum for allowing

oxygen diffusion from vessels. Neoangiogenesis

supplies a tumor with oxygen and nutrients, while

the newly formed endothelial cells appear to stimulate

the growth of adjacent tumor cells by secreting

numerous active peptides [21]. It has also been

Attia et al.


documented that the tumor cells themselves emit

proteins that stimulate neoangiogenesis and this

process is correlated with the diffusion of metastases

[22]. Elevated levels of circulating Ang-2 have

been linked with angiogenesis in malignancies

such as angiosarcoma [23], breast cancer [24], and

multiple myeloma and CML [14] . In analogy, high

levels of Ang-2 in AML may contribute to angiogenesis

in the BM in the presence of mitogenic mediators,

thereby facilitating leukemic blast proliferation

and disease progression. However, Schliemann et

al. [13] reported that microvessel counts did not

show any association with clinical outcome, and

they suggested a potential alternative mechanism

independent from angiogenesis, which might have

greater impact on prognosis in AML patients intensively

treated with chemotherapy.

The findings of our results are not in line with

Loges et al. [15], who stated that cellular Ang-2

expression could be identified as an independent

predictor of favorable prognosis in AML patients.

However, this discrepancy can be explained by the

fact that intracellular levels of angiogenic factors

may not reflect their blood levels, and circulating

Ang-2 most likely originates not only from leukemic

blasts but also from other cell types such as endothelial

cells. It is possible that a predominant proportion

of circulating Ang-2 could be secreted by

activated BM endothelial cells given the increased

angiogenic activity in the leukemic BM. Ang-2 could

also be released by the endothelium of the normal

vasculature upon dysregulated stimulation by leukemic

blasts [19]. Therefore, different prognostic

values may be observed when investigating either

cell-associated or circulating Ang-2 separately.

Previous studies have documented that Ang-1

mediates vascular stability while Ang-2 induces vascular

instability by overriding Ang-1-mediated Tie-2

activation [25]. Thus, the balance between Ang-1

and Ang-2 determines the grade of endothelial Tie-2

phosphorylation. Ang-1 appears to be the dominant

Tie-2 ligand in normal BM. This balance strongly

shifts towards Ang-2 during leukemic transformation.

Reversal of the normal angiopoietin balance in

favor of Ang-2 acting in concert with other angiogenic

inducers may be essential for BM angiogenesis

in AML [13].

In conclusion, a better understanding of the precise

functions of angiopoietin-signaling pathways in

AML may open new options of therapeutic interventions,

and modulation of the autocrine angiopoietin/

Tie-2 axis may be a promising approach to improve

the outcome in AML patients. Furthermore, circulating

Ang-2 and Tie-2 may represent attractive therapeutic

targets when introducing anti-angiogenic

strategies into the treatment of AML.

Finally, measurement of circulating Ang-2 and Tie-2

concentrations at disease presentation may find its

way into clinical routine as an additional prognostic

tool in the risk-stratified management of AML.






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8. Fiedler W, Graeven U, Ergün S, Verago S, Kilic N,

Stockschläder M, Hossfeld DK. Vascular endothelial

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H, Buchner T. Expression of angiopoietins and their

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L, D`Angelo A. Differential levels of soluble angiopoietin-2

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Case Report

289

Acquired pure megakaryocytic aplasia

successfully treated with cyclosporine

Siklosporin ile baarl ekilde tedavi edilen kazanlm saf

megakaryositik aplazi

Halima El Omri 1 , Firyal Ibrahim 2 , Ruba Yasin Taha 1 , Riham Hassan Negm 1 , Aisha Al Khinji 1 ,

Mohammed Yassin 1 , Ibrahim Al Hijji 1 , Hanadi El Ayoubi 1 , Hussein Baden 2

1Department of Laboratory Medicine and Pathology, Al Amal Hospital, Doha, Qatar

2Department of Hematology and Bone Marrow Transplant, Al Amal Hospital, Doha, Qatar

Abstract

Acquired pure megakaryocytic aplasia is a rare hematological disorder characterized by thrombocytopenia

with absent or markedly reduced megakaryocytes in the bone marrow. We report a case of a

25-year-old male diagnosed as acquired pure megakaryocytic aplasia. Treatment with prednisone and

intravenous immunoglobulin failed, but he was successfully treated with cyclosporine, with complete

remission after 90 days and normal platelet count maintained thereafter.

(Turk J Hematol 2010; 27: 289-93)

Key words: Acquired amegakaryocytic thrombocytopenia, steroids, cyclosporine

Received: May 27, 2009 Accepted: July 31, 2009

Özet

Kazanlm saf megakaryositik aplazi, kemik iliinde namevcut ya da anlaml ölçüde azaltlm megakaryosit

içeren trombositopeni ile karakterize, nadir hematolojik bir hastalktr. Kazanlm saf megakaryositik

aplazi tehisi konmu, prednizon ve intravenöz immunoglobulin ile tedavisi baarsz olmu

ve siklosporin ile baarl biçimde tedavi edilerek 90 gün sonra tam remisyona ulaan ve ondan sonra

platelet saym normalleen 25 yanda erkek bir olgunun raporu sunulmutur.

(Turk J Hematol 2010; 27: 289-93 )

Anahtar kelimeler: Kazanlm amegakaryositik trombositopeni, steroidler, siklosporin

Geli tarihi: 27 Mays 2009 Kabul tarihi: 31 Temmuz 2009

Address for Correspondence: M.D. Halima El Omri, Department of Hematology and Bone Marrow Transplant Al Amal Hospital - Hamad

Medical Corporation P.O. Box 3050 Doha, Qatar Phone: +974- 4397857 E-mail: helomri@hmc.org.qa

doi:10.5152/tjh.2010.51

290

Omri et al.

Acquired amegakaryocytic thrombocytopenia treated with cyclosporine Turk J Hematol 2010; 27: 289-93

Introduction

Megakaryocytic aplasia, especially acquired pure

megakaryocytic aplasia (APMA), is a rare hematologic

disorder. APMA is characterized by severe

thrombocytopenia resulting from marked decrease

or absence of megakaryocytes in the marrow in the

presence of otherwise normal erythropoiesis and

granulopoiesis. APMA can be either idiopathic or

caused by a variety of conditions, such as acquired

clonal cytogenetic abnormalities, drug sensitivity,

toxin exposure, infectious diseases such as viral

infection [1,2], immune diseases such as lupus erythematosus

[3], systemic sclerosis [4], eosinophilic

fasciitis [5], and malignancy [6,7]. Patients with

acquired amegakaryocytic thrombocytopenia may

have additional hematological abnormalities such

as macrocytosis or dyserythropoiesis, abnormalities

which may indicate potential future progression to

aplastic anemia or myelodysplasia [8-10].

Case Report

A 25-year-old male from Nepal presented in

August 2008 with a one-week history of headache,

gum bleeding and epistaxis. There was no history of

trauma, arthralgia, weight loss, drug intake, alcohol

consumption, or fever and no family history of

bleeding diathesis. He was not known to have any

chronic disease. Physical examination showed multiple

ecchymoses and petechiae all over the body

with bilateral retinal hemorrhage. He had no hepatosplenomegaly

or lymphadenopathy.

A

Complete blood count showed white blood cells

(WBC) 9.7x10 9 /L with normal differential, hemoglobin

(Hb) 9.1 g/dl, mean corpuscular volume (MCV)

102 fl, reticulocytes 7.2% (total 210x10 9 /L), and

platelet (PLT) count 5x10 9 /L. Peripheral blood

smears revealed normochromic normocytic red

cells with polychromasia and markedly decreased

PLT. Direct and indirect Coombs tests were negative;

prothrombin time, partial thromboplastin time,

liver and renal function tests, serum iron, transferrin

saturation, serum B12, red cells, and serum folate

were all normal. Autoimmune screen including

anticardiolipin, antinuclear antibodies, rheumatoid

factor, and C3 and C4 were negative. Serological

markers for infections like hepatitis virus (A, B & C)

and antibodies against human immunodeficiency

virus (HIV), rubella, cytomegalovirus, varicella, herpes

simplex virus types 1& 2, Epstein-Barr virus

(EBV) and Toxoplasma gondii were all negative;

serological test for parvovirus B19 was not done.

The magnetic resonance imaging (MRI) of the

brain showed multiple subcentrimetric foci of hemorrhagic

lesions in the left and right parietal regions

and in the frontal region. Computed tomography

scan of the chest, abdomen and pelvis was normal.

Bone marrow aspiration revealed complete

absence of megakaryocytes in an otherwise normocellular

marrow with active erythro- and granulopoiesis,

with no dysplastic features or abnormal cell

infiltrates. Iron stores were depleted. The histology

of the biopsy and immunohistochemistry using

CD61 antibody (GPIIIa) confirmed the isolated

megakaryocytic aplasia (Figure 1). Cytogenetic

analysis of the bone marrow showed a normal male

B

Figure 1. A. Peripheral smear showing severe thrombocytopenia (Wright stain X1000). B: Bone marrow biopsy shows mixed cellular

hemopoietic cells with absent megakaryocytes before treatment (H&E stain X400)

Omri et al.

Turk J Hematol 2010; 27: 289-93 Acquired amegakaryocytic thrombocytopenia treated with cyclosporine 291

A

B

Figure 2. A. Peripheral smear after treatment with cyclosporine (after 3 months) showing normalization of the platelet count (Wright stain

X1000). B: Bone marrow biopsy shows active marrow with adequate number of megakaryocytes (H&E X400)

200 Cyclosporine

5 mg/kg

180 500mg IV 1 mg/kg/day

Steroids

160

tapering

Platelets Count (10 3 / L)

140

120

100

80

60

40

20

IV Ig

1 g/kg/day

X2 days

platelets transfusion

0

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time after admission (in weeks)

Figure 3. Clinical course with treatment given and changes in platelet count

karyotype. Based on the clinical and laboratory

results, a diagnosis of APMA was made.

In view of the widespread hemorrhage and MRI

findings, the patient was treated initially with PLT

transfusion and methylprednisolone 5 mg/kg/day

intravenous (IV) for 3 days followed by oral dose of

prednisone 1 mg/kg/day that was continued for 2

weeks and then tapered over 4 weeks until discontinuation.

On Day 5 of steroid, no response was

obtained, so empirical intravenous immunoglobulin

(IVIG) 1 g/kg/day for 2 days was added, but

again no significant response was achieved. Oral

cyclosporine 5 mg/kg/day was started on Day 12 of

treatment, and 2 weeks later the PLT count began

to rise and transfusions were no longer required.

The patient was discharged with a PLT count of

60x10 9 /L and was followed as an outpatient. Three

months later, complete blood count normalized,

PLT count was 148x10 9 /L with Hb of 14 g/dl, and

the bone marrow follow-up was cellular with a

good number of megakaryocytes and well-represented

erythropoiesis and granulopoiesis (Figure 2).

The patient is currently under follow-up with maintenance

of a normal PLT count (Figure 3) on the

same dose of cyclosporine, with satisfactory therapeutic

levels and normal renal function. The plan

is to continue cyclosporine for up to one year with

gradual tapering of the dose before stopping.

Waiver consent is available at Hamad Medical

Corporation Research office.

292

Omri et al.

Acquired amegakaryocytic thrombocytopenia treated with cyclosporine Turk J Hematol 2010; 27: 289-93

Discussion

Isolated thrombocytopenia and megakaryocytic

aplasia, inconsistently described as APMA or

acquired amegakaryocytic thrombocytopenia purpura

(AATP), is a rare disease in the field of hematology.

The exact prevalence is unknown and the

available literature comprises case reports and

small case series. It is possible that the incidence

rate is higher than what is reported and that many

of the cases are underdiagnosed or misdiagnosed

as immune thrombocytopenia [10]. The clinical

course of this rare disease seems to be variable. In

some patients, it progresses rapidly to aplastic anemia

[8] or myelodysplasia [9]. The usual clinical

presentation of APMA is with bruising and bleeding

with the absence of splenomegaly.

The exact pathogenesis behind this disease is

still uncertain; several studies suggest an immune–

mediated process. Benedetti et al. [11] showed

cell-mediated immunosuppression of megakaryocytes

by demonstrating a marked increase in

T-activated suppressor cells (CD8+/DR+) in association

with AATP. A role for humoral immunity was

also proposed in the pathogenesis of AATP when

Katai et al. [12] showed significant suppression of

megakaryocyte colony formation of normal marrow

cells with the addition of AATP patient serum to

marrow cultures. Antibodies against thrombopoietin

have been described to cause this disorder

[13], as have antibodies against the TPO receptor,

the c-mpl [3,14]. Chromium-tagged survival studies

in patients with APMA have shown normal results,

ruling out PLT destruction or sequestration [15].

Due in part to the heterogeneous nature of the

syndrome and the variety of the pathogenic mechanisms,

no standard treatment has been established;

however, several empirical therapies are

used in patients with AATP and include the administration

of corticosteroids , IVIG, cyclophosphamide,

vincristine, cyclosporine, anti-thymocyte

globulin (ATG), splenectomy [15-18], allogenic

bone marrow transplantation [19], and recently,

mycophenolate mofetil [20].

The administration of corticosteroids, IVIG, cyclophosphamide,

vincristine, androgens, and mycophenolate

mofetil are transiently effective in occasional

patients with AATP [1,15,16,20]; however, the

administration of cyclosporine alone or in combination

with ATG was shown to be very effective in the

treatment of AATP [10,15,17,18].

Our patient represents a typical case of APMA

with severe thrombocytopenia and absent marrow

megakaryocytes. Predictors that indicate the disease

progression such as clonal cytogenetic abnormalities,

macrocytosis or dyserythropoiesis were

not present; the anemia was explained by the significant

mucocutaneous bleeding or autoimmune

mechanism. No obvious cause of the APMA could

be found in this patient. There was no history of

exposure to chemicals or drugs, and clinical examination

and investigations excluded collagen diseases,

infections, malignancies, and congenital

anomalies like absent radius. Unfortunately, tests

for TPO and c-MpI antibodies are not available in

our center.

The response to immunosuppressive treatments,

especially the cyclosporine, would suggest an

immune-mediated pathogenetic mechanism.

Normalization of the PLT count was achieved 90

days after the start of treatment. As of the preparation

of this report, the patient is well, with a PLT

count of 198x10 9 /L and Hb of 14 g/dl, maintained on

5 mg/kg/day cyclosporine (for 7 months from the

initiation of treatment), with continuing monitoring

to ensure response and to detect any progression to

aplastic anemia or myelodysplastic syndrome.

In conclusion, this case report supports the effectiveness

of cyclosporine at the prescribed dose of 5

mg/kg/day in the management of APMA.

Conflict of Interest





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Turk J Hematol 2010; 27: 289-93 Acquired amegakaryocytic thrombocytopenia treated with cyclosporine 293

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294 Case Report

Syngeneic peripheral blood stem cell

transplantation with immunosuppression for

hepatitis-associated severe aplastic anemia

Hepatite bal iddetli aplastik anemi için immunosüpresyonla

singeneik periferik kan kök hücre transplantasyonu

Aleksandar Savic 1 , Bela Balint 2 , Ivana Urosevic 1, Nebojsa Rajic 1 , Milena Todorovic 3 ,

Ivanka Percic 1 , Stevan Popovic 1

1Clinic of Hematology, Clinical Center of Vojvodina, Novi Sad, Serbia

2Institute of Transfusiology, Military Medical Academy, Belgrade, Serbia

3Clinic of Hematology, Clinical Center of Serbia, Belgrade, Serbia

Abstract

Hepatitis-associated aplastic anemia occurs in up to 10% of all aplastic anemia cases. Syngeneic bone

marrow transplantation is rare in patients with severe aplastic anemia and usually requires pre-transplant

conditioning to provide engraftment. We report on a 29-year-old male patient with hepatitisassociated

severe aplastic anemia who had a series of severe infectious conditions before transplantation,

including tracheal inflammation. Life-threatening bleeding, which developed after bronchoscopy,

was successfully treated with activated recombinant factor VII and platelet transfusions. Syngeneic

peripheral blood stem cell transplantation using immunosuppressive treatment with antithymocyte

globulin and cyclosporin A without high-dose pre-transplant conditioning was performed, followed by

complete hematologic and hepatic recovery. (Turk J Hematol 2010; 27: 294-8)

Key words: Aplastic anemia, hepatitis, peripheral blood stem cell transplantation, trachea, infection,

bleeding

Received: September 27, 2009 Accepted: Juanuary 25, 2010

Özet

Hepatite bal aplastik anemi, tüm aplastik anemi olgularnn %10’undan daha az bir oranda meydana

gelir. iddetli aplastik anemili hastalarda singeneik kemik ilii transplantasyonu nadir olup, genellikle

hibritlemeyi salamak için nakil öncesi artlandrma gerektirmektedir. Transplantasyondan önce,

trakea enflamasyonu da dahil olmak üzere iddetli enfeksiyonlar geçiren hepatite bal iddetli aplastik

anemili 29 yanda erkek bir hastaya ilikin bir rapor sunulmutur. Bronkoskopiyi takiben gelien

yaam tehdit eden kanama, aktive edilmi rekombinant faktör VII ve platelet transfüzyonlaryla

baarl ekilde tedavi edilmitir. Yüksek doz nakil öncesi artlandrma olmakszn, antitimosit globu-

Address for Correspondence: M.D. Aleksandar Savic, Hajduk Veljkova 1-3 21000 Novi Sad, Serbia

Phone: +381 21 484 3963 E-mail: asavic@uns.ac.rs

doi:10.5152/tjh.2010.52

Savic et al.

Turk J Hematol 2010; 27: 294-8 Syngeneic transplantation for hepatitis-associated aplastic anemia 295

lin ve siklosporin A ile immunosüpresif tedavi yöntemiyle singeneik periferik kan kök hücre transplantasyonu

gerçekletirilmi ve ardndan tam hematolojik ve hepatik iyileme gözlenmitir.

(Turk J Hematol 2010; 27: 294-8)

Anahtar kelimeler: Aplasti Anemi, hepatit, periferik kan kök hücre transplantasyonu, trake, enfeksiyon,

kanama

Geli tarihi: 27 Eylül 2009 Kabul tarihi: 25 Ocak 2010

Introduction

Hepatitis-associated aplastic anemia is a welldescribed

disease that occurs in 2 to 5% of aplastic

anemia cases in the West, and in 4 to 10% in the

Far East [1]. Hepatitis of the hepatitis-associated

aplastic anemia does not appear to be caused by

any of the known hepatitis viruses [1,2]. Syngeneic

bone marrow transplantation in patients with

severe aplastic anemia (SAA) is rare, and usually

requires pre-transplant conditioning to provide

engraftment [3,4].

We report a patient with hepatitis-associated SAA

who was successfully treated with syngeneic

peripheral blood stem cell (PBSC) transplantation

after a series of infectious and bleeding complications.

To our best knowledge, this is only the second

published case of syngeneic PBSC transplantation

in hepatitis-associated SAA [5]. The patient gave

informed consent for review of his medical records

and publication of this case.

Case Report

A 29-year-old male patient presented with fatigue,

fever and skin and mucosal bleeding at the end of

January 2008. He has a twin brother.

Initial laboratory findings showed severe pancytopenia

and elevation of bilirubin and liver enzymes

(Table 1, Day -116). Acquired idiopathic SAA was

diagnosed following bone marrow biopsy, which

indicated less than 10% cellularity. A series of tests

were performed including hepatitis C virus (HCV),

HBV, human immunodeficiency virus (HIV), antinuclear

antibody (ANA), and Ham and Hartman

test, which were all negative. Cytogenetics evaluation

was normal. The patient was completely

dependent on platelet and packed red blood cell

(RBC) transfusions in order to control the bleeding

and anemia.

There was a severe deterioration in the patient’s

liver condition, with development of jaundice and

an increase in blood liver enzyme concentrations,

at the beginning of March 2008 (Table 1, Day -77). In

mid-March, the patient received mycophenolate

mofetil 750 mg twice daily, together with 5 g/kg/d

of recombinant human granulocyte colony-stimulating

factor (G-CSF) and 1 mg/kg/day of methylprednisolone.

The patient developed diabetes mellitus,

which required regular short-acting insulin

treatment and necessitated discontinuation of corticosteroid

treatment. Combined mycophenolate

mofetil and G-CSF treatment caused elevation of

granulocytes to 1.3 x 10 9 /L in the second half of

April, but showed no improvement in the platelet

count (Table 1, Day -30). The improvement in RBC

was the result of RBC transfusions. From the beginning

of the disease until the end of April, the patient

suffered two episodes of enterococcal sepsis and

pneumonia and four febrile episodes.

HLA typing was performed after repeated

attempts. His twin brother was HLA identical (PCR-

SSP A*01-, B*08 B*35, DRB1*03-). We decided to

perform syngeneic PBSC transplantation using antithymocyte

globulin (ATG) and cyclosporin A as an

immunosuppressive regimen without high-dose

chemotherapy conditioning.

The transplantation procedure was postponed

because the patient developed fever, hemoptysis

and a sensation of a foreign body in his throat at the

initial day of the planned conditioning regimen in

April. Inspiratory stridor was auscultated over the

trachea. Indirect laryngoscopy showed no signs of a

laryngeal disease. A computed tomography (CT)

scan showed a thickening of the anterior wall of the

trachea, with internal air pockets, mucosal erosion

and intraluminal soft tissue formations (Figure 1a).

The patient received liposomal amphotericin and

antibiotics. A bronchoscopy was performed with a

support of platelet transfusions and fresh frozen

plasma, because of slightly prolonged prothrombin

time. This caused extensive bleeding from the trachea,

which was life-threatening given the concurrent

development of alveolar hemorrhage and

global respiratory insufficiency (Figure 1b). The

patient received one dose of recombinant factor

296

Savic et al.

Syngeneic transplantation for hepatitis-associated aplastic anemia Turk J Hematol 2010; 27: 294-8

(rF) VIIa 90 g/kg and platelet transfusion, after

which the bleeding was markedly reduced. A week

later, the fever stopped and the respiratory insufficiency

was eliminated, but the patient still suffered

from hemoptysis. A decision was made to start the

transplantation procedure. Mycophenolate mofetil

and G-CSF were discontinued before transplantation.

The patient was still severely pancytopenic

before transplantation, and exhibited hepatitis with

some signs of improvement (Table 1, Day -4).

G-CSF was given as 10 g/kg/day to mobilize the

donor’s PBSCs. A large volume apheresis specimen

was obtained using peripheral vein access with a

Cobe-Spectra cell separator on Day 5 of mobilization.

The cell harvest, with a total of 7.8 x 10 8 /kg of

MNC, 4.37 x 10 8 /kg of CD3+, 11.7 x 10 6 /kg of CD34+,

and 12.9 x 10 6 /kg of CD133+ cells, was infused on

Day 0, following immunosuppression, as follows:

8.5 mg/kg/day of ATG (Fresenius) for 4 days (from

Day -4 to -1) and 5 mg/kg/day of cyclosporin A intravenous

(i.v.) in two equal doses for 7 days (from Day

-1 to Day 6), followed by a switch to oral cyclosporin

A. Mycophenolate mofetil was added on Day +14 as

1 g twice daily due to persistent thrombocytopenia

and anemia. The patient received G-CSF as 5 g/kg

after PBSC infusion until white blood cell (WBC)

Table 1. Laboratory findings for blood and liver function

Parameters*

Day#

-116 -77 -30 -4 +8 +48 +250

WBC [x10 9 /L] 0.2 0.345 3.15 1.83 4.2 6.09 6.81

Neutrophils [x10 9 /L] 0.069 0.107 1.3 0.354 1.57 2.43 2.66

Hb [g/L] 46 80.4 79.9 90.6 78.9 102 117

Hct [l/l] 0.13 0.215 0.218 0.237 0.222 0.27 0.32

Plt [x10 9 /L] 15.6 19.2 11.9 23.9 13 106 176

Rtc [%] 0.3 - 0.3 0.3 0.2 4.2 1.68

AST [U/L] 67 835 460 283 32 40 22

ALT [U/L] 197 1835 1360 1099 27 49 13

Gamma–GT [U/L] 95 166 449 285 34 35 13

Total bilirubin [mol/L] 25 157 51 56 18 26 17

Direct bilirubin [mol/L] 7.2 68.4 39.9 14.3 4.9 6.7 3.9

*upper normal values: AST 37 U/L, ALT 40 U/L, gamma–GT 55 U/L, Total bilirubin 21 mol/L, direct bilirubin 4.2 mol/L

# – before transplantation, + after transplantation

Figure 1. a) The neck CT scan shows a thickened anterior wall of the trachea, with internal air pockets, mucosal erosion and intraluminal

soft tissue formations; b) The chest CT scan shows alveolar hemorrhage, particularly in the right lung

Savic et al.

Turk J Hematol 2010; 27: 294-8 Syngeneic transplantation for hepatitis-associated aplastic anemia 297

recovery. He also received standard prophylactic

antibacterial, antifungal and antiviral treatments.

The transplant took place without any febrile episodes

or other complications. Neutrophil engraftment

was rapid. The granulocytes were over 1.0 x

10 9 /L on Day +8 (Table 1). The final RBC transfusion

was on Day +16, and the final platelet transfusion

on Day +27. The platelet number reached 100 x

10 9 /L on Day +48 (Table 1), whereas RBC reached

100 g/L on Day +79. The patient was discharged

from the hospital on Day +34. A follow-up CT scan

of the trachea showed resolution of the lesions,

with minor scarring. A complete resolution of the

patient’s liver disease was established. Diabetes

mellitus was well controlled through diet. A followup

bone marrow biopsy showed normal findings.

Mycophenolate mofetil was discontinued, while

cyclosporin A was gradually tapered. The patient is

well and without signs of rejection 15 months after

transplantation.

Discussion

In summary, this case demonstrates concomitant

hepatitis of unknown origin and SAA. The

patient has a twin brother, but the transplantation

was delayed for several reasons, such as severe

hepatitis, unusual life-threatening tracheal complications,

most likely of infectious origin, and due to

repeated, unsuccessful HLA testing.

Inflammation of the tracheal wall is rarely reported

in aplastic anemia, and it is usually caused by

aspergillosis [6,7]. The situation was further complicated

by life-threatening bleeding provoked by

bronchoscopy, which was successfully treated by

rFVIIa and platelet transfusions. This complication

has not been previously reported in aplastic anemia.

Successful off-label use of rVIIa has been

reported for bleeding in cases of liver disease and in

severely injured trauma patients [8,9]. It has been

used rarely for bleeding complications in aplastic

anemia [10].

Ultimately, we conducted a successful syngeneic

transplantation using immunosuppressive treatment

without high-dose pre-transplant conditioning,

which is rarely reported in hepatitis-associated

SAA [4]. Conditioning regimens with cyclophosphamide

± ATG, or with fludarabine, cyclophosphamide

± ATG, are widely used in allogeneic settings

with high rates of sustained engraftment and survival

[11,12]. In the settings of syngeneic transplantation

in aplastic anemia, application of cyclophosphamide

in the conditioning regimen provides sustained

engraftment, but increases early mortality

[3]. Syngeneic transplantation without conditioning

is followed by a high rate of graft failure without the

adverse effects on overall survival [3]. The concept

of syngeneic transplantation without conditioning

or with ATG conditioning alone, as in our case, is

therefore feasible [4,13]. Hepatitis usually precedes

aplastic anemia [1,2]. In this case, hepatitis was

present at the time of diagnosis and it severely deteriorated

during the course of SAA. We believe that

the immunosuppressive treatment may have had

an important role both for the hematologic condition

and for liver improvement [14]. The liver

improvement was rapid and complete following

ATG and cyclosporin A treatment applied during the

transplantation procedure. In all likelihood, both

SAA and hepatitis were probably caused by immune

reactions [1,2,14,15]. In light of other studies that

have explored the role of stem cells in liver injury, it

is even possible that the stem cell transplantation

may have contributed, to some extent, to the resolution

of the patient’s liver disease [16]. Although

hematopoietic stem cell transplantation with highdose

conditioning is generally a safe and successful

treatment procedure in hepatitis-associated aplastic

anemia [1,2,17], there is a risk of liver disease deterioration

[18].

This case confirms that rFVIIa, combined with

platelet transfusion, may be effective in controlling

tracheal bleeding in rare situations of tracheal infection

in hepatitis-associated SAA. It also demonstrates

the possibility of rapid liver disease resolution

and sustained engraftment after syngeneic

PBSC transplantation using immunosuppressive

treatment without high-dose pre-transplant conditioning

in hepatitis-associated SAA.






References

1. Brown KE, Tisdale J, Barrett AJ, Dunbar CE, Young NS.

Hepatitis-associated aplastic anemia. N Engl J Med

1997;336:1059-64.

2. Safadi R, Or R, Ilan Y, Naparstek E, Nagler A, Klein A,

Ketzinel-Gilaad M, Ergunay K, Danon D, Shouval D,

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Galun E. Lack of known hepatitis virus in hepatitisassociated

aplastic anemia and outcome after bone

marrow transplant. Bone Marrow Transplant

2001;27:183-90.

3. Hinterberger W, Rowlings PA, Hinterberger-Fischer M,

Gibson J, Jacobsen N, Klein J, Kolb HJ, Stevens D,

Horowitz MM, Gale RP. Results of transplanting bone

marrow from genetically identical twins into patients

with aplastic anemia. Ann Intern Med

4. Hwang WL, Yang Y, Chen GR, Tsai CS, Jour JH.

Syngeneic peripheral blood stem cell transplantation

with brief immunosuppression for severe aplastic anemia.

Bone Marrow Transplant 2000;25:337-9.

5. Niki T, Nakao S, Ueda M, Tsuchiya H, Nakatsumi T,

Shiobara S, Mori T, Matsuei K, Matsuda T, Tachibana Y.

Incomplete marrow recovery associated with hepatitis

after syngeneic bone marrow transplantation for aplastic

anaemia: successful treatment with second marrow

transplantation without preconditioning. Br J

Haematol 1990;75:285-6.

6. Drury AE, Allan RA, Underhill H, Ball S, Joseph AE.

Calcification in invasive tracheal aspergillosis demonstrated

on ultrasound: a new finding. Br J Radiol

2001;74:955-8.

7. Vail CM, Chiles C. Invasive pulmonary aspergillosis:

radiologic evidence of tracheal involvement. Radiology

1987;165:745-6.

8. Ramsey G. Treating coagulopathy in liver disease with

plasma transfusions or recombinant factor VIIa: an

evidence-based review. Best Pract Res Clin Haematol

2006;19:113-26.

9. Boffard KD, Riou B, Warren B, Choong PI, Rizoli S,

Rossaint R, Axelsen M, Kluger Y; NovoSeven Trauma

Study Group. Recombinant factor VIIa as adjunctive

therapy for bleeding control in severely injured trauma

patients: two parallel randomized, placebo-controlled,

double-blind clinical trials. J Trauma 2005;59:8-15.

10. Blatt J, Gold SH, Wiley JM, Monahan PE, Cooper HC,

Harvey D. Off-label use of recombinant factor VIIa in

patients following bone marrow transplantation. Bone


11. Storb R, Blume KG, O’Donnell MR, Chauncey T,

Forman SJ, Deeg HJ, Hu WW, Appelbaum FR, Doney K,

Flowers ME, Sanders J, Leisenring W. Cyclophosphamide

and antithymocyte globulin to condition patients with

aplastic anemia for allogeneic marrow transplantations:

the experience in four centers. Biol Blood


12. Srinivasan R, Takahashi Y, McCoy JP, Espinoza-Delgado

I, Dorrance C, Igarashi T, Lundqvist A, Barrett AJ, Young

NS, Geller N, Childs RW. Overcoming graft rejection in

heavily transfused and allo-immunised patients with

bone marrow failure syndromes using fludarabinebased

haematopoietic cell transplantation. Br J

Haematol 2006;133:305-14.

13. Anderlini P, Riggs SA, Koerbling M, Champlin R.

Syngeneic blood stem cell transplantation for infectious

mononucleosis-related aplastic anaemia. Br J

Haematol 1999;106:159-61.

14. Osugi Y, Yagasaki H, Sako M, Kosaka Y, Taga T, Ito T,

Yamamoto M, Ohara A, Sato T, Mimaya J, Tsukimoto I,

Kojima S. Antithymocyte globulin and cyclosporine for

treatment of 44 children with hepatitis associated

aplastic anemia. Haematologica 2007;92:1687-90.

15. Young NS, Maciejewski J. The pathophysiology of

acquired aplastic anemia. N Engl J Med 1997;336:

1365-72.

16. Dalakas E, Newsome PN, Harrison DJ, Plevris JN.

Hematopoietic stem cell trafficking in liver injury.

FASEB J 2005;19:1225-31.

17. Locasciulli A, Bacigalupo A, Oneto R, Bruno B, Marsh

J, Tichelli A, Socie G, Passweget J. Hepatitis-associated

aplastic anaemia: epidemiology and treatment results

obtained in Europe. A Report of the EBMT Aplastic

Anemia Working Party. Blood 2010;149:890-5.

18. Van Dam J, Farraye FA, Gale RP, Zeldis JB. Fulminant

hepatic failure following bone marrow transplantation

for hepatitis-associated aplastic anemia. Bone Marrow

Transplant 1990;5:57-60.

Case Report

299

A case of acute lymphoblastic leukemia with

additional chromosomes X and 5 associated with

a Philadelphia chromosome in the bone marrow

Kemik iliinde ekstra kromozom 5 ve X’e ilave Philadelphia

kromozomu içeren akut lenfoblastik lösemi olgusu

Burak Durmaz 1 , Asude Alpman Durmaz 1 , Emin Karaca 1 , Güray Saydam 2 ,

Özgür Çoulu 1 , Ferda Özknay 1

1Department of Medical Genetics, Ege University Faculty of Medicine, Izmir, Turkey

2Department of Internal Medicine, Ege University, Faculty of Medicine, Izmir, Turkey

Abstract

We report herein a very rare case of acute lymphoblastic leukemia having a chromosomal constitution of

48,XY,+X,+5,t(9;22)(q34;q11) in the bone marrow. A patient with additional chromosomes X and 5

with a Philadelphia chromosome has not been reported previously. However, no abnormal karyotype was

obtained from the lymphocytes in our patient, and he did not have the characteristics of Klinefelter syndrome.

He achieved a complete remission with IDA-FLAG and dasatinib therapy. The mechanism of

trisomy 5 or any other chromosomal aneuploidy in the pathogenesis of leukemogenesis remains unclear.

Further studies involving the genes affected by this karyotype and their products may lead to strategies

to further increase the understanding of drug-resistant acute lymphoblastic leukemia and may represent

the next frontier in the targeted therapy of those patients. (Turk J Hematol 2010; 27: 299-302)

Key words: ALL, X chromosome, chromosome 5, Philadelphia chromosome

Received: December 24, 2009 Accepted: April 30, 2010

Özet

Bu makalede, kemik iliinde çok nadir bir kromozom yaps olan 48,XY,+X,+5,t(9;22)(q34;q11)’e

sahip bir olgu sunulmaktadr. Ekstra kromozom 5 ve X’e ilave olarak Philadelphia kromozomunu

tayan bir olguya literatürde rastlanmamtr. Hastamzn lenfositlerden elde edilen karyotipi normal

olarak deerlendirilmi olup, Klinefelter Sendromu klinii bulunmamaktayd. IDA-FLAG tedavisi

sonucunda tam remisyon saland. Trizomi 5 veya dier kromozomal anöploidilerin lökomogenez

üzerindeki rolü net deildir. Karyotipte saptanm olan bölgelerdeki genler ve ürünlerinin yaplacak

çalmalarla incelenmesi, ilaca dirençli akut lenfoblastik lösemiyi anlamamzda ve bu hastalarn hedefe

özgün tedavi seçeneklerinde yardmc olabilecektir. (Turk J Hematol 2010; 27: 299-302)

Anahtar kelimeler: ALL, X kromozomu, kromozom 5, Philadelphia kromozomu

Geli tarihi: 24 Aralk 2009 Kabul tarihi: 30 Nisan 2010

Address for Correspondence: M.D. Burak Durmaz, Ege University Faculty of Medicine, Department of Medical Genetics, 35100 Bornova,

Izmir, Turkey Phone: +90 232 390 39 61 E-mail: burak.durmaz@ege.edu.tr

doi:10.5152/tjh.2010.53

300

Durmaz et al.

ALL case with 48,XXY,+5,t(9;22)(q34;q11) Turk J Hematol 2010; 27: 299-302

Introduction

Chromosomal abnormalities have been reported

in 60-85% of patients with acute lymphoblastic leukemia

(ALL) [1]. Cytogenetic aberrations are not

only important in the evaluation of prognosis but are

also useful in classifying patients into risk groups

[2]. Numerical chromosomal changes such as

hypodiploidy are associated with poor outcome,

while hyperdiploidy is shown to be associated with

a better prognosis [3]. Even though aneuploidies

usually occur in autosomal chromosomes, sex

chromosome aneuploidies may also be associated

with hyperdiploidy in ALL. Trisomy 5 and sex chromosome

aneuploidies in high-hyperdiploid karyotypes

have been found in 20% of ALL patients,

which revealed poor prognosis [4]. Compared with

all chromosomal abnormalities, less is known about

patients with trisomies as a sole numerical chromosomal

abnormality. Since single trisomies are

extremely rare, with an incidence of 1%, their prognostic

value and relevant clinical features have not

been established in ALL patients [5]. Trisomy 5 is a

very rare chromosomal abnormality, with only few

cases in the literature, and it is shown to be associated

with other chromosomal abnormalities [6]. For

instance, an additional X chromosome is a rare

finding observed in the hematological malignancies

[7]. Besides numerical abnormalities, Philadelphia

(Ph) chromosome is the most common cytogenetic

abnormality, occurring in almost 25% of ALL adults

[8]. Here, we present an unusual ALL case with a

full karyotype showing an additional X chromosome,

chromosome 5 and a Ph chromosome in the

bone marrow, and we discuss the outcomes and

the management of these rare chromosomal abnormalities.

Case Report

An 18-year-old male patient was referred to our

hospital with fatigue, weakness and weight loss. On

physical examination, massive splenomegaly was

recorded (total vertical length: 180 mm). The complete

blood count (CBC) showed white blood cell

(WBC) count 30000/L, hemoglobin (Hb) 10 g/dl,

hematocrit (Hct) 30%, and platelet (PLT) count

23000/mm 3 . Peripheral blood smear (PS) showed

leukocytosis and the presence of 90% myeloblastic

cells, which were peroxidase- and periodic acid-

Schiff-(PAS)-negative. Biochemical parameters

were within normal limits except lactate dehydrogenase

(LDH) of 788 U/L. Bone marrow aspiration

and biopsy specimen were consistent with the diagnosis

of B-cell ALL showing 90% myeloblastic cells

infiltration. Hoelzer protocol phase I treatment was

started [9]. At diagnosis, the karyotype obtained

from the bone marrow revealed trisomies of chromosomes

X and 5 with a Ph chromosome and was

reported as 48,XY,+X,+5,t(9;22)(q34;q11) (Figure 1).

In the meantime, molecular BCR/ABL fusion gene

was detected by molecular analysis. Regarding the

Ph positivity, 400 mg/day imatinib-mesylate combination

was added to his chemotherapy regimen.

After continuing the treatment for three months, he

was hospitalized due to deterioration in his condition

and bone pain. Bone marrow aspiration showed

more than 90% peroxidase- and PAS-negative blastic

infiltration. Since a very high level of BCR/ABL

fusion gene expression was detected by reverse

transcription-polymerase chain reaction (RT-PCR),

he was considered to be relapsed Ph (+) ALL, and

IDA-FLAG protocol (idarubicin 12 mg/m 2 /day 3

days, fludarabine 30 mg/m 2 /day 5 days, AraC 2 g/m 2

5 days) was started. At the time of relapse, cytogenetic

analysis from his bone marrow aspiration

material showed a full karyotype of

48,XY,+X,+5,t(9;22)(q34;q11), the same as at the

time of diagnosis. However, physical examination

was not consistent with the features of Klinefelter

syndrome, and the cytogenetic evaluation of his

peripheral blood showed a normal karyotype.

Failure to achieve complete remission led us to add

another tyrosine kinase inhibitor, dasatinib (70 mg/

day), to the treatment protocol. After one month of

the treatment, his bone marrow aspiration showed

4% blastic infiltration with normal immunohistochemical

staining and flow cytometry. During follow-up,

cytogenetic analysis of bone marrow aspiration

material revealed a normal karyotype, indicating

a complete cytogenetic remission. He was

considered to be in remission, and he is still under

IDA-FLAG and dasatinib treatment. Written informed

consent was obtained from the patient's family.

Discussion

Trisomy 5 usually occurs in a high-hyperdiploid

karyotype or secondary to structural abnormalities,

in particular t(9;22)(q34;q11.2), t(12;21)(p13;q22)

and t(1;19)(q23;p13). Those conditions are more

Durmaz et al.

Turk J Hematol 2010; 27: 299-302 ALL case with 48,XXY,+5,t(9;22)(q34;q11) 301

A

1

2

3

4 5

6

7

8

C

9 10

11

12

13

14

15

16

17

18

19

20

21

22

B

X

Y

Figure 1. The karyotype of the patient obtained from bone marrow, showing trisomy 5 (A), an extra chromosome X (B) and the Philadelphia

chromosome (C)

frequent than the existence of sole or primary chromosomal

abnormality [7]. The mechanism of trisomy

5, or any other chromosomal aneuploidy, in the

pathogenesis of leukemogenesis remains unclear.

Simple gene-dosage effect and the duplication of a

mutation are considered to be two possible results

of the gain of a whole chromosome [6]. It has been

previously reported that dasatinib, multi-targeted

tyrosine kinase inhibitor of BCR-ABL and SRC family

kinases, plays an important role in the effective

treatment of imatinib-resistant or intolerant

Ph-positive ALL patients [10]. In our patient, dasatinib

treatment resulted in complete remission. After

treatment, bone marrow aspiration biopsy showed

normal immunohistochemical staining, flow cytometry

and cytogenetic results. Although the influence

of additional aberrations on the result of the treatment

with tyrosine kinase inhibitors in Ph-positive

ALL requires large patient series, we think that our

patient demonstrates a pioneer achievement during

the course of ALL management.

Genes affected by chromosomal alterations tend

to be involved in the pathways that control cell

growth or development. Functional studies of these

gene products may reveal the complex mechanisms

playing a role in the pathogenesis of leukemogenesis

and may help in the design of new

therapeutic strategies.






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Niemann - Pick disease associated with

hemophagocytic syndrome

Hemofagositik sendrom ve Niemann-Pick hastal birliktelii

Serap Karaman 1 , Nafiye Urganc 2 , Günsel Kutluk 3 , Feyzullah Çetinkaya 3

1Department of Pediatric Hematology, Sisli Etfal Training and Research Hospital, Istanbul, Turkey

2Department of Pediatric Gastroenterology, Sisli Etfal Training and Research Hospital, Istanbul, Turkey

3Clinics of Pediatrics, Sisli Etfal Training and Research Hospital, Istanbul, Turkey

Abstract

Hemophagocytic lymphohistiocytosis (HLH) is a disease characterized by phagocytosis of blood cells

by macrophages within the lymphoreticular tissue. It can develop secondary to some diseases or be

familial as a result of genetic mutations. Niemann-Pick disease (NPD) is a very rare lipid storage disease.

A three-month-old girl presented with high fever (39°C), abdominal distension and paleness. The

parents were consanguineous. The liver and spleen were palpable 10 cm and 11 cm below the costal

margins, respectively. Bicytopenia (Hb: 5.5 g/dl, platelet: 77000/mm 3 ), hypertriglyceridemia (351 mg/

dl), hyperferritinemia (>1500 ng/dl) and hypofibrinogenemia (120 mg/dl) were detected. Bone marrow

aspiration demonstrated foam cells and hemophagocytosis by macrophages and Niemann-Pick

cells. Lysosomal sphingomyelinase activity was 0.24 nmol/h/mg/protein (normal: 0.86-2.8). Due to the

parents’ refusal of further evaluation, the nature of HLH as primary or secondary could not be determined.

To the best of our knowledge, this is the first case of NPD associated with HLH and the first

demonstration of hemophagocytosis by Niemann-Pick cells. (Turk J Hematol 2010; 27: 303-7)

Key words: Niemann-Pick disease, hemophagocytic syndrome, hemophagocytosis by Niemann-

Pick cell, hemophagocytosis, hemophagocytic lymphohistiocytosis, HLH

Received: May 19, 2010 Accepted: July 26, 2010

Özet

Hemofagositik lenfohistiositoz (HLH), lenforetiküler dokudaki makrofajlarn kan hücrelerini fagosite

etmesiyle karakterize olan, genetik olarak mutasyonlara bal ailevi veya baz hastalklara ikincil

gelien bir hastalktr. Nieman-Pick hastal ise bir lipid depo hastal olup son derece nadir görülmektedir.

Üç aylk kz hasta yüksek ate (39°C), karn ilii ve solukluk nedeni ile bavurdu.

Ebeveynler arasnda ikinci derecede akrabalk vard. Karacier 10 cm, dalak 11 cm ele geliyordu.

Hastada bisitopeni (Hb: 5,5 gr/dl, trombosit: 77000/mm 3 ), hipertrigliseridemi (351 mg/dl), hiperferritinemi

(>1500 ng/dl), hipofibrinojenemi (120 mg/dl) tesbit edildi. Kemik ilii aspirasyonunda köpük

hücresi, makrofajlar ve Niemann-Pick hücreleri tarafndan yaplan hemofagositoz görüldü. Lizozomal

Address for Correspondence: M.D. Serap Karaman, Yeni Mahalle Derya Sokak No: 9/4, Küçükçekmece, stanbul, Turkey

Phone: +90 212 624 46 07 E-mail: drkaramans@yahoo.com

doi:10.5152/tjh.2010.54

304

Karaman et al.

Hemophagocytosis and Niemann-Pick Turk J Hematol 2010; 27: 303-7

sfingomyelinaz aktivitesi 0.24 nmol/hr/mg/ptn (normal:0.86-2.8) bulundu. Hastadaki HLH’nin primer

veya sekonder olduu, ailenin tetkiki kabul etmemesi nedeni ile incelenemedi. Olgumuz literatürde

Niemann-Pick ile birlikte HLH’nin saptand ilk vakadr ve Niemann-Pick hücresinin de hemofagositoz

yapt ilk kez gösterilmitir. (Turk J Hematol 2010; 27: 303-7)

Anahtar kelimeler: Niemann-Pick Hastal, hemofagositik sendrom, Niemann-Pick hücresinin

hemofagositozu, hemafagositoz, hemofagositik lenfohistiositoz, HLH

Geli tarihi: 19 Mays 2010 Kabul tarihi: 26 Temmuz 2010

Introduction

Niemann-Pick disease (NPD) is an autosomal

recessive metabolic disorder characterized by

sphingomyelin accumulation in certain tissues. Six

subtypes of NPD have been described: type A-acute

neuronopathic form, type B-visceral form, type

C-chronic neuronopathic form, type D-Nova Scotia

variant, which is mostly prevalent in the Nova Scotia

region of west Canada, type E - adult form, and type

F-Sea Blue histiocyte disease. All subtypes are

inherited as autosomal recessive traits and display

variable clinical features. Foam-cell infiltration and

visceromegaly are common features in all forms,

but neurologic involvement occurs only in types A

and C and not in type B. Type A is the most common

type and occurs in infants. It is characterized by

jaundice, an enlarged liver, and profound brain

damage. Children with this type rarely live beyond

18 months. The clinical presentation and course in

patients with type B disease are variable. The condition

is diagnosed in most patients in infancy or

childhood when enlargement of the liver, spleen, or

both is detected during routine physical examination.

At diagnosis, patients with NPD type B also

have evidence of mild pulmonary involvement, usually

detected as a diffuse reticular or finely nodular

infiltration on chest radiography. Foam-cell infiltration

and visceromegaly are common features in all

forms, but in types A and B, sphingomyelinase

enzyme activity is deficient, whereas in types C and

D, enzyme levels are almost normal [1-4].

Hemophagocytic lymphohistiocytosis (HLH) is

characterized by sustained fever, spleen enlargement,

cytopenias, hypertriglyceridemia and/or

hypofibrinogenemia, hyperferritinemia, decrease or

absence of natural killer (NK) cell activity, elevated

levels of soluble CD 25, and hemophagocytosis in

bone marrow, spleen and lymph node [5,6]. This

syndrome may be primary, as a familial form, or it

may develop secondary to viral, bacterial, fungal

and parasitic infections, and collagen vascular,

rheumatic and malignant diseases [7,8].

We report the case of a three-month-old girl suffering

from pallor and abdominal distension. She had

lipid-laden foamy cells and hemophagocytosis in the

bone marrow simultaneously. To our knowledge, this

is the first concomitant occurrence of two separate

diseases: HLH associated with Niemann-Pick.

Case Report

A three-month-old girl with a complaint of progressive

abdominal distension and agitation was

admitted to our hospital. According to the family

history, she was delivered by cesarean section in

the 38 th gestational week following a normal pregnancy,

weighing 3200 g. During the neonatal period,

she had no problems such as jaundice or feeding

difficulties but her family noted pallor and abdominal

distension after the first month. Her parents

were second-degree relatives and she was their first

child. She had no lost siblings but two uncles of the

patient died from an unknown disease in the first

three months of life.

On the initial physical examination, her weight

and height were in the 50 th percentile. She was

pale, agitated, tachypneic (respiration rate: 50/min)

and tachycardic (heart rate: 140/min, rhythmic and

no murmur). She also had mild jaundice and

marked hepatosplenomegaly, which caused

abdominal distension; the liver was firmly enlarged

10 cm below the right costal margin, and the spleen

was palpated 11 cm below the left margin.

Initial biochemical investigations revealed the

following: hemoglobin 6.3 g/dl, hematocrit 18.8%,

white blood cell 9950/mm 3 , neutrophil 4200/mm 3 ,

platelet count 103000/mm 3 , mean corpuscular volume

(MCV) 72 fl, red cell distribution width (RDW)

16%, mean corpuscular hemoglobin concentration

(MCHC) 32 g/dl, alanine transaminase 142 U/L,

aspartate transaminase 346 U/L, total bilirubin 3 mg/

dl, conjugated bilirubin 2.29 mg/dl, and serum lactate

dehydrogenase (LDH) 2005 U/L levels were

elevated. Renal function tests and electrolyte levels

were in normal limits, but serum albumin level was

Karaman et al.

Turk J Hematol 2010; 27: 303-7 Hemophagocytosis and Niemann-Pick 305

2.2 g/dl. Clotting tests were mildly deranged.

Prothrombin activity was 56%, international normalized

ratio (INR) was 4.2 and activated partial thromboplastin

time (aPTT) was 38.2 sec. Alpha-1 antitrypsin,

hepatitis B markers, anti-HAV (hepatitis A

virus), immunoglobulin M, and TORCH (toxoplasmosis,

rubella, cytomegalovirus, and herpesvirus)

immunoglobulin M values were interpreted as negative.

Blood, urine and throat culture were also

negative. Peripheral blood smear demonstrated

normal red and white blood cell morphology; there

were no atypical lymphocytes, and platelets were

clustered. The chest radiograph revealed bilateral

reticulonodular infiltration of the lungs. Abdominal

ultrasonography revealed enlarged liver and spleen

with increased echogenicity in the parenchyma.

Diffuse ascites was also noted. A bone marrow aspiration

showed foamy macrophages with significantly

vacuolated cytoplasm, called Niemann-Pick

cells. These findings led us to NPD, and the diagnosis

was confirmed by the measurement of the lysosomal

sphingomyelinase activity: 0.24 nmol/h/mg/

protein (normal: 0.86-2.8). During the follow-up, the

patient had lethargy and high fever four days later.

On physical examination, her general appearance

was not well, and her rectal temperature was 39°C.

Laboratory studies revealed the following: hemoglobin

5.5 g/dl, white blood cell 3300/mm 3 , platelets

77000/mm 3 , serum ferritin (>1500 ng/ml) and triglycerides

(351 mg/dl) were significantly elevated

and fibrinogen concentration was 120 mg/dl.

Repeated bone marrow aspiration showed

Niemann-Pick cells (Figures 1-4) and macrophages

(Figures 5, 6) with marked erythrophagocytic and

Figure 1. Lipid storaged macrophage and erytrocyte hemophagocytosis

Figure 3. Lipid storaged macrophage, erythrocyte and thrombocyte

hemophagocytosis

Figure 2. Lipid storaged macrophage and erytrocyte hemophagocytosis

Figure 4. Lipid storaged macrophage, erythrocyte and thrombocyte

hemophagocytosis

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Hemophagocytosis and Niemann-Pick Turk J Hematol 2010; 27: 303-7

Figure 5. Erythrocyte and thrombocyte hemophagocytosis

thrombophagocytic activity. Hemophagocytic syndrome

associated with NPD was considered based

on these findings. The criteria of the Histiocyte

Society were used for the diagnosis of HLH [7]. We

could not study the NK cell activity or levels of CD25,

but our case fulfilled the other criteria.

Treatment was initiated with intravenous immunoglobulin

(IVIG) 0.8 mg/kg, and transfusions of

fresh frozen plasma and erythrocyte suspension

were administered. We also decided to proceed

with HLH-2004 protocol as immunosuppressive

therapy. However, the parents declined further

evaluation and treatment, so the patient was discharged

without any treatment while clinical findings

of the disease continued. It was learned that

the patient died 10 days after the diagnosis.

Written informed consent was obtained from the

patient's family.

Discussion

Niemann-Pick disease (NPD) refers to a group of

inherited metabolic disorders known as the leukodystrophies

or lipid storage diseases, in which

harmful quantities of a fatty substance (lipids) accumulate

in the spleen, liver, lungs, bone marrow, and

the brain. This lipid storage disorder results from the

deficiency of a lysosomal enzyme, acid sphingomyelinase.

NPD is a very rare autosomal recessive

disease. Our patient had marked hepatosplenomegaly,

reticulonodular infiltration in the lungs,

foam-cell infiltration in the bone marrow, and

sphingomyelinase enzyme deficiency, but she had

Figure 6. Erythrocyte and thrombocyte hemophagocytosis

no neurologic findings, so we assumed that she was

more likely to be NPD type B [1,2].

On the other hand, hemophagocytic syndrome is

a mostly fatal condition of severe hyperinflammation

caused by the uncontrolled proliferation of

activated lymphocytes and histiocytes secreting

high amounts of inflammatory cytokines. As mentioned

previously, HLH may develop secondary to

viral, bacterial, fungal and parasitic infections, or

collagen vascular, rheumatic and malignant diseases.

It can also occur in the course of metabolic diseases.

Although an association between amino acid

metabolism disorders such as lysinuric protein

intolerance and HLH was reported, there is no

documented case in the literature about HLH secondary

to a lipid storage disease [9,10].

Both primary and secondary HLH are more common

in our country than elsewhere [11-14]. As the

disease shows autosomal recessive inheritance, it is

probable that the present case, with parental consanguinity,

might have had a primary HLH.

Unfortunately, the patient died within a short period

of time. Due to the parents’ refusal of further evaluation,

it could not be determined whether the HLH

was primary or secondary in this patient. The

patient was lost within 10 days most probably

because of HLH since she did not receive chemotherapy.

Although early deaths can also be seen in

some types of NPD, the death of the patient immediately

following the onset of high fever led us to

believe that the cause may have been due to HLH.

Persistence of severe disease despite IVIG therapy is

consistent with primary HLH since most of the secondary

HLH cases except in some disorders such as

Karaman et al.

Turk J Hematol 2010; 27: 303-7 Hemophagocytosis and Niemann-Pick 307

infectious mononucleosis show a good response to

IVIG therapy [14]. On the other hand, in the present

case, the consanguinity of the parents, early death

within 10 days with HLH and other infant deaths in

the family may suggest the presence of familial

HLH. If this case had a familial HLH, there should be

a severe mutation. In previous studies from our

country, it was reported that the perforin gene

W374X mutation was responsible for familial HLH

cases presenting within six months of life. This is

also the most common mutation in our country and

leads to death within a very short time after the presentation

of the disease [11]. Since the genetic

analysis could not be done, we can not comment

on whether this case is a Niemann-Pick associated

with familial HLH or a secondary case. In the present

study, it is interesting that Niemann-Pick cells

also exhibited hemophagocytosis. As far as we

know, phagocytosis by Niemann-Pick cells has not

been described until now.

In conclusion, NPD and HLH are within the group

of rare diseases. It is not surprising to diagnose this

association, since both are autosomal recessive disorders

in our country where consanguineous marriages

are frequent. Clinicians must be aware of HLH

developing in NPD. Clinical and laboratory findings of

Niemann-Pick such as hepatosplenomegaly and

cytopenia may mask the diagnosis of HLH.






References

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Behrman R, Jenson H, editors. Nelson Textbook of

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3. Watts RWE. Lysosomal storage disease. In: Weatherall

DJ, Warrell DA, editors. Oxford Textbook of Medicine,

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5. Lipton J. Histiocytosis syndromes. In: Lanzkowsky P,

editor. Manual of Pediatric Hematology and Oncology,

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8. Gurgey A, Secmeer G, Tavil B, Ceyhan M, Kuskonmaz B,

Cengiz B, Ozen H, Kara A, Cetin M, Gumruk F. Secondary

hemophagocytic lymphohistiocytosis in Turkish children.

Pediatr Infect Disease J 2005;24:1116-7.

9. Doireau V, Fenneteau O, Duval M, Perelman S, Vilmer

E, Touati G, Schlegel N, Ogier de Baulny H. Lysinuric

dibasic protein intolerance: characteristic aspects of

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10. Duval M, Fenneteau O, Doireau V, Faye A, Emilie D,

Yotnda P, Drapier JC, Schlegel N, Sterkers G, de Baulny

HO, Vilmer E. Intermittent hemophagocytic lymphohistiocytosis

is a regular feature of lysinuric protein

intolerance. J Pediatr 1999;134:236-9.

11. Balta G, Okur H, Unal S, Yarali N, Gunes AM, Unal S,

Turker M, Guler E, Ertem M, Albayrak M, Patiroglu T,

Gurgey A. Assessment of clinical and laboratory presentations

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patients with homozygous W374X mutation. Leuk

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12. Gurgey A, Unal S, Okur H, Orhan D, Yurdakok M.

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2008;30:871-6.

13. Okur H, Balta G, Akarsu N, Oner A, Patiroglu T, Bay A,

Sayli T, Unal S, Gurgey A. Clinical and molecular

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308 Case Report

Autoimmune hemolytic anemia and giant cell

hepatitis: Report of three infants

Otoimmun hemolitik anemi ve dev hücreli hepatit birliktelii olan üç

olgunun sunumu

ule Ünal 1 , Bar Kukonmaz 1 , Necati Balamtekin 2 , Gökhan Baysoy 2 , Selin Aytaç

Elmas 1 , Diclehan Orhan 3 , Gülsev Kale 3 , Aysel Yüce 2 , Figen Gürakan 2 , Fatma Gümrük 1 ,

Mualla Çetin 1

1Division of Pediatric Hematology, Hacettepe University Faculty of Medicine, Ankara, Turkey

2Division of Pediatric Gastroenterology, Hacettepe University Faculty of Medicine, Ankara, Turkey

3Division of Pediatric Pathology, Hacettepe University Faculty of Medicine, Ankara, Turkey

Abstract

Giant cell hepatitis associated with direct Coombs’ test-positive hemolytic anemia is a rare condition of

childhood and the pathogenesis remains unclear. An autoimmune activation and loss of self-tolerance in

these patients may be the underlying pathology related to the response of some of the patients to immunosuppressive

treatment. Herein, we report the clinical presentation and course of three consecutive patients

with this rare condition. We conclude that serum ferritin at diagnosis may be used for prediction of the

outcome. (Turk J Hematol 2010; 27: 308-13)

Key words: Giant cell hepatitis, immune hemolytic anemia, direct Coombs’ test, rituximab

Received: May 5, 2010 Accepted: August 19, 2010

Özet

Direk Coombs’ testi pozitif hemolitik anemi ve dev hücreli hepatit birliktelii çocukluk çanda patogenezi

tam olarak aydnlatlmam nadir bir durumdur. Bu hastalarn bir ksmnn immünosupresif tedaviye

cevap veriyor olmas otoimmün aktivasyon ya da kendi antijenlerine kar tolerans kaybnn patogenezde

sorumlu olabileceini düündürmektedir. Bu makalede bu nadir duruma sahip üç hastann bavuru

özellikleri ve takibi sunulmutur. Bavuru anndaki serum ferritin deerinin hastann prognozunu belirlemede

kullanlabilecei önerilmitir. (Turk J Hematol 2010; 27: 308-13)

Anahtar kelimeler: Dev hücreli hepatit, immün pozitif hemolitik anemi, direk Coombs’ testi, rituximab

Geli tarihi: 5 Mays 2010 Kabul tarihi: 19 Austos 2010

Address for Correspondence: M.D. ule Ünal, Hacettepe University, Division of Pediatric Hematology 06100, Ankara, Turkey

Phone: +90 312 305 11 70 E-mail: suleunal@hacettepe.edu.tr

doi:10.5152/tjh.2010.55

Ünal et al.

Turk J Hematol 2010; 27: 308-13 Autoimmune hemolytic anemia-giant cell hepatitis 309

Introduction

Giant cell hepatitis (GCH) associated with direct

Coombs’ test-positive autoimmune hemolytic anemia

(AIHA) is a rare, often lethal condition of early

childhood with unknown pathogenesis. GCH-AIHA

association is a distinct disorder from the more

common GCH diagnosed in the neonatal period in

association with infections, metabolic disorders

and cholestasis [1-4]. Management of these patients

is a challenge, carries poor response to immunosuppressive

therapy and often progresses to fatal

liver disease [1,5]. Tissue autoantibodies have been

negative in most cases; however, these patients

usually fail to respond to orthotopic liver transplantation

compared with other types of GCH found in

children and have a high rate of GCH recurrence in

the transplanted liver. This may suggest the presence

of an as-yet undetermined circulating autoantibody

[1,2,6].

To our knowledge, 24 cases of GCH with AIHA

have been reported [1-17]. The AIHA usually precedes

the diagnosis of GCH by 1 week to 15 months

(usually 1-2 months), and the hepatitis presents

more insidiously [5,11]. Herein, we present the

clinical presentation and course of three consecutive

patients with this rare condition with possible

implications as to the etiopathogenesis.

Case Reports

Table 1 summarizes the clinical and laboratory

characteristics of the study group.

Case 1

A two-month-old girl, the first child of parents

with first-degree consanguinity, who presented with

pallor was found to have direct Coombs’ test-positive

hemolytic anemia and was placed on steroid

treatment (2-10 mg/kg/day) and intravenous immunoglobulin

(1 g/kg/day, for 2 days; 3 courses) in

another center. The patient was transiently responsive

to attacks; however, she was found to have

cytomegalovirus (CMV) IgM and CMV polymerase

chain reaction (PCR) positivity (8800 copy/ml) at

eight months of age and was placed on ganciclovir

treatment. By the 14 th day of antiviral treatment, she

developed hypertransaminasemia and was referred

to our center for further evaluation.

Physical examination revealed a jaundiced girl

with body weight <3 rd percentile and height 50-75 th

percentile. Liver and spleen were palpable 4 cm

and 2 cm below the costal margins, respectively.

The hemogram revealed hemoglobin (Hb): 10.3 g/

dl, hematocrit (Hct): 28.9%, white blood cells

(WBC): 31.4x10 9 /L, thrombocyte: 244x10 9 /L, mean

corpuscular volume (MCV): 94.1 fl, and red cell distribution

width (RDW): 26.3. Reticulocytes were

8.6% and peripheral blood smear exhibited polychromasia,

spherocytosis, anisocytosis, poikilocytosis,

and normoblastemia. The liver function tests

revealed aspartate aminotransferase (AST): 2147

(<37 IU/L), alanine aminotransferase (ALT): 2117

(<41 IU/L), gamma-glutamyl transpeptidase (GGT)

83.3 (5-36 IU/L), alkaline phosphatase (ALP): 949

(<281 IU/L), total bilirubin: 62.7 (0.1-1.2 mg/dl), and

conjugated bilirubin 62.1 (0-0.3 mg/dl). Serum IgA

level was 116 mg/dl (8-80), IgG: 774 mg/dl (220-900)

and IgM: 140 mg/dl (35-125). Viral serologies for

hepatitis A, hepatitis B and hepatitis C were negative.

CMV IgM and IgG were positive and CMV PCR

was 951 copy/ml. Funduscopic examination for

CMV infection was negative. Ganciclovir was continued

until the result of the liver biopsy was available,

which revealed giant cell transformation, portal

fibrosis, focal hepatocyte necrosis, lymphocytic

infiltration, and hemosiderin accumulation in hepatocytes

and Kupffer cells, without any evidence of

CMV involvement. The patient was diagnosed to

have GCH-AIHA condition, and anti-nuclear antibody,

anti-smooth muscle antibody, anti-liver kidney

microsomal antibody, and anti-cardiolipin and

anti-phospholipid antibodies were found to be

negative. Initial serum ferritin was measured as

32078 ng/ml, and subsequently dropped to 4858 ng/

ml by the second week of admission. Serum triglyceride

was 547 (<200 mg/dl), cholesterol 132 (<200

mg/dl) and plasma fibrinogen 360 mg/dl. The bone

marrow aspiration revealed bi- and tri-nucleated

normoblasts indicating erythroid hyperactivity and

dyserythropoiesis with increased histiocytes without

any evidence of hemophagocytosis.

Treatment was continued with 2 mg/kg methylprednisolone,

and azathioprine (1 mg/kg) was initiated,

in addition to cholestyramine for cholestasis

and omega-3 for hyperlipidemia. She developed

pneumonia with ground-glass appearance on thorax

computed tomography and CMV positivity

310

Ünal et al.

Autoimmune hemolytic anemia-giant cell hepatitis Turk J Hematol 2010; 27: 308-13

recurred; ganciclovir was re-initiated. During the

course, hepatic failure progressed with portal hypertension

and ascites, and rituximab (anti CD20) was

initiated at 375 mg/m 2 /week, concomitant to ganciclovir.

The patient died after the 3 rd dose of rituximab

treatment, with intervening sepsis and hepatic

coma, at 17 months of age.

Case 2

A six-month-old boy presented to our center with

pallor and jaundice. The personal history revealed a

similar admission to a local hospital at the first

month of life. He was thought to have prolonged

jaundice and was transfused with packed red blood

cells, and a second admission to the same hospital

at three months of age with dark stools revealed

disturbed liver function tests and prolonged coagulation

tests. Antibiotics, fresh frozen plasma and

vitamin K were initiated and the patient was referred

to our center at six months of age because of refractory

illness.

The patient was the only child of a first-degree

consanguineous couple, without history of any sibling

death. The physical examination revealed a

jaundiced boy with hepatosplenomegaly, palpable

8 cm and 6 cm below the costal margins, respectively.

Body weight and height were 3-10 p. The

hemogram revealed Hb: 9 g/dl, Hct: 24.7%, WBC:

6.7x10 9 /L, thrombocyte: 102x10 9 /L, MCV: 87.5 fl,

and RDW: 16.6. Reticulocyte count was 4.2%. Direct

Coombs’ test was positive. The liver function tests

revealed AST: 309 IU/L, ALT: 92 IU/L, GGT: 29.9 IU/L,

total bilirubin: 25.5 mg/dl, direct bilirubin 15.7 mg/dl,

total protein: 5.86 g/dl, and albumin: 3.67 g/dl.

Activated partial thromboplastin time (aPTT) and

prothrombin time (PT) (international normalized

ratio [INR]) were 46.6 sec (27.9-38.1) and 1.64 (0.86-

1.2), respectively. Serum IgA was 287 mg/dl (8-80),

IgG: 1100 mg/dl (220-900) and IgM: 408 mg/dl (35-

125). Viral serologies for hepatitis A, hepatitis B and

hepatitis C were negative. CMV IgM and IgG were

positive and CMV PCR was 330 copy/ml, and ganciclovir

was initiated. Funduscopic examination for

CMV infection was normal. The liver biopsy revealed

giant cell formation with canalicular and hepatocellular

cholestasis, without any evidence of CMV

involvement. Immunohistochemical examination

with MDR3 staining excluded the diagnosis of progressive

familial intrahepatic cholestasis type 3. The

patient was diagnosed to have GCH-AIHA condition,

and anti-nuclear and anti-liver kidney microsomal

antibodies were found to be negative. Serum ferri-

Table 1. Characteristics of patients with GCH and AIHA

Case 1 Case 2 Case 3

Onset of AIHA (mo) 2 1 11

Onset of hepatitis (mo) 8 3 15

CMV status + + -

Ganciclovir therapy + + -

Serum 32078, 325 93

ferritin (ng/ml) (4858)*

Treatment Methylprednisolone Ursodeoxycholic Methylprednisolone

IVIG acid Azathioprine

Azathioprine Cholestyramine Rituximab

Rituximab

Ursodeoxycholic acid

Cholestyramine

Omega-3

Additional problem High serum IgA High serum IgA, Transient

and IgM levels IgG and IgM levels; hypogammaglobulinemia

G6PDH deficiency

of infancy

Outcome Died at 17 mo Died at 8 mo Alive at 35 mo

* Initial serum ferritin was measured as 32078 ng/ml and subsequently dropped to 4858 ng/ml by the second week of admission.

AIHA: Autoimmune hemolytic anemia; mo: month; CMV: Cytomegalovirus; IVIG: Intravenous immunoglobulin; G6PDH: Glucose 6-phosphate dehydrogenase.

Ünal et al.


tin was measured as 325 ng/ml. Interestingly, the

patient was found to have additional glucose 6-phosphate

dehydrogenase deficiency. He was placed on

ursodeoxycholic acid and cholestyramine and was

discharged for close follow-up; however, it was

learned that he died in the 2 nd month of discharge

in a local hospital.

Case 3

An 11-month-old male presented initially to

another center with pallor and was found to have

Hb of 4.1 g/dl with 4+ direct Coombs’ test. It was

learned that methylprednisolone 10 mg/kg/day had

been initiated and the dose was tapered off subsequently;

however, he had two more attacks during

periods of dose reduction of steroid treatment and

needed doses as high as 20 mg/kg/day for hemolysis

control. At 15 months of age, while on 2 mg/kg/

day methylprednisolone treatment, he developed

vomiting and diarrhea and presented to our center.

The family history was unremarkable excluding the

second- degree consanguinity between parents. He

had three healthy older siblings. The physical examination

revealed a jaundiced boy with liver palpable

2 cm subcostally on the midclavicular line. The

hemogram revealed Hb: 7.9 g/dl, Hct: 23.1%, WBC:

12.1x10 9 /L, thrombocyte: 147x10 9 /L, MCV: 93.7 fl,

and RDW: 18.6. Reticulocytes were 4%. Direct

Coombs’ test was positive. The liver function tests

revealed AST: 770 IU/L, ALT: 1225 IU/L, GGT: 141

IU/L, total bilirubin: 3.95 mg/dl, direct bilirubin 2.36

mg/dl, total protein: 5.31 g/dl, and albumin: 3.33 g/

dl. aPTT and PT (INR) were 30.0 sec (25-40) and

0.96 (0.75-1.5), respectively. Serum haptoglobulin

level was <5.83 mg/dl (36-195). Viral serologies for

Epstein-Barr virus (EBV), hepatitis A, hepatitis B,

hepatitis C, hepatitis E, human immunodeficiency

virus (HIV) and parvovirusB19 were negative,

whereas the patient was immune for hepatitis B

related to prior immunization, and he was IgM

negative and IgG positive for CMV infection. Serum

ferritin was 93 ng/ml. Anti-mitochondrial, antismooth

muscle and anti-liver kidney microsomal

antibodies were negative. Serum IgA, IgG and IgM

were measured as 81 mg/dl (30-107), 420 mg/dl

(605-1430) and 96 mg/dl (66-228), respectively, and

the low IgG level for age was considered as transient

hypogammaglobulinemia of infancy.

Ursodeoxycholic acid was initiated and steroid

dose was increased up to 10 mg/kg/day. Liver biopsy

revealed fibrosis in the portal areas, giant cell

transformation of hepatocytes, intracellular cholestasis,

sinusoidal dilatation, and focal areas of

extramedullary hematopoiesis, with no evidence of

hemosiderin or copper accumulation. Azathioprine

was initiated in addition to steroid treatment. During

the course, because of the frequent attacks of

Coombs’-positive hemolytic anemia, rituximab (375

mg/m 2 /week, for four weeks) was started at the 6th

month of azathioprine, at the age of 24 months.

Steroid was gradually tapered off and azathioprine

was continued. This patient is currently alive, free of

transfusion, with negative direct Coombs’ test and

normal liver function tests, after a follow-up of 18

months and completion of four weekly doses of

rituximab.

Written informed consent was obtained from

patients.

Discussion

Although GCH-AIHA association was reported

more commonly among infants, there are rare

reports of late-onset cases, such as in an 18-year-old

male who survived after orthotopic liver transplantation

[3]. In our series of cases, the patients presented

between 1 and 11 months of age with AIHA,

which preceded hepatitis by 2-6 months. Case 1

and 3 received immunosuppressive treatment prior

to the development of overt hepatitis, which could

not prevent the development of the subsequent disease.

The presence of consanguinity between parents

of all three patients may indicate an underlying

inherited immunological defect predisposing these

children to this peculiar association.

Giant cell hepatitis (GCH) associated with AIHA

has been reported to have mortality rates as high as

approximately 50% [13], and this high mortality has

been attributed not only to the severe liver failure or

uncontrollable anemia, but also to sepsis related to

the aggressive use of immunosuppressive drugs

[6,8,13,17]. As our cases and the literature data indicate,

although this condition carries a high mortality,

the clinical spectrum shows some diversity in

severity and may respond to steroid alone or in

combination with azathioprine [9,14,15]. However,

there are other reports in which the cases were

unresponsive to immunosuppressive therapies and

312

Ünal et al.

Autoimmune hemolytic anemia-giant cell hepatitis Turk J Hematol 2010; 27: 308-13

required orthotopic liver transplantation. The results

of liver transplantation also show variable outcomes,

including very early (even within weeks)

recurrence of the disease in the transplanted liver

[13,17] as well as survival after transplantation [2,3].

The post-transplant survivors may also have benefited

from the immunosuppressants used during the

post-transplantation period.

Recently, there have been promising results with

monoclonal antibodies including rituximab or alemtuzumab

therapy, which may decrease the requirement

for transplantation [1,4,5]. The good results

with rituximab and recurrence of disease after liver

transplantation in the transplanted liver may indicate

a circulating as-yet undetermined antibody in

these patients. Additionally, the serum immunoglobulin

abnormalities in our study group may also

indicate an immune dysregulation in these patients.

All three of our patients were negative for the autoimmune

markers. Two of the patients were CMV

PCR-positive and received ganciclovir treatment;

however, no accompanying viral agent could be

demonstrated in Case 3. This finding may indicate

an autoimmune activation and loss of self-tolerance

in these patients related to some infections.

Camerero Salcés et al. [16] also reported associated

CMV infection with this condition.

The presence of high conjugated bilirubinemia,

in addition to a less prominent unconjugated bilirubinemia

in these patients may be attributed more to

the hepatitis than hemolytic anemia in these

patients. Additionally, the unconjugated bilirubin

may have caused inspissated bile syndrome, which

could also explain the conjugated bilirubinemia in

these patients.

Both Cases 1 and 3 had received less than 10

packed erythrocyte transfusions prior to serum ferritin

measurement and liver biopsy. The serum ferritin

level was extremely high in Case 1 (Table 1)

and the liver biopsy also exhibited hemosiderin

accumulation. The rapid decline in serum ferritin

from 32078 ng/ml to 4858 ng/ml after intensification

of immunosuppressive treatment may indicate that

the high serum ferritin level in this patient reflects

the severity of inflammation. Hyperferritinemia may

also be related to the prior transfusions and may

also exacerbate the underlying hepatitis in the presence

of iron accumulation in the liver. The serum

ferritin level of Case 3 was 93 ng/ml, without any

evidence of hemosiderin accumulation in the liver

biopsy, and this patient was the only one to survive.

Serum ferritin was measured as 325 ng/ml in Case

2, without any evidence of iron accumulation in the

liver. The previously reported case of Akyildiz et al.

[3] was reported to have a pre-transplantation

serum ferritin level of 3236 ng/ml and their patient

survived after liver transplantation. These findings

may indicate that lower serum ferritin levels can

predict the responsiveness to immunosuppressive

treatment, and as a consequence, the better outcome

in some patients; it may also explain the

clinical heterogeneity of the GCH-AIHA. Case 1, who

had very high serum ferritin levels, had a poor

course in spite of immunosuppression, whereas

Case 3, who had lower initial serum ferritin levels,

responded well to treatment. However, Case 2, who

had a moderate increase in ferritin, did not have the

opportunity to receive immunosuppressive treatment;

thus, it is not possible to draw a conclusion

about the relationship between his serum ferritin

and the treatment response-outcome. Additionally,

CMV infection might also have contributed to the

higher serum ferritin levels and poorer outcomes in

Cases 1 and 2, compared to the CMV-negative Case

3. While the sample size is limited for making a

definitive conclusion, if this observation is confirmed

with further studies with increased numbers

of patients, serum ferritin may be used to predict

which patients may benefit from immunosuppressives

and those which may require more aggressive

approaches, including earlier transplantation. This

would also help to decrease the deaths related to

the prolonged use of immunosuppressives.






References

1. Gorelik M, Debski R, Frangoul H. Autoimmune hemolytic

anemia with giant cell hepatitis: case report and

review of the literature. J Pediatr Hematol Oncol

2004;26:837-9.

2. Melendez HV, Rela M, Baker AJ, Ball C, Portmann B,

Mieli-Vergani G, Heaton ND. Liver transplant for giant

Ünal et al.


cell hepatitis with autoimmune hemolytic anemia.

Arch Dis Child 1997;77:249-51.

3. Akyildiz M, Karasu Z, Arikan C, Nart D, Kilic M.

Successful liver transplantation for giant cell hepatitis

and Coombs-positive hemolytic anemia: a case report.

Pediatr Transplant 2005;9:630-3.

4. Rovelli A, Corti P, Beretta C, Bovo G, Conter V, Mieli-

Vergani G. Alemtuzumab for giant cell hepatitis with

autoimmune hemolytic anemia. J Pediatr Gastroenterol

Nutr 2007;45:596-9.

5. Miloh T, Manwani D, Morotti R, Sukru E, Shneider B,

Kerkar N. Giant cell hepatitis and autoimmune hemolytic

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6. Perez-Atayade AR, Sirlin SM, Jonas M. Coombs-positive

autoimmune hemolytic anemia and post-infantile giant

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7. Hartman C, Berkowitz D, Brik R, Arad A, Elhasid R,

Shamir R. Giant cell hepatitis with autoimmune hemolytic

anemia and hemophagocytosis. J Pediatr

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8. Kehrli ME Jr, Goff JP, Stevens MG, Boone TC. Coombs

positive giant cell hepatitis—a new feature of Evans’

syndrome. Arch Dis Child 1998;78:397-8.

9. Weinstein T, Valderrama E, Pettei M, Levine J. Early

steroid therapy for the treatment of giant cell hepatitis

with autoimmune hemolytic anemia. J Pediatr

Gastroenterol Nutr 1993;17:313-6.

10. Imgrueth M, Wagner HP, Pipczynski-Suter K, Beguin L,

Wyss M, Pfister AM, Stock HK, Oetliker O.Plasma

exchange: an important part of the therapeutic procedure

in a small child with autoimmune hemolytic

anemia. Acta Paediatr Scand 1986;75:1037-41.

11. Kashyap R, Sarangi JN, Choudhry VP. Autoimmune

hemolytic anemia in an infant with giant cell hepatitis.

Am J Hematol 2006;81:199-201.

12. Choulot JJ, Parent Y, Etcharry F, Saint-Martin J, Mensire

A. Giant cell hepatitis and autoimmune hemolytic anemia:

efficacy of splenectomy on hemolysis. Arch

Pediatr 1996;3:789-91.

13. Vajro P, Migliaro F, Ruggeri C, Di Cosmo N, Crispino G,

Caropreso M, Vecchione R. Life saving cyclophosphamide

treatment in a girl with giant cell hepatitis and

autoimmune haemolytic anaemia: case report and upto-date

on therapeutical options. Dig Liver Dis

2005;38:846-50.

14. Bernard O, Hadchouel M, Scotto J, Odièvre M, Alagille

D. Severe giant cell hepatitis with autoimmune hemolytic

anemia in early childhood. J Pediatr 1981;99:

704-11.

15. Brichard B, Sokal E, Gosseye S, Buts JP, Gadisseux JF,

Cornu G. Coombs-positive giant cell hepatitis of infancy:

effect of steroids and azathioprine therapy. Eur J

Pediatr 1991;150:314-7.

16. Camarero Salcés C, Vasconez Muñoz F, San José

Hernando D, Morales P, Suárez Cortina L, Parra Vázquez

C, Escobar Castro H, Muñoz Villa A. Autoimmune

hemolytic anemia associated with severe giant cell

hepatitis. An Esp Pediatr 1991;34:149-51.

17. Pappo O, Yunis E, Jordan JA, Jaffe R, Mateo R, Fung J,

Demetris AJ. Recurrent and de novo giant cell hepatitis

after orthotopic liver transplantation. Am J Surg Pathol

1994;18:804-13.

314 Letter to the Editor

Mega-dose methylprednisolone (MDMP) for

chronic idiopathic myelofibrosis

Kronik idiopatik miyelofibrosis için yüksek doz metilprednizolon (YDMP)

inasi Özsoylu

To the Editor,

I would like to make few comments on Balc and

her colleagues’ paper entitled “Complex cytogenetic

findings in the bone marrow of a chronic idiopathic

myelofibrosis patient” which was published in the

recent issue of the Journal. [1].

Although the authors’ cytogenetic findings looks

original, it should be considered that chronic idiopathic

myelofibrosis (CIF) is a colonal disorder like

chronic myeloid leukemia (CML), polycythemia

vera, etc.

Among them only Philadelphia chromosome

(Ph1) is specific for CML, but mutations are present

in all of these clonal disorders. I believe since the

cytogenetic changes are not pathognomic or specific,

these kind of studies should be carried only on

research basis. In addition to radiation as mentioned

by the authors makes the interpretation of

their findings more questionable.

On this occasion, I would specifically like to

emphasize the MDMP treatment for these patients

which seems to be the only approach in the treatment

of this disorder. In addition to some stem cell

transplantation reports, MDMP is cheap, safe and

easily applicable in all conditions.

Although we first reported patient with CIF in

1957 from this country [2], we treated almost a

dozen of CIF cases without major complications in

children and adults since 1983 [3-13]. MDMP treatment

was mentioned in several occasions in this

journal, we sorry that despite of its international

consideration, except ITP treatment,my colleagues

did not take it seriously [14].

References

1. Balc TB, Yüksel MK, Ylmaz Z, ahin FI. Complex cytogenetic

findings in the bone marrow of a chronic idiopathic

myelofibrosis patient. 2010;27:113-6.

2. Erman M, Özsoylu S. Sur un cas d’anemie splenique

erythromyeloide de l’adulte. La Presse Medicale

1957;65:1309-11.

3. Özsoylu , Ruacan . High dose bolus methylprednisolone

treatment for primary myelofibrosis. Eur J Pediatr

1983;140:810.

4. Özsoylu , Ruacan . High-dose intravenous corticosteroid

treatment in childhood idiopathic myelofibrosis.

Acta Haematol 1986;75:49-51.

5. Özsoylu . Myelofibrosis with myeloid metaplasia.

Blood 1986;67:253.

6 Özsoylu . High-dose intravenous methylprednisolone

for idiopathic myelofibrosis. Lancet 1988;1:766.

7. Özsoylu . High-dose intravenous methylprednisolone

for idiopathic myelofibrosis. Brit J Haematol

1988;70:388-9.

8. Özsoylu . Mega dose methylprednisolone (MDMP)

treatment. Turk J Pediat 2004;46:292.

9. Özsoylu . Treatment of primary myelofibrosis. Turk J

Hematol 2009;26:211-2.

10. Özsoylu . High dose intravenous methylprednisolone

(HIVMP) in hematologic disorders. Hematology

Reviews 1990;4:197-207.

Address for Correspondence: M.D. inasi Özsoylu, Altunehir Sitesi No.30, Beysukent, Ankara, Turkey

Phone: +90 312 203 55 55 E-mail: sinasiozsoylu@hotmail.com

doi:10.5152/tjh.2010.56

Özsoylu et al.

Turk J Hematol 2010; 27: 314-5 MDMP for chronic idiopathic myelofibrosis 315

11. Özsoylu . Myelofibrosis in children. Pediatr Hematol

Oncol 1994;11:337-40.

12. Özsoylu . Myelofibrosis. Acta Hematol 1995;94:218.

13. Megadose metilprednizolon; resyonal kortikosteroid

kullanm (Eds.Y Karaaslan, A Kadayfç) 2004;70:70-4.

14. Bernini JC, Carrillo JM, Buchanon GR. High-dose intravenous

methylprednisolone therapy for patient with

Diamond-Blackfan anemia refractory to conventional

doses of prednisone. J Pediat 1995;127:654-9.

Reply

Dear Editor,

We read the comments of Dr. Özsoylu about our

recently published case report titled “Complex cytogenetic

findings in the bone marrow of a chronic

idiopathic myelofibrosis patient”. We would like to

thank Dr. Özsoylu very much for reminding us the

importance of especially megadose methyl prednisolon

for the treatment of different hematologic

diseases.

Idiopathic myelofibrosis that is seen in adults is

an incurable disease without allogeneic stem cell

transplantation [1,2]. Many kinds of therapies such

as chemotherapeutics [3,4] including busulfan,

hydroxyurea, chlorambucil, 2-chlorodeoksiadenosamine;

biologic response modifier regimens [5];

corticosteroids, anabolic steroids [5,6]; thalidomide

[7, 8], lenalidomide [9] have been used but none of

them has changed the biology of the disease and

survival rates of patients [10]. Since there is no optimal

therapy, the choice of the drug depends on the

patient’s characteristics and the physician’s experience.

Hydroxyurea was the drug of choice by the

doctor who first saw him. When we met the patient,

he was refractory to that therapy and had a very

painful large spleen. Because of his comorbid conditions,

splenic irradiation had been used to palliate

his symptoms. The response was transient, as

expected, and after a short period of time he died

due to progressive disease.

As we have mentioned in the case report, most

patients with primary myelofibrosis have karyotypic

abnormalities at diagnosis and some of them have

been associated with an adverse prognosis. A correlation

between the presence of multiple chromosomal

deletions and poor survival has been reported

[10]. The reason for the development of new

chromosomal abnormalities in this patient is a difficult

question to answer. As you have mentioned it

may be the radiation he received or the immunologic

stimuli or the chemical agents and/or industrial

solvents he had been exposed [10]. Whatever

the reason, we have shown the abnormality and

wanted to share this new knowledge with our colleagues

studying in the same area.

References

1. Deeg HJ, Gooley TA, Flowers ME, Sale GE, Slattery JT,

Anasetti C, Chauncey TR, Doney K, Georges GE, Kiem HP,

Martin PJ, Petersdorf EW, Radich J, Sanders JE, Sandmaier

BM, Warren EH, Witherspoon RP, Storb R, Appelbaum

FR. Allogeneic hematopoietic stem cell transplantation

for myelofibrosis. Blood. 2003;102:3912-8.

2. Rondelli D, Barosi G, Bacigalupo A, Prchal JT, Popat U,

Alessandrino EP, Spivak JL, Smith BD, Klingemann HG,

Fruchtman S, Hoffman R; Myeloproliferative Diseases-

Research Consortium. Blood.2005;105:4115-9.

3. Lofvenberg E, Wahlin A,Roos G, Ost A.Reversal of

myelofibrosis by hydroxyurea. Eur JA

Haematol.1990;44:33-8.

4. Faoro LN, Tefferi A, Mesa RA. Long term analysis of the

palliative benefit of 2-chlorodeoxyadenosine for myelofibrosis

with myeloid metaplasia. Eur J Haematol.

2005;74:117-20.

5. Barosi G, Hoffman R. Idiopathic myelofibrosis. Semin

Hematol.2003; 42:248-58.

6. Cervantes f, Hernadez- Boluda JC, Alvarez A, Nadal E,

Montserrat E. Danazol treatment of idiopathic myelofibrosis

with severe anemia. Haematologica.2000;85-59 5-599.

7. Mesa RA, Steensma DP, Pardanani A, Li CY, Elliott M,

Kaufmann SH, Wiseman G, Gray LA, Schroeder G,

Reeder T, Zeldis JB, Tefferi A. A phase 2 trial of combination

low dose thalidomide and prednisone for the treatment

of myelofibrosis with myeloid metaplasia. Blood

2003; 101:2534-41.

8. Barosi G, Grossi A, Comotti B, Musto P, Gamba G,

Marchetti M. Safety and efficacy of thalidomide in

patients with myelofibrosis with myeloid mataplasia.Br

J Haematol.2001; 114:78-83.

9. Tefferi A, Cortes J, Verstovsek S, Mesa RA, Thomas D,

Lasho TL, Hogan WJ, Litzow MR, Allred JB, Jones D,

Byrne C, Zeldis JB, Ketterling RP, McClure RF, Giles F,

Kantarjian HM. Lenalidomide therapy in myelofibrosis

with myeloid metaplasia. Blood 2006; 108:

1158-64.

10. Hoffman R,Ravandi-Kashandi F. diopathic myelofibrozis.

In: Hoffman R, Benz EJ Jr, Shattil SJ, et al eds.

Hematology: Basic Principles and Practices. 4th edition.

Philedelphia, PA: Elsevier Scientific; 2005:1255-1257.

Corresponding author:

M.D. Feride Iffet Sahin,

Department of Medical Genetics, Baskent University

Faculty of Medicine, Kubilay Sokak No:36 06570,

Maltepe Ankara, Turkey

Tel: +90 312 232 44 00 - 302

Fax: +90 312 231 91 34


Pancreatitis in a child with acute lymphoblastic

leukemia after Erwinia asparaginase:

Evaluation of ultrasonography and

computerized tomography as diagnostic tools

Akut lenfoblastik lösemili bir çocukta Erwinia asparaginazdan sonra

olan pankreatit: Tan araçlar olarak ultrasonografi ve bilgisayarl

tomografi ile deerlendirme

Fatih Mehmet Azk 1 , Gonca Klç 2

1Division of Pediatric Haematology, Dkap Children’s Hospital, Ankara, Turkey

2Department of Pediatrics, Dkap Children’s Hospital, Ankara, Turkey

To the Editor,

Acute pancreatitisis is a well described

complication of L-asparaginase therapy [1,3].

Complications include hemorrhage, pseudocyst

formation, pancreatic insufficiency, sepsis, and

respiratory distress due to pulmonary edema or

pleural effusion [2,5]. An 8 year old boy with ALL,

who was on St. Jude Total XIII-high risk continuation

therapy, tolerated E-coli asparaginase treatments

well until he developed anaphylaxis reaction during

E-coli asparaginase administration at 8 th week of

continuation therapy. According to protocol the

next L-asparaginase treatment was four weeks

later. We switched E-coli asparaginase to Erwinia

asparaginase, and he received vincristine, and

prednisone at the 12 th week of continuation

treatment. One week after first Erwinia asparaginase

administration the patient was admitted to hospital

with vomiting, diffuse abdominal pain and

decreased oral intake. Physical examination

revealed a pale, ill appearing child in acute distress

with a diffuse sensitivity of upper abdomen but no

rebound tenderness or guarding. Laboratory

findings showed an amylase level of 477 mg/dl

(normal <125 mg/dl), pancreatic lipase 292 U/L

(0-60 U/L), pancreatic amylase 81 U/L (17-115 U/L).

The most possible diagnosis was drug induced

pancreatitis. Repeated laboratory evaluation

showed a gradual increase in the serum amylase to

775 mg/dl by hospital day two. On the second day

of admission, ultrasonography of the abdomen

showed the pancreas was diffusely enlarged and

heteroechogen. Although hypoechoic texture is

expected in pancreatitis, ultrasonographic

evaluation was heteroechogen in our patient. On

the third day of admission abdominal computed

tomography (CT) study was done to explain

heteroechogenity in ultrasonography. Computed

tomography scanning with intravenous contrast

Address for Correspondence: M.D. Fatih Mehmet Azk, Division of Pediatric Haematology, Dkap Children’s Hospital, Ankara, Turkey

Phone: +90 312 317 99 89 E-mail: mfatihazik@yahoo.com

doi:10.5152/tjh.2010.57

Azk et al.

Turk J Hematol 2010; 27: 316-7 Pancreatitis after Erwinia asparaginase 317

was normal. Initial management included

nasogastric tube placement with suctioning, bowel

rest, and analgesics. Intravenous hyper-alimentation

was also begun. The following day serum amylase

level was decreased to 172 mg/dl. The patient was

discharged home on hospital day seven, tolerating

a regular diet, free of abdominal pain, and with an

amylase of 128 mg/dl. The rest of the L-asparaginase

treatment was omitted. Imaging techniques confirm

the diagnosis of pancreatitis, and sometimes

identify the cause, and also assess complications

such as pseudocyst [3]. Among the most useful and

frequently used are ultrasound and CT. The two

major sonographic findings are increased

pancreatic size and decreased pancreatic

echogenecity [4]. However, a normal gland can be

observed in mild cases [3]. Changes in pancreatic

size, contour, and echotexture are appreciated best

with ultrasonography, as is the presence of dilated

ducts, pseudocysts, abscesses, ascites and

associated gallstone disease. While pancreatic

enlargement and hypoechogenicity are usually

diagnostic of acute pancreatitis, because of the

variability of the size of the normal pancreas,

enlargement is often difficult to assess. Thus,

abnormality is based more often on the relative

echogenicity of the pancreas rather than its size.

On the other hand, the pancreas in normal children

is equal or more echodense than the left lobe of the

liver owing to the increased fibro fatty content of

the pancreas [4]. Computed tomography often is

used to help manage the complication of

pancreatitis, such as providing guidance in the

aspiration and drainage of an abscess, phlegmon,

or pseudocyst or prior to surgical intervention.

Computed tomography scanning is gaining favor

despite the common notion that CT is not useful in

children due to their having less retroperitoneal fat

[5,6]. It is not clear how soon the full extent of the

necrotic process will occur, but it is at least four

days after the onset of symptoms and early CT may

therefore under estimate the final severity of the

disease [7]. Kearney et al, reported 39% of children

with clinical and chemical pancreatitis did not have

abnormalities detected by ultrasound; therefore,

the diagnosis should not be dependent on

radiographic confirmation. There was a high

concordance between ultrasound and CT in

detecting abnormalities, and so it may be reasonable

to reserve the use of CT for those patients with

persistence or worsening of clinical symptoms or

laboratory findings in the setting of non-diagnostic

ultrasonography [8]. While ultrasound findings

were compatible with acute pancreatitis, abdominal

CT (on the third day of admission) in our patient

was normal. This might be related with mild

pancreatitis or having less retroperitoneal fat in

children.

Written informed consent was obtained from

the patients.






References

1. Underwood TW, Frye CB. Drug-induced pancreatitis.

Clin Pharm 1993;12:440-8.

2. Eden OB, Shaw MP, Lilleyman JS, Richards S. Nonrandomised

study comparing toxicity of Escherichia

coli and Erwinia asparaginase in children with leukaemia.

Med Pediatr Oncol 1990;18:497-502.

3. Elmas N. The role of diagnostic radiology in pancreatitis.

Eur J Radiol 2001;38:120-32.

4. Cox KL, Ament ME, Sample WF, Sarti DA, O’Donnell M,

Byrne WJ. The ultrasonic and biochemical diagnosis

of pancreatitis in children. J Pediatr 1980;96: 407-11.

5. Mader TJ, McHugh TP: Acute pancreatitis in children.

Pediatr Emerg Care 1992;8:157-61

6. Ziegler DW, Long JA, Philippart AI, Klein MD.

Pancreatitis in childhood. Experience with 49 patients.

Ann Surg 1988;207:257-61.

7. Beger HG, Bittner R, Block S, Büchler M. Bacterial

contamination of pancreatic necrosis. A prospective

clinical study. Gastroenterology 1986;91:433-8.

8. Kearney SL, Dahlberg SE, Levy DE, Voss SD, Sallan SE,

Silverman LB. Clinical course and outcome in children

with acute lymphoblastic leukemia and asparaginase-associated

pancreatitis. Pediatr Blood Cancer.

2009;53:162-7.


Thrombosis and risk factors

Tromboz ve risk faktörleri

Nejat Akar

TOBB Economy and Technical University Hospital, Ankara, Turkey

To the Editor,

I read the three papers related to thrombosis,

appeared in the recent issue of the journal with

great interest [1-3].

Yoku et al. described their patients with cerebral

vein thrombosis and evaluated the risk factors for

thrombosis. Especially they emphasize the MTHFR

677 C-T alteration. They stated that none of the

MTHFR homozygote had high homocysteine levels.

And also the two patients with high homocysteine

levels did not have 677 T polymorphism [1]. lhan et

al’s patient was heterozygous for 677 T with normal

homocysteine level [2]. These reports indicated a

very interesting point.

Yoku et al. stated that association between the

MTHFR 677 T polymorphism and vascular disease is

a matter of debate. Their data does not support the

common view that TT genotype of MTHFR 677 CT is

an influencing factor on homocysteine levels.

Recently, we reported that MTHFR 677 T has an

influence on homocysteine levels in our population

but also we found another possible MTHFR 677 TT

haplotype, which does not have an effect on homocysteine

levels [4].

These reports and our data indicated a very

important point. Only homocysteine levels should

be routinely analyzed and not the MTHFR 677 T

polymorphism.

Further, Uz et al. reported a patient with portal

vein thrombosis (PVT) secondary to Klebsiella oxytoca

bacteriemia [3]. They omitted Prothrombin

20210A mutation and FVIII levels of their patient. It

was hypothesized that evaluation of these two risk

factors in PVT is needed [5-8]. Although the patient

is septic and FVIII may increase during infection, it

would be an interesting finding. Moreover, although

PT mutation is present mainly in cirrhotic patients

[5] and not present in endemic areas of PVT like

India [9,10], it worth analyzing both risk factors in

Turkish population with PVT.


Author of this paper has no conflict of interest,




References

1. Yoku O, Balçk Ö, Albayrak M, Ceran F, Dada S,

Ylmaz M,Özet G. Evaluation of risk factors for thrombophilia

in patients with cerebral venous thrombosis.

2010;27:162-7.

Address for Correspondence: M.D. Nejat Akar, Koru Mah. 2621. Sokak Uyum Sitesi 18 Çayyolu, 06100, Ankara,Turkey

Phone: +90 312 241 39 80 E-mail: nejatakar@hotmail.com

doi:10.5152/tjh.2010.58

Akar et al.

Turk J Hematol 2010; 27: 318-9 Portal vein thrombosis secondary to bacteriemia 319

2. Gül lhan, Selami K. Toprak, Neslihan Andç, Sema

Karaku. Idiopathic thrombocytopenic purpura with

venous thrombosis: A case report. 2010;27:209-10.

3. Uz E, Alc Ö, Balçk Ö, Kanbay M, Ik A, Uz B, Kaya

A, Koar A. Portal vein thrombosis secondary to

Klebsiella oxytoca bacteriemia. 2010;27:213-5.

4. Koç YL, Akar N. Single nucleotide polymorphisms that

affect homocysteine levels in Turkish population. Clin

Appl Thromb Hemost. 2009;15:701-4.

5. Erkan O, Bozdayi AM, Disibeyaz S, Oguz D, Ozcan M,

Bahar K, Karayalcin S, Ozden A, Bozkaya H, Yurdaydin

C, Uzunalimoglu O. Thrombophilic gene mutations in

cirrhotic patients with portal vein thrombosis. Eur J

Gastroenterol Hepatol. 2005;3:339-43.

6. Hirmerova J, Liska V, Mirka H, Chudacek Z, Treska V.

Portal and mesenteric vein thromboses in a patient

with prothrombin G20210 mutation, elevated lipoprotein

(a), and high factor VIII. Clin Appl Thromb Hemost.

2008;14:481-5.

7. Koshy A, Jeyakumari M. High FVIII level is associated

with idiopathic portal vein thrombosis in South India.

Am J Med. 2007; 120:552.e9-11.

8. Akar N. Idiopathic portal vein thrombosis and FVIII

levels. Thromb Res. 2007;120:141.

9. Sharma S, Kumar SI, Poddar U, Yachha SK, Aggarwal R.

Factor V Leiden and Prothrombin gene G20210A mutations

are uncommon in portal vein thrombosis in India.

Indian J Gastroenterol. 2006;25:236-9.

10. Koshy A, Jeyakumari M. Prothrombin G20210A gene

variant is not associated with idiopathic portal vein

thrombosis in an area endemic for portal vein thrombosis.

Ann Hematol. 2006;85:126-8.


Laboratory diagnosis of acute leukemia in Iraq,

the available options

Irak'ta akut löseminin laboratuvar tans, uygun seçenekler

Abbas Hashim Abdulsalam

Hematology Unit, Teaching Laboratories Department, al-Yarmouk Teaching Hospital, Iraq

To the Editor,

Diagnosis of acute leukemia in Iraq is mainly

dependent on the personal experience of the laboratory

physician. Local guidelines in this field were

never proposed and the international guidelines are

very difficult to apply as the only available techniques

include morphology of peripheral blood and

bone marrow specimens plus very limited immunohistochemistry

CD markers and PCR testing for

BCR-ABL oncogene only, therefore the aim for diagnosis,

classification and subclassification of acute

leukemia in this country should be that of diagnosis

and lineage assignment that serves a clear therapeutic

goal.

Having been working in the field of laboratory

hematology since 2003 in the major teaching hospitals

in Baghdad, I found that the following scheme

is the available useful option:

Acute leukemia should be classified on the basis

of FAB group, but using a cut-off point of 20% blast

cells, as proposed in the WHO classification [1].

Acute myeloid leukemia

With Romanowsky stain morphology AML- M2,

M3, M4, M5b and M6 can be recognized readily.

By adding few special stains such as Sudan black

B (SBB) [2] (and not myeloperoxidase as SBB has a

little more sensitivity in detecting myeloblasts which

is the crucial point), plus a non-specific esterase

stain as -naphthyl acetate esterase it becomes

possible to recognize AML-M1 and most cases of

AML-M5a [3].

The AML cases that cannot be distinguished by

morphology and cytochemistry, specifically M0 and

M7, for which the presence of myeloid dysplasia in

the former and the cytoplasmic blebs in the latter

may give a hint for the probable diagnosis, however

there is still the need for more positive diagnostic

technique and as the flow cytometry immunophenotyping

is not available then the use of a limited

number of CD markers study by immunohistochemistry

to identify the lineage of acute leukaemia

is the option, these include CD33, anti-myeloperoxidase

and CD41.

Rare types of AML like M5c require high degree

of morphology experience, in which malignant

cells appearance is reminiscent of tissue histiocytes

[4].

There is still a small proportion of cases that

would be only certainly diagnosed after the response

to treatment as in rare forms of AML-M3v [5].

Address for Correspondence: M.D. Abbas Hashim Abdulsalam, Iraq-Baghdad-al-Yarmouk Teaching Hospital 964 Baghdad, Iraq

Phone: 964 7904 188690 E-mail: dr.abbas77@yahoo.com

doi:10.5152/tjh.2010.59

Abbas Hashim Abdulsalam.

Turk J Hematol 2010; 27: 320-1 Laboratory diagnosis of acute leukemia 321

Acute lymphoblastic leukemia

Consideration of clinical as well as hematological

features permits a strong presumptive diagnosis

of ALL.

Morphologically if a case of acute leukaemia has

the cytological feature of ALL-L1 then it is highly

likely that it does represent ALL [6]. Also if a patient

with an acute leukemia showing heterogeneous

blasts that has no morphological or cytochemical

markers of myeloid differentiation with unavailability

of further differentiating procedures then it may

be treated as ALL-L2, as statistically speaking it

would be much more possible than AML-M0 [7].

ALL-L3 diagnosis would be obvious by morphology

alone.

The negative result in staining with SBB is very

helpful, also the addition of the special stain PAS

would improve the chances of the correct diagnosis

of ALL.

Clinical setting may presumptively aid in differentiating

between B- and T- ALL, however, using

immunohistochemistry antibodies including CD79a

for B lineage and CD3 for T lineage are necessary.

Rare cases of ALL-L2 that are confused with leukemic

phase of large cell lymphoma can be differentiated

through the use of TdT immunohistochemistry

typing on bone marrow biopsy slide, which

would be positive in ALL but not in lymphoma.

After setting the diagnosis of B-ALL, having performed

immunohistochemistry CD20 typing and

ordering PCR for BCR-ABL fusion gene would affect

the treatment options.

Conclusion

In Iraq it is essential at this time where the diagnostic

resources are very limited to establish guidelines

that are simple and practical in developing

cost-effective diagnostic protocols for conditions for

which the treatment is available, plus leaving the

door wide open for future improvements, as the

introduction of newer techniques and added procedures

to the already available ones once a newer

therapeutic agent has been introduced.

Also it is always a realistic option to seek a more

precise diagnosis with genetic study and lineage

specification outside this country for those who can

afford it.






References

1. Barbara JB, David MC, Irvin A, Bridget SW. Bone marrow

pathology. 3rd ed. Blackwell science, 2001.

2. Abbas HA, Nafila S. Unusually large granules in M1

AML. Slide atlas, BloodMed, British Society of

Haematology; Blackwell publishing, 2009.

3. Barbara JB. Blood cell, a practical guide. 4th ed.

Blackwell publishing, 2006.

4. Abbas HA, Nafila S. Acute myeloid leukemia with histiocytic

differentiation. American Society of Hematology

image bank. 2009;9:80.

5. Abbas HA, Nafila S. Acute promyelocytic leukaemia.

Slide atlas, BloodMed, British Society of Haematology;

Blackwell Publishing, 2009.

6. Barbara JB. Leukaemia diagnosis. 3rd ed. Blackwell

publishing, 2003.

7. Abbas HA: Chemotherapeutic trial for acute leukemia

in Iraq. Letter to the editor. Turkish Journal of

Hematology. 2009;264:216.


The incidence of alpha-thalassemia in Setif, Algeria

Cezayir, Setif'te alfa-talasemi skl

Ayenur Öztürk 1 , Bakhouche Houcher 2 , Nejat Akar 1

1Department of Pediatric Genetics, Ankara University Faculty of Medicine, Ankara, Turkey

2Department of Biology, Faculty of Sciences, University of Setif, Setif, Algeria

To the Editor,

Thalassemias are hereditary anemia syndromes

occurring due to erroneous producing of globin

chain of hemoglobin. Thalassemia syndromes are

named according to the type of the affected globin

chain. The most common types of thalassemia are

alpha thalassemia and beta thalassemia. In

-thalassemia, -globin chain production is either

by decreased or completely disappeared. The gene

encoding the -globin chain localized on the short

arm of chromosome 16 (16p13.3). In normal adults,

alpha-globin genes are found in four copies. One

copy of each 1 and 2 genes are located in cis

position on DNA chain [1]. The alpha-globin gene

deletions constitute 80-90% of the mutations. The

carriers for -thalassemia have either three (-/)

or two (--/) genes. The most common clinical

form is HbH disease and the most frequently

encountered genotype of Hb H disease is --/- and

rarely Hb Bart’s hydrops fetalis in which all four

genes are deleted. In Mediterranean region, - 3.7 ,

- 4.2 , -- MED and - 20.5 deletions are the most prevalent

molecular defects [2].

The mutations of -thalassemia have been

reported as - 3.7 , -- MED , - 20.5 and Hph I in Algerian

population [3,4] and - 3.7 was determined as the

most frequent haplotype [3]. In a recent study, in

addition to these mutations, Nco I was shown and

-thal allele frequency found 4.6%, with the - 3.7

haplotype being 2.9% in randomly selected blood

donors in Algiers, the capital city of Algeria which is

located at the Mediterranean Sea coast [5].

On the other hand, Setif province is located in

the high plateau of north-east Algeria, approximately

100 kilometers from the Mediterranean Sea. This

is the first study for the molecular characterization

of the -thal gene frequency in healthy individuals

from the Setif region.

The study group included 153 unrelated healthy

individuals from Setif. Informed consent was

obtained from all the participants. Blood samples

were collected with EDTA-containing tubes, transferred

and DNA was extracted from peripheral

blood leukocytes according to phenol-chloroform

method. Genomic DNA was tested for the - 3.7 ,

- 4.2 , -- MED and - 20.5 deletions using multiplex-polymerase

chain reaction (PCR) according to described

methods [6,7].

The prevalence of alpha thalassemia trait was

found to be 6.5% in the study group. The molecular

characterization of the -thal defects in these subjects

revealed - 3.7 allele frequency as 3.3%. We

have not found any other individual of carrying

Address for Correspondence: M.D. Nejat Akar, Koru Mah. 2621.Sok. Uyum Sitesi 18 Çayyolu, 06100, Ankara,Türkiye

Phone: +90 312 241 39 80 E-mail: akar@medicine.ankara.edu.tr

doi:10.5152/tjh.2010.60

Öztürk et al.

Turk J Hematol 2010; 27: 322-3 Alpha-Thalassemia in Setif 323

alpha 4.2 del, MED or 20.5 deletions in our study

group.

The frequency difference between the two

regions can be explained by the location of the two

cities. As Algiers is located at the Mediterranean Sea

coast, it is expected to have high -thal frequency

because of the probable malaria infection. This is

not the case for Setif as it is located far from the

coast.






References

1. Deisseroth A, Nienhuis A, Turner P, Velez R, Anderson

WF, Ruddle F, Lawrence J, Creagan R, Kucherlapati R.

Localization of the human -globin structural gene to

chromosome 16 in somatic cell hybrids by molecular

hybridization assay. Cell 1977;12:205-18.

2. Kattamis AC, Camaschella C, Sivera P, Surrey S, Fortina

P. Human alpha-thalassemia syndromes: detection of

molecular defects. Am J Hematol 1996;53:81-91.

3. Henni T, Morlé F, Lopez B, Colonna P, Godet J.

-Thalassemia haplotypes in the Algerian population.

Hum Genet 1987;75:272-6.

4. Leclerc T, Guetarni D, Bernet A, Colonna P, Godet J,

Morlé F. Identification of three different -thalassemic

haplotypes: -3.7, (--)MED, Hph I in the same

Algerian family. Hum Mutat 1995;5:182-3.

5. Molecular basis of alpha-thalassemia in Algeria.

Mesbah-Amroun H, Rouabhi F, Ducrocq R, Elion J.

Hemoglobin. 2008;32:273-8.

6. Oron-Karni V, Filon D, Oppenheim A, Rund D. Rapid

detection of the common Mediterranean alpha-globin

deletions/rearrangements using PCR. Am J Hematol.

1998;58:306-10.

7. Tan AS, Quah TC, Low PS, Chong SS. A rapid and reliable

7-deletion multiplex polymerase chain reaction

assay for alpha-thalassemia. Blood. 2001;98:250-1.

324 Images in Hematology

FDG-PET in mantle cell lymphoma involving skin

Deri tutulumlu mantle hücreli lenfomada FDG-PET

Mehmet Sönmez 1 , Ümit Çobanolu 2 , Sava Karyaar 3 , Bircan Sönmez 4

1Department of Hematology, School of Medicine, Karadeniz Technical University, Trabzon, Turkey

2Department of Pathology, School of Medicine, Karadeniz Technical University, Trabzon, Turkey

3Department of Nuclear Medicine, Trabzon Numune Training and Research Hospital, Trabzon, Turkey

4 Department of Nuclear Medicine, School of Medicine, Karadeniz Technical University, Trabzon, Turkey

A 61-year-old female patient presents to the hospital

with fatigue and weakness. Physical examination

was unremarkable except for pallor. White blood cell

count (WBC) was 40.500/L with 62% blast.

Hemoglobin (Hgb) level was 7.4g/dl and platelet (Plt)

count was 146.000/L. Bone marrow examination

revealed hypercellular marrow and infiltration with

blast cells (Figure 1). Blastic cells expressed CD45,

CD79a, CD19, CD20, CD5, and cyclin D1 with no

expression of CD23. Other laboratory investigations

including urea, electrolytes and liver function tests

were all in normal limits. No abnormality was detected

by computed tomography (CT) scans of neck,

thorax, abdomen, and pelvis except splenomegaly

(150 mm). Based on the findings, the diagnosis of

blastoid variant of mantle cell lymphoma (MCL) was

established. She received the CHOP chemotherapy

regimen (Cylophosphamide-Doxorub- icin-Prednisone-Vincristine)

combined with rituximab and

achieved complete remission. Ten months later,

under routine follow-up, multiple reddish nodules on

the back and upper extremities, and trunk were

observed (Figure 2). Laboratory parameters, bone

marrow examination, and CT scans were normal.

Skin lesion biopsy revealed MCL (Figure 3).

Fluorodeoxyglucose-positron emission tomography

(FDG-PET) scan, which was performed to determine

the stage of the skin involvement, showed multiple

areas of abnormal uptake in skin (Figure 4).

MCL commonly involves extranodal sites. Skin

involvement occurs in only 2%-6% of all cases of MCL

but is seen in 17% of stage IV patients. The blastoid

types MCL are more aggressive than MCL and associ-

Figure 1. Atypical blastic cell infiltration in the bone marrow

biopsy (inset; cyclin D1 positivity)

Address for Correspondence: M.D. Mehmet Sönmez, Department of Hematology, School of Medicine, Karadeniz Technical University,

61080 Trabzon, Turkey Phone: +90 462 377 58 48 E-mail: mesonmez@yahoo.com

doi:10.5152/tjh.2010.39

Turk J Hematol 2010; 27: 324-5

Sönmez et al.

FDG-PET in mantle cell lymphoma

325

Figure 2. Appearance of reddish nodules on upper extremity

Figure 4. Appearance of multiple abnormal uptake on FDG-PET

Figure 3. Histopathological appearance of mantle cell lymphoma

in the skin biopsy (inset; cyclin D1 positivity)

ated with a worse clinical outcome. Only a few cases

with blastoid variant MCL and skin involvement have

been described in English literature. In MCL, FDG-

PET detects more disease sites, nodal as well as

extranodal, than conventional imaging methods,

resulting in a higher sensitivity [1-3].


patient.






References

1. Hutchings M, Specht L. PET/CT in the management of haematological

malignancies. Eur J Haematol. 2008;80:369-80.

2. Estrozi B, Sanches JA Jr, Varela PC, Bacchi CE. Primary

cutaneous blastoid mantle cell lymphoma-case report.

Am J Dermatopathol. 2009;31:398-400.

3. Motegi S, Okada E, Nagai Y, Tamura A, Ishikawa O. Skin

manifestation of mantle cell lymphoma. Eur J Dermatol.

2006;16:435-8.

326 Images in Hematology

Spinal cord involvement of multiple myeloma

detected by F-18 FDG PET/CT Scan

F-18 FDG PET/CT tarama ile saptanan multiple myeloma’nn spinal

kord tutulumu

lknur Ak Sivrikoz 1 , Havva Üsküdar Teke 2 , Zafer Gülba 2

1Department of Nuclear Medicine, Eskiehir Osmangazi University, School of Medicine, Eskiehir, Turkey

2Department of Haematology, Eskiehir Osmangazi University, School of Medicine, Eskiehir, Turkey

F-18 2-fluoro-2-deoxy-D-glucose positron emission

tomography/computed tomography (F-18 FDG

PET/CT) images of a patient with spinal cord involvement

of light chain (Lambda type) multiple myeloma

(MM) is reported. A 56-year-old man had a

15-months history of light chain MM and have got

out of hand after a chemotherapy regimen with only

two cycles (melphalan, prednisone) was admitted

to hospital because of weakness and lower back

pain. To assess the systemic involvement of disease

F-18 FDG PET/CT scan was performed. PET/CT

images revealed a diffuse F-18 FDG uptake on spinal

cord in the spinal canal along the thoracic 9th,

10th and 11th vertebrae (Fig 1). In addition, there

were multiple active myeloma lesions and bone

marrow involvement. At this time, bone marrow

aspiration biopsy showed 30-50% of atypical plasma

cells with a plasmablastic morphology (CD138 and

CD56 positive by immune staining). Serum lactate

dehydrogenase level was in normal range, alkaline

phosphatase level was high (468 U/L). There was no

plasma cell in peripheral blood smear. A lumbar

puncture revealed a Cerebrospinal Fluid (CSF) content

of 200 nucleated cells/L. Flow cytometric

analysis of CSF confirmed the spinal cord involvement

of MM, the percentage of both CD38 and

CD138 positve cells was %19.46 by flow cytometry

(Fig 2). Although spinal cord involvement, there was

no symptoms related spinal cord involvement such

as limb weakness, paraparesis. To the best of our

knowledge, it is the first report of spinal cord

involvement of MM imaged by F-18 FDG PET/CT.

Extramedullary spread of MM may occur either

at diagnosis or during the course of the disease. The

involvement of central nervous system (CNS) occurs

in approximately1 % of patients [1-3] and may present

either with localized cerebral lesions or with

meningeal myelomatosis, defined by the presence

of monoclonal plasma cells in the CSF. The clinical

presentation of CNS involvement include a wide set

of neurological signs and symptoms, but headache,

confusion, and limb weakness are the most common

[3-5]. However, even in patients without circulating

plasma cells, the haematogenous spread of

their lymphoid progenitors has been postulated as a

possible mechanism for CNS involvement [6].

Active myeloma is FDG-positive for focal and diffuse

abnormalities. It has been reported that FDG

Address for Correspondence: M.D. lknur Ak Sivrikoz, Department of Nuclear Medicine, Eskiehir Osmangazi University, School of

Medicine, Eskiehir, Turkey Phone: +90 222 239 29 79 E-mail: ilknur_ak@yahoo.com

doi:10.5152/tjh.2010.61

Sivrikoz et al.

Turk J Hematol 2010; 27: 326-8 Spinal cord involvement on FDG PET/CT 327

400 600 800 1000

CD38 PE

10 0 10 1 10 2 10 3 10 4

10 0 10 1 10 2 10 3 10 4

CD 138 FITC

200

0

0

200

400 600

FSC-H

800

1000

Quad % Gated X Mean Y Mean

UL 3.11 10.39 1194.91

UR 19.46 144.44 166.16

LL 24.90 3.53 7.18

LR 52.53 281.98 3.06

Figure 1. Three plane PET images (CT, PET and fused PET/CT in axial, sagittal and coronal projections) shows an intense F-18 FDG uptake

with a maximum standard uptake value (SUVmax) of 5.7 in the thoracic 9th, 10th and 11th vertebrae and FDG avid masses in sternum

and left 9 th rib

Figure 2. Flow cytometric analysis of CSF showing CD38 and CD 138 positive cells on upper riggt quadrant of the two-parameter histogram

(%19.46 of the cells)

328

Sivrikoz et al.

Spinal cord involvement on FDG PET/CT Turk J Hematol 2010; 27: 326-8

PET can significantly contribute to an accurate

whole-body evaluation of multiple myeloma

patients due to the ability to visualise highly energyconsuming

cells such as tumour cells [7, 8]. In addition,

the limited anatomical resolution of PET can

be overcome by co-registration of functional PET

images with morphological CT data with an integrated

PET/CT system.


patient.






References

1. Beksaç M, Delforge M, Richardson P. The evolving

treatment paradigm of multiple myeloma: From past

to present and future. Turk J Hematol 2008;25:60-70.

2. Fassas AB, Ward S, Muwalla F, Van Hemert R,

Schluterman K, Harik S, Tricot G. Myeloma of the central

nervous system: strong association with unfavorable

chromosomal abnormalities and other high-risk

disease features. Leuk Lymphoma. 2004;45:291-300.

3. Nieuwenhuizen L, Biesma DH: Central nervous system

myelomatosis: Review of the literature. Eur J Haematol.

2008;80:1-9

4. Petersen S, Wagner A, Gimsing P: Cerebral and meningeal

multiple myeloma after autologous stem cell

transplantation: A case report and review of the literature.

Am J Hematol. 1999;62:228-33.

5. Patriarca F, Zaja F, Silvestri F, Sperotto A, Scalise A, Gigli

G, Fanin R. Meningeal and cerebral involvement in multiple

myeloma patients. Ann Hematol. 2001;80:758-62

6. Warner TF, Krueger RG: Circulating lymphocytes and

the spread of myeloma: Review of the evidence.

Lancet 3;1:1174-6.

7. Lütje S, de Rooy JW, Croockewit S, Koedam E, Oyen

WJ, Raymakers RA. Role of radiography, MRI and FDG-

PET/CT in diagnosing, staging and therapeutical evaluation

of patients with multiple myeloma. Ann Hematol.

2009;88:1161-8.

8. lknur Ak, Zafer Gülba. Nuclear Medicine Applications

in Hemato-Oncology. Turk J Hematol 2003;20:89-211.

Obituary

Prof. Hüseyin Sipahiolu (1925-2010)

A Turkish hematologist, Professor Hüseyin Sipahiolu, passed away September 11, 2010. He

made several contributions to Turkish hematology including filiariasis, thalassemia, G6PD deficiency.

In 1947, he graduated from stanbul University Faculty of Medicine and had his Internal

Medicine residency at Vakf Gureba. He worked at Istanbul University and Kayseri Gevher Nesibe

University Medical School, respectively. He was the dean of Gevher Nesibe Medical School

between 1978-1980 and then he was the rector of Kayseri Erciyes University between 1980-1982.

He retired in 1982. Sipahiolu had over 200 published articles (14 international) and 5 printed

books including his memories.

Nejat Akar, MD,Prof.

Ankara University, Turkey

Advisory Board of This Issue (December 2010)

Akif Yeilipek, Turkey

Alessandro M. Vannucchi, Italy

Ali Turhan, France

Ali Uur Ural, Turkey

Alicia Rovo, Switzerland

Alp Can, Turkey

Ayegül Ünüvar, Turkey

Aytemiz Gürgey, Turkey

Bela Telek, Hungary

Betül Tavil, Turkey

Burhan Turgut, Turkey

Can Boa, Turkey

Celalettin Üstün, USA

Cengiz Beyan, Turkey

Dilber Talia leri, Turkey

Emel Özyürek, Turkey

Emin Kürekçi, Turkey

Erdal Karaöz, Turkey

Evren Özdemir, Turkey

Fahir Özkalemka, Turkey

Fatma Gümrük, Turkey

Feride Duru, Turkey

Ferit Avcu, Turkey

Fevzi Altunta, Turkey

Filiz Vural, Turkey

Görgün Akpek, USA

Güray Saydam, Turkey

Güçhan Alanolu, Turkey

Gülersu rken, Turkey

Hakan Göker, Turkey

Hilmi Apak, Turkey

Insu Kuzu, Turkey

brahim Barta, Turkey

dil Yenicesu, Turkey

nci Alacacolu, Turkey

smet Aydodu, Turkey

Kaan Kavakl, Turkey

Klara Dalva, Turkey

Lale Olcay, Turkey

Mehmet Ali Erkurt, Turkey

Mehmet Ertem, Turkey

Meral Beksaç, Turkey

Mine Hekimgil, Turkey

Muhit Özcan, Turkey

Mustafa Nuri Yenerel, Turkey

Mutlu Arat, Turkey

Muzaffer Demir, Turkey

Namk Özbek, Turkey

Nazan Sarper, Turkey

Nee Yaral, Turkey

Nurdan Taçyldz, Turkey

Önder Arslan, Turkey

Özden Pikin, Turkey

Pervin Topçuoglu, Turkey

Reyhan Diz Küçükkaya, Turkey

Selami Koçak Toprak, Turkey

Sema Anak, Turkey

Sema Karaku, Turkey

ule Ünal, Turkey

Tansu Sipahi, Turkey

Teoman Soysal, Turkey

Tiraje Celkan, Turkey

Türkan Patrolu, Turkey

Ülker Koçak, Turkey

Vefki Gürhan Kadköylü, Turkey

Yener Koç, Turkey

Yurdanur Klnç, Turkey

Yusuf Baran, Turkey

Zahit Bolaman, Turkey

Zühre Kaya, Turkey

Announcements

5-6 February 2011

Turkish School of Hematology 10 - Transfusion Medicine

Antalya

17-21 February 2011

2011 BMT Tandem Meetings

Honolulu Hawaii

24-25 February 2011

13th International Symposium on Febrile Neutropenia

Nice, France

4-6 March 2011

Update on Hematology - Post-Graduate Hematology

Education

Antalya

25-27 March 2011

Diagnosis and Treatment Workshop on Benign Hematology

Antalya

3-6 April 2011

EBMT-European Group for Blood and Marrow

Transplantation

Paris, France

11-14 May 2011

3. International Congress of Leukemia Lymphoma Myeloma

stanbul

18-21 May 2011

17th Annual ISCT Meeting

Rotterdam, Netherlands

18-21 May 2011

11th International Symposium on Myelodysplastic

Syndromes

Edinburgh, Scotland, United Kingdom

3-7 June 2011

2011 ASCO Annual Meeting

Chicago, USA

9-12 June 2011

16th Congress of the European Hematology Association

London, United Kingdom

24-26 June 2011

Turkish School of Hematology 11 – Practical Hemostasis

Thrombosis Laboratory Course 2

Sivas

9-11 September 2011

Turkish School of Hematology 12 – Stem Cell Course

19-22 October 2011

37. Turkish National Hematology Congress

10-13 December 2011

53rd ASH Annual Meeting and Exposition

San Diego, USA

23-25 December 2011

Practical Basic Hematology Laboratory Course 1

27 th Volume Index

27. Cilt Dizini

SUBJECT INDEX - KONU DZN

March 2010 - December 2010

Mart 2010 - Aralk 2010

Acute Leukemia / Myelodysplastic Syndrome / Akut Lösemi /

Miyelodisplastik Sendrom

High-dose methylprednisolone / Yüksek doz metilprednisolon, 1

Differentiation / Farkllama, 1

Apoptosis / Apoptosis, 1

Acute myeloblastic leukemia / Akut myeloblastik lösemi, 1

Myelodysplastic syndrome / Myelodisplastik sendrom, 1

Children / Çocuklar, 1

FLT3 - ITD positive acute lymphocytic leukemia, does it impact on

disease´s course? / FLT3 - ITD pozitif akut lenfositik lösemi hastalnn

gidiatn etkiliyebilir mi?, 133

Stem Cell Transplantation / Thalassemia / Kök Hücre Nakli / Talasemi

Thalassemia major / Talasemi majör, 8

Mixed chimerism / Kark tip kimerizm, 8

Pediatrics / Pediatri, 8

Chimerism / Kimerizm, 8

Hematopoietic stem cell transplantation / Hematopoetik kök hücre

transplantasyonu, 8

Non-malignant / Non-malign, 8

Hypercoagulability / Molecular Hematology / Hiperkoagülabilite /

Moleküler Hematoloji

Lower FXII activity is not a risk factor / Düük FXII aktivitesi bir risk

faktörü olmamakla birlikte, 15

It simply represents a risk marker for thrombosis / yalnzca tromboza

yönelik bir risk göstergesini temsil etmektedir, 15

Infection / Chemotherapy

Immunocompromised patients / mmunsupresif hasta, 20

Hospital-acquired pneumonia / Hastane kökenli pnömoni, 20

Survival / Sürvi, 20

Laboratory Medicine

Splenomegaly / Splenomegali, 25

Ultrasonography / Ultrasonografi, 25

Prevalence of accessory spleen / Aksesuar dalak skl, 25

Lymphomas / Lenfomalar

Non-Hodgkin’s lymphoma / Hematolojik hastalklar, 29

Bone / Kemik, 29

Lymphoma / Lemfoma, 29

Primary cutaneous diffuse large B-Cell lymphoma, leg type / Primer

kutanöz diffüz büyük B hücreli lenfoma, bacak tipi, 46

Primary uterine lymphoma of the uterine cervix in advanced age / leri

yata uterus serviksinde oluan primer uterus lenfomas, 219

Infection / Acute Leukemia / Enfeksiyon / Akut Lösemi

Leukemia / Lösemi, 34

Pediatric / Pediatrik, 34

Chest pain / Göüs ars, 34

Fungal infection / Fungal enfeksiyon, 34

Pneumothorax / Pnömotoraks, 34

Hemophagocytosis / Bone marrow failure / Hemafagositoz / Kemik lii

Yetmezlii

Hemophagocytosis / Hemofagositoz, 38

Aplastic anemia / Aplastik anemi, 38

Hepatitis / Hepatit, 38

Acute Leukemia / Akut Lösemi

Granular ALL / Granüler ALL, 43

Basophilic leukemia / Bazofilik lösemi, 43

Toluidine blue / Toluidin mavisi, 43

Azurophilic granules / Azurofilik granüller, 43

Acute tumor lysis syndrome secondary to a single-dose

methylprednisolone in acute lymphoblastic leukemia / Akut lenfoblastik

lösemide tek doz metilprednizolona sekonder olarak gelien akut tümör

lizis sendromu, 55

Mumps / Kabakulak, 117

Parotitits / Parotit, 117

Dactylitis / Daktilit, 117

Childhood / Çocukluk ça, 117


Acute leukemia / Akut lösemi, 156

Hepatitis B vaccine / Hepatit B as, 156

Hepatitis B immunoglobin / Hepatit B immunoglobin, 156


Aplastic anemia / Aplastik anemi, 168

Posterior reversible leukoencephalopathy syndrome / posterior geri

dönüümlü lökoensefalopati sendromu, 168

Tumor lysis syndrome / tümör lizis sendromu, 168

Laboratory diagnosis of acute leukemia in Iraq, the available options /

Irak’ta akut löseminin laboratuvar tans, uygun seçenekler, 320

Ang-2 / Ang-2, 282

Tie-2 / Tie-2, 282

AML / AML, 282

ELISA / ELISA, 282

Red Cell Enzyme / Eritrosit Enzimleri

A note on oseltamivir treatment in a boy with G6PD deficiency / G6PD

eksiklii olan bir erkek çocukta oseltamivir tedavisine ilikin bir not, 48

Oseltamivir and G6PD deficiency / Oseltamivir ve G6PD eksiklii, 132

Hemophagocytosis / Malignancy / Hemafagositoz / Malignite

Malignancy-associated hemophagocytosis in children / Çocuklarda

malignite ile ilikili hemofagositoz, 49

Leukocytosis, thrombocytosis and hypercalcemia as a triple

paraneoplastic syndrome in a patient with squamous cell carcinoma of

the renal pelvis / Renal yass hücreli karsinomda multiple pareneoplatik

sendrom olarak lökositoz, trombositoz ve hiperkalsemi, 51

Malignancy / Malignite

Leukocytosis, thrombocytosis and hypercalcemia as a triple

paraneoplastic syndrome in a patient with squamous cell carcinoma of

the renal pelvis / Renal yass hücreli karsinomda multiple pareneoplatik

sendrom olarak lökositoz, trombositoz ve hiperkalsemi, 51

Radioactive iodine therapy / Radioaktif iyod tedavi, 171

Complete blood count / Tam kan saym, 171

Thyroid cancer / Tiroid kanseri, 171

Thalassemia / Talasemi

Nitric oxide in beta-thalassemia minor: what factors contribute? / Beta

talasemi minörde nitrik oksit: hangisi katk salar?, 53

Plasma Cell Diseases / Plazma Hücre Hastalklar

Solitary plasmacytoma / Soliter plazmasitom, 57

Radiotherapy / Radyoterapi, 57

Multiple myeloma / Multipl miyelom, 57

Management / Tedavi yaklam, 57

IgA lambda oligoclonal gammopathy in multiple myeloma / Oligoklonal

gammopati ile prezante olan bir Ig A multipl miyelom olgusu, 126

Multiple myeloma / Multipl miyelom, 182

uPA / uPA, 182

CD87 (uPAR) / CD87 (uPAR), 182

Flow cytometry / Akm sitometri, 182

Pleural fluid plasmacytosis in a patient with plasma cell leukemia /

Plazma hücreli lösemi hastasnda plevral sv plazmasitozu, 135

Spinal cord involvement of multiple myeloma detected by F-18 FDG

PET/CT Scan / F-18 FDG PET/CT tarama ile saptanan multiple

myeloma’nn spinal kord tutulumu, 326

Chemotherapy / Experimental Hematology / Kemoterapi / Deneysel

Hematoloji

Amifostine / Amifostin, 62

Acute cardiotoxicity / Akut kardiyotoksisite, 62

Mitoxantrone / Mitoksantron, 62

Lipid peroxidation / Lipid peroksidasyonu, 62

Molecular Hematology / Moleküler Hematoloji

Ankaferd / Ankaferd, 70

Proteomics / Proteomiks, 70

Hemostasis / Hemostaz, 70

Bleeding / Kanama, 70

Cytotoxic T lymphocyte antigen-4 (CTLA-4) / Sitotoksik T lenfosit

antijen-4 (CTLA-4), 78

A49G polymorphism / A49G polimorfizmi, 78

Autoimmunity / Otoimmünite, 78

Iidiopathic thrombocytopenic purpura / diopatik trombositopenik

purpura, 78

Autoimmune hemolytic anemia / Otoimmün hemolitik anemi, 78

Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 78

ALL / ALL, 299

X chromosome / X kromozomu, 299

Chromosome 5 / Kromozom 5, 299

Philadelphia chromosome / Philadelphia kromozomu, 299

Autoimmune Disorders / Molecular Hematology / Otoimmün

bozukluklar / Moleküler Hematoloji

Cytotoxic T lymphocyte antigen-4 (CTLA-4) / Sitotoksik T lenfosit

antijen-4 (CTLA-4), 78

A49G polymorphism / A49G polimorfizmi, 78

Autoimmunity / Otoimmünite, 78

Iidiopathic thrombocytopenic purpura / diopatik trombositopenik

purpura, 78

Autoimmune hemolytic anemia / Otoimmün hemolitik anemi, 78

Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 78

Chronic Myeloproliferative Disorders

Essential thrombocythemia / Esansiyel trombositemi, 82

Platelet activation / Platelet aktivasyonu, 82

Adhesion / Adezyon, 82

Thrombosis / Tromboz, 82

Platelet assay / Platelet tayini, 82

Stem Cell Transplantation / Kök Hücre Nakli

Allogeneic hematopoietic stem cell transplantation / Allogeneik

hematopoietik kök hücre nakli, 91

Sclerodermatous graft-versus-host disease / Sklerodermatoz Graft-

Versus-Host Hastal, 91

Stimulation index / Stimülasyon indeksi, 263

Mixed lymphocyte culture / Mikst lenfosit kültür, 263

Hematopoietic stem cell transplantation / Hematopoetik kök hücre

nakli, 263

Cytokines / Sitokinler, 263

Platelet Disorders / Trombosit Bozukluklar

Platelets / Trombositler, 99

Hyperlipidemia / Hiperlipidemi, 99

Clopidogrel / Klopidogrel, 99

Apolipoprotein A1 / Apolipoprotein A-I, 99

Apolipoprotein B / Apolipoprotein B, 99

Oxidative stres / Oksidatif stres, 99

Nitrites / Nitrite, 99

Hosphatidylserine / Fosfatidilserin, 99

P-selectin / P-selektin, 99

Glycoproteins IIb/IIIa / Glikoprotein IIb/IIIa, 99

Cyclic thrombocytopenia / Siklik trombositopeni, 196

Idiopathic thrombocytopenic purpura / diyopatik trombositopenik

purpura, 196

Hepatitis B / Hepatit B, 196

Case report / Olgu sunumu, 196

Acquired amegakaryocytic thrombocytopenia / Kazanlm

amegakaryositik trombositopeni, 289

Steroids / Steroidler, 289

Cyclosporine / Siklosporin, 289

Plasma Cell Diseases / Cytogenetics / Plazma Hücre Hastalklar /

Sitogenetik

Myeloma / Miyelom, 109

Pulmonary involvement / Akcier tutulumu, 109

Prognostication / Prognoz, 109

Cytogenetics / Sitogenetik, 109

Molecular genetics / Moleküler genetik, 109

Del(13q) / Del(13q), 109

Hypodiploidy / Hipodiploidi, 109

Cytogenetics / Bone Marrow Failure / Sitogenetik / Kemik lii Yetmezlii

Chronic idiopathic myelofibrosis / Kronik idiopatik myelofibrozis, 113

Cytogenetics / Sitogenetik, 113

Hemoglobinopathies / Hemoglobinopatiler

Hemoglobinopathy / Hemoglobinopati, 120

Abnormal hemoglobin / Anormal hemoglobin, 120

Hb Tunis / Hb Tunis, 120

HbQ-India / Hb Q India, 200

HbQ / HbQ, 200

Hemoglobin variants / Hemoglobin varyantlar, 200

Laboratory Hematology / Laboratuvar Hematolojisi

Ribosomal protein S19 - 631 insertion is an Africanoriginated mutation /

Afrika kökenli toplumlarda ribozomal protein S19- 631 insersiyonu, 123

Calabash chalk / Kalaba tebeir, 177

Blood parameters / Kan parametreleri, 177

Wistar rat / Wistar sçan, 177

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

Türkiye’de Van bölgesinde Faktör V 1691G-A mutasyonunun prevalans, 211

Stem cell / Kök hücre, 134

Cord blood / Kordon kan, 134

LTC-IC / LTC-IC, 134

Limiting dilution / Kstl dilüsyon, 134

CFU assay / CFU kültürü, 134

Cytometry / Akan hücre ölçer, 134

Cell cycle / Hücre siklusu, 142

Heparin / Heparin, 142


Thrombosis / Tromboz

Lower FXII activity and thrombosis: a comment / Düük FXII aktivitesi

ve tromboz: bir yorum, 125

Thrombosis and risk factors / Tromboz ve risk faktörleri, 318

Transfusion / Transfüzyon

Blood transfusion services in Iraq; an unfortunate field / Irak’ta kan

transfüzyon hizmetleri: talihsiz bir alan, 128

Blood donation / Kan ba, 275

University / Üniversite, 275

Student / Örenci, 275

Hematologic Disorders / Hematolojik Hastalklar

Mega-dose methylprednisolone in hematologic and non-hematological

disorders / Hematolojik ve hematolojik olmayan hastalklarda yüksek

doz metilprednizolon, 130

Molecular Hematology / Iron Disorders / Moleküler Hematoloji / Demir

Hastalklar

Iron / Demir, 137

Iron transport / demir tanm, 137

Mitochondria / Mitokondri, 137

Mitochondrial iron / Mitokondriyal demir, 137

Bleeding Disorders / Kanama Bozukluklar

Immune thrombocytopenic purpura / mmun trombositopenik

purpura, 147

Children / Çocuk, 147

Clinical course / Klinik seyir, 147

Chronic / Kronik, 147

Recurrent / Rekürren, 147

Outcome / Prognoz, 147

Acute Leukemia / Infection / Akut Lösemi / Enfeksiyon


Hepatitis B vaccine / Hepatit B as, 156

Hepatitis B immunoglobin / Hepatit B immunoglobin, 156

Hypercoagulability / Hiperkoagülabilite

Cerebral venous thrombosis / Serebral venöz tromboz, 162

Thrombophilia / Trombofili, 162

Thrombophilic defects / Trombofilik defektler, 162

Idiopathic thrombocytopenic purpura with venous thrombosis: A case

report / Venöz trombozlu idiopatik trombositopenik purpura: Bir olgu

sunumu, 209

Storage Disorders / Depolama Bozukluklar

Gaucher disease / Gaucher hastal, 190

Glucosylceramidase / Glukoserebrosidaz, 190

Treatment / Tedavi, 190

Anemia / Anemi

Rhesus hemolytic disease / Rh hemolitik hastal, 204

Late hyporegenerative anemia / Geç hiporejeneratif anemi, 204

Transfusion-related hepatic iron overload / transfüzyona ikincil karacier

demir birikimi, 204

Chelation therapy / elasyon tedavisi, 204

Cobalamin deficiency / Kobalamin eksiklii, 250

Autonomic dysfunction / Otonom bozukluk, 250

Dysautonomia / Disotonomi, 250

Autonomic neuropathy / Otonom nöropati, 250

Serum cytokine levels / Serum sitokin düzeyi, 250

Hypercoagulability / Thrombosis / Hiperkoagülabilite / Tromboz

Idiopathic thrombocytopenic purpura with venous thrombosis: A case

report / Venöz trombozlu idiopatik trombositopenik purpura: Bir olgu

sunumu, 209

Infection / Thrombosis / Enfeksiyon / Tromboz

Portal vein thrombosis secondary to Klebsiella oxytoca bacteriemia /

Klebsiella oxytoca bakteriyemisine sekonder portal ven trombozu, 213

Metabolic Disorders / Metabolik Hastalklar

Late onset of isovaleric acidemia presenting with bicytopenia /

Bisitopeni ile birliktelik gösteren geç balangçl izovalerik asidemi, 216

Immunologic Disorders Leishman-Donovan (LD) bodies in bone

marrow biopsy of an adult male with AIDS / AIDS hastas yetikin erkein

kemik ilii biyopsisinde Leishmania Donovan (LD) cisimcii, 221

Hematologic Disorders / Infection / Hematolojik Hastalklar / Enfeksiyon

Children / Çocuk, 124

Hematologic findings / Hematolojik bulgular, 124

Parvovirus / Parvovirus, 124

Treatment / Tedavi, 124

Hemophagocytosis / Laboratory Hematology / Hemafagositoz /

Laboratuvar Hematolojisi

Primary hemophagocytic lymphohistiocytosis / Primer hemofagositik

lenfohistiositozis, 257

Clinical and laboratory findings / Klinik ve laboratuvar veriler, 257

Stem Cell Transplantation / Bone marrow failure / Kök Hücre Nakli /

Kemik lii Yetmezlii

Aplastic anemia / Aplasti anemi, 294

Hepatitis / Hepatit, 294

Peripheral blood stem cell transplantation / Periferik kan kök hücre

transplantasyonu, 294

Trachea / Trake, 294

Infection / Enfeksiyon, 294

Lymphomas / Lenfomalar

FDG-PET in mantle cell lymphoma involving skin / Deri tutulumlu

mantle hücreli lenfomada FDG-PET, 324

Chemotherapy / Acute Leukemia / Kemoterapi / Akut Lösemi

Pancreatitis in a child with acute lymphoblastic leukemia after Erwinia

asparaginase: Evaluation of ultrasonography and computerized

tomography as diagnostic tools / Akut lenfoblastik lösemili bir çocukta

Erwinia asparaginaz dan sonra olan pankreatit: Tan araçlar olarak

ultrasonografi ve bilgisayarl tomografi ile deerlendirme, 316

Chronic Myeloproliferative Disorders / Bone Marrow Failure / Kronik

Miyeloproliferatif Hastalklar / Kemik lii Yetmezlii

Mega-dose methylprednisolone (MDMP) for chronic idiopathic

myelofibrosis / Kronik idiopatik miyelofibrosis için yüksek doz

metilprednizolon (YDMP), 314

Storage Disorders / Hemophagocytosis / Depolama Bozukluklar /

Hemafagositoz

Niemann-Pick disease / Niemann-Pick Hastal, 303

Hemophagocytic syndrome / Hemofagositik sendrom, 303

Hemophagocytosis by Niemann-Pick cell / Niemann-Pick hücresinin

hemofagositozu, 303

Hemophagocytosis / Hemafagositoz, 303

Hemophagocytic lymphohistiocytosis / Hemofagositik lenfohistiositoz, 303

HLH / HLH, 303

Autoimmune Disorders / Otoimmün bozukluklar

Giant cell hepatitis / Dev hücreli hepatit, 308

Immune hemolytic anemia / mmün pozitif hemolitik anemi, 308

Direct Coombs’ test / Direk Coombs’ testi, 308

Rituximab / rituximab, 308

27 th Volume Index

27. Cilt Dizini

AUTHOR INDEX - YAZAR DZN

March 2010 - December 2010

Mart 2010 - Aralk 2010

Abbas Hashim Abdulsalam, 128,

221, 320

Naseem Adil Salman, 135

Rishu Agarwal, 43

lknur Ak Sivrikoz, 326

Nur Akad Soyer, 29

Nejat Akar, 48, 70, 123, 318, 322,

235

Nisa Bengü, 91

Akay Akbayram Sinan, 211

Hakan Akbulut, 126

Arzu Akçay, 263

Cihangir Akgün, 211

ahika Zeynep Ak, 250

Aye Aksoy, 142

Sema Akta, 234

Faruk Aktürk, 78

Ibrahim Al Hijji, 289

Aisha Al Khinji, 289

Fatin Al Yassin, 221

Fereidun Ala, 15

Murat Albayrak, 109, 162, 182

Füsun Alehan, 168, 216

Bülent Aliolu, 168

Asude Alpman Durmaz, 299

Mokutima Amarachi Eluwa, 275

Neslihan Andç, 209

Olu Api, 219

Mutlu Arat, 91, 190

Nur Arslan, 204

Esra Arun Özer, 147

Berna Atabay, 147

Ayfer Atalay, 120

Erol Ömer Atalay, 120

Mohamed A. Attia, 282

Zekai Avc, 168, 216

Hülya Aybek, 62

Selin Aytaç Elmas, 34, 124, 308

Fatih Mehmet Azk, 316

Hussein Baden, 289

Bahadr Anzel, 120

Sameer Bakhshi, 43

Necati Balamtekin, 308

Yasemin Ik Balc, 117

Bela Balint, 196

Muharrem Balkaya, 62

Giorgio Ballerini, 57

Esra Baskn, 168

Theresa Bassey Ekanem, 275

Moses Bassey Ekong, 275

Ali Bay, 257

Mahmut Bayk, 234

Gökhan Baysoy, 308

Mehmet Selçuk Bekta, 211

Rahul Bhargava, 156

Hülya Bilgen, 263

Carsten Bokemeyer, 133

Zahit Bolaman, 62

Aye Bora Tokçaer, 250

Hakan Bozcuk, 51

Tülin Budak Alpdoan, 234

Tuçe Bulakba Balc, 113

Sadullah Bulut, 219

Abdullah Büyükçelik, 126

Gamze Can, 269

Cengiz Canpolat, 257

Tiraje Celkan, 38, 49, 257

Bülent Cengiz, 117

Funda Ceran, 109, 162, 182

Cengiz Ceylan, 25

Erman Cilsal, 117

Hasan enol Cokun, 51

Özge Cumaoullar, 123

Seçkin Çargan, 29

ule Çalayan Sözmen, 204

Ebru Çakr Edis, 20

Ümran Çalkan, 257

Mahmut Çarin, 263

Hasniye Çelik, 219

Özcan Çeneli, 250

Mualla Çetin, 34, 308

Mustafa Çetin, 275

Feyzullah Çetinkaya, 303

Betül Çevik Küçük, 250

Ümit Çobanolu, 324

Özgür Çoulu, 299

Simten Dada, 109, 162, 182

Klara Dalva, 8

Muzaffer Demir, 20

Süleyman Demir, 62

Funda Demira, 109

Fatih Demirciolu, 204

Sanem Demirtepe, 120

Reyhan Diz Küçükkaya, 196

smail Doan, 269

Murat Doan, 211

Öner Doan, 49

Ayhan Dönmez, 29

Fulden Dönmez, 168

Burak Durmaz, 299

kbal Durak, 147

Feride Duru, 257

Alper Eker, 20

Hanadi El Ayoubi, 289

Halima El Omri, 289

Solaf Elsayed, 123

Ezzat Elsobky, 123

Ela Erdem, 49

Erol Erduran, 142, 257

Andreas C. Eriksson, 82

Mehmet Ertem, 8, 257

Bülent Eser, 275

Salwa A. Essa, 282

Tunç Fgn, 257

Walter Fiedler, 133

Yusuf Gedik, 142

Ritu Gupta, 43

Zafer Gülba, 326

Aye Gültekingil, 117

Fatma Gümrük, 34, 117, 308

Figen Gürakan, 308

Aytemiz Gürgey, 117

Mehmet Gürtekin, 263

Mithat Halilolu, 34

Veysel Sabri Hançer, 78

Alev Hasanolu, 216

Osman Nuri Hatipolu, 20

brahim C. Haznedarolu, 70

Sahar M. Hazzaa, 282

Gönül Hiçsönmez, 1

Bakhouche Houcher, 322

Samuel Ibok Ofon, 177

Firyal Ibrahim, 289

Aye Ik, 213

Fikri çli, 126

Talia leri, 8

Gül lhan, 209

Osman lhan, 91

Gülersu rken, 204

Mohammad Jazebi, 15

Gökhan Kabaçam, 190

Gülah Kabaçam, 190

Vefki Gürhan Kadköylü, 62

Mete Kalak, 62

Sevgi Kalayolu Bek, 263

Gülsev Kale, 308

Nausheen Kamran, 135

Mehmet Kanbay, 213

Tuphan Kanti Dolai, 156

Emin Karaca, 299

Sema Karaku, 209

Serap Karaman, 49, 303

Ayegül Karg, 51

Sava Karyaar, 324

Arif Kaya, 213

Leylagül Kaynar, 275

Ahmad Kazemi, 15

Turay Kevser Yardmc, 99

Gonca Klç, 316

Nerbil Klç, 133

Tülay Klçaslan Ayna, 263

Sebastian Kobold, 133

Esengül Koçak, 57

Toprak Selami Koçak, 126

Pelin Koçyiit, 91

Ali Koar, 213

Hasan Koyuncu, 120

Aylin Köseler, 120

Abdullah Kumral, 204

Fatih Kurnaz, 275

Reha Kuruolu, 250

Bar Kukonmaz, 34, 117, 308

Günsel Kutluk, 303

Ayça Esra Kuybulu, 55

Insu Kuzu, 190

Reyhan Küçükkaya, 78

Kourosh Lotfi, 82

Manoranjan Mahapatra, 156

Bar Malbora, 168, 216

brahim Meteolu, 62

Dilsa Mzrak, 126

Pravas Mishra, 156

Bhaskar Mitra, 46

Bushra Moiz, 135

Krishnendu Mukherjee, 46

Mehmet Mutlu, 142

M. Murat Naki, 219

Riham Hassan Negm, 289

Rabia Oba, 99

Serkan Ocakç, 29

Emel Aye Onal, 263

Diclehan Orhan, 308

Akpantah Amabe Otoabasi, 177

Faruk Öktem, 55

Haldun Öniz, 147

Hale Ören, 204

ebnem Örgüç, 25

Namk Özbek, 137, 168, 216, 257

Akif Özdemir, 257

Nagehan Özdemir, 219

Mustafa Özdoan, 51

Duygu Özel Demiralp, 70

Pnar Özen, 29

Erdener Özer, 204

Gülsüm Özet, 109, 162, 182

Ferda Özknay, 299

Alc Özlem, 213

Derya Özsavc, 99

inasi Özsoylu, 130, 132, 314

Mahmut Özahin, 57

Ayenur Öztürk, 123, 322

Onur Öztürk, 120

Yüksel Pabuçcu, 25

Türkan Patrolu, 257

Ivanka Percic, 294

Nedim Polat, 49

Stevan Popovic, 294

Pati Hara Prasad, 156

Nebojsa Rajic, 294

Parisa Rasighaemi, 15

Shyam Rathi, 156

Niranjan Rathod, 156

Farnaz Razmkhah, 15

Usha Rusia, 200

Raihan Sajid, 135

Deniz Sargn, 263

Burhan Sava, 51

Aleksandar Savic, 294

Renu Saxena, 156

Güray Saydam, 29, 299

Hülya Saylan en, 263

John Scharlau, 133

Mahmoud F. Seleim, 282

Selim Serter, 25

Tulika Seth, 156

Atul Sharma, 43

Ankit Shrivastav, 46

Tansu Sipahi, 55

Ender Soydan, 91

Bircan Sönmez, 269, 324

Mehmet Sönmez, 269, 324

Necdet Süt, 20

Özlem ahin Balçk, 109, 162,

182, 213

Feride ffet ahin, 113

Hatice anl, 91

Azize ener, 99

Hayriye entürk Çiftçi, 263

Serdar vgn, 177

Abdullah umnu, 294

Ruba Yasin Taha, 289

Özlem Tansel, 20

Betül Tavil, 34

brahim Tek, 126

Yavuz Tekeliolu, 142

Emre Tekgündüz, 20

Milena Todorovic, 294

Murat Tombulolu, 29

Pervin Topçuolu, 8, 190

Selami K. Toprak, 209

Murat Tuncer, 117

Özüm Tunçyürek, 25

Hüseyin Tutkak, 126

Meral Türker, 147

Duygu Uçkan Çetinkaya, 34

Fikriye Uras, 99

Nafiye Urganc, 303

Ivana Urosevic, 294

Güngör Utkan, 126

Zümrüt Uysal, 8

Burak Uz, 213

Ebru Uz, 213

Ali Ünal, 177

Elif Ünal nce, 8

Orhan Ünal, 219

ule Ünal, 34, 117, 308

Havva Üsküdar Teke, 326

Canan Vergin, 257

Filiz Vural, 29

Sema Vural, 49

Per A. Whiss, 82

Viroj Wiwanitkit, 53, 125

Amit Kumar Yadav, 200

Münci Yac, 250

Bülent Yalçn, 126

Gülderen Yankkaya Demirel,

99, 134

In Yaprak, 147

Nee Yaral, 257

Mohammed Yassin, 289

Canan Yavruolu, 269

Mehmet Yay, 177

Çidem Yenisey, 62

Sevgi Yetgin, 124

lker Ylmam, 20

Deniz Ylmaz Karapnar, 257

Mesude Ylmaz, 162, 182

ebnem Ylmaz, 204

Zerrin Ylmaz, 113

Osman Yoku, 109, 162, 182

Aye Yüce, 308

Meltem Yüksel, 113

Abderrahim Zouhair, 57

Revlimid 215x280 ilan_cn.fh11 10/1/10 6:53 PM Page 1

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2010-4

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