UNIVERSITI PUTRA MALAYSIA

HAEMATOLOGICAL PARAMETERS OF MALAY PATIENTS WITH

COINHERITANCE OF SOUTHEAST ASIAN OVALOCYTOSIS AND

THALASSAEMIA HAEMOGLOBI-NOPATHY TRAITS IN KLANG VALLEY,

MALAYSIA

SHAHRZAD RIAHI

FPSK(m) 2013 6

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HAEMATOLOGICAL PARAMETERS OF MALAY PATIENTS WITH COINHERITANCE OF SOUTHEAST ASIAN OVALOCYTOSIS AND THALASSAEMIA HAEMOGLOBI-

NOPATHY TRAITS IN KLANG VALLEY, MALAYSIA

By

SHAHRZAD RIAHI

Thesis submitted to the School of Graduate Studies of Universiti Putra Malaysia in the fulfilment of the requirements for the Degree of Master of Science

February 2013

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DEDICATION

This thesis is dedicated to

My dearest Father and Mother

The understanding and encouragement they provided during all the these years

of the study

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Abstract of thesis presented to the senate of Universiti Putra Malaysia in the fulfilment of the requirement for the degree of Master of Science

HAEMATOLOGICAL PARAMETERS OF MALAY PATIENTS WITH COINHERITANCE OF SOUTHEAST ASIAN OVALOCYTOSIS AND THALASSAEMIA HAEMOGLOBI-

NOPATHY TRAITS IN KLANG VALLEY, MALAYSIA

By

Shahrzad Riahi

February 2013

Chairman: Sabariah Md Noor, MD (UKM), Mpath (UKM)

Faculty: Medicine and Health Science

Southeast Asian Ovalocytosis (SAO) and thalassaemia-haemoglobinopathies are com-

mon inherited red blood cell (RBC) disorders in Southeast Asian countries. SAO, a RBC

membrane defect is clinically asymptomatic. It is characterized by normal RBC parame-

ters by in full blood count (FBC) with presences of oval stomatocytic RBCs in peripheral

blood film. Thalassaemia-haemoglobinopathy, a globin defect of RBC is one of the ma-

jor public health problems in Malaysia. Unlike SAO, thalassaemia-haemoglobinopathies

is generally accompanied by hypochromic microcytic RBC parameters. Thalassaemia-

haemoglobinopathies screening programme is mainly depends on evaluation of haema-

tology (RBC) parameters. Therefore, its evaluations in the presence of those co-

inheritance disorders are important to ensure the screening for thalassaemia-

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haemoglobinopathies is optimum. Objective: To determine the prevalence of co-

inheritance of SAO and thalassaemia-haemoglobinopathies in the Malay population and

to evaluate the RBC parameters in those patients. Method: Prevalence of co-

inheritance of SAO and thalassaemia-haemoglobinopathies was determined by recruit-

ing 150 respondents among Malay volunteers’ blood donors in UPM. Their blood sam-

ple were analysed for FBC, blood film, Hb analysis and serum ferritin. A total of 132 Ma-

lay patients with thalassaemia-haemoglobinopathies, SAO and co-inheritance of both

were identified through samples for thalassaemia-haemoglobinopathies screening sent

to laboratory haematology, Kuala Lumpur Hospital. These patients were consented and

recruited to involve in this study. Their blood samples also were analysed for similar

tests as blood donors. The DNA analysis was performed for respondents suspected of

alpha thalassaemia (hypochromic microcytic RBC indices with normal serum ferritin and

HPLC) and SAO (presence of stomatocytes in blood film). Single–tube multiplex PCR

was used for confirmation of α-thalassaemia while PCR carried out to detect SAO. One-

way ANOVA test using SPSS version 19 was used to analyse these data where p-value

of < 0.05 was considered as statistically significant. Results: Prevalence of thalassae-

mia–haemoglobinopathies and SAO among blood donors like Hb E trait, β-thalassaemia

trait, α3.7-thalassaemia, α4.2-thalassaemia, αSEA-thalassaemia and SAO were 2.66%,

0.66%, 7.33%, 0.66% and 0.66% and 4.66% respectively. None of these donors was

with co-inheritance of SAO and thalassaemia-haemoglobinopathies. All the participants

in this study were divided into normal and anaemic groups according to their Hb level.

The mean of RBC parameters in co-inheritance of SAO and Hb E such as Hb, MCV,

MCH, MCHC, and RDW were 4.5 x106/µL, 10.33 g/dL, 66.33 fl, 22.5 pg, 33.96 g/dL,

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18.3%, respectively. Among all the RBC indices, the mean MCV were significantly lower

in co-inheritance cases as compared to respondents with only Hb E trait alone. The

mean of RBC parameters in co-inheritance of SAO and β- thalassaemia trait such as

RBC count, Hb, MCV, MCH, MCHC and RDW were of 3.98 x106/µL, 9.41 g/dL, 73 fl,

23.81 pg, 32.68 g/dL, 21.25%, respectively. Among these parameters, the RDW, MCV,

and MCH were significantly higher and RBC count was significant lower in co-

inheritance cases as compared to respondents with only β-thalassaemia trait alone.

Discussion: This study showed respondents with co-inheritance of thalassaemia-

haemoglobinpathies and SAO have lower MCV and MCH values as compared to refer-

ence standard in thalassaemia screening programme. The difference of the MCV value

was significantly lower in co-inheritance of SAO and Hb E trait. The mean MCH in both

groups of in co-inheritance was 22.5 pg and 23.8 pg, that has also lower value as com-

pared to the references cut–off point, which were, being used for thalassaemia-

haemoglobinopathies screening programme in Malaysia. Conclusion: Co-inheritance

of SAO and thalassaemia-haemoglobinopathies does not have significant effect on cut-

off values of RBC parameters which is, fundamental for thalassaemia-

haemoglobinopathies screening.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

PARAMETER HEMATOLOGI DIKALANGAN PESAKIT YANG MEMPUNYAI KEDUA-DUA MASALAH KETURUNAN SEL DARAH MERAH, SOUTHEAST ASIAN

OVALOCYTOSIS DAN PEMBAWA PENYAKIT TALASEMIA-HEMOGLOBINOPATI DI LEMBAH KLANG, MALAYSIA

Oleh

Shahrzad Riahi

Februari 2013

Pengerusi: Sabariah Md Noor, MD (UKM), Mpath (UKM)

Fakulti: Perubatan dan Sains Kesihatan

Southeast Asian Ovalocytosis (SAO) dan talasaemia-hemoglobinopati adalah penyakit

keturunan melibatkan keabnormalan sel darah merah (SDM) yang biasa di kalangan

penduduk di negara-negara Asia Tenggara. SAO, penyakit yang melibatkan keabnor-

malan pada membran SDM tidak mempunyai sebarang masalah klinikal. Pesakit SAO

di cirikan oleh parameter SDM melalui ujian “full blood count” (FBC) yang normal , na-

mun mempunyai SDM yang berbentuk stomatosit pada ujian calitan darah periferi. Ta-

lasaemia-hemoglobinopati yang diakibatkan oleh keabnormalan rantai globin pada SDM

merupakan salah satu lagi masalah kesihatan awam yang ketara di Malaysia.Tidak

seperti SAO, pesakit talasaemia-hemoglobinopati di cirikam oleh parameter SDM yang

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secara kebiasaannya menunjuk SDM yang hipokromik mikrositik.Analisa parameter

hematologi (SDM) merupakan ujian yang terpenting dalam ujian saringan talasemia-

hemoglobinopati.Oleh yang demikian, kajian pada kesan parameter SDM dikalangan

pesakit yang mewarisi kedua-dua masalah keturunan SDM ini secara bersama adalah

amat penting bagi memastikan ujian saringan talasemia di Malaysia adalah ditahap

yang optima.Objektif: Kajian ini bertujuan untuk mengetahui prevalens pesakit yang

mengidapi kedua-dua penyakit keturunan SDM secara bersama dan seterusnya melihat

kesan pada parameter SDM dikalangan mereka berbanding dengan peserta yang men-

gidap talassemia-hemoglobinopati sahaja. Kaedah: Prevalens pesakit yang mengidapi

kedua-dua masalah keturunan SDM iaitu SAO dan talasemia-hemoglobinopati secara

bersama ditentukan dikalangan 150 peserta yang direkrut dikalangan penderma darah

berbangsa Melayu di Universiti Putra Malaysia (UPM). Sampel darah penderma dianali-

sa untuk ujian FBC, calitan darah periferi, ujian analisa hemoglobin dan tahap serum

ferritin. Seramai 132 daripada pesakit berbangsa Melayu yang mengidap penyakit ta-

lasemia-hemoglobinopati, SAO dan kedua-duanya secara bersama telah dikenalpasti

melalui ujian saringan talasemia yang di hantar ke Laboratori Hematologi, Hospital

Kuala Lumpur. Pesakit yang memberi kebenaran dan bersetuju untuk turut serta dalam

kajian ini telah direkrut. Sampel darah pesakit-pesakit ini telah dianalisa untuk ujian-

ujian yang sama seperti yang dilakukan dikalangan penderma darah. Ujian DNA telah

dilakukan pada semua peserta yang disuspek mengidapalfa-talasemia (parameter SDM

yang hipockromik mikrositik tetapi mempunyai paras serum ferritin yang normal) dan

SAO (calitan darah periferi menunjukkan terdapatnya sel stomatositik). Penyakit α-

talasemia telah disahkan menggunakan kaedah “Single–tube multiplex PCR” manakala

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SAO pula menggunakan kaedah PCR. Ujian ANOVAmenggunakan change to SPSS 19

Version telah digunakan untuk menganalisa data kajian dimana nilai-p yang <0.05 akan

di anggap sebagai digit bermakna secara statistik. Keputusan: Prevalens penyakit ta-

lasemia-hemoglobinopati dan SAO dikalangan penderma darah di UPM untuk Hb E

trait, β-thalassaemia trait, α3.7-thalassaemia, α4.2-thalassaemia, αSEA-thalassaemia dan

SAO adalah masing-masing seramai 2.66%, 0.66%, 7.33%, 0.66% , 0.66%. dan 4.66%.

Tiada seorang pun dikalangan penderma darah yang dikenalpasti telah mengidap ta-

lasemia-hemoglobinopati dan SAO secara bersama.Peserta kajian ini telah dibahagi

kepada kumpulan normal (tahap hemoglobin (Hb) yang normal) dan kumpulan anemia

(tahap Hb yang kurang dari normal). Peserta yang dikenalpasti mempunyai masalah

Hb E dan SAO secara bersama pula menunjukkan parameter SDM seperti RBC count,

Hb, MCV, MCH, MCHC dan RDW adalah masing-masing 4.5 x106/µL, 10.33 g/dL,

66.33 fl, 22.5 pg, 33.96 g/dL, 18.3%. Purata MCV adalah lebih rendah dalam kumpulan

masalah SAO dan talasemia-hemoglobinopati secara bersama berbanding dengan

kumpulan pembawa Hb E sahaja. Peserta yang dikenalpasti mempunyai masalah β-

thalassaemia trait dan SAO secara bersama pula menunjukkan parameter SDM seperti

RBC count, Hb, MCV, MCH, MCHC dan RDW adalah masing-masing 3.98 x106/µL,

9.41 g/dL, 73 fl, 23.81 pg, 32.68 g/dL dan 21.25%. Kumpulan ini menunjukkan berbeza

statistic yang bermakna pada nilai parameter RDW, MCV dan MCH dimana ianya lebih

tinggi dan nilai RBC count yang lebih rendah berbanding dengan kumpulan pembawa

β-thalassaemia sahaja.Perbincangan:Kajian ini menunjukkan responden yang mewari-

si secara bersama talasemia haemoglobinpathies dan SAO mempunyai nilai MCV dan

MCH yang lebih rendah berbanding nilai parameter tersebut yang digunakan untuk ru-

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jukan standard dalam program saringan talasemia. Perbezaan nilai MCV adalah jauh

lebih rendah dikalangan pesakit yang mewarisi penyakit talasemia-hemoglobinopati dan

SAO secara bersama berbanding dengan kumpulan pembawa Hb E.Purata MCH

dikalangan kedua-dua kumpulan yang mengidap talasemia-hemoglobinopati dan SAO

secara bersamaadalah masing-masing 22,5 pg dan 23,8 pg, dimana nilai ini adalah

lebih rendah berbanding dengan rujukan potong titik, yang digunakan untuk tujuan

saringan talasemia-haemoglobinopathies di MalaysiaKesimpulan: Penyakit keturun-

anSAO dan talasemia–hemoglobinopati yang diwarisi secara bersama tidak mempunyai

sebarang kesan yang ketara pada nilai rujukan parameter SDM yang menjadi teras da-

lam ujian saringan talasemia di Malaysia.

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ACKNOWLEDGEMENTS

First and foremost I wish to convey my sincere thanks to God Almighty who continually

gives me strength and perseverance that He has bestowed upon me during my study.

I owe a depth of gratefulness to everyone without whom this work would not have been

possible. Specially, I offer my sincerest gratitude to my supervisor; Dr. Sabariah Md

Noorwho supported and helped me from the very beginning of my studies. I would nev-

er have been able to finish my dissertation without the guidance of my committee mem-

bers Prof. Dr. Elizabeth George, Dr. Lai Mei I and Dr. Faridah binti Idris. They spend

their valuable time in reading and correcting mistakes in the earlier drafts.

I would also like to thank the many people who have given their suggestions and ideas

during the progress of this project and to all my friends and course-mates. My acknowl-

edgement also goes to all the technicians and office staffs of UPM Haematology labora-

tory.

I would also like to thank all the staff of the National Blood donation Centre of Malaysia

for their cooperation to collect the samples and all the personnel of Haematology la-

boratory of HKL specially, Dr Ida Marihainis Isahak, Dr Siti Aaisyah Ismail and Puan

Masakmah bt Hj.A.Kadir.

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Last but not least, my deepest gratitude goes of my family for their unflagging love and

support throughout my life. Even they are not with me during these years, but they are

with me in spirit. They always give me words of encouragement and believe that en-

gaged me to finish this research.

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I certify that a Thesis Examination Committee has met on 1/2/2013 to conduct the final examination of Shahrzad Riahi on his thesis entitled “haematological parameters of malay patients with co-inheritance of Southeast asian ovalocytosis (SAO) and thalassaemia-haemoglobinopathies trait in Klang Valley " in accordance with the Universities and University Colleges Act 1971 and the Constitution of the University Pu-tra Malaysia [P.U. (A)] 15 March 1998. The Committee recommends that the Student be awarded the Master of Haematology. Members of the Thesis Examination Committee are as follows:

Chairman,

Professor Madya Dr. Hairuszahbinti Ithnin

Faculty of medicine and Health Science

Universiti Putra Malaysia

(Chairman)

Examiner1, PhD DR. Eusni Rahayu binti Mohd.Tohit Faculty of medicine and Health Science Universiti Putra Malaysia (Internal Examiner) Examiner2, PhD Dr. Norshariza binti Nordin Faculty of medicine and Health Science Universiti Putra Malaysia (Internal Examiner) External Examiner, Professor Mayda Dr. Wan Zaidah Abdullah School of Medical Sciences Universiti Sains Malaysia (External Examiner)

_____________________________

PROF. DR. SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Sabariah Md Noor, MD (UKM), M path (UKM) Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member) Elizabeth George, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member) Faridah Binti Idris, MD (UM), M path (UKM) Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member) Lai Mei I, PhD Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member)

BUJANG BIN KIM HUAT, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date

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DECLARATION

I declare that the thesis is my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institutions

SHAHRZAD RIAHI

Date: 1 February 2013

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TABLE OF CONTENTS

Page

DEDICATION ii ABSTRACT III ABSTRAK VI ACKNOWLEDGEMENTS x APPROVAL XII DECLARATION xiv LIST OF TABLES xviii LIST OF FIGURES xix LIST OF ABBREVIATIONS/ NOTATIONS/ GLOSSARY OF TERMS xxi CHAPTER

CHA1PTER INTRODUCTION AND OBJECTIVE 1

1.1 Introduction 1 1.2 Objective 4

1.2.1 General objective 4 1.2.2 Specific Objectives 5

1.3 Hypothesis 6 CHA2PTER LITERATURE REVIEW 7

2.1 Red blood cell (RBC) 7 2.2 Red blood cell membrane 10

2.2.1 RBC membrane proteins 11 2.2.1.1 Integral proteins 12 2.2.1.2 Cytoskeleton proteins 14

2.3 Deficiency of membrane proteins 15 2.3.1 Southeast Asian Ovalocytosis (SAO) 15

2.3.1.1 Geographical distribution and prevalence 16 2.3.1.2 Genetic defect of SAO 17 2.3.1.3 Clinical significant of SAO 19 2.3.1.4 SAO Diagnosis 21

2.3.1.4.1 Peripheral blood smears 21 2.3.1.4.2 Osmotic fragility test 22 2.3.1.4.3 DNA analysis 22

2.4 Red blood cell haemoglobin 23 2.5 Haemoglobin disorders 24

2.5.1 Thalassaemia 25

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2.5.1.1 Beta –thalassaemia 26 2.5.1.1.1 Beta –thalassaemia trait 28 2.5.1.1.2 Beta –thalassaemia intermedia 29 2.5.1.1.3 Beta –thalassaemia major 29

2.5.1.2 Alpha –thalassaemia 31 2.5.1.2.1 Alpha –thalassaemia minor 32 2.5.1.2.2 Alpha-thalassaemia intermedia 33 2.5.1.2.3 Alpha –thalassaemia major 35

2.5.2 Other haemoglobin variants 36 2.5.2.1 Hemoglobin E 36

2.5.3 Thalassaemia screening 38 2.5.3.1 Laboratory tests in thalassaemia-haemoglobinopathies38

2.5.3.1.1 Full blood count 39 2.5.3.1.2 Peripheral blood film 40 2.5.3.1.3 High performance liquid chromatography (HPLC) 40 2.5.3.1.4 Hb electrophoresis 41 2.5.3.1.5 DNA analysis 42

2.6 Co-inheritance of thalassaemia and SAO 43

CHA3PTER MATERIALS AND METHODS 44

3.1 Ethical approval 44 3.2 Study period and population 44 3.3 Location 46

3.3.1 Sample collection in UPM 46 3.3.2 Samples collection in HKL 48

3.4 Experimental flow 49 3.5 Methods 52

3.5.1 Full Blood Count (FBC) 52 3.5.2 Peripheral blood film (Leishman’s stain) 52 3.5.3 Serum ferritin measurement 53 3.5.4 High performance liquid chromatography (HPLC) 54 3.5.5 DNA Extraction 54 3.5.6 DNA quantitation 56 3.5.7 Single –tube multiplex Polymerase chain reaction (PCR) 57 3.5.8 PCR for detection of Southeast Asian Ovalocytosis (SAO) 61 3.5.9 Statistical analysis 63

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CHP4PTER RESULTS 64

4.1 Prevalence of co-inheritanpce 64 4.2 The differences in RBC parameters 68

4.2.1 Malay respondents with Hb E trait 69 4.2.2 Malay respondents with β-thalassaemia trait 70 4.2.3 Malays respondents with α-thalassaemia trait 71 4.2.4 Respondents with SAO 75 4.2.5 Respondents with co-inheritance of SAO and thalassaemia- haemoglobinopathies trait 77

4.2.5.1 Respondents with co-inheritance of SAO and Hb E 78 4.2.5.2 Respondents with co-inheritance of SAO and β- thalassaemia trait 83

CHA5PTER DISCUSSION 87

5.1 Prevalence of SAO, thalassaemia-haemoglobinopathies trait and co- inheritance of them 87 5.2 The difference in RBC parameters 89

5.2.1 RBC parameters in Hb E trait 90 5.2.2 RBC parameters in β-thalassaemia trait 94 5.2.3 RBC parameters in α-thalassaemia trait 98 5.2.4 RBC parameters in SAO 102 5.2.5 RBC parameters in co-inheritance of SAO and thalassaemia- haemoglobinopathies trait 103

5.2.5.1 RBC parameters in co-inheritance of SAO and Hb E 103 5.2.5.2 RBC parameters in co-inheritance of SAO and β-thalassaemia trait 104

5.3 Conclusion 105 5.4 Limitation of study 106 5.5 Recommendations 107

5.5.1 For using in the haematology laboratory 107 5.5.2 Future studies 108

REFERENCES 110 APPENDICES 122 BIODATA OF STUDENT 136