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Introduction 1
(i)
Section D: IgA Monoclonal Gammopathies 32
(ii)
Section G: Hypogammaglobulinemia and 69
Hypergammaglobulinemia
Case # 1 Polyclonalgammopathy 69
Case # 2 Hypogammaglobulinemia 72
Case # 3 Apparent hypogammaglobulinemia 75
with monoclonal gammopathy
Section J: Cryoglobulins 87
1
Track # 1 Serum Protein Electrophoresis-six fractions
Track # 2 Immunoglobulin G
Track # 3 Immunoglobulin A
Track # 4 Immunoglobulin M
Track # 5 Kappa Chains
Track # 6 Lambda Chains
2
Selection of Patients for Case Studies: We performed serum protein
electrophoresis on over 5000 patients over a period of nine months. All the sera
having either abnormal band or atypical pattern were sequestered. The patient’s
medical record was reviewed to obtain clinical information. For each case we have
presented in this compendium, a brief medical history, prior diagnosis and the
diagnosis after hospitalization (if applicable), and any other information that may be
helpful in the understanding of the electrophoretic patterns. First the serum protein
electrophoretic (agrose gel) scan, and serum immunofixation (agarose gel) picture
were presented. We also presented a picture of the electrophoretic separation on the
agarose gel. The five fractions of serum (albumin, alpha-1, alpha-2, beta, and
gamma globulin) in gram/dL along with the quantified immunoglobulins were also
provided. An enlarged version of the serum protein CZE scan was also presented
separately for interpretative purposes. Finally, the serum protein electrophoresis
pattern obtained from the automated CZE method (Sebia CAPILLARYS) was
presented six different times on a separate page. One of these scan was used as a
reference. On the remaining five scans the electrophoretic pattern after the antigen-
antibody pretreatment reaction of the serum with each of the five immunoglobulins
(IgG, IgA, IgM, kappa and lambda chains) was electronically superimposed for the
indentification of monolclonal bands. In some cases the identification of the mini-
monoclonal band was facilitated by the electronic magnification of the area under
study of the electrophoretic pattern. Comments were made in some cases in order to
explain the laboratory results otherwise the scans of protein electrophoresis,
immunofixation and immunotyping were obvious to interpret.
3
In order to familiarize with the salient features of electrophoresis, immunofixation
electrophoresis, immunotyping based on CZE either after immunosubtraction using
antibodies coated to Sepharose beads (Beckman Coulter Paragon CZE 2000), or
after immunosubtraction using liquid phase antigen-antibody reaction (Sebia
CAPILLARYS), the reader is advised to review the first three chapters of the “Protein
Electrophoresis in Clinical Diagnosis” by David F. Keren, MD , 2003 Edition (Arnold,
a member of the Holder Headline Group, Great Britain), ISBN 0340 812133.
First, let us examine the symmetrical changes in the shape of the electrophoretic
curve after the immunosubtraction step in a normal person. Please see the
hypothetical figure No A.
4
The difference between the two curves (green minus red) is plotted in violet color.
This violet colored curve is plotted only to exhibit symmetrical polyclonal
immunoreduction process in a normal person, and one will never notice it on the
immunotyping scans of any of the five tracks (anti-IgG, anti-IgA, anti-IgM, anti-kappa,
and anti-lambda). A smooth and symmetrical shaped curve after the
immunoreduction step (red curve) indicates a polyclonal reduction of the
immunoglobulins from the serum. This kind of smooth and symmetrical shaped curve
(red color) is most commonly seen in a normal person after the liquid phase reaction
with anti-IgG. Uniform immunoreduction in the green curve, Fig No. A, cannot be
construed as a monoclonal band, as polyclonal immunosubtraction results in the
reduction of the gamma globulin fraction in a symmetrical manner.
In a normal person the serum IgA is lower than the serum IgG, therefore a very small
difference is observed between the serum CZE scan and the serum CZE scan after
the anti-IgA reaction. This immunoreduction after reaction with anti-IgA is primarily
witnessed in the beta-globulin region. Similarly in a normal person the serum IgM is
even lower than IgA, therefore again a very little reduction without change of the
symmetry of the fraction is observed (virtually mirror image of each other).
5
The situation in case of kappa and lambda chains is slightly different. Kappa chains
are normally present in an approximate 2:1 ratio to lambda chains. Therefore in a
normal person after liquid phase reaction with anti-kappa, one should expect 2/3
reduction in the gamma region. Obversely, in a normal person after liquid phase
reaction with anti-lambda, one should expect 1/3 reduction in the gamma region. The
magnitude of the reduction in the gamma globulin region for both the kappa and
lambda chains is very low as compared to the IgG, and this is obviously due to the
high concentration of serum IgG as compared to kappa and lambda chains in serum.
It is pointed out that in this hypothetical case of monoclonal band detected in the
gamma globulin region from CZE (green color, Fig No. B), there is also a
symmetrical shaped immunoreduction of the gamma globulin region after the
reaction with anti-IgG (red curve, Fig No. B). The difference between the green and
the red curve is illustrated by violet colored curve, thus indicating a sharp monoclonal
6
peak. It is again pointed out that in actual practice the violet colored curve will
never be depicted on the CZE scan after the immunosubtraction step.
Generally speaking (except in a very rare case of heavy chain disease associated
with gamma, alpha, or mu chains), any abnormal / unsymmetrical disappearance of
the immunoglobulin fraction (IgG, IgA, IgM) in the liquid phase immunosubtraction
step followed by CZE must be associated with a concomitant abnormal /
unsymmetrical disappearance of either kappa or lambda chains or both.
Also the position (electrophoretic mobility) of the abnormal / unsymmetrical heavy
chain and the light chain must be the same on the CZE scan (after
immunosubtraction step) in order to correctly assign the monoclonal band. There are
rare cases in which abnormal / unsymmetrical disappearance of the light chain is
observed after the immunosubtraction step without any concomitant subtraction of
the IgG, IgA, and IgM. In these cases the laboratory must rule out the possibility of
either IgD or IgE monoclonal gammopathy, and also the possibility of light chain
gammopathy.
7
Selected Case Studies:
Hint: The presence of fibrinogen in serum due to any reason, e.g. patient on
heparin therapy, improper processing of specimen, etc., can mimic the
pattern of monoclonal gammopathy.
8
9
10
Case # 2 Restricted band superimposed on C3 complement band
Comments: The intense band detected in both the agarose gel and capillary zone
electrophoresis (superimposed on the C3 complement band position) is an artifact,
perhaps due to fibrinogen, as no monoclonal band was detected either by
immunofixation or immunotyping. We did not confirm it by reacting the patient’s
specimen with anti-fibrinogen. In view of the high intensity of this band, we believe
that the patient sample was most likely plasma and not serum. A follow up inquiry
confirmed that indeed it was plasma specimen.
11
12
NOTE: The apparent increase in β-2 globulin was persistent as indicated by an
arrow, perhaps due to fibrinogen. Similar bands in the β-2 globulin region as an
artifact due to fibrinogen were reported in the CZE (Xavier Bossuyt, et at, Automated
serum protein electrophoresis by Capillarys, Clin Chem Lab Med 2003; 41:704-710).
13
Case # 3 Atypical shape of the Beta1-Beta2 region in the capillary zone
electrophoresis, and heavy staining of the beta1 region (transferrin) in
agarose gel electrophoresis.
14
15
NOTE: IgA-Kappa monoclonal band after the immunoreduction step is indicated by
an arrow.
16
Section B: Acute-Phase Reaction Pattern
Comments: The serum electrophoretic pattern by both the agarose gel and the
capillary zone electrophoresis depicts features of acute-phase reaction pattern. In
view of the fact that gamma globulin concentration though not increased but greater
than albumin in serum, this pattern suggested active, chronic inflammation.
17
18
NOTE: The difference between the red and green curve for the IgM and Kappa
chain is not symmetrical (indicated by an arrow), thus suggesting monoclonal band.
19
Section C: Double Gammopathy and Biclonal Gammopathy
The presence of the heavy chain can be the same or different in either
the double or biclonal gammopathy.
It is pointed out that the clinical course and treatment is the same for
biclonal and double gammopathies.
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21
IgG
IgM
22
Case # 2 Biclonal gammopathy (IgM-Kappa and IgM-Lambda)
61 years old male admitted for urinary tract infection with renal
dysfunction, and was discharged with the diagnosis of monoclonal
gammopathy of undetermined significance (MGUS).
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24
IgM-Kappa IgM-Lambda
25
Case # 3 Biclonal gammopathy (IgM-Lambda and IgG-Kappa)
26
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Case # 4 Double Gammopathy (IgA1 – Kappa and IgA2 – Kappa)
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Section D: IgA Monoclonal Gammopathies
32
Case # 1 IgA monoclonal band masking the beta globulin region
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Case # 2 IgA monoclonal band cathodal to C3 comlement
65 years old male with multiple medical problems (chronic renal failure,
hypertension, coronary artery disease, non-ST elevation myocardial
infarction, anemia, osteomyelitis, diabetes mellitus, GI bleed,
hepatitis C) was admitted because of the shortness of breath and lower
lobe pneumonia.
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38
Section E: IgM Monoclonal Gammopathies:
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Case # 2 IgM monoclonal gammopathy in malignant lymphoma (IgM-Kappa)
42
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Case # 3 IgM monoclonal gammopathy (MGUS) without any clinical symptoms
(IgM-Kappa)
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Section F: IgG Monoclonal Gammopathies
The following cases are selected to show the various shapes of the
electrophoretic pattern that are linked to monoclonal gammopathies.
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Case # 2 IgG-Kappa monoclonal gammopathy in prostate cancer patient with
bone metastasis
79 years old male with known prostate cancer was admitted with
severe anemia (hemoglobin 6.1), renal failure, and hypertension.
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Case # 3 IgG-Lambda monoclonal gammopathy of undetermined significance
(MGUS)
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Case # 4 IgG-Lambda monoclonal gammopathy of undetermined significance
(MSUG)
48 years old male was admitted to the hospital for a viral illness, and
was placed on acylclovir but with no relief of his hyperpyrexia. After the
antiviral therapy the patient was administered antibiotics, and the
pulmonary symptoms were resolved. Several serological tests were
performed during hospitalization, but all of them were negative.
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Case # 5 IgG-Kappa monoclonal gammopathy with persistent anemia
and elevated serum ferritin
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Case # 6 IgG-Kappa monoclonal gammopathy without manifestation of
myeloma from the bone marrow and clinical symptoms
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65
Case # 7 IgG-Lambda monoclonal gammopathy (MGUS)
66
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Section G Hypogammaglobulinemia and Hypergammaglobulinemia
Case # 1 Polyclonalgammopathy
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Case # 2 Hypogammaglobulinemia
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Case # 3 Apparent Hypogammaglobulinemia with monoclonalgammopathy
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Section H: Oligoclonal Bands in Serum Gammaglobulin Region
The term “oligo” means few (more than one). The gammaglobulin
region of the serum protein electrophoresis very rarely exhibits
oligoclonal bands of different intensities spaced at equal distance or
crowded very close to each other. Sometimes one band is very
prominent as compared to the other bands. In general there is
polyclonal increase in the serum gammaglobulins, and the
concomitant presence of oligoclonal bands are observed in few clinical
conditions, e.g. chronic infection, autoimmune diseases and less
frequently in lymphoproliferative processes. In this situation
immunotyping and/or immunofixation is helpful in the interpretation of
these bands. In cases the clinical condition of the patient requires the
diagnosis of light chain multiple myeloma, it is suggested to perform
urine protein electrophoresis and immunofixation or alternatively the
assay of free kappa and lambda chains in serum.
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Section I: Light Chain Multiple Myeloma
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Case #2 Kappa Chains Myeloma
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Section I: Case #2
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Section J: Cryoglobulins
Agarose gel serum protein electrophoresis is not the procedure for the detection of
the cryoglobulins, however in some cases due to the employment of cooler
temperatures, one might observe a precipitate at the application point / abnormal
electrophoretic pattern. This formation of the precipitate or abnormal pattern triggers
further investigation, e.g., serum immunofixation. It is the observation of several
lanes with precipitate upon imuunofixation (and repeat immunofixation upon dilution
of the serum and also replacement of one of the antisera with buffer or saline), that
alerts the laboratorian about the possibility of cryoglobulins. It is emphasized that the
confirmation of cryoglobulins in serum requires establishment of the cryocrit, and
immunofixation of the washed cryoglobulin. The assay for Hepatitis C virus antibody,
rheumatoid factor and complement C4 for other diagnostic reasons are also
recommended. Conversely the CZE and the immunotyping by the Sebia System is
performed at 35.5o C, thus the cryoglobulins remain dissolved and do not precipitate,
thus the presence of cryoglobulins are eluded. Quantitative analysis for IgG, IgA,
igM, Free κ, Free λ were performed at 37o C, therefore accurate results were obtained.
87
Comments: The serum protein electrophoresis by agarose gel electrophoresis
(HYDRASYS) indicated three restrictions. Serum immunofixation indicated
precipitate lanes in all five sectors (serum protein electrophoresis, IgG, IgA, IgM,
kappa, and lambda). Repeat serum immunofixation after 1:5 dilution of the serum
again indicated the precipitate lanes in all the five areas. The CZE at 35.5o C
indicated no such phenomenon and the elctrophoretic pattern indicated a
monoclonal band in the gamma globulin region. Immunotyping indicated a distinct
IgM-Kappa monoclonal band.
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