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。等電聚焦電泳可減少需要進行免疫固定樣品數量。

viii

Chapter 1.

hitroduction

Monoclonal immunoglobulin is comprised of intact or fragmented immunoglobulin

molecules, which are homogenous in structure and originate from a single clone of B

cells. The identification of monoclonal immunoglobulin is important in the diagnosis

and management of some B cell tumors (e.g. myeloma). 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. In this chapter, the history of identification of

monoclonal protein, the current knowledge of physiology of immunoglobulin, the

pathology of monoclonal immunoglobulin and the diseases associated with the

monoclonal immunoglobulin are reviewed. It also reviews the different techniques for

detecting this protein. Finally, the aim of the present study is described.

1 • 1 History

In 1845, Bence -Jones protein was found in the urine of a patient with severe back

pain. Dr Henry Bence Jones made the earliest description of abnormally occurring

protein in urine, associated with myeloma, which preceded recognition of the disease,

in a lecture given to the Royal College of Physicians in 1846. Another patient with

bone disease was studied in Amsterdam twenty -two years later and the urine of this

patient contained the same type of urinary protein described by Bence Jones [1, 2].

1

There was little progress in the further characterization of this protein until Tiselius

in 1937 separated serum globulins by electrophoresis into three components: alpha,

beta, and gamma globulins [7]. Apitz in 1940 introduced the term paraprotein to

describe the abnormal proteins in blood, urine and tissues that are produced by

myeloma cells [2，48]. Electrophoresis on a substrate of paper (by the 1950s) or starch

gel[l] and later on cellulose acetate and agarose, greatly advanced the study of the

disease. The term ’ M-protein , was proposed by Riva in 1957 to designate the sharp

peak or band of protein of homogenous isoelectric point that appeared in the serum

protein electrophoresis of patients with myeloma and the letter 'M' agreed with the

later determination that these proteins were monoclonal [1].

Grabar and Williams in 1953 devised the technique of immunoelectrophoresis which

was later adapted to detect monoclonal immunoglobulin [14]. Immunofixation which

was first used by Ritchie and Smith in 1976 [3，4] is now a common method in

identifying monoclonal proteins with greater sensitivity and specificity than

immunoelectrophoresis. Immunoisoelectric focusing was developed by Sinclair et al

in 1983 and become the most sensitive method in detecting monoclonal protein [5, 6].

2

1.2 Immunoglobulins

1.2.1 Structure

It has long been recognized that those immunoglobulins, sometimes referred to as

"gamma globulins" migrate in the beta and alpha-2 mobility regions as well as in the

gamma region. The basic monomeric unit of immunoglobulins is an Y-shaped

molecule consisting of two identical heavy chains and two identical light chains (Fig.

1) [2, 7, 84]. There are five classes of heavy chains designated by Greek small lower-

case letters (y, a， i, 5，e) occurring in IgG, IgA, IgM, IgD, and IgE respectively and

two types oflight chains (K and X).

In any given immunoglobulin molecule, there is only one type of heavy chain and

only one type of light chain. Heavy and light chains are composed of related

'domains'; both have a single variable region domain and the light chain has a single

constant region domain while the heavy chain has three or four constant region

domains depending the class. Disulphide bonds link usually four polypeptide chains.

The name of the immunoglobulin is taken from the combination of the heavy and

light chain designations. The properties of the five major immunoglobulin molecules

are listed in Table 1 [7, 8].

There are approximately 110 residues at the amino terminal ends of each light and

heavy chain constituting a region where the amino acid sequences differ considerably.

This is the site called the variable or 'V' region that determines the 'idiotype' or unique

3

Fig. 1 The basic monoclonal unit of immunoglobul ins [ref 7]

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4

Table 1 Properties of plasma immunoglobulins [ r e f 2 , 7]

Properties IgG IgA IgM IgD IgE

Molecuhr weight 150000 170000 900000 180000 196000 % total plasma Ig 73 19 7 1 0.001 Subcksses 4(Ig 14) 2(IgA 1-2) None None None Complement Yes Yes Yes No No activation Sedimentation 6.7S 7.1S 19S 7.0S 8.0S coefficient LightK:hain isotype icand X K and X Kand X K and \ Kand X Placental transfer Yes No No No No Half-iife 21 days 5.8days 5.1 days 2.8 days 2.3 days Approx. mean 10 2.0 1.0 0.0003 0.03 normal adult conc. (g/Hter) Daily synthetic 33 24 6.7 0.4 0.02

5

antigen binding ability of the antibody. But each variable residue is not involved

equally in the process of antigen binding and three subregions stand out as being

'hypervariable'. The hypervariable regions of light and heavy chains are the regions

specifically responsible for the binding of the antigen, for that individual

immunoglobulin molecule. The remainder of the molecule, which is not involved in

antigen recognition, is named the 'constant region' and is identical to other

immunoglobulin molecules of the same class, subclass, and allotype. The region

between the antigen binding part of the immunoglobulin and the constant part is the

hinge region. The length of the hinge region varies between the immunoglobulin

classes and subclasses.

In light chains, there are two intrachain disulphide bridges and there are four of such

bridges in heavy chains. A peptide loop of 60-70 amino acid residues is enclosed in

each bridge, and a high degree of sequence homology between sections of peptide

chains is contained within the disulphide bridged loops. These regions are indicated

by a specific name which is 'homology' regions. Each homology region is folded in a

compact globular structure or domain and each domain plays a particular biological

role. Both IgM and IgA have the propensity to form polymers.

Two-thirds of serum light chains are K and one-third X. They have a molecular

weight of22000 Daltons and 210-220 amino acids and contain a constant and variable

region. The variable region extends from the amino terminal for approximately 110

amino acids and is responsible for the unique thermal solubility and antigen binding

properties [7].

6

Light chains may play a role in immunoregulation of the immune response and they

are catabolized by the kidney. Clyne postulated that nephrotoxicity is related to the

isoelectric point in 1979 with cationic light chains being more nephrotoxic than

anionic chains [9，10，11]. The ratio offree light chains KiX in the serum may play an

important role in monitoring and provides earlier warning of myeloma

progression[76].

1.2.2 Properties of immunoglobulins

(i) IgG

There are four subclass of normal human IgG. IgGl and IgG3 fix complement by

the classical pathway, while IgG2 and IgG4 do not. Other varying properties of the

IgG subclass include the presence of the receptor for macrophages (IgGl and IgG3)

and the failure to react with staphylococcal A protein (IgG3). IgGl antibodies are

against isoagglutinins and viruses, while IgG2 antibodies are detected against

polysaccharides. IgG4 antibodies act in the circulating anticoagulants against

coagulation factors viii and ix. All classes of IgG cross the placenta and are

responsible for passive immunity in the newborn. IgG interacts with the Fc receptors

on neutrophils, monocytes, and macrophages. The plasma IgG level rises slightly later

in response to soluble antigens such as bacterial toxins. The IgG molecules can

diffuse fairly freely into the interstitial fluid and act in the tissue against infection.

They may be detected in a raised level and as a diffusely increased y band on the

electrophoresis strip within a few weeks of the initial infection.

7

(ii) IgM

The characteristic structure ofIgM is shown in Fig. 2. There are five H2L2 subunits

in the IgM molecule which is a pentamer shape. IgM antibodies are the first to appear

in the immune response and are quite effective in the activation of complement by the

classical pathway. They have low molecular weight and low concentrations in normal

serum. They are almost confined to the intravascular compartment since they are

large. This makes them the first line of defense amongst immunoglobulins against

infection. IgM is the major protein found on the surface ofB-lymphocytes.

(iii) IgA

IgA is produced adjacent to secretary surfaces such as intestinal, respiratory tracts,

sweat glands on the skin, etc. It is affected more than other immunoglobulins in

disease of the gastrointestinal and respiratory tracts. Most IgA is monomeric, although

approximately 15 per cent of serum IgA circulate as a dimer linked by a joining or J

chain (Fig. 3). The J chain combines covalently with the H chain of IgA (and IgM)

and is structurally unrelated to heavy and light immunoglobulin chains. Secretory IgA

differs from serum IgA in its association with a peptide chain called the secretary

component. The role of this secretory component is to increase the resistance of the

IgA molecule to the proteolytic digestion in secretions such as those in the digestive

system. Secretory IgA has a molecular weight of 380000 and the complete molecule

is a dimer of IgA together with a J chain and secretary component. Secretory IgA has

antibacterial and antiviral activity and prevents microorganisms from penetrating the

mucosal pathway.

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(iv) IgD

Rowe and Fahey first described IgD in a patient with myeloma in 1965. It is found

on the surface ofmost B lymphocytes, although present in normal serum in very small

amounts (less than 1 per cent of the total serum Ig ). Ninety per cent of the normal

serum IgD, and IgD myeloma paraproteins are of X light-chain isotype. Surface IgD is

usually found in association with IgM and, in this situation, both molecules have the

same VH and VL regions [12,13]. The role of IgD in normal immune regulation is

still unknown.

(v) IgE

The serum level of IgE is low because of a very low synthetic rate and short

intravascular half-life. IgE is synthesized by plasma cells beneath the mucosae of the

gastrointestinal and repiratory tracts and by those in the lymphoid tissue of the

nasopharynx. It is the reaginic antibody of man and these antibodies mediate the

wheal and flare reactions and are often present in high allergic individuals. It is

present in nasal and bronchial secretions. Combination of antigen results in the cell

releasing mediators and accounts for immediate hypersensitivity reaction such as

occur in hay fever. The level of IgE is raised in several disease with an allergic

component such as in some cases of asthma, eczema and parasitic infestation.

11

1.3 Monoclonal proteins and monoclonal gammopathies

(1.3.1) Monoclonal proteins

Monoclonal proteins result from the over-production of immunoglobulin molecules

by plasma cells and lymphocytes and are the hallmark of multiple myeloma and

related conditions [7]. They may arise from malignancies of B cell origin or result

from hyperstimulation of one or a few normal clones giving rise to single

(monoclonal) bands on electrophoresis. The clinical laboratory importance of

monoclonal proteins lies in their use as tumor markers for the diagnosis and

monitoring of malignancies o f B cell origin [44, 59]. In malignant diseases, they are

usually associated with myeloma, lymphoma and chronic lymphocytic leukaemia

[53]. However, the presence of a monoclonal protein per se is not only a marker of

malignancy, and several benign conditions, such as collagen vascular disease, can be

associated with monoclonal immunoglobulin production. Monoclonal proteins can

occur in the elderly apparently without malignant significance (e.g. MGUS or the

benign paraproteins). It is important to distinguish malignant paraproteins from those

ofabenign nature which are the products of static or slowly growing clones of a well

differentiated type. Although some of these diseases are benign, monoclonal protein

charactererisation and level is required in follow-up investigation because they may

become to malignant [54, 68-69，71], e.g. 17% at ten years and 33% at twenty years

ofMGUS eventually progress to malignant, with an annual risk rate of 0.8%[14, 56,

63], and the presence of kappa light chain is thought to be a risk factor for malignant

transformation[66].

1 2

Monoclonal B cell proliferations in which B lymphocyte maturation is blocked in the

final stages of the differentiation cycle lead to monoclonal gammopathies [81]. The

heavy chain gene will be rearranged first during the B lyphopoiesis without antigen,

and then the K gene. The K rearrangement is abortive in some cells and a X

rearrangement occurs. Then the cell is committed to a unique variable region

specificity (the idiotype) and light chain type. It is possible to select different heavy

chain constant regions so that a cell may change from the synthesis ofIgM to another

class during maturation, a process called class switching. Immunoglobulin resulting in

the absence ofantigen is bound on the membrane ofB cell. Antigen may choose those

B cell clones able to recognize the antigen. After the proliferation of clones, some of

those B cells are turned into plasma cells while others become memory cells. Plasma

cells are non-proliferative end cells producing large amounts of secreted

immunoglobulin. In the high level ofcell proliferation that occurs on contact ofB cell

with antigen, antibody diversity exists due to the accumulated somatic mutations in

the variable regions. During immune reaction, in a low antigen concentration, somatic

mutations resulting in higher affinity of the antibody for antigen will be selected.

A polyclonal antibody response may due to a large number of different idiotypes

produced by many different clones in response to even a single antigen results in

microheterogeneity amongst the immunoglobulin. If there is predominant

proliferation of a single clone of B cells, the antibody product will be confined to a

1 3

single heavy and light chain type and to a single idiotype, leading to a monclonal

immunoglobulin or paraprotein[2].

(1.3.2) Monoclonal gammopathies

Monoclonal gammopathies are disorders characterized by the proliferation of a

single clone of cells producing a monoclonal protein (M-component, M-protein,

paraprotein) [14, 49]. The most common plasma cell disorder is the benign

monoclonal gammopathy (BMG) or monoclonal gammopathy of undetermined

significance (MGUS), although multiple myeloma is the most comment malignancy

in monoclonal gammopathies [14, 17, 78]. A current classification of monoclonal

gammopathies is shown in Table 2 [14].

To make an accurate diagnosis for the patients with monoclonal gammopathies is

very important. Some of them don't need the treatment while others need to be treated

immediately [18]. A wrong diagnosis will lead to unnecessary cost or delay of

treatment. Monoclonal immunoglobulin is the characteristic of these diseases. The

follow-up investigation of M-protein is necessary even in those benign monoclonal

gammopathies.

In monoclonal gammopathies, all plasma cells produce a slight excess of light chains

which may precipitate in the renal tubules causing renal disease [75]. Malignant B cell

clones often secrete considerably more than their benign counterparts, resulting in

detectable amounts of Bence Jones protein in the urine. Bence Jones protein is

occasionally seen in the serum due to renal failure of filtration by the kidney or in

cases where very large amounts are produced by the tumor. Monoclonal

1 4

Tab le 2. C lass i f i ca t ion of monoc lona l gammopa th ies [ref 1]

MGUS 5 ^

Mul t ip le

Myeloma 18%

Amylo idos is 10%

Lymphoma 5%

SMM 4%

Sol i tary ^

CLL ^

Macro ^

15

immunoglobulins may deposit in the body and develop to monoclonal Ig deposition

disease (MIDD)[50]. Discrete changes in V region sequences are the major cause in

tissue deposition of human L chains [65]. The presentations are variable due to the

difference deposition places for paraproteins, e.g. respiratory insufficiency may

happen due to accumulation of IgG-kappa paraprotein in the alveolar space[70];

deposition of paraprotein in small vessels is a cause of skin ulcers in Waldenstrom's

macroglobulinemia[72] and deposition in kidney leads to renal lesions [75]. The

precipitation of monoclonal immunoglobulin is also related to the type I and type II

cryoglobulinemia because monoclonal IgM, IgG, and IgA may be shown to

cryoprecipitate when they are exposed below 37°c [58], Neuropathy was found in

some monoclonal gammopathies associated with IgG, IgA and IgM [67, 79].

Paraproteins were detected even in systemic capillary leak syndrome [74].

The number of clone cells in the blood of patients with monoclonal gammopathies is

different, e.g. clonal circulating cells of MGUS patients is less than those with

myeloma [64], Then serum concentration of monoclonal proteins often relates to the

tumor cell mass in myeloma, Waldenstrom's macroglobulinaemia and alpha-heavy

chain disease (alpha HCD) and thus may be used for detecting and monitoring these

disorders [47]. The level of monoclonal proteins is not as useful a prognostic marker

as the serum beta 2-microglobulin (beta 2-M) level because beta 2-M reflects both

tumor cell production and the failure of excretion which results from the renal damage

which has long been known to be an important prognostic feature in myeloma [19].

Although it is not always a very good prognostic marker, monoclonal proteins do give

useful information [18]. For example: type of paraprotein may influence treatment;

level of monoclonal protein may influence whether therapy is necessary.

1 6

1.4 Laboratory investigation of monoclonal immunoglobulin

1.4.1 The current procedure of investigation in this laboratory

The laboratory investigation of monoclonal immunoglobulin is summarized in Fig. 4

[41]

Serum and urine protein electrophoresis is requested when an abnormal protein is

suspected. Electrophoresis is the first step for detecting monoclonal immunoglobulin

since it is the simplest and most reliable method. It is widely used in clinical

chemistry laboratories and became a routine test in the measurement of proteins. But

it is only the first line of investigation in paraprotein and not adequate for identifying

the paraprotein [20]. Five main groups of proteins, albumin and the al- , a2-, p-, and

y-globulins, may be distinguished after staining and compared with those in a normal

control serum. Each group contains several proteins. Some of the abnormal

electrophoretic patterns are characteristic of a group of related disorders, while others

show non-specific pathological processes. Most immunoglobulin species run in y

range or move into P or a2 range. Electrophoresis may suggest whether a raised

immunoglobulin concentration represents a monoclonal increase and also demonstrate

immune suppression, especially of IgG, which usually accompanies malignant

paraproteinaemia. This is shown by a sharp band representing the monoclonal protein

that is accompanied by a corresponding decrease in the intensity of staining for the

remainder of the y region.

1 7

Fig. 4 Laboratory investigation of monoclonal immunoglobulins

Electrophoresis

V

Suspicious abnormality

Immunoelectrophoresis or

Immunofixation z \ Paraprotein No paraprotein \ z

Report

1 8

Further investigation should be employed to confirm the presence of monoclonal

protein and to distinguish the immunoglobulin class. There are several electrophoresis

techniques generally used in the detection, identification/characterization and

monitoring of gammopathies. The current methods are immunoelectrophoresis and

immunofixation [4，19，22, 35]. Both of these methods are the next step for

confirming a monoclonal immunoglobulin and identifying its type. And the third step

is quantification of paraprotein by densitometry from the electrophoresis strip [24，25,

28，30, 36，33, 37，77].

Thus the strategy to diagnose monoclonal gammopathies includes electrophoresis of

serum protein on agarose gel or cellulose acetate; immunoelectrophoresis or

immunofixation; and quantification of paraprotein [21,26, 51, 80],

1.4.2 Problems in identifying monoclonal immunoglobulin

An oligoclonal pattern results if more than one clone of cells in an individual are

producing significant amounts of "monoclonal" immunoglobulin [52]. It is sometimes

difficult to distinguish between oligoclonal bands and small monoclonal bands by the

techniques mentioned above. This is even more of a problem in Hong Kong than

many other areas because of the high incidence of cirrhosis which is one of the

conditions associated with oligoclonal bands [55, 84].

19

1.5 Comparison of different techniques

1.5.1 Immunoelectrophoresis (IEP)

Immunoelectrophoresis is a common method in identification of the monoclonal

protein. Electrophoresis on agarose gel or cellulose acetate separates the various

proteins. When this has occurred, antibody is applied alongside the full length of the

electropherogram. Usually, the sample is loaded a number of times on the same gel at

regular intervals. This allows different antiserum to be applied such as IgG, IgA, IgM,

K and X between the samples. The precipitin lines or arcs are formed along where the

antigen and the antibody meet. Most precipitin arcs form within 24 hours. The arcs

become diffuse and exaggerated if the diffusion is continued for more than 24 hours.

This will make the interpretation difficult. The immunoelectrophoretic pattern of

monoclonal protein is shown in Fig.5. The presence of a monoclonal protein is

indicated by the distortion (thickening or bowing) of an arc caused by the presence of

a relatively larger amount of protein in one portion of the gel owing to the uniform

isoelectric point of the monoclonal protein [1].

However, it is difficult to monitor by immunoelectrophoresis if monoclonal Ig is

presence in a low concentration and/or it has a diffuse electrophoresis mobility. IEP is

not good in detecting oligoclonal Ig since the second dimension in IEP is a diffusion

step, and not only do the antigens diffuse toward the antiserum, but also diffuse

towards each other. And this loses much of the resolution obtained during the

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f

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

antibody antigen precipitation

J^ _^^0^ channel for antiserum

^ • ^ ― ^m-^

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21

electrophoretic step [15], The ,umbrella efFect' which is caused by the polyclonal Ig,

may increase the difficulties in identification of the small monoclonal components in

immunoelectrophoresis[3 8].

1.5.2 Immunofixation

The method employs electrophoresis in agarose gel or cellulose acetate followed by

application of specific antibody by overlay of a cellulose acetate strip soaked with the

specific antibody [16]. The strip is laid on the gel so that it covers the zone in which

the antigen of interest may be located. After an incubation of 1 h, the strip is removed

and the gel washed and stained. Antigen and antibody that have formed a precipitate

are not removed during the washing step, whereas antigens that have not reacted with

the antibody are soluble and will be removed [23], As for immunoelectrophoresis, the

sample is usually loaded a number of times to allow fixation with several antibodies

on a single gel, and one portion of gel may be stained without the immunofixaton step

to give an electrophoretic pattern for comparison. This allows serum from a patient to

be characterized with several different antibodies on a single plate. A template may be

used to apply the antiserum instead of cellulose acetate strips. Fig.6. shows a

monoclonal protein by immunofixation.

Immunofixation is a good technique to identify monoclonal protein with greater

sensitivity and specificity than immunoelectrophoresis [32，39，40]. It may identify

bands of protein seen on electrophoresis but which are lost on immunoelectrophoresis

because of diffusion. It also may identify small bands, IgA and IgM [31], which are

2 2

Fig. 6 lmmunof ixat ion electrophoresis

. S P G A M K 人 一

^ 3 5 ^ ^ 二： * ^ ^ antibody antigen precipitation band % { ^

* — -.. wjr^ ^ • •‘. <Qf ,

^ ?

十

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1

23

impossible to see on immunoelectrophoresis[ 15]. The technique of immunofixation is

easier than immunoelectrophoresis[3 8]. The Paragon JFE system which is made by

Beckman Instruments Inc is the method used in our laboratory and it is good for

detecting small monoclonal proteins.[21]

Oligoclonal bands may still be difficult to distinguish from monoclonal bands even

using immunofixation although it is a very good technique in identifying paraproteins.

The method is expensive as more antiserum is used than with immunoelectrophoresis.

Antigen-excess in immunofixation requires the examination to be repeated with

several dilutions of serum or antiserum [43].

1.5.3 Isoelectric focusing and immunoisoelectric focusing

In isoelectric focusing, ampholytes, which contain molecules of different pH, are

added to the agarose or acrylamide gels. When the current is turned on the ampholytes

migrate until they reach the pH of their isoelectric point [1, 36] and a pH gradient is

created. Thus the gel consists of a pH increase from anode to cathode in the range of

ampholytes used in our experiment 3-10. Similarly proteins from the sample migrate

to their isoelectric points. If they diffuse in either direction they gain or lose a charge

and move back to their isoelectric points. This results in narrow bands with no

problem of diffusion and it "concentrates" the sample so it is a more sensitive

technique than electrophoresis.

2 4

Isoelectric focusing is a technique of high resolution capable of resolving proteins

which differ in isoelectric point by as little as 0.001 pH units.

A single monoclonal band despite the name may show microheterogeneity (multiple

bands) in isoelectric focusing [15, 34，62, 73], see over 1.3.1. This is due to post-

synthetic modification, and is due to carbohydrate heterogeneity, especially in heavy

chain disease proteins and changes in the oxidation state of amino acids, particularly

those containing sulphur, and metal ions resulting in recognizable patterns of bands

know as the spectrotype. Generally microheterogeneity cannot be detected in

electrophoresis and immunoelectrophoresis. The width of the band on electrophoresis

or degree ofcurvature of the arc on immunoelectrophoresis is related to the number of

bands in the spectrotype. Typically, "narrow" bands have 5-7 lines, and "broad" bands

can have in excess of 20 lines in isoelectric focusing. Fig.7 shows the monoclonal

proteins by isoelectric focusing.

Immunoisoelectric focusing is the most sensitive electrophoretic method described

in identifying monoclonal proteins [1, 42，61]. This method is about 10-40 times more

sensitive than immunoelectrophoresis [45]. For monoclonal IgA, IgM, and IgD,

immunoisoelectric focusing is much more sensitive than either

immunoelectrophoresis or isoelectric focusing. In Immunoisoelectric focusing, the

tracks are overlaid with strips of cellulose acetate membrane soaked in specific

antiserum for 2 h at 37°c after focusing mentioned above similar to

immunoelectrophoresis is [45]. It is also sensitive in detecting oligoclonal bands [29].

As with the immunofixation, there is a high cost in this technique since the antiserum

is quite expensive.

2 5

Fig. 7 Isoelectr ic focus ing

* 麵 ’ ： - 〜 . 1 ST' (.. _ : t ' -r- 1

ifif 11 f p _ 丨 一 _ 譽

I麗編M k I 發 v

丫， wedge shape pattern of monoclonal IgG

1

26

1.6. Aim of the present study

The current method for investigating samples with abnormal bands is to perform

immunofixation. Also it is a good method for identifying monoclonal bands, but does

not always distinguish clearly between monoclonal and oligoclonal bands. Another

method, as stated above with high sensitivity and specificity for detecting abnormal

IgG bands is isoelectric focusing, although it is not so good at detecting IgA and IgM

paraproteins [27]. It cannot therefore be used as the initial screening technique but is a

good method for identifying oligoclonal and monoclonal IgG. The cost of isoelectric

focusing should be cheaper than immunofixation because the antiserum is not used.

The aim of this project was to see if isoelectric focusing without immunofixation

could help to distinguish between monoclonal and oligoclonal bands, thus reducing

the number of immunofixations to those patients with monoclonal bands.

1.7. Design ofExperiments

50 serum samples were screened by routine electrophoresis. Some of the samples

had monoclonal bands and others did not.

The abnormal samples were analysed further by the immunofixation method used in

the laboratory and the monoclonal proteins were distinguished from those non-

monoclonal proteins which included oligoclonal or polyclonal proteins.

2 7

The results ofthese samples were unknown to me until after I had made a diagnosis

independently by isoelectric focusing. Then the results of the two techniques were

compared.

The design of experimental is shown in Fig.8.

2 8

Fig.8 Design of experiment

50 samples

V electrophoresis

. 人 immunofixation isoelectric focusing

,' I result result \ /

comparison

29

Chapter 2

Materials and method

1. Study subjects

i) Samples

Samples were selected by the Scientific Officer from the Protein Unit of the

laboratory. They were selected with and without monoclonal bands, particularly those

samples which gave inconclusive results on routine protein electrophoresis and

required fixation for a definitive diagnosis.

ii) Storage

The samples were stored in the freezer with the temperature below 0° C.

2. Apparatus

i) Home made ffiF chamber (Fig. 9)

ii) Power supply

(200Z Power Supply, LKB.)

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iii) Corning oven for the drying gel

(75amp)

iv) Containers for fixative, staining, destainer.

V) Shaker

3. Reagents and materials

i) Templates (Fig. 10)

Templates were obtained from the Coming ACI electrophoresis system. They were

modified with "Dymo" tape to provide a template measuring 75xll5x0.75mm as

described in ref[86]. There are eight wells for samples allowing eight samples to be

run at the same time.

ii) Mylar film (FMC lMC BioProducts)

Gel Bone TM 127mm wide 0.2mm thick and cut to 80mm length. The properties

were different on both sides of the film. One side has affinity to water, while the other

side has not. It is important to distinguish these sides correctly.

iii) ffiF grade agarose (Pharmacia Biotech)

3 2

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3 3

iv) Carrier ampholytes

(Pharmalytes pH 3-10，Pharmacia Biotech)

V) Filter paper, absorbent paper.

vi) Microsample applicators

vii) Fixative reagent

A) The "Beckman's" reagent which is used in inmmunofixation.

B) The reagent was 5% TCA, 35% SSA, 30% Methanol. 50g trichloracetic acid

and 3.5g sulphosalicylic acid were dissolved in approximately 500ml distilled water.

300ml methanol was added and made up to lL with distilled water[85-90].

viii) Staining reagent

A) The "Beckman's" reagent which is used in immunofixation.

B) 0.3% Coomassie Brilliant Blue R in methanolic acetic acid. 0.3g Coomassie

Brilliant Blue R250 was dissolved in 50ml methanol and 10% glacial acetic was

added to 500ml with distilled water [60, 85-90],

ix) Destainer

A) The Beckman's reagent which is used in immunofixation.

B) 100ml glacial acetic acid was diluted to 500ml with distilled water and 500ml

methanol was added [86].

x) Distilled water

3 4

4.Preparation ofIEF gel

The gels were prepared as in reference [85-90] but with the following minor

modifications.

i) 0.4g agarose was added in 38.0ml distilled water to prepare 6 gels and the weight

was recorded.

ii) It was heated in a microwave oven until completely dissolved. It took about 40

seconds and the temperature was nearly 100 degrees. It was weighed again and

distilled water was added to make up for water lost by evaporation.

iii) It was cooled to 55-65^C in a water bath for 10 minutes at least.

iv) The water bath was 55-65^C, and was also used to warm 2.0ml carrier

ampholytes, 6 templates and the tubes too.

V) The carrier ampholytes were added to the agarose solution and swirled to mix

thoroughly avoiding the creation ofbubbles.

vi) The agarose solution was distributed into 6 clean tubes. One tube of solution was

poured onto a template. Immediately the mylar film was applied, hydrophilic surface

down, to the molten gel, spreading the gel evenly over the template and avoiding

3 5

entrapment ofair bubbles. The excess agarose solution was allowed to exude from the

corners. The other tubes were poured in a similar manner.

vii) The gels were cooled and stored in sealed plastic bags at 4®C. They were

protected from direct light. Several drops of water were added into the plastic bag to

protect them from drying. Best results were achieved if the gels were allowed to

mature for approximately 16 hours, but if necessary they may be used 30 minutes

after pouring [86]. Gel may be stored for up to one month [86-90].

5. ffiF procedure

i) The mylar film was pulled away from the template starting from one corner. The

gel adhered to the mylar film.

ii) The gel was placed on a dark level surface.

iii) Samples (0.5-1.0ul serum) were applied to the first to sixth sample wells. In the

eighth channel a haemolysed sample was added in the sample well and a drop was

added on the other side of the gel. The haemoglobin was used as a visual marker of

ffiF. In the seventh channel, a known sample with a monoclonal band was added as a

control to show the ampholytes were working.

iv) The gel was inverted and placed directly onto the electrodes with the sample wells

close to the anode.

3 6

V) It was focused for 210 v.hr at 1.0-1.2watt/gel. The formula, which is used to

describe the relation of electric power, resistance and current, is: electric power

(watt)= resistance (voltage) x current (ampere). To keep the electric power in the

same range, the higher the voltage, the lower the ampere. The initial voltage should be

150-260v, with a relatively high initial current 7.0-7.5mA to keep the wattage in the

range. Final voltage approximately increases to 500v and final current approximately

decreases to 2mA.

vi) The power pack was turned off allowing the voltage to drop to zero.

vii) The gel was then acid fixed.

6. Acid fixation and staining

i) The gel was placed in fixative for 10 minutes.

ii) The gel was washed in distilled water for 10 minutes.

iii) Filter paper was laid onto the gel, avoiding entrapment of air bubbles. Several

layers of absorbent paper were applied on top, covered with a glass plate and a weight

of approx. 1 kg was applied for 2 minutes.

iv) The gel was dried completely by oven for 30-60 minutes.

3 7

V) It was washed in destainer for 5 minutes.

vi) Then it was placed in stain for at least 10 minutes.

vii) Finally it was washed in destainer until background was clear (it took nearly 10

minutes), avoiding excessive destaining since this may elute stain from some proteins.

7. Technical factors affecting results

7.1 The temperature of adding ampholytes

The ampholytes may be destroyed if the temperature of the agarose is too high

when the ampholytes are added into the agarose. We made several pieces of gels and

their temperature was increased to 100°c after adding ampholytes. The same samples

were added both in the 'normal' gel (ampholytes added in 55-65°c) and abnormal' gel

(ampholytes added in 100°c). Both of the results were compared after isoelectric

focusing.

7.2 The factors in staining

(i) Reagents

We used two kinds of staining reagents: Beckman's immunofixation reagents and

0.3% Comassie Brilliant Blue in methanolic acetic acid.

3 8

(ii) Ampholytes

Some gels were not washed and dried thoroughly after ffiF to investigate if the

ampholytes affect the result in staining.

7.3 Time

(i) Time ofIEF

The time required for focusing was determined by two methods: 1, following the

number of volt hours the method stated; 2，by watching the focusing of the drop of

Hb. The time was increased to see if the patterns improved. We did the ffiF in 1.5hr

and l.Ohr respectively.

(ii) Time for destainer

Both fresh and old reagents were used in destainer. The time for destain was

recorded to have a comparison.

7.4 The position of the electrode

The method stated to place the gel with the sample wells near the anode. The gel

was also placed with the sample wells near the cathode to see if it gives a different

result.

7.5 Watt, Voltage and Ampere

In difference reports, the watt for isoelectric focusing has two difference ranges:

1.0- 1.2watt/gel and 1.0-1.5watt/gel. We ran the same samples in both these two

range.

3 9

Chapter 3

Results

1. Interpretation of the results in ffiF

The ampholytes distribute regularly in the track with an increase pI from cathode

to anode [85-90]. The interpretation of the results does not depend on the exact

pIs of the bands but their own characteristic appearance.

1.1 Normal Patterns (Fig. 11)

The range of the normal patterns (track 1) from the anode to cathode is: al-

antitrypsin in pI 4.2-4.7; albumin in pI 4.7-5.0 and transferrin in pI 5.2.

Fibrinogen focuses in pI 4-5.5 but is usually not present in a serum sample.

Polyclonal bands (track 2-7) emerge as a diffuse zone from pH 5-9.5. The pI

range was selected specifically to give a good separation ofIgG.

1.2 Monoclonal IgG (Fig. 12)

The character of monoclonal IgG (track 2, 8) is a wedge-shaped pattern of very

regularly spaced bands. There is always a wedge with a group of reducing

intensity bands towards the lower pH region or a 'bidirectional' wedge reducing

intensity of banding in both directions. Monoclonal IgG may be detected

anywhere from pI 5 to 9.5. But most of them could be located in the range 7-9.5.

Residual IgG may be normal, depressed or not detected.

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1.3 Monoclonal IgA (Fig.l3)

Monoclonal IgAs (track 4) were shown as patterns consisting of large numbers

of very regular, closely spaced, sharp bands. However, they will also found in

precipitates (track 6) or partially focused and partially precipitated since they may

precipitate before reaching their pIs. Residual immunoglobulins were usually

depressed or not detected, but can be normal.

1.4 Monoclonal IgM (Fig.l4)

The monoclonal IgMs (track 3) were characterized by invariably precipitated

protein before their true pIs were reached. This is caused by not focusing. The pI

range in which monoclonal IgMs appear as zones of precipitated protein is 4.5-

7.0. The pI range of albumin is 4.7-5.0. Then monoclonal IgM could be on the top

of albumin and low levels of monoclonal IgMs may not be seen. They may appear

as: 1, early precipitation in which the whole track looks severely distorted; 2,

partially focused and 3, as a diffuse zone.

1.5 Monoclonal free light chains (Fig.l5)

The pI range for the monoclonal free light chains is 4-10. And their character

bands (track 5) were sharp and variable. They may appear as a pair of bands

widely spaced or very closely spaced.

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The pI range of oligoclonal IgG (track 3, 4) is 5-9.5. It is commonly in the 7-

9.5 range. There were several groups of monoclonal IgGs composed in

oligoclonal IgG. Each group of them has the characteristic wedge-shaped of

monoclonal IgGs. The overlapping bands between these groups form a track

which is full of multiple sharp bands.

2.Factors

2.1 The temperature of adding ampholytes

The results were found to be not so good in some of tracks. The patterns were

not focused well in these tracks. But the others were all right (Fig.l7). Maybe

only some of the ampholytes were destroyed when the temperature is too high.

The best temperature condition should be 55-65°c when the ampholytes is added.

2.2 The factors in staining

(i) Reagents

We used the Beckman's immunofixation reagents to fix and stain several gels.

It was found that the monoclonal bands could be shown clearly. But the pattern of

albumin could not be found (Fig.l8). That means that immunofixation reagent

may not ‘fix，albumin although it will 'fix' less soluble proteins such as globulins.

The group B reagents were found satisfactory for fixing both the albumin and

golbulins.

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'-、’

*‘

人.*

>

. ••

•

.•,

.'-i

g

6^

ck

nA

n

^

‘'Mk

Fig

.1 8

Pa

tte

rns

used

B

eck

ma

n's

im

mu

no

fixa

tio

n re

ag

en

ts

in

IEF

(ii) Ampholytes

The ampholytes affected the staining procedure greatly if they were not

washed and dried thoroughly in the oven after isoelectric focusing. Ampholytes

stained strongly and make the background so blue that you can't interpret the

result (Fig.l9). The background will not turn back to clear even when destained

for a long time once this does happen. Drying it again in the oven does not work

either.

2.3 Time

(i) Time ofIEF

The proteins will not be focused well if there is not enough time for them to

run. It needs at least l.Ohr for all the proteins to reach their pI. The patterns

were not better when we prolonged the time to 1.5hr. The suitable time for ffiF

is l.Ohr.

(ii) Time for destainer

The time for destainer is not a constant. It will take a long time in destainer if

the reagent had been used for several times. The time it took could be vary from

0.5hr to 24hr according to the times the reagent had been used. It needs only 5

minutes if the reagent is used for the first time.

2.4 The position of the electrode

In theory, it should make no difference with the technique of isoelectric

focusing in whether the samples are placed near the cathode or anode. It was

5 1

^^m

jM^^

IB

M

iPP^

2^

^

""''

^''^

^^-'

^^^^

^^B

^HH

fifc

k'

|H

:-

二一“

’”

«^W

MH

r .

^F

1『

Fig

.19

Am

ph

oly

tes

aff

ect

ing

the

IEF

pa

tte

rns

in s

tain

ing

found that there was no big difference in the results in practice either. However

there is smooth space for immunoglobulins to run when the samples are placed

near the anode. The patterns of immunoglobulins will be interfered by the

application point if samples are placed near the cathode (Fig.20).

2.5 Watt,Voltage and Ampere

We compared the results in two ranges. The patterns in the range of 1.0-

1.2watt slightly were near clear than in 1.0-1.5watt.

3.Comparison of the results between isoelectric focusing(without antiserum

fixation) and immunofixation electrophoresis (Table.3). Oligoclonal bands were

clearly distinguished from monoclonal bands by EEF. There was one case with

monoclonal IgM missed by isoelectric focusing. Its results of routine

electrophoresis, immunofixation, and isoelectric focusing are shown on Fig.21

(track 4), 22, and 23 (track 5). •

•

5 3

s a m p > l e a p > p l i c a t i o n

t

1 t ’ 0

. f f 4 l -

! o

3 ) / • _

m f

c e

4 f

j , \ i —

f • f J

1 r 少 # . t f L

f L

2 i ^ i 0 _ a

\ . 。

^ i , F

3 § * 二 書

V

U O T : l s J l ; d d l ? f w

5 4

Table 3. Comparison of the results beween isoelectric focusing and immunofixation electroporesis

paraprotein

. Heavy Chains BJP no. paraprotein

IgG IgM IgA J r e e f r ^ & b b Kappa Lambda

Immunofixation ^ 2i 4 7 2 0 ‘ Electrophoresis

Isoelectric Focusing without immunofixation 19 (4 are

oligoclonal 21 3 7 2 � bands)

55

Fig.21 Routine electrophoresis of the missed monoclonal IgM

• _ * **

-•*-•‘ 一 - • .^fe^^___ _ j i ^

• _ 棚 ^^^^^^ *" ••—— , _1,.攀,.. ^n||^ ^ BppvivpHi ^p i ^

_ ;v-m .

‘ ^ ¾ ^ .

m m • # g l g g , sHBBp|ff B^ ^ ' ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ V ；

. ^ ¾ ^ ¾ ¾ ^^H^^^^^IPP,-

j m ^ ^ ^ ^ ^ [ ^ j i H ^ ^ g ^ ^ g ^ ^ggj^ lg l l l l l l l^�

1 ^ ^ ^ ^ S H H ^ H I ^ V w ^ w M , ^ H P ^ * i P /

. ^ B | ^ ' . . 急 』

1 ^ . m . j y | ; .

^ ^ ^ ^ p ^ ^ ^ ^ H ^ ^ P ^ ^ P r

^ ^ ^ ^ v M H p

3 , 4 5 1.

•

� . / _ • •

5 6

Fig.22 CFE ofthe missed monoclonal IgM

a g o n ' I F E G e l = 广 ^ C K N f D I

I I I b I f~ •: _ _ • • • - • • -

2 2 • _ _ - - “ • _ - •

3 3

_ . • • • • •- .睡 一 •/ 4 4 1减.勃.丨 ^

- • • P | • • \ : \ • • : : 5 5 ^ m

• .6 • � • e B ' “ • _ m m _ _ ^ ^ W P _ _ _ m • •

一 一 i _ 7 7 _ _

\ ^ * • J 狭 明

4 5 6 * j • 1 2 | ^ f i i 1 ^ I 1 H ® ^ J > f c b 、

5 7

Fig.23 ffiF ofthe missed monoclonal IgM

» ‘

1 ^ 5 | r ^ I

l P f Mj>

II fl|| • £ '

r 9 W\ umL J K

_ l ^ ^ l m^^m ^ ^ l ^ ^ ^ V * ^ M ^ m • •丨 i : ^ H • ™ » I ^^m ^ ^ B , : : 動 ^ : f l B : 龐” 々.；r i . 濰: i ^ - t ^

*、 5 8

》::,> 麵/.

Chapter 4

Discussion

The identification of monoclonal immunoglobulins is important in the

diagnosis and management of some B cell tumours. It is necessary to detect

monoclonal proteins in the follow-up investigation of some diseases even

including the benign monoclonal gammopathies [55,63-66]. But the monoclonal

proteins cannot be detected clearly when the concentration is low in serum and

about two-thirds of patients with monoclonal immunoglobulins have low

concentration in serum (<5 g/L)[41]. In this condition, it is difficult to distinguish

between oligoclonal bands and small monoclonal bands by routine protein

electrophoresis. The ability to distinguish these two types ofbands is necessary in

the clinical laboratory because they indicate different diseases and prognosis

respectively [55]. The monoclonal proteins are associated with some B cell tumor

(e.g. myeloma) while oligoclonal proteins commonly relate to infection and other

causes of chronic immune stimulation such as cirrhosis, rheumatoid arthritis and

collagen vascular diseases (see 1.3.1).

Distinguishing small monoclonal from oligoclonal bands is even more of a

problem in Hong Kong than many other countries because of a high incidence of

cirrhosis that is one of the conditions associated with oligoclonal bands[55,82].

The incidence ofoligoclonal bands in Hong Kong is about three times higher than

the figure of3% quoted by Kyle[55,69].

5 9

In the clinical laboratory, the current methods for identifying and typing the

monoclonal immunoglobulin after the routine electrophoresis are

immunoelectrophoresis and immunofixation electrophoresis [41]. However,

immunoelectrophoresis is not used in many of laboratories because of its low

sensitivity and specificity, although it was the early technique used in identifying the

monoclonal proteins. And the most common method used now is immunofixation

electrophoresis. Immunofixation has high sensitivity and specificity in identifying

monoclonal proteins but antiserum and instruments are quite expensive. Isoelectric

focusing is another method with high sensitivity and specificity for detecting abnormal

IgG bands. But this method is not sensitive for detecting abnormal IgA and IgM bands

and therefore cannot be used for initial screening. We roughly calculated each

specimen cost in ffiF and IFE. The cost of one specimen in ffiF includes agarose and

ampholytes while the cost of one specimen in JFE includes the gel and antiserum. The

prices of the two methods were compared (Table 4). Obviously, DEF is cheaper than

immunofixation because expensive antiserum is not required.

By comparing the results in these two methods, there was agreement between

the two methods as to which electrophoretic patterns showed no monoclonal band

ofIgG. There were monoclonal IgG, IgA, IgM and free light chains in samples we

studied. However it was found that isoelectric focusing distinguished more clearly

between oligoclonal and monoclonal IgG than routine electrophoresis and

immunofixation electrophoresis. There was only one case in which isoelectric

focusing missed a monoclonal IgM detected by immunofixation. In this case, the

6 0

Table 4. Compar ison of the pr ices between IEF and IFE

Price (HK^) [ f P l f E “

Agarose/geU 4 9 . 4 ‘ “ “ •

(8 specimens)

Ampholytes/gel 1 2 9 . 6 “ ~ ~ ‘ “

(8 specimens) •

Ant iserum/gel — 3 8 5

(1 specimen)

Pr ice/specimen 2 2 . 3 7 5 3 3 5

61

concentration of paraprotein was low (1.5 g/L) and it was detected only by

immunofixation, not protein electrophoresis. The sample was fixed because of

oligoclonal IgG bands present, not because of the IgM bands which was not

detected.

There are several classes of monoclonal immunoglobuins including monoclonal

IgG IgM IgA, IgD, IgE and Bence-Jones proteins. Proportions of each class of

paraprotein are not equal. Monoclonal IgG has the largest proportion. IgA and

IgM have a small proportion. There is about 57-59% of patients with myeloma

which have an monoclonal IgG in the serum, 21-23% have a monoclonal IgA, 1%

has IgD, and 18% are light chain disease [49,93], In our results, monoclonal IgG

was the largest proportion of paraproteinaemia and monoclonal IgA and IgM

relatively had a small proportion. Thus the major paraprotein detected in the

clinical laboratory is monoclonal IgG which is detected well by isoelectric

focusing.

Routine electrophoresis is a good technique to detect all kinds of paraproteins.

But it's not always good in differentiating oligoclonal from monoclonal bands.

Therefore immunofixation is necessary and its cost is quite expensive because of

the antiserum. Isoelectric focusing has a high sensitivity and specificity in

identifying monoclonal IgG and it distinguishes well between oligoclonal and

monoclonal bands. Although it is not good in detecting monoclonal IgM and IgA,

it may be used as a further technique in investigation of paraprotein because

monoclonal IgG is the most common paraprotein present in serum. Expensive

instruments and antiserum are not required in isoelectric focusing and the cost is

6 2

far lower than immunofixation. Immunofixation may then be as a complementary

test only on patients with a definite monoclonal band. This would avoids the

waste oflarge sums ofmoney on antiserum and satisfies the clinical requirements

at the same time.

I would propose the following scheme (Fig. 24) for investigation abnormal

bands based in my results.

6 3

Fig.24 A new scheme for laboratory investigation of monoclonal

protein based on our results.

Electrophoresis

/ \ Obvious abnormal Suspicious abnormal

V

Isoelectric focusing

/ \ Monoclonal protein Oligoclonal protein

i 丄 / I m m u n o f i x a t i o n /

\ / Report

64

Chapter 5

Conclusion

Isoelectr ic focusing is a technique with high sensit ivi ty and

specif ic i ty in detecting monoclonal IgG. It dis t inguishes well

between monoclonal and oligoclonal bands and Its cost is low.

We recommended this as a cheap method for fur ther

invest igat ing bands which do not have a typical appearance of a

monoclonal band. It can reduce the number of samples which

require immunofixat ion.

6 5

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