Serum Protein Electrophoresis and Immunofixation

(04-30-2008 11:19A.M.
)
INTRODUCTION
The detection and identification of monoclonal proteins in serum/urine, for the

diagnosis of multiple myeloma and other diseases by solid phase zone
electrophoresis and immunofixation has been traditionally a slow and labor intensive
laboratory procedure. In view of the recent availability of the automated capillary
zone electrophoresis (CZE) instruments for the identification of monoclonal proteins
(immunotying) in serum, the laboratory results are provided to the physician more
rapidly than before. There are two automated capillary zone electrophoresis
instruments that are currently available, i.e. Paragon CZE 2000 (Beckman Coulter,
USA), and CAPILLARYS (Sebia, France). We have assembled this compendium of
selected cases for the educational purposes of the medical technologists, residents
in pathology, pathologists, oncologists, and clinical chemists. The objective is to
familiarize the reader with the salient features of the results of the automated method
(Sebia CAPILLARYS) so that an accurate interpretation is made for diagnostic
purposes, especially when the laboratory is switching from the manual/semi-
automated solid phase electrophoretic to the liquid phase automated procedure.
We have briefly described the semi-automated electrophoretic and

immunofixation methods, and the automated methods used for the detection and
identification of the monoclonal bands in serum. Serum immunoglobulins (IgG, IgA,
IgM) were quantified using Beckman Coulter IMMAGE nephelometer.
Serum Protein Electrophoresis: Sebia Hydragel Beta1-Beta2 kit was used

to separate serum proteins on alkaline buffered agarose gels (pH 8.5) into six
fractions, i.e., albumin, alpha-1 globulin, alpha-2 globulin, beta-1 globulin, beta-2
globulin, and gamma globulin. The kits were used in conjunction with the semi-
automated HYDRASYS instrument (Sebia). Amidoblack was used to stain the gels,
and the percent fractions were quantified using the scanner.
Serum Protein Immunofixation: Sebia HYDRAGEL IF kit was used first to

separate the proteins into six fractions (see above) in six different lanes or tracks.
The reference track was overlaid with fixative reagents and each of the five
additional tracks were overlaid with different antisera (IgG, IgA, IgM, kappa and
lambda). The immunoglobulin-antiserum complex was trapped in the agarose, and
then stained with amidoblack for visual examination of the protein electrophoresis
pattern and only bound specific polyclonal or monoclonal immunoglobulins in each
track. Any protein restriction (monoclonal band) was thus determined.
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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
Capillary Zone Electrophoresis: The proteins in serum were separated into

six fractions using a liquid-based system (Sebia CAPILLARYS), without the use of a
solid support medium i.e., agarose gel. In this method a fused silica capillary (into
which a small volume of serum is aspirated) with strong negative charge on the
interior of the capillary provided a large negative surface area. Under the conditions
of electrophoresis (movement of protein molecules under charge) and endosmotic
flow of cations toward the cathode, the proteins were separated into six fractions,
and quantified by an ultraviolet detector using the absorbance of the amide band of
the proteins at 200-215 nm. All the serum CZE scans wherever illustrated in this
compendium are in green color.
Immunotyping of Serum Proteins: The monoclonal protein present in

serum was detected and identified by an antigen-antibody reaction in liquid phase
followed by CZE using the Sebia CAPILLARYS. In this automated methodology the
specific immunoglobulin and the antisera react to provide an antigen-antibody
complex (pretreatment step). The pretreated samples (reference, IgG, IgA, IgM,
kappa and lambda) were electrophoresed simultaneously in six capillaries. The
antigen-antibody complexes migrated slowly than an unbound immunoglobulin, i.e.,
migrated anodically to albumin, or migrated as an increased baseline co-migrating
with albumin, alpha-1 and/or alpha-2 globulin. This procedure is obverse of
immunofixation (see above), as the monoclonal proteins were removed and thus not
detected on the electropherogram after CZE. The interpretation (identification of the
monoclonal band) was made by the “overlay” of the untreated reference
electrophoretic curve on each of the five electrophoretic curves of the pretreated
samples. All the serum CZE scans after the each pretreatment step
(imunosubtraction process with anti-IgG, anti-IgA, anti-IgM, anti-kappa, and
anti-lambda) wherever illustrated in this compendium are in red color.
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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.
Detection and Identification of Monoclonal Bands: The majority of the

readers of this compendium are familiar with the interpretation of the serum protein
electrophoresis and immunofixation patterns. The interpretation of the serum protein
electrophoresis by CZE is virtually identical to the agarose gel serum protein
electrophoresis, and thus needs no further comments.
The detection of protein bands with restricted mobility by immunotyping needs

careful evaluation of the five individual CZE scans of the pretreated samples ( anti-
IgG, anti-IgA, anti-IgM, anti-kappa, and anti-lambda) in conjunction with CZE scan of
the neat serum.
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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.
The interpretation of the immunotyping patterns requires the visualization of the

subtle changes in the shape of the electrophoretic scans after the antigen-antibody
reaction. A large monoclonal band (M-protein greater than 100 mg/dL) can be
accurately identified without much difficulty. It is the minimonoclonal bands (M-
protein less than 25 mg/dL) that could not be identified easily due to not so obvious
large changes in the shape of the electrophoretic curve, especially if the
minimonoclonal band is either embedded with a polyclonal gamma globulin,
transferrin, complement C3, or associated with an artifact (fibrinogen, contrast
media, etc.).
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.
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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).
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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.
Let us consider another hypothetical case with a protein restriction (monoclonal

band) in the gamma globulin region (Fig No. B).
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
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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.
Assignment of monoclonal bands: The first step is the visual examination

of serum CZE scan to note the presence of, a) sharp peak, b) unsymmetrical band
(from the alpha-2 to the farthest cathode end of gamma globulin region), c) distortion
(similar to electrical noise of a signal) of the shape of any of the alpha-2, beta-1,
beta-2, and gamma globulin bands, and d) decreased gamma globulin. A decreased
gamma globulin band also suggests the electrophoretic migration of the possible
restricted immunoglobulin towards the anode, therefore one might observe
unsymmetrical bands for any of the alpha-2, beta-1, and beta-2 globulin bands. The
location of the abnormal / unsymmetrical band on the CZE scan must be kept in
mind while comparing it with the five pretreated (anti-IgG, anti-IgA, anti-IgM, anti-
kappa, and anti-lambda) CZE scans.
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.
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Selected Case Studies:
Section A: Case # 1 Normal Person

Case # 2 Restricted band superimposed on
C3 complement band
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
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.
Case # 1 Normal Person
Serum of a 28 years old male employee, presented for annual physical

examination. No abnormality observed in the laboratory tests and
examination by the physician.
Total Protein: 7.5 g/dL (6.4-8.3 g/dL) Immunoglobulins:

Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 4.5 g/dL (3.8-5.8 g/dL) IgG: 902 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 208 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 81 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.2 g/dL (0.5-1.3g/dL)
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Case # 2 Restricted band superimposed on C3 complement band
51 years male admitted with hypotension, acute renal failure,

protein malnutrition, and was diagnosed with staphylococcus aureus
septicemia during hospitalization.

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.
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NOTE: The apparent increase in -2 globulin was persistent as indicated by an
arrow, perhaps due to fibrinogen. Similar bands in the -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).
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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.
76 years old female with severe symptomatic anemia (hemoglobin of

5.3%) due to gastrointestinal bleeding, bleeding gastric ulcer,
obstructive pulmonary disease, coronary artery disease, atrial
fibrillation, congestive heart failure, and diabetes mellitus Type II
complicated with peripheral neuropathy. Cord compression secondary
to malignant lymphoma involving the spine.

Albumin: 3.1 g/dL (3.8-5.8 g/dL) IgG: 701 mg/dL(751-1560 mg/dL)
Comments: The serum protein electrophoresis by agarose gel indicated increased

concentration of the beta1 globulin region (suggesting increased transferrin due to
serum iron deficiency as the patient is severely anemic). Another possibility of
increased transferrin may be double transferrin bands due to genetic reasons in
some Afro-Americans. The third possibility is of embedded monoclonal band under
the transferrin band. The serum protein electrophoresis by capillary zone
electrophoresis indicated asymmetrical beta1-beta2 globulin region with a minor
restriction on the cathode side of the C3 complement band. It is obvious from the
study of Cases # 2- 4 that fibrinogen can be the possible cause of the atypical
electrophoretic pattern. A monoclonal IgA band is known to migrate between alpha-2
globulin to any place in the gamma globulin region. Indeed immunofixation and
immunotyping indicated the presence of monoclonal IgA-kappa in serum of this
patient. Therefore it is imperative to perform immunotyping or immunofixation of
serum, whenever a minor restriction band or atypical shape of the fractions are
observed on electrophoresis in the area of beta 1-beta 2 globulin region.
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NOTE: IgA-Kappa monoclonal band after the immunoreduction step is indicated by
an arrow.
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Section B: Acute-Phase Reaction Pattern
The serum protein electrophoresis is a useful tool to detect acute

tissue damage ( infection, tissue injury, tumor necrosis), with or
without inflammation. In general a group of hepatocyte-derived
proteins are increased, and these proteins are labeled as acute
phase reactants. In a classical acute-phase serum protein
electrophoresis pattern, one detects, a) decreased albumin,
b) increased alpha-1 globulin, c) increased alpha-2 globulin,
d) decreased transferrin if there is no concomitant decrease in
serum iron, e) and small band in the gamma globulin region due
to C-reactive protein (which can be confused with a mini-monoclonal
band). It must be pointed out that C-reactive protein band is never
detected with capillary zone electrophoresis. Fibrinogin is increased
within 24 hour of the injury, and C3 complement elevations without
consistency are noticed within a week after acute tissue damage. In
clinical practice the acute–phase reaction pattern of serum protein
electrophoresis is not used for making any differential diagnosis,
however if detected it can be useful in a variety of clinical conditions,
e.g. predicting infections in patients, etc.
Case # 1 Acute-phase reaction pattern with monoclonal gammopathy
63 years old male with a known history of anemia, nephrotic

syndrome, hypomagnesemia, edema, hypertension, neuropathy,
deep venous thrombosis was admitted to the hospital and a colon
cancer diagnosis with metastasis to the liver was made.

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.
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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.
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Section C: Double Gammopathy and Biclonal Gammopathy
The presence of two distinct bands of restricted mobility on the

serum electrophoretic pattern are rarely observed in the laboratory.
Biclonal gammopathy: If the light chains identified by either

immunofixation or immunotyping differ, i.e. both kappa and lambda
chains are detected then it is called “biclonal gammopathy” as these
light chains originate from two separate clones.
Double gammopathy: If the light chains identified by either

immunofixation or immunotyping are the same, i.e. both are either
“kappa” or “Lambda” then it is called “double gammopathy” as these
light chains originate from a same clone.
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.
Case # 1 Double gammopathy (IgG-Lambda and IgM-Lambda)
63 years old female under chemotherapy for a previously known case

of multiple myeloma, residing at the nursing home was routinely
checked by the oncologist for several laboratory tests including serum
protein electrophoresis and quantitative immunoglobulins.

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IgG
IgM
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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 (MSUG).

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IgM-Kappa IgM-Lambda
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Case # 3 Biclonal gammopathy (IgM-Lambda and IgG-Kappa)
58 years old male with a past medical history of monoclonal B-cell

lymphoproliferative disorder (Waldenstrom’s macroglobulinemia).

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Case # 4 Double Gammopathy (IgA1 – Kappa and IgA2 – Kappa)
55 years old unconscious female admitted to the hospital for liver

cirrhosis, asthma, hypertension, etc. Her kidney function was normal.
Serum creatinine was 0.6 mg/dL (0.7 – 1.5 mg/dL), and the calculated
glomerular filtration rate (GFR) was 105 (> 60 ml/min/1.73 m2 ).
Total Protein: 6.3 g/dL Immunoglobulins:

Albumin: 2.9 g/dL (3.8 – 5.8 g/dL) IgG: 1300 mg/dL (751 –1560 mg/dL)
Alpha-1 Globulin: 0.1 g/dL (0.1 – 02 g/dL) IgA: 1640 mg/dL (82 – 453 mg/dL)
Alpha-2 Globulin: 0.6 g/dL (0.4 – 0.9 g/dL) IgM: 105 mg/dL (46 - 304 mg/dL)
Beta Globulin: 1.4 g/dL (0.5 – 1.1 g/dL) Free  37.6 mg//L (3.3 – 19.4 mg/L)
Gamma Globulin: 1.3 g/dL (0.5 – 1.3 g/dL) Free  20.7 mg/L (5.7 – 26.3 mg/L)
Free / 1.82 (0.26 – 1.65)
Comments: Serum protein electrophoresis (Sebia, HYDRASIS) showed a dark

staining for C3 band, and another monoclonal band adjacent to the C3 band
(cathode side). According to Feinstein et al (Nature 212: 1496-1498, 1966), IgA in
human beings is composed of two antigenically different subclasses IgA1 and IgA2,
and approximately 80-90% is IgA1 in serum (Gray et al., Exp. Med. 128: 1233-1236,
1969).
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Section D: IgA Monoclonal Gammopathies
The monoclonal bands due to IgA migrate in the agarose gel

anywhere from alpha-2 region to the gamma globulin region, however
in the capillary zone electrophoresis (Sebia) the IgA restrictions were
mostly observed in the beta globulin region. The identification of these
monoclonal bands is complicated by the superimposed bands of C3
complement and lipoproteins. In normal population the twin bands of
C3 complement and transferrin (beta globulin region) are very
symmetrical in shape. The shape and the concentration of transferrin
in serum deviates in several clinical conditions, notably serum iron
deficiency (increased transferrin), acute phase reactions (decreased
transferrin), etc. Similarly the shape and the concentration of C3
complement band are altered in some clinical conditions. Serum
samples accidentally exposed to elevated temperatures (above
refrigeration) for a long period of time commonly exhibit lower
concentration (decreased height of the band) of C3 complement.
Upon visual examination of the electrophoretic pattern, if any distortion

of the beta globulin region is detected, it is advised to perform
immunotyping or immunofixation.
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Case # 1 IgA monoclonal band masking the beta globulin region
67 years old male admitted to hospital and was diagnosed with

anemia, acute renal failure, urinary tract infection, congestive heart
failure, coronary artery disease, and gastrointestinal hemorrhage.
Positive for Bence Jones protein in urine.

Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 43 mg/dL (46-304 mg/dL)
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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.
Total Protein: 5.6 g/dL (6.4-8.3 g/dL) Immunoglobulins

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Section E: IgM Monoclonal Gammopathies:
IgM monoclonal gammopathies when detected are generally

associated with either plasma cell proliferations (IgM myeloma) or a
low-grade B-cell lymphoplasmacytic disorder (Waldenstrom’s
macroglobulinemia). In both the diseases the bone marrow is involved,
however one major characteristic of the Waldenstrom’s
macrglobulinemia is the absence of lytic skeletal lesions.
Case # 1 IgM monoclonal gammopathy in multiple myeloma
92 years old male with several clinical disorders was on

chemotherapy, and the treatment was monitored by serum protein
electrophoresis and quantitative IgM. In view of the hyperviscosity
usually associated with increased concentration of the large molecule
IgM in serum (1770 mg/dL; Normals: 46-304 mg/dL), other clinical
problems may be present, e.g. cryoglobulinemia either with or without
hepatitis C.

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Case # 2 IgM monoclonal gammopathy in malignant lymphoma (IgM-Kappa)
60 years old male with a past medical history of malignant lymphoma

was examined after first round of chemotherapy and radiation for bone
marrow and flow cytometric tests.

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Case # 3 IgM monoclonal gammopathy (MGUS) without any clinical symptoms
(IgM-Kappa)
72 years old female with a history of hypothyroid and ovarian cancer

without any prior cardiac history was admitted for chest pain. Stress
test and heart catheterization was performed, but no evidence of
coronary artery disease was found. The left ventricular ejection volume
was normal.

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Section F: IgG Monoclonal Gammopathies
The gamma globulin region of the electrophoretic pattern consists of

five immunoglobulins (IgG, IgA, IgM, IgD, and IgE), and in normal
person the shape of this is symmetrical akin to half moon. IgG is the
major component of these five immunoglobulins in a normal person.
Several diseases are associated with either the increase or decrease
in serum IgG. It is also known that more than 50% of the monoclonal
bands in serum are linked to IgG. The IgG monoclonal bands can
migrate any place from beta globulin to the most cathodal area of
gamma globulin region. Therefore, anytime a deviation of the
symmetrical shape of the band (beta/gamma globulin) is observed, a
possibility of monoclonal band must be ruled by immunofixation or
immunotyping. The presence of large monoclonal band (IgG
concentration greater than1.0 gram/dL) can be easily detected,
however it is the mini-monoclonal bands that requires follow up, e.g.
free kappa and lambda chain studies in serum.
The following cases are selected to show the various shapes of the
electrophoretic pattern that are linked to monoclonal gammopathies.
Case # 1 IgG-Kappa multiple myeloma
73 years old female with a past medical history of multiple myeloma

was examined during her treatment and laboratory tests were
performed.

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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)
85 years old female, known case of dementia was admitted to the

hospital for multiple disorders (septicemia, renal failure, anemia,
respiratory failure, etc.).

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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
61 years old female with persistent anemia, weakness, multiple falling

episodes, refractory lumbosacral pain, hypothyroidism, hypertension,
pelvic ramus fracture in 2003 and sacral fracture in 2003 was
examined by an oncologist, but no morphologic cause for the patient’s
anemia was demonstrated. The mini-monoclonal band detected
(indicated by the shaded area) at the most cathode part of the
electrophoretic pattern was very faint in both the agarose and the
capillary zone electrophoresis (approximately 0.08 gram/dL).

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Case # 6 IgG-Kappa monoclonal gammopathy without manifestation of
myeloma from the bone marrow and clinical symptoms
56 years old male with IgG-Kappa monoclonal gammopathy was

examined for bone marrow aspirate, however the bone marrow
features and other hematopathology assays did not meet the criteria of
multiple myeloma.

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Case # 7 IgG-Lambda monoclonal gammopathy (MGUS)
74 years old female was presented at the emergency department with

complaints of dizziness, weakness, and inability to walk. She had lost
weight of over thirty pounds during the last six months. Preliminary
work-up diagnosed cerebrovascular accident. Final diagnoses
included neuropathy secondary to diabetes mellitus Type II, renal
tubular acidosis with chronic kidney disease, chronic obstructive
pulmonary disease, anemia, and hypertension.

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Section G Hypogammaglobulinemia and Hypergammaglobulinemia
A decrease or increase in the gamma globulins noticed from serum

protein electrophoresis requires a careful evaluation of the over all
electrophoretic pattern. Both of these situations may be associated
with some clinical condition. The hypogamma globulinemia pattern in a
person over 50 years old and with no aberration in other bands of the
protein electrophoresis pattern may suggest Bence Jones proteinuria.
This requires urine protein electrophoresis and immunofixation to
detect free light chains in urine or alternatively assay of free kappa and
free lambda chains in serum. In certain cases it is possible that there is
decrease in the gamma globulin fraction, however some other fraction,
e.g., the beta1-beta2 globulin fraction is increased due to the migration
of the monoclonal immunoglobulin in this region. This situation also
dictates a follow-up of the patient with immunotyping, etc. The
hypergammaglobulinemia pattern of the serum electrophoresis must
be examined for a superimposed (embedded) monoclonal band, and
thus immunotyping is required to rule out mini-monoclonal
gammopathy.
Case # 1 Polyclonalgammopathy
68 years old female admitted for rectal bleeding, anemia

and infection

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Case # 2 Hypogammaglobulinemia
84 years old female with several previously known clinical conditions

was admitted to the hospital for acerbating weakness and chronic
obstructive pulmonary disease.

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Case # 3 Apparent Hypogammaglobulinemia with monoclonalgammopathy
65 years old female previously diagnosed for multiple myeloma was

checked for clinical response for chemotherapy, and serum protein
electrophoresis along with quantitative immunoglobulins assay was
performed on out-patient basis.

Comment: In this patient though the gammaglobulin region appears to be low, it is an

aberration as the monoclonal gammaglobulin band (IgG-Kappa) migrates with C3
complement. In this manner the concentration of C3 complement and the total
beta1-beta2 region appears to be increased, which in fact is not.
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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.
Case # 1 Oligoclonal bands without any clinical significance
79 years old female with known peripheral neuropathy was evaluated

to rule out lumbar radiculopathy (EMG was negative for radiculopathy).
She was having some gait dysfunction and parathesia in her feet. The
bone X-ray and CT scan were negative. A few laboratory tests were
slightly abnormal (BUN, creatinine, PTT, WBC, and Vitamin B12), but
were not diagnostic of any settled clinical condition.
Comments: The oligoclonal bands (indicated by dashed lines) were detected in

both the agarose gel and capillary zone electrophoresis, however, they were not
present on the immunofixation plate and the immunotyping scan. The laboratory
report was signed off (without any prior access to the clinical and other laboratory
data), a) oligoclonal bands present perhaps due to immune complex formation, and
b) Suggest urine protein electrophoresis to rule out Bence Jones proteinuria.
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Section I: Light Chain Multiple Myeloma
Case #1 Lambda Chains Myeloma
72 years old male (previously known case of light chain

multiple myeloma for more than three years), was admitted
to the hospital due to renal failure, fever, and urinary
tract infection. In the most recent serum specimen, there
was no evidence of a monoclonal band from agarose gel
electrophoresis. The shape of the C3 complement band
was not symmetrical, but diffuse and slanted, and this triggered
serum immunofixation studies. The initial laboratory interpretation
provided to the physician was: " Acute inflammation pattern,
possibility of monoclonal band." "Final interpretation to
follow after immunofixation studies."
Serum immunofixation studies indicated a monoclonal band

due to free lmbda chains, and no precipitation band was
detected for any of the heavy chains (IgG, IgA, IgM, IgD,
and IgE). The immunofixation results using the anti-IgD and
anti-IgE are not displayed on next page.

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Case #2 Kappa Chains Myeloma
69 years old male brought to the emergency room of the

hospital with abdominal pain, ileus, fever, anemia, and weakness in lower
extremities. Patient was admitted and oncologist was consulted. Serum
protein electrophoresis (agarose gel) indicated hypogammaglobulinemia
and a very faint monoclonal band (identified as free kappa chains from
immunofixation). There was no evidence of monoclonal heavy chains
(lgG, lgA, lgM, lgD, and lgE) from immunofixation studies. Bone marrow
examination showed 42% plasma cells.

Free Kappa = 901 mg/L (3.3 – 19.4 mg/L)

Free Lambda = 15.0 mg/L (5.7 – 26.3 mg/L)
Kappa/Lambda Ratio = 60 (0.3 – 1.2)
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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 , Free  were performed at 37o C, therefore accurate results were obtained.
Case # 1 Brouet Type I
68 year old male, with a known history of several clinical disorders,

e.g., hypentension, anemia, weakness in the legs, deep venous
thrombosis, etc. was admitted to the hospital. Negative for Hepatitis C.
Total Protein: 7.7 g/dL Immunoglobulins:

Albumin: 4.2 g/dL (3.8 – 5.8 g/dL) IgG: 1480 mg/dL (751 –1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1 – 02 g/dL) IgA: 160 mg/dL (82 – 453 mg/dL)
Alpha-2 Globulin: 0.8 g/dL (0.4 – 0.9 g/dL) IgM: 943 mg/dL (46 - 304 mg/dL)
Beta Globulin: 1.2 g/dL (0.5 – 1.1 g/dL) Free  56.9 mg//L (3.3 – 19.4 mg/L)
Gamma Globulin: 1.4 g/dL (0.5 – 1.3 g/dL) Free  31.3 mg/L (5.7 – 26.3 mg/L)
Free / 1.82 (0.26 – 1.65)
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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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Section K : Seven Cases for Interpretation
In this section we have presented seven cases and the reader is

requested to provide the interpretation of the laboratory results.
Case # 1 74 years old male was admitted from the emergency department with
complaints of dizziness and lightheadedness, difficulty in walking and
near syncopal episode. He had a history of atrial fibrillation. The
patient had a history of seizure disorder and was on dilantin. The
patient had a history of coronary artery disease and had angioplasty in
the past. Chest x-ray showed cardiomegaly. Echocardiogram showed
left ventricular ejection fraction of 40%. The patient was seen by a
neurologist and a psychiatrist. The dilantin dose was adjusted in the
therapeutic range, and the patient was sent to the nursing home, and
as a follow-up advised to see the primary physician in two weeks.

Hint: A band of restricted mobility is present between alpha-1 globulin and alpha-2
globulin in both the agarose gel electrophoresis and the capillary zone
electrophoresis. Neither the immunofixation and nor the immunotyping by capillary
zone electrophoresis indicated any monoclonal band. Sometimes in the agarose gel
electrophoresis system (Sebia, HYDRASYS), the beta-lipoprotein band migrates
between the alpha-1 and alpha-2 globulin.
Interpretation please:
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Case # 2 68 years old female, resident of a nursing home complained of severe
abdominal pain, and was brought to the emergency department. Acute
pancreatitis was the admitting diagnosis. The discharge diagnoses
were pleural effusion, chronic obstructive pulmonary disease,
pneumonia, congestive heart failure, anemia and arteriosclerotic heart
disease, etc.
Test Result Normal Value
Amylase 290 50 – 130 U/L

Lipase 913 23 – 300 U/L
Iron 14 37 – 170 ug/dL

Ferritin 381 8 – 120 ng/mL
WBC 25.6 4.0 – 11.0 k/cumm
C-Reactive Protein 10.9 Less than 1.0 mg/dL
Haptoglobin 379 16 – 200 mg/dL

(component of alpha-2 globulin)

Hints: Apparent increase in transferrin is due to low serum iron in a patient that also
has high acute phase protein (C-Reactive Protein). Another acute phase protein
(haptoglobin) was elevated, however there was no evidence of intracellular
hemolysis or elevation of LD-1 isoenzyme. Patient had lower lobe pneumonia and
was treated with antibiotics.
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Case # 3 86 years old female came to the emergency department from the
nursing home in view of ataxia and slurred speech. She had been
diagnosed for atherosclerotic heart disease and a permanent
pacemaker was inserted past coronary artery bypass graft. She suffers
from degenerative arthritis and chronic joint pain. A final diagnosis of
cerebrovascular accident was made.
Bone marrow examination in 1998 and 2000 were essentially similar

(normocellular with normoblastic erythropoiesis, maturing
granulopoieses and megakaryopoiesis).

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Case # 4 59 years old male with a past medical history of thrombocytopenia was
admitted for bone marrow examination to rule out myelodysplasia and
assess the megakarocyte population. Intravenous gamma globulin
was administered during hospitalization along with the higher dose of
prednisone.

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Case # 5 80 years old female during routine check-up at her primary physician’s
office presented pale, with hemoglobin of 6.9 gram/dL. She was
admitted on the basis of profound anemia with probable GI blood loss
versus bone marrow hypoplasia-neoplasia. She had a history of more
than ten other diseases. Bone marrow did not demonstrate any
evidence of monoclonality, acute leukemia or non-Hodgkin’s
lymphoma. No diagnostic morphologic evidence of myelodysplastic
syndrome.

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Case # 6A and 6B We have only presented the electrophoretic, immunofixation,
and immunotyping data for these two patients.
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Serum Protein Electrophoresis and Immunofixation

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(04-30-2008 11:19A.M.

The detection and identification of monoclonal proteins in serum/urine, for the

We have briefly described the semi-automated electrophoretic and

Serum Protein Electrophoresis: Sebia Hydragel Beta1-Beta2 kit was used

Serum Protein Immunofixation: Sebia HYDRAGEL IF kit was used first to

Capillary Zone Electrophoresis: The proteins in serum were separated into

Immunotyping of Serum Proteins: The monoclonal protein present in

Detection and Identification of Monoclonal Bands: The majority of the

The detection of protein bands with restricted mobility by immunotyping needs

The interpretation of the immunotyping patterns requires the visualization of the

Let us consider another hypothetical case with a protein restriction (monoclonal

Assignment of monoclonal bands: The first step is the visual examination

Section A: Case # 1 Normal Person

Case # 1 Normal Person

Serum of a 28 years old male employee, presented for annual physical

Total Protein: 7.5 g/dL (6.4-8.3 g/dL) Immunoglobulins:

51 years male admitted with hypotension, acute renal failure,

Total Protein: 2.9 g/dL (6.4-8.3 g/dL) Immunoglobulins:

76 years old female with severe symptomatic anemia (hemoglobin of

Total Protein: 5.8 g/dL (6.4-8.3 g/dL) Immunoglobulins:

Comments: The serum protein electrophoresis by agarose gel indicated increased

The serum protein electrophoresis is a useful tool to detect acute

Case # 1 Acute-phase reaction pattern with monoclonal gammopathy

63 years old male with a known history of anemia, nephrotic

Total Protein: 4.3 g/dL (6.4-8.3 g/dL) Immunoglobulins:

The presence of two distinct bands of restricted mobility on the

Biclonal gammopathy: If the light chains identified by either

Double gammopathy: If the light chains identified by either

Case # 1 Double gammopathy (IgG-Lambda and IgM-Lambda)

63 years old female under chemotherapy for a previously known case

Total Protein: 6.4 g/dL (6.4-8.3 g/dL) Immunoglobulins:

Total Protein: 8.4 g/dL (6.4-8.3 g/dL) Immunoglobulins:

58 years old male with a past medical history of monoclonal B-cell

Total Protein: 7.0 g/dL (6.4-8.3 g/dL) Immunoglobulins:

55 years old unconscious female admitted to the hospital for liver

Total Protein: 6.3 g/dL Immunoglobulins:

Comments: Serum protein electrophoresis (Sebia, HYDRASIS) showed a dark

The monoclonal bands due to IgA migrate in the agarose gel

Upon visual examination of the electrophoretic pattern, if any distortion

67 years old male admitted to hospital and was diagnosed with

Total Protein: 6.8 g/dL (6.4-8.3 g/dL) Immunoglobulins:

Total Protein: 5.6 g/dL (6.4-8.3 g/dL) Immunoglobulins

IgM monoclonal gammopathies when detected are generally

Case # 1 IgM monoclonal gammopathy in multiple myeloma

92 years old male with several clinical disorders was on

Total Protein: 7.1 g/dL (6.4-8.3 g/dL) Immunoglobulins:

60 years old male with a past medical history of malignant lymphoma

Total Protein: 6.7 g/dL (6.4-8.3 g/dL) Immunoglobulins:

72 years old female with a history of hypothyroid and ovarian cancer

Total Protein: 7.1 g/dL (6.4-8.3 g/dL) Immunoglobulins:

The gamma globulin region of the electrophoretic pattern consists of

Case # 1 IgG-Kappa multiple myeloma

73 years old female with a past medical history of multiple myeloma

Total Protein: 7.2 g/dL (6.4-8.3 g/dL) Immunoglobulins:

Total Protein: 6.8 g/dL (6.4-8.3 g/dL) Immunoglobulins:

85 years old female, known case of dementia was admitted to the

Total Protein: 6.2 g/dL (6.4-8.3 g/dL) Immunoglobulins:

Total Protein: 5.9 g/dL (6.4-8.3 g/dL) Immunoglobulins:

61 years old female with persistent anemia, weakness, multiple falling

Total Protein: 5.0 g/dL (6.4-8.3 g/dL) Immunoglobulins: