comparison of isoelectric focusing and immunofixation ... · electrophoresis to distinguish...
TRANSCRIPT
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
y ^ ^ V j^ymLSKS Ai;vyanN®\
fi/^ ~ \ 4 [ g • iar 9 I ] J
勉 岁
. T 1
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]
W " 0 ^ / 1 謹 < ^ \ V A ^ " ' / ^ / > Antigen ^
V l V A - ¾ ^ < ^ ^ ^ ' ^ ^ Binding . -
c . < X A A A > 画 \ , HlNGE J ~ |
-S-S- . REGION 8io(og-.ca( -S-S- Activrcy g
^
p u ^ ⑴ 出 ^"2 w cb Complement °
binding ^ <
CH3 r ~ | ^ ~ ] fc receptor | binding 0
- S - S - COO" disulphide
Oridges
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.
8
9
乂 J
^
r
,,^¾!!!!!!
^y/
^^
^
lllil
7:
#illrte
^
f
%iggilil
趣
M-
% Jn^^—
^
麵麵
J^II3S
j
么二
e H_
h
J
T
2 ^
n
\
一
llllil=llflllllllllll_llll=llllll!^^^
9
三|=三三三二一1 =
|三==||二||一1
= |一二|二一
s^^r 5s
o
OS
f
i
£# 一
s^\
=|三三|三三|三三三二三2一1||=一二33
^-^
|1|_謹|一||1_|_謹11^^^
fz
lt
o
(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
2 0
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-^
m ^ m ^ ^ ^ p s ^ ^ ^ m m ^ s m
^B、
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
I
I
]
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.)
3 0
|
^^j^
~:—
iji
' l^
g^H
|g^^
^^^^
^H^^
^||^
^^^^
^^^^
^^^|
^^^|
|||^^^
m^^
^^^^
^H|^
^^^^
^H|^
^^^^
^^H
mB
^,
^^
..
..
..
^-
Hl
^^
^^
^^
^^
^^
^^
^^
^^
^^
^^
^^
^^
^^
l |
K^
k .
, ,
^Z
£H
^^
^H
^^
^^
^^
^^
^^
^^
^^
|
w^t^
m
|M
.
KB
^HKi
F
ig.
9 H
om
e-m
ad
e ch
am
be
r
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
a i i
I H I H i
i ^ ^ ^ ^ ^ ^ ^ ^ ^ H n l ^ ^ H I
l ^ ^ ^ ^ ^ ^ ^ : 9 m :
_ H H H _ _ K f f ^
B F £ I
__•_
• B % . . A m m f . ^ ^ t ^ v i i ^ A ^ I • i i ^ s ^ ^ H
^ ^ ^ H
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.
4 0
1 2
3 4
5 6
7 8,
•
^黌
禱麵
•冊
•1
1^
- i
4
~k
Fig
11
No
rma
l p
att
ern
s m
IE
F
d3l
u! o
6|
|eu0
|30U
0[A
I ^
r6ij
CN
货
a
. 攀
\眷
j|
«^
S«
p_
--
8 L
9 9
fr
e z
i . 急
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.
4 3
8 7
6 5
4 3
2 1
/«—- - -,
..
^^
^3
_^^
.
• . ‘
'*^^
M>
.參,.W
*-
^^^^
^^^
释雾
f^t 1具
等泰
t ^^ |
i .効
m
•
7
Fig
.13
M
on
ocl
on
al
lgA
m
IEF
TS
5 論書
^ ljL
dm
^ a
^ r
1^
9p
i •!
I f
胃
暦參
‘J
H
二
45»
tn
Fig
.14
Mo
no
clo
na
l lg
M 丨门
IE
F
\
.- »
^‘
- •-, • *
--W
_.
.,•
一
--. .._
_
8 7
6 5
4 3
2 i
« ‘
• M
^ M
M
3 «
一 ^
J^
iipi;
麗,
尋“
^/
^ ^
^ %
^ ^
^ '
0i
;麗
】
•
娜
•…
-^
..
iM
I .%
^ ..
^ 0)
Fig
.15
Fre
e lig
ht
cha
ins 丨门
lE
F
1.6 Oligoconal IgG(Fig.l6)
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.
4 7
7 6
5 4
3 2
1
•i _1
-一
Hf i
冒 i f
I
’ »
m
. •
iS
P
4
.麵
\ ‘妄
k •
:§
= ^
^ ,
• z
H\
_ A
F
ig.1
6 O
lig
ocl
on
al
IgG
in
IE
F
...-,
. -
.
, — "
~^
wi
...I
::..
;
,
: ”
八•
‘.
is
ik
flH
i,,'
'、-
^mm
mg
-
—一
^
〔
——
‘ ^
S^
,
.|'.:v'
^^^^^^
...
-J.
.:.•-»•
•
I W
? f管置置_
"_t
_Jilfl
f
f"、
:邏
昼_“
學
-3
一 誦
•
M
# ",
么
CD
;•?••
�
f
-i
•• F
ig.1
7 P
atte
rns
of a
mp
ho
lyte
s ad
ded
at
1 00°
c a
ga
rose
‘’,.•
::: ;jf'
'
n ^
"7
^ —
“
~r
i •
j ,�
• 1
--
身方
各'汝
一".
*^
^^
t^
^^
^M
\
.
mt
UK
^*
^ ‘
:办
、
>^-
^iib
•画
1兴
• •—<•
_ 1"
Bii
r>i^
,<^
^<
rt^
[|^
g
:4
\
M
B
&
^f
^
.「
::
、.
7、
^
r !
/、
\^
^^
丨 “
[
.,‘
:’
/ .,
/ \
Wrv
[
+_
1 .
>�
. •
-�
•�
•.
�
一
氣
-,.-
•
%
’
^ ,
. ^
、
^ '
-.
, ,’
*
"^
f?^
=�
, '•
.
• ' .
- .
/%^
:.
« ‘
;-
m
-:$
^ ‘
:
‘ :
,j
,
. ' _
::r
y
„ -,
丨
丨.
j^
•
~*
» 學
•
•^
~*
w^
m
K^
^H
Mg
MH
^^
Mi
^ 1
III
-'
ni
f^
""
‘‘ ‘
J^
H
-.^
BB
lg*
'^^
"^
fti
^^
* i
iJ
mJ
S^
^M
BB
^B
B^
^^
ft
fc
^^
^i
ZS
dm
^1
.^>
^¾,^
^-
” -^
" ‘ ^
fj5"'<
ikj I
.fc
;cr-
^^
^^
^*
™*
^^
'*^
^^
^^
^^
^^
^ ;
'-、’
*‘
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
Reference
1. 0. Ross McIntyre. Laboratory investigation of myeloma. James S. Malpas, Daniel
E. Bergsagel and Robert A. Kyle. Myeloma biology and management. OXFORD
MEDICAL PUBLICATIONS, 1995. pl91-221.
2. William J. Marshall and Stephen K. Bangert. Clinical Biochemistry.
P.Riches:Paraproteinaemias. Pp493-506. New York, Churchill Livingstone,1995.
3. Richie, R.F. and Smith, R. (1976a). Immunofixation. I. General principles and
application to agarose electrophoresis. Clinical chemistry,22,487-9
4. Richie, R.F. and Smith, R. (1976b). Immunofixation. III. Application to the study
ofmonoclonal proteins. Clinical chemistry,22,1982-85
5. Sinclair, D.,Kumararatne, D.s.,Forrester, J.b. Lamont, A. and Stott, D.I. (1983).
The application of isoelectic focusing to routine screening for paraproteinmia.
Journal ofClinical Pathology, 37,225-62.
6. Sinclair, D.,Kumararatne, D.S., and Stott, D.I.(1984). Detection and identification
of serum monoclonal immunoglobulin by immunoisoelectric focusing. Limits of
sensitivity and use during relapse of multiple myeloma. Journal of Clinical
Pathology, 37, 255-62
6 6
7 D. E. Joshua. Immunoglobulins. James S. Malpas, Daniel E. Bergsagel and Robert
A. Kyle. Myeloma biology and management. OXFORD MEDICAL
PUBLICATIONS, 1995, p3-29
8. Joan F. Zilva, Peter R. Pannall, Philip D. Mayne: Clinical Chemistry in Diagnosis
and Treatment. Edward Arnorld, 1988.
9. Clyne, D.H., Amadeo, J.P., and Tompson, R.E. Nephrotoxicity of Bence-Jones
proteins in the rat: importance of protein isoelectric point. Kidney International,
1979, 16,345.
10. Meclion, C., Mougenot, B, Baudouin, B., Ronco, P., Moulongvet-Doleris, L.,
Vanhille, P., et al. Renal failure in myeloma: relationship with isoelectric point of
immunoglobulin light chains. ClinicalNephrology, 1984,22, 138.
11.McIntyre, O.R., Kochwa, S., and Propert, K.J.. Prognostic value of light-chain
isoelectric point (IEP) in myeloma. Proceedings of the American Society of
Clinical Oncology, 1988, 7,8844.
12 Vitella E.A. and Uhr, J.W. Immunoglobulin receptors revisited. A model for the
differentiation of bone marrow derived lymphocytes is described. Science, 189,
964, 1975
6 7
13. Tu, S.M., Winchester, R.J., and Kunkel, H.G. Occurrence ofsurface IgM, IgD and
free light chains in human lymphocytes. Journal ofExperimental Medicine, 1974,
139,451.
14. Joan Blade and Robert A. Kyle. Monoclonal gammopathies of undetermined
significance. James S. Malapas, Daniel E. Bergsagel and Robert A. Kyle.
Myeloma biology and management. OXFORD MADICAL PUBLICATIONS.
1995, P433-476.
15. F.N. Cornell, R. Mc Lachlan. Idsoelectric focusing in the investigation of
gammopathies. The clinical biochemistry, 31-37, September, 1985.
16. Ritchie, R.F. and Smoth, R.. Immunofixation. I. General principles and
application to agarose electrophoresis. Clinical Chemistry, 22,p497-9
17. Barlogie B: Plasma cell myeloma, in Beutler E, Lichtman MA, Coller BS, et
al(eds): William's Hematology (ed5). Batilmore,MD, McGraw-Hill, 1995, p
1109-1126
18. P. R. Greipp. Monoclonal gammopathies: new approaches to clinical problems in
diagnosis and prognosis. Blood reviews (1989) 3, p222-236
19. j T Whicher, j Calvin, P Riches and C Warren. The laboratory investigation of
paraproteinaemia. Ann Clin Biochem 1987; 24; pl 19-132
6 8
20. J T Whicher and C E Spence. Serum protein zone electrophoresis--an outmode
test? Ann Clin biochem 1987; 24: 133-139
21. Samuel Y. Chu, Janet E. Macleod, Lewis Bocci, and Mabel Monteith.
Characterization of small monoclonal protein bands with Beckman's "Paragon"
immunofixation system. Clinical Chemistry, Vol.33, No.4, 1987,p617
22. J T Whicher, M. Wallage, and R. Fifield. Use of immunoglobulin heavy- and
light-chain measurement compared with existing techniques as a means of typing
monoclonal immunoglobulins. Clin Chem. 33/10,1771-1773(1987)
23. A. Myron Johnson, M. D. Immunofixation following electrophoresis or isoelectric
focusing for identification and phenotyping of proteins. Annals of clinical and
laboratory science, Vol. 8,No. 3,195-200, 1978
24. Devery A. Howerton, Irene J. Check, and Robert L. Hunter. Densitometric
quantitation of high resolution agarose gel protein electrophoresis. Am J Clin
Pathol, February 1986,213-218
25. David Stemerman,Christine Papadea, David Martino-Saltzman, A. Christine
Connel, Barbara Demaline, and Garth E. Austin. Precision and reliability of
paraprotein determinations by high-resolution agarose gel electrophoresis.
A.J.C.P. April 1989,435-440
69
26. David F. Keren, Jeffrey S. Warren, and John B. Lowe. Strategy to diagnose
monoclonal gammophathies in serum: high-resolution electrophoresis,
immunofixation, and yJ% quantification. Clin. Chem. 34/11,2196-2201(1988)
27. David E. Normansell. Comparison of five methods for the analysis of the light
chain type of monoclonal serum IgM proteins. Am J Clin Pathol 1985; 84: 469-
475
28. P M Dennis, B Biegler and R Papas. Improved measurement of monoclonal
paraproteins in serum using agarose gel electrophoresis. Ann Clin Biochem 1987;
24; 73-76
29. James B. Peter, Karen L. McKeown, and Melkon S. Agoplan. Assessment of
different method to detect increased autochthonous production of
immunoglobulins in multiple sclerosis. Am J Clin Pathol, June,1992, 858-868
30. Tsieh Sun, Shiu-Kee Chan, and Stanley Gross. Evaluation of a high-resolution
electrophoresis system. Am J Pathol 67: 247-250, 1977
31. Sidney N. Kahn and Mahin Bina. Sensitivity of immunofixation electrophoresis
for detecting IgM paraprotein in serum. Clin Chem, Vol. 34/8, 1633-1635(1988)
32. Julia E. C. Guinan, David F. Kenny and Paul A. Gatenby. Detection and typing of
paraproteins: Comparison of different methods in a routine diagnostic laboratory.
Pathology (1989),21, pp35-4
7 0
33. Arnold L. Schultz and Louis M. Fink. What is the most efficient way to evaluate
immunoglobulins? Clin. Chem. 32/2, 391-2 (1986)
34. UH Paluch, G Keir, S Moyle, EJ Thompson. Quantification ofbands produced by
isoelectric focusing using immunoperoxidase. J Clin Pathol 1984; 37: 1172-1176
35. Frank J. Fasulio, Jr., Herbert A. Fritsche, Jr., Frank J. Liu, and Robert G.
Hamilton. IgG heavy-chain typing of myeloma by isoelectric focusing immunoblot
analysis. Clin. Chem. 35/3,364-368 (1989)
36. Teitz NW. Fundamentals of clinical chemistry. Philadelphia, PA: WB Saunders
Company, 1987.
37. Fritsche HA, De Leon E. The determination of serum immunoglobulins by
automated nephelometric analysis. Am J Med Technl, 1975; 41: 19-28
38. Giampaolo Merlini, Paola Piro, Franco Pavesi, Renzo Epis, and Francesco
Aguzzi. Detection and identification of monoclonal components:
immunoelectrophoresis on agarose gel and immunofixation on cellulose acetate
compared. Clin. Chem. 27/11,1862-1865 (1981)
39. Cheryl M. Reichert, Donald F. Everett Jr., Paul I. Nadler, and Nicholas M.
Papadopoulos. High-resolution zone electrophoresis, combined with immunofixation,
7 1
in the detection of an occult myeloma paraprotein. Clin. Chem. 28/11, 2312-2313
(1982)
40. Keshgegian, A. A., and Peiffer, P., Immunofixaton as an adjunct to
immunoelectrophoresis in characterization of serum monoclonal immunoglobulins.
Clin. Clhim. Acta 110,337-340 (1981)
41. Sidney N. Kahn. Strategy for diagnosis of monoclonal gammopathies in serum.
Clin. Chem.35/3,508-509(1989)
42. T Sheehan, D Sinclair, P Tansey,JR 0 , Donnell. Demonstration of serum
monoclonal immunoglobulin in a case of non-secretory myeloma by
immunoisoeIectric focusing. J Clin Pthol 1985; 38: 806-809
43. Anthony G. W. Norden, Leah M. Fulcher, and Anthony D. Heys. Rapid typing
serum paraproteins by immunoblotting without antigen-excess artifacts. Clin. Chem.
33/8, 1433-1436 (1987)
44. David Sinclair, John H Dagg, Allan McI Mowat, Delphine MV Parrott, David I
Stott. Serum paraproteins in chronic lymphocytic leukaemia. J Clin Pathol 1984; 37:
463-466
45. Sheehan T, Sinclair D, Tansey P,and 0'Donnell JR. Demonstration of serum
monoclonal immunoglobulin in a case of non-secretory myeloma by
immunoisoeIectric focusing. J Clin Pathol, 1985 Jul, 38:7, 806-9.
7 2
46. David Stemerman, B.A., Christine Papadea, David Martino-Saltzmam, A.
Christine 0'Connell, Barbara Demaline, and Garth E. Austin. Precision and reliability
of paraprotein determinations by high-resolution agarose gel electrophoresis. Am J
Clin Pathol, April 1989,435-440
47. Rudolf H. Zubler. Key differentiation steps in normal B cells and in myeloma
cells. Seminars in Hematology, 34/1 1997; 13-22
48. Pamela G Richies. Pitfalls in investigating proteins-how to avoid them. Chemical
pathology/immunology.
49. Robert A. Kyle, Philip R. Greipp. Laboratory medicine series on clinical testing 3.
The laboratory investigation of monoclonal gammopathies. Moyo Clin Proc 53: 719-
739, 1978
50. Editorial review. Monoclonal immunolgobulin deposition disease (Randall type)
relationship with structural abnormalities of immunoglobulin chains. Kidney
International, Vol. 46 (1994),965-975
51. Laboratory investigation of paraproteinaemia. Association of Clinical
Pathologists; Broadsheet 118: July 1998,776-785
7 3
52. Martin E. Gore, Pamela G. Riches, and J. Kohn. Identification ofthe paraproteins
and clinical significance of more than one paraprotein in serum of56 patients. Journal
ofClinical Pathology, 1979,32, 313-317
53. D. Sinclair, J. H. Dagg, A. E. Dewar, A. McI. Mowat, D. M. V. Parrott, G.
Stockdill and D. I. Stott. The incidence, clonal origin and secretory nature of serum
paraproteins in chronic lymphocytic leukaemia. British Journal of Haematology,
1986,64,725-735
54. Robert A. Kyle. Monoclonal gammopathy of undetermined significance, natural
history in 241 cases. The American Joumal ofMedicine,May 1978,Vol 64, 814-826
55. Yvette I. Lolin, John Chow, and Nicholas W.R. Wickham. Monoclonal
gammopathy of unknown significance and malignant paraproteinemia in Hong Kong.
Am J Clin Pathol,October 1996,449-456
56. Regis Bataille. New insights in the clinical biology of multiple myeloma.
Seminars in Hematology, 34/1, 23-28 (1997)
57. Peter A. Miescher,Y. P. Huang, and S. Izui. Type II cryoglobulinemia. Seminars
inHematology, 32/1, 80-85 (1995)
58. M. Pascual, S. Mach-Pascual,and JA Stiffer. Paraproteins and complement
depletion: pathogenesis and clinical syndromes. Seminars in Hematology, 43/1, 40-48
(1997)
7 4
59. Walters MT, Stevenson FK, Herbert A, Crawly MI, Smith JL. Lymphoma in
Sjogren's syndrome: urinary monoclonal free light chains as a diagnostic aid and a
means of tumor monitoring. Scand J Rheumatol Suppl, 1986,61: 114-7
60. Gallo P, Braco F,Tavolato B. Detection of IgG oligoclonal bands in
unconcentrated CSF by means ofagarose isoelectric focusing, double immunofixation
peroxidase staining and avidin-biotin amplification. Ital J Neurol Sci, 1985 Sep, 6:3,
275-82
61. Sheehan T,Sinslair D, Tansey P, 0'Donnell JR. The potential value of immuno-
isoelectric focusing in the diagnosis and management of solitary plasmacytoma. Clin
Lab Haematol, 1985,7:4, 375-7
62. Schipper HI, Bertram G, Kaboth U. Microheterogeneity of paraproteins. I.
Diagnostic value of isoelectric focusing followed by immunoblotting. Clin Chim
Acta, 1988 Feb 15, 171:2-3, 271-8
63. Pasqualetti P, Festuccia V,Collacciani A, Casale R. The natural history of
monoclonal gammopathy of undetermined significance. A 5- to 20-year follow-up of
263 cases. Acta Hematol (Switzerland) 1997 97(3) pl74-9
64. Billadeau D, Van Ness B, Kimlinger T,Kyle Ra, Terneau TM, Greipp PR. Clonal
circulating cells are common in plasma cell proliferative disorders: a comparison of
7 5
monoclonal gammopathy of undetermined significance, smoldering multiple
myeloma, and active myeloma. Blood,Jul 1996 88(1) P289-96
65. Khamlichi AA, Rocca A, Touchard G, Aucouturier P, Preud'homme JL, Cogne M
.Role of light chain variable region in myeloma with light chain deposition disease:
evidence from an experimental model. Blood, Nov 15 1995 86(10) p 3655-9
66. van de Poel MH, Coebergh JW, Hillen HF. Malignant transformation of
monoclonal gammopathy of undetermined significance among out-patients of a
community hospital in southeastern Netherlands. Br J Haematol Sep 1995 91 (1)
pl21-5
67. Barbieri S, Sandroni P, Nobile-Orazio E, Cappellari A, Cavestro C. Small fibre
involvement in neuropathy associated with IgG, IgA and IgM monoclonal
gammopathy. Electromyogr Clin Neurophysiol, Jane-Feb 1995 35(1) p39-44
68. Simmons Z,Albers JW, Bromberg MB, Feldman EL. Long-term follow-up of
patients with chronic inflammatory demyelinating polyradiculoneuropathy, without
and with monoclonal gammopathy. Brain, April 1995 118 (Pt 2) p359-68
69. Kyle RA. Monoclonal gammopathy ofundetermined significance. Blood Rev, Sep
1994 8 (3)pl35-41
7 6
70. Meijer WG, Van Marwijk Kooy M, Ladde BE. A patient with multiple myeloma
and respiratory insufficiency due to accumulation of paraprotein in the alveolar space.
Br J Haematol, Jul 1994 87(3) p663-5
71. Kampe CE, Hart S, Miller RA, Lichtenstein A, Kyle RA, Berenson JR.
Expression of shared idiotypes by paraproteins from patients with monoclonal
gammopahty of undetermined significance. Br J Haematol, Aug 1994 87(4) p719-24
72. Wagner DR, Eckert F, Gresser U, Landthaler M, Middeke M, Zollner N. Deposits
of paraprotein in small vessels as a cause of skin ulcers in Waldenstrom's
macroglobulinemia. Clin Investig, Dec 1993 72(1) p46-9
73. Harrison HH, Miller KL, Abu-Alfa A, Podlasek SJ. Immunoglobulin clonality
analysis. Resolution of ambiguities in immunofixation electrophoresis results by high-
resolution, two-dimensional electrophoretic analysis of paraprotein bands eluted from
agarose gel. Am J Clin Pathol, Nov 1993 100 (5) p550-60
74. Zhang W, Ewan PW, Lachmann PJ. The paraproteins in systemic capillary leak
syndrome. Clin Exp Immunol, Sep 1993 93(3) p424-9
75. Sanders PW, Herrera GA. Monoclonal immunoglobulin light chain-related renal
diseases. SeminNephrol, May 1993 13(3)p324-41
7 7
76. Nelson M, Brown RD, Gibson J, Joshua DE. Measurement of free kappa and
lambda chains in serum and the significance of their ratio in patients with multiple
myeloma. Br J Haematol, Jun 1992 81(2) o223-30
77. Kyle RA. Diagnostic criteria of multiple myeloma. Hematol Oncol Clin North
Am, Apr 1992 6(2) p347-58
78. Choo-Kang E, Campbell M. Biochemical abnormalities in multiple myeloma.
West Indian Med J, Dec 1991 40(4) pl70-2
79. Bleasel AF, Hawke SH, Pollard JD, McLeod JG. IgG monoclonal
paraproteinaemia and peripheral neuropathy. J Neurol Neurosurg Psychiatry, Jan
1993 56(l)p52-7
80. MacNamara EM, Whicher JT. electrophoresis and densitometry of serum and
urine in the investigation and significance of monoclonal immunoglobulins.
Electrophoresis, May 1990 11(5) p376-81
81. Ricci C,Cascio G, Canavera R, Basso M, Baldassi R. Immunoregulation and
differentiation markers in monoclonal gammopathies. Acta Haematol, 1987 78 Suppl
1 p50-5
82. Gibson JB; Wu Pc; Ho Jc; Lauder IJ. Hepatitis B surface antigen, hepatocellular
carcinoma and cirrhosis Hong Kong: a necropsy study:1963-1976. Br J Cancer, 1980
Sep, 42:3, 370-7
7 8
83. Steven M. Rosen, Joel N. Buxbaum, and Blass Frangione. The structure of
immunoglobulins and their genes, DNA rearrangement and B cell differentiation,
molecular anomalies of some monoclonal immunoglobulins. Seminars in Oncology,
13/3 (Sep), 1986 p260-274
84. Guinan JE, Kenny DF, Gatenby PA. Detection and typing of paraproteins :
comparison of different methods in a routine diagnostic laboratory. Pathology, 1989
Jan,21:l,35-41
85. Detection of oligoclonal IgG in CSF by immunoblotting following isoelectric
focusing. The Clinical Biochemist, November 1989, p70-l
86. Isoelectric focusing. The Clinical Biochemist, Novemberl989, p64-5
87. McLachlan R. Isoelectric focusing of immunoglobulins- an altas of patterns. Clin
Biochem Monogr, Sep 1985; p38-52
88. Cornell FN, McLachlan R. Isoelectric focusing in the investigation of
gammopathies. In: Monograph advanced electrophoretic techniques for protein
investigation in clinical diagnosis. The clinical Biochemist, Sep 2985, p31-7
89. Isoelectric focusing-Principles and Methods. Pharmacia Fine Chemicals
Handbook.
7 9
90. Righetti PG. Isoelectric focusing: theory, methodology and applications. Work TS
and Burdon RH eds. Laboratory Techniquea in Biochemistry and Molecular Biology
Vol. II. Amsterdam, Elsevier Biomedical, 1983.
8 0
{
. . ) , . . .:...�,-.i:ii:^i^ii^i^:^^:
C U H K L i b r a r i e s
_ _ l _ l l DD37D5Dbl