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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