Comparison of Isoelectric Focusing and Immunofixation ... Electrophoresis to Distinguish Oligoclonal

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  • Comparison of Isoelectric Focusing and Immunofixation

    Electrophoresis to Distinguish Oligoclonal from Monoclonal

    Immunoglobulin Bands

    THESIS

    SUBMITTED BY

    LIU DAN

    in partial fulfilment ofthe requirement for the degree of

    Master of Science in Clinical Biochemistry in

    The Chinese University ofHong Kong

    March 1998

    DEPARTMENT OF CHEMICAL PATHOLOGY

    THE CmNESE UNIVERSITY OF HONG KONG

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  • Page

    CONTENTS i

    LIST OF TABLES iii

    LIST OF FIGURES iv

    LIST OF A B B R E V I A T I O N S v

    A C K N O W L E D G E M E N T S vi

    A B S T R A C T yii

    Chapter 1 I N T R O D U C T I O N 1

    1.1 H i s t o r y 1

    1.2 I m m u n o g l o b u l i n s 3

    1.2.1 S t r u c t u r e 3

    1.2.2 P r o p e r t i e s of i m m u n o g l o b u l i n s 7

    1.3 M o n o c l o n a l p r o t e i n s and m o n o c l o n a l g a m m o p a t h i e s 12

    1.3.1 M o n o c l o n a l p r o t e i n s 12

    1.3.2 M o n o c l o n a l g a m m o p a t h i e s 14

    1.4 L a b o r a t o r y i n v e s t i g a t i o n of m o n o c l o n a l i m m u n o g l o b u l i n 17

    1.4.1 The c u r r e n t p r o c e d u r e of i n v e s t i g a t i o n in l a b o r a t o r y 17

    1.4.2 P r o b l e m s in i d e n t i f y i n g m o n o c l o n a l i m m u n o l g o b u i n 19

    1.5 C o m p a r i s o n of d i f f e r e n t t e c h n i q u e s 20

    1.5.1 I m m u n o e l e c t r o p h o r e s i s 20

    1.5.2 I m m u n o f i x a t i o n e l e c t r o p h o r e s i s 22

    1.5.3 I s o e l e c t r i c f o c u s i n g and i m m u n o i s o e l e c t r i c f o c u s i n g 24

    1.6 Aim of the p r e s e n t s tudy 27

    1.7 D e s i g n of e x p e r i m e n t 27

    C h a p t e r 2 M A T E R I A L S AND M E T H O D S 30

    2.1 S tudy s u b j e c t s 30

    2.2 A p p a r a t u s 30

    i

  • 2.2 A p p a r a t u s 3 0

    2.3 R e a g e n t s and m a t e r i a l s 32

    2.4 P r e p a r a t i o n of ge l s 35

    2.5 I s o e l e c t r i c f o c u s i n g p r o c e d u r e 36

    2.6 Acid f i x a t i o n and s t a i n i n g 3 7

    2.7 T e c h n i c a l f a c t o r s a f f e c t i n g r e s u l t s 38

    Chapter 3 R E S U L T S 40

    3.1 I n t e r p r e t a t i o n of r e s u l t s in i s o e l e c t r i c f o c u s i n g 40

    3.2 A f f e c t i n g f a c t o r s 47

    3.3 C o m p a r i s o n of the r e s u l t s b e t w e e n IEF and IFE 53

    C h a p t e r 4 D I S C U S S I O N 59

    C h a p t e r 5 C O N C L U S I O N 65

    R e f e r e n c e s 66

    ii

  • LIST OF TABLES

    TABLE Page

    1. The p roper t i e s of f ive major immunoglobul ins 5

    2. C lass i f i ca t ion of monoclonal gammopath ies 15

    3. Compar ison of the resul t s between IEF and IFE 55

    4. Compar i son of the pr ices between IEF and IFE 61

    iii

  • LIST OF FIGURES

    Page

    1. The b a s i c m o n o m e r i c u n i t of i m m u n o g l o b u l i n s 4

    2. The s t r u c t u r e of IgM 9

    3. The s t r u c t u r e of IgA 10

    4. The l a b o r a t o r y i n v e s t i g a t i o n of m o n o c l o n a l i m m u n o g l o b u l i n 18

    5. I m m u n o e l e c t r o p h o r e s i s 21

    6. I m m u n o f i x a t i o n e l e c t r o p h o r e s i s 23

    7. I s o e l e c t r i c f o c u s i n g 26

    8. D e s i g n of e x p e r i m e n t 29

    9. H o m e - m a d e IEF c h a m b e r 31

    10. T e m p l a t e f o r IEF 33

    1 1 . N o r m a l p a t t e r n s in IEF 41

    1 2 . M o n o c l o n a l IgG in IEF 42

    1 3 . M o n o c l o n a l IgA in IEF 44

    1 4 . M o n o c l o n a l IgM in IEF 45

    15. F r e e l i g h t c h a i n s in IEF 46

    16. O l i g o c l o n a l IgG in IEF 48

    17. P a t t e r n s of a m p h o l y t e s a d d e d at 100°c a g a r o s e 49

    18. P a t t e r n s u s e d B e c k m a n ' s i m m u n o f i x a t i o n r e a g e n t s in IEF 50

    19. A m p h o l y t e s a f f e c t i n g t h e IEF p a t t e r n s in s t a i n i n g 52

    20. P a t t e r n s of d i f f e r e n t e l e c t r o d e p o s i t i o n 54

    21. R o u t i n e e l e c t r o p h o r e s i s of t h e m i s s e d m o n o c l o n a l IgM 55

    2 2 . I F E of t h e m i s s e d m o n o c l o n a l IgM 56

    23. IEF of t h e m i s s e d m o n o c l o n a l I g M 57

    2 4 . A new s c h e m e of l a b o r a t o r y i n v e s t i g a t i o n 58

    of m o n o c l o n a l p r o t e i n b a s e d on our r e s u l t s

    iv

  • LIST OF ABBREVIATIONS

    IFE: immunof ixa t ion e lec t rophores i s

    IEF: i soe lec t r ic focus ing

    MGUS: monoclonal gammopath ies of undetermined s ign i f i cance

    CLL: chronic lymphocyt ic leukaemia

    SMM: smoulder ing mul t ip le myeloma

    Macro: macrog lobu l inaemia

    Ig: immunoglobu l in

    V

  • Acknowledgements

    I am grateful to Professor N.M. Hjelm, Chairman, Department of Chemical

    Pathology, the Chinese University of Hong Kong, for accepting me as an MSc

    student.

    My sincere thanks to my course work research supervisor, Dr. Ann Read,

    Adjunct lecturer, Department of Chemical Pathology, the Chinese University of

    Hong Kong, for her superior guidance and valuable suggestions during the course

    of this work.

    I am indebted to Dr. C.W.K. Lam and Dr. N.S. Panesar, Senior lecturers,

    Department of Chemical Pathology, the Chinese University of Hong Kong, for

    their continuous encouragement and support.

    I would like to thank Dr Robert Cheung, Adjunct lecturer, Department of

    Chemical Pathology, the Chinese University of Hong Kong, for preparing the

    major instruments to me.

    I wish to acknowledge my many thanks to Mr. Fung Siu Fan, Medical

    Technologist, Clinical Biochemistry unit, the Chinese University of Hong Kong,

    for collecting speciments under his care in Prince ofWales hospital.

    My appreciation is expressed to Mr. Fung Loi Mo, MLT H, Department of

    Chemical Pathology, the Chinese University of Hong Kong, for his

    encouragement and lending the shaker to me.

    Lastly, I must thank my family, for their understanding, patience and

    continuous support during my study.

    vi

  • Abstract

    The identification of monoclonal immunoglobulins is important in the diagnosis and

    management ofsome B cell tumours. It is difficult to distinguish between oligoclonal

    bands and small monoclonal bands by routine protein electrophoresis.

    This is even more of a problem in Hong Kong than many other countries because of

    a high incidence of cirrhosis which is one of the conditions associated with

    oligoclonal bands. Immunofixation electrophoresis (ff"E) can be used to help

    distinguish these bands but antiserum is quite expensive. Another method with high

    sensitivity and specificity for detecting abnormal IgG bands is isoelectric focusing.

    This method is not sensitive for detecting abnormal IgA and IgM bands and therefore

    cannot be used for initial screening. It is cheaper than immunofixation because

    expensive antiserum is not required.

    Fifty samples which had been found to have monoclonal, oligoclonal or polyclonal

    bands by protein electrophoresis and immunofixation were reanalysed by EEF without

    knowing the reported result. There was agreement between the two methods as to

    which electrophoretic patterns showed no abnormality of IgG. However it was found

    that ffiF distinguished more clearly between oligoclonal and monoclonal IgG than

    IFE. We recommend this as a cheap method for further investigating bands which do

    not have a typical appearance of a monoclonal band. It can reduce the number of

    samples which require immunofixation.

    vii

  • 摘 要

    單克隆免疫球蛋白的確定對於某些B細胞腫瘤的診斷和治療都非常重要. 但常規的蛋白電泳難以區分寡克隆及微小的單克隆球蛋白帶。

    這個問題在香港尤爲顯著。因爲香港是一個肝硬化高發地區,而肝硬化是寡

    克隆帶出現的原因之一。免疫固定電泳可用於區分這些免疫球蛋白帶,但其抗血

    淸試劑則非常昂貴。另一種方法-等電聚焦電泳,能高度敏感,高度特異地測定

    IgG帶。由於這種方法對於測定異常IgA和IgM不敏感,故不能用於第一步的 飾選。等電聚焦電泳不需用昂貴的抗血淸試劑,所以它比免疫固定電泳便宜。 ’

    我們總共分析了 50個樣品。經過常規蛋白電泳和免疫固定電泳的檢測,確定

    它們含有單克隆,寡克隆或多克隆免疫球蛋白。在結果保密的情況下,我們用等電

    聚焦電泳重新分析這些樣品o在未見異常IgG的免疫球蛋白帶中,這兩方法的結 果是一致的。但等電聚焦電泳比免固定電泳更淸晰地區分寡克隆與單克隆IgG。 我們以爲,當非典型的單克隆免疫球蛋白帶出現時,等電聚焦電泳是一個便宜的

    方法去進行深入觀察。等電聚焦電泳可