compendium-second-edition-may-2011-Dr-Zia

Protein Electrophoresis, Immunofixation, and
Immunodisplacement in Clinical Diagnosis
A Compendium of Selected Cases Employing
the Procedures and Instruments of
Helena Laboratories, USA, and Sebia, France
by
Zia Uddin, Ph.D.
&
Christopher R. McCudden, Ph.D.
First Edition: September 2010
Second Edition: May 2011

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This compendium is dedicated to our
teachers, to whom we would like to
express our heartfelt undying gratitude
for their relentless effort, sincerity,
dedication, and virtuous contribution
towards our education.

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Contents
Preface 1
Acknowledgements 3
Introduction 5
Procedures and Instruments 6
Selection of Patients for Case Studies 8
Presentation of Clinical Cases and the Electrophoretic Data 9
Detection and Identification of Monoclonal Bands 10
Assignment of Monoclonal Bands 14
Section A: Monoclonal Gammopathy of 15
Undermined Significance
Case #1 IgG-Lambda Monoclonal Gammopathy: Low Risk 18
Case #2 IgA-Kappa Monoclonal Gammopathy: 23
Low Intermediate Risk
Case #3 IgA-Kappa Monoclonal Gammopathy: 28
High Intermediate Risk
Case #4 IgG-Kappa Monoclonal Gammopathy: Low Risk 33
Section B: Acute Phase Reaction Pattern 38
Case#5 Acute Phase Reaction without Serum Iron Deficiency 40
Case#6 Acute Phase Reaction (Bacterial Meningitis) with 46
Concomitant Serum Iron Deficiency
Case#7 Post Acute Phase Reaction: Monoclonal Band 51
Superimposed on C3 Band (Elevated Fibrinogen)
(i)

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Section C: Double and Biclonal Gammopathy 56
Case#8 Double Gammopathy: a) IgA-Lambda (major band) 57
b) IgG-Lambda (minor band)
Case#9 Double Gammopathy: a) IgM-Kappa (major band) 62
b) IgG-Kappa (minor band)
Case#10 IgM-Kappa and IgM-Lambda Biclonal Gammopathy 67
Section D: Polyclonal Gammopathy 73
Case#11 Polyclonal Gammopathy: Increased IgG, IgA, and IgM 73
with Beta-Gamma Globulin Bridging
Case#12 Polyclonalgammopathy: Pancytopenia with Bone 78
Marrow Containing <5% Plasma Cells
Section E: Hypogammaglobulinemia/Light Chain 83
Multiple Myeloma
Case #13 Hypogammaglobulinemia Secondary to Chemotherapy 83
for Non-Hodgkin’s Lymphoma: Anaphylactic Reaction
in Response to Intravenous Immunoglobulin Infusion
Case#14 Simultaneous Adenocarcinoma of Lung and Light Chain 89
(Kappa) Multiple Myeloma
Case#15 Hypogammaglobulinemia and Light Chain (Lambda) 94
Multiple Myeloma
Case#16 Light Chain (Lambda) Multiple Myeloma with normal 99
Gammaglobulins
Section F: Multiple Myeloma and other B-Cell Dyscrasia 104
Case#17 IgM-Lambda Multiple Myeloma 104
Case#18 IgG-Lambda Multiple Myeloma 110
Case#19 IgD-Lambda Multiple Myeloma 115
Case#20 IgE-Lambda Multiple Myeloma 120
Case#21 Light Chain (Lambda) Multiple Myeloma 122
(ii)

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Case#22 Smoldering Myeloma vs Symptomatic Myeloma 128
Case#23 Autologous Stem Cell Transplantation 134
Case#24 Waldenstrom’s Macroglobulinemia 137
Case#25 Hodgkin’s Lymphoma 143
Case#26 Plasma Cell Leukemia 148
Case#27 Solitary Plasmacytoma of Bone 153
Section G: Rheumatic Diseases 158
Case#28 Felty’s Syndrome: Advanced Arthritis 158
with Splenomegaly
Section H: Unknown Cases 164
Case#29 Unknown Diagnosis: Please make the diagnosis 164
on the basis of the information provided.
(iii)

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Preface
Four years ago, Frank N. Bever, MD, Medical Director of Laboratories, St.
John Detroit Riverview Hospital, Detroit, Michigan, asked me (Zia Uddin) to assay
the serum of few clinically significant patients for serum protein electrophoresis
(SPE) and immunofixation (IF) employing the agarose gel on the semi-automated
HYDRASYS system (Sebia, France); and this was compared with the SPE and
immunotyping results obtained from the capillary zone electrophoresis (CZE) using
the semi-automated CAPILLARYS system (Sebia, France).
Permission for the research project (Study # SJ 1207-06) was obtained from
the Institutional Review Board (IRB) of St. John Health System, Detroit, Michigan,
and a formal authorization was issued by Peter Nickles, MD, Chairperson, IRB.
One of the objectives of our research was to familiarize for educational
purposes our laboratory staff with the electrophoretic patterns obtained by two
distinct techniques. The data collected was put into a compendium, under the
caption “Comparison of Agarose Gel Serum Protein Electrophoresis and
Immunofixation with Capillary Zone Electrophoresis and Immunotyping.” This
compendium was put together for the internal use of associates in the St. John
Health System, Detroit, but the word about its existence leaked out via the grape
vine, and we started receiving requests for its copy (nationally and internationally).
Eventually, I decided to make this compendium available worldwide via the Internet
at no charge on April 6, 2009:
http://www.scribd.com/doc/14114106
It is to be emphasized that I (Zia Uddin) am not associated with the
manufacturers of the instruments and reagents used for the completion of the
project. Neither have I accepted any financial support or honorarium. All the
associated expenses were borne by me personally.
In 2009, David G.Grenache, Ph.D. (an expert in this field) and I discussed
the possibility of putting together a similar compendium comparing the two most
commonly used agarose gel systems for SPE and IF of Sebia, France
(HYDRASYS) and Helena Laboratories, USA (SPIFE 3000). We also decided to
compare the SPE and immunotyping (IT) results of CZE (CAPILLARYS System,
Sebia, France) with the SPE and immunodisplacement (ID) results of a recently
introduced CZE system (V8) of Helena Laboratories, USA. In view of his joining
ARUP Laboratories, Salt Lake City, Utah, where he no longer directed this service
(SPE, IF, immunodisplacement, etc.), David G. Grenache, Ph.D. recommended the
name of his associate Christopher R. McCudden, Ph.D., Department of Pathology
and Laboratory Medicine, University of North Carolina School of Medicine, Chapel
Hill, NC, to collaborate with me on this project. A formal approval for this project was
obtained by Christopher R. McCudden, Ph.D. from the University of North Carolina
(Chapel Hill) Human Research and Ethics Board (IRB No. 09-2209).

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During the course of our work on this new compendium we consulted more
than one hundred internists, oncologists, pathologists, hematologists, surgeons, etc.
to provide their expertise in understanding of the laboratory results and the eventual
clinical diagnosis. We are highly indebted to all these professionals for their
contribution in this compendium. As per the instructions of the IRB, we are not
allowed to disclose the names of these physicians (HIPPA), as this might
inadvertently disclose the identity of the patient, in view of the clinical diagnosis
associated with a particular patient and his or her physician.
The clinical cases of the compendium were selected by us. Again, we
emphasize that we are not associated with any of the manufacturers mentioned in
the compendium, and have not accepted any monetary benefit or honorarium from
any organization for the purposes of this compendium. Our goal is education.
We intend to add more clinically useful cases in future as deemed necessary.
We hope that this compendium is helpful to its readers. We welcome your
comments for incorporation in future editions.
Zia Uddin, Ph.D. Christopher R. McCudden, Ph.D.
St. John Macomb –Oakland University of North Carolina,
Hospital Laboratories School of Medicine, Chapel Hill, NC
Warren, Michigan 48093, USA cmccudde@unch.unc.edu
zi.uddin@stjohn.org
September 2010
Several readers contacted us since the release of the first edition of this
compendium, and made comments and suggestions. In the second edition, we
have tried to accommodate them.
Zia Uddin, Ph.D. Christopher R. McCudden, Ph.D.
May 2011

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Acknowledgements
During the past year several physicians at St John Providence Health
System, Detroit, Michigan, and the University of North Carolina, School of Medicine,
Chapel Hill, North Carolina have helped us establish the clinical diagnosis of the
patients by interpreting data from bone marrow aspirates, flow cytometry,
cytogenetics, and imaging as well as in the formatting of this compendium.
Unfortunately their names cannot be acknowledged here individually due to the
“IRB Guidelines” of our institutions. We are highly indebted to all of them for their
contribution.
We are grateful to all the medical technologists (who performed the
electrophoretic experiments and immunochemistry procedures) at St. John
Providence Health System, Detroit, Michigan, and University of North Carolina,
Hospital Laboratories, Chapel Hill, North Carolina. In particular, we would like to
thank Ms. Shirley Hainsworth for taking the time to analyze the many specimens
included in this study.
The Medical Records and Medical Informatics staff at St. John Providence
Health System, Detroit, Michigan played a significant role in providing us the data of
more than 2000 patients reviewed for this compendium. We sincerely appreciate
their contribution in this project.
The technical staff (Ruthann Stewart and Leela Tansamrit) at the Research &
Development Division of Helena Laboratories, Beaumont, Texas, performed the
experiments on the Helena SPIFE 3000 and Helena CZE System (V8). Mr. John
O’Keefe, Manager, Immunodiagnostics and Electrophoresis, Helena Laboratories,
Beaumont, Texas, played a pivotal role in coordinating the laboratory aspects of the
data collection related to SPIFE 3000 and CZE System (V8), and we appreciate
their efforts.
We are highly appreciative of and thankful to Mr. Tipton Golias, President &
CEO, Helena Laboratories, Beaumont, Texas, for his encouragement and approval
of this project, and providing us the instruments, reagents and the facilities for the
collection of data presented in this compendium. Without the indispensible help of
Mr. Tipton Golias, this project was not feasible.

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Finally we would like to thank the following persons at our institutions for facilitating
our work:
Adrian J. Christie, MD, Medical Director of Laboratories,
St. John Macomb-Oakland Hospital, Warren, Michigan
Anoop Patel, MD, Assistant System Medical Director,
St. John Providence Health System Laboratories
Kenneth F. Tucker, MD, Webber Cancer Center
St. John Macomb-Oakland Hospital, Warren, Michigan
Jessica Poisson, MD, Resident Physician
Department of Pathology, University of North Carolina,
Chapel Hill, NC
Stephanie Mathews, MD, Assistant Professor,
Chapel Hill, NC
Yuri Fedoriw, MD, Associate Director Core Laboratory & Hematopathology
Chapel Hill, NC

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Introduction
The objective of this compendium is to provide real examples of common
and rare findings encountered in routine serum protein electrophoresis testing and
immunofixation/immunodisplacement. We show patient results using 4 different
methods (Table 1) to contrast the types of patterns that one might encounter using
different methods. The compendium was not designed and is NOT intended as a
method comparison study, but to illustrate the salient features of interesting cases
to the naïve interpreter using multiple methodologies. Accordingly, the data we
show is not exhaustive, but illustrative (e.g. the V8 has free kappa and lambda, and
IgD/IgE, but data is not shown). The reader is encouraged to consider the title and
point of each case and to seek out the references provided.
In our first compendium (http://www.scribd.com/doc/14114106) we compared
the results of agarose gel SPE and IF (HYDRASYS System, Sebia, France) with the
SPE and immunotyping results obtained from CZE (CAPILLARYS System, Sebia,
France).
In 2009, Helena Laboratories, USA introduced their CZE system for SPE and
immunosubtraction.
In June 2009, we decided to put together another compendium of selected
cases that compared the SPE, IF, and immunotyping (immunodisplacement) results
of the most common and popular electrophoretic systems of Sebia, France and
Helena Laboratories, USA:
Test Electrophoretic System
SPE and IF Agarose gel
(HYDRASYS, Sebia France)
SPE and IF Agarose gel
(SPIFE 3000, Helena Laboratories, USA)
SPE and Immunotyping CZE
(CAPILLARYS, Sebia, France)
SPE and Immunodisplacement CZE
(V8, Helena Laboratories, USA)
An additional benefit of this compendium was to familiarize the laboratory
personnel with the distinct features of the automated liquid phase technology (CZE)
of these two separate manufacturers, which might help them if they are planning to
switch from the agarose gel method to the CZE method.

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Procedures and Instruments:
We advise the reader of this compendium to contact the manufacturer
directly for details concerning procedures, reagents, supplies and instruments. We
have provided a brief description.
Helena Laboratories, USA
P.O. Box 752, Beaumont, Texas 77704-0752, USA
e-mail: helena@helena.com
Tel: 409-842-3714
www.helena.com
Helena Bioscience Europe
Queensway South, Team Valley Trading Estate
Gateshead, Tyne and Wear, NE11 OSD, United Kingdom
Tel: 44 (0) 191 482 8440
e-mail: v8@helena-biosciences.com
www.helena-biosciences.com
Sebia, France
Parc Technologique Leonard de Vinci
CP 8010 LIsses-91008 EVRY Cedex-France
e-mail: sebia@sebia.com
Tel: 33(0) 1 69 80 80
Sebia, USA
1705 Corporate Drive
Norcross, GA 30093-2991
Tel: 770-446-3707
Fax: 700-446-5511
http://www.sebia-usa.com
Serum Protein Electrophoresis (Sebia): Sebia Hydragel Beta1 – Beta2 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 System (Sebia, France). Amidoblack was used to stain the
SPE gels, and the percent fractions were quantified using the scanner.
Serum Protein Electrophoresis (Helena): Helena SPIFE 3000 (serum protein
immunofixation electrophoresis system 3000) Spilt Beta SPE System employed
agarose gel in a tris-barbital MOPS buffer with calcium lactate for the separation of
serum protein upon electrophoresis into six fractions. The semi-automated SPIFE
3000 system employed for electrophoresis also performed the tasks of staining,

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destaining, and drying of the gels. The six fractions after SPE were quantified using
the Helena scanner (QuickScan 2000).
Serum Immunofixation (Sebia): Sebia HYDRAGEL IF kit was used first to
separate the proteins into six fractions (see above) in six different lanes and tracks.
The reference track was overlaid with fixative reagents and each of the five
additional tracks were overlaid with different anti-sera (IgG, IgA, IgM, kappa and
lambda). The immunoglobulin-antiserum complex was trapped in the agarose, and
then stained with acid-violet 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 in Tracks 2-6.
Track # 1 Serum protein electrophoresis-six fractions
Track # 2 Immunoglobulin G
Track # 3 Immunoglobulin A
Track # 4 Immunoglobulin M
Track # 5 Total Kappa Chains
Track # 6 Total Lambda Chains
Serum Immunofixation (Helena): Helena SPIFE 3000 was used for the qualitative
identification of monoclonal gammopathies, and this procedure consists of first SPE
(see above), then followed by IF. Here again there were six tracks as employed by
the Sebia IF procedure (see above).
Capillary Zone Electrophoresis (Sebia): Sebia’s liquid based capillary zone
electrophoresis system (CAPILLARYS) separated the serum proteins into six
fractions, without the use of a solid support medium, i.e. agarose gel. In this
procedure 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 towards anode) and endosmotic flow of cations toward the
cathode, the proteins were separated into six fractions. The quantification of the six
fractions was achieved by an ultraviolet detector using the absorbance of the amide
bond of proteins at 200 nm. All the serum CZE scans whenever illustrated in
this compendium are in green color.
Capillary Zone Electrophoresis (Helena): The principle of the Helena's liquid
based CZE system (V8) is similar to that of Sebia, France.
Immunosubtraction of serum proteins (Sebia and Helena): It must be pointed
out at the very outset that Sebia uses the term “Immunotyping” and Helena uses
the term “Immunodisplacement” for the same process (immunosubtraction of serum

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proteins). Basically, the term immunosubtraction means the removal of specific
individual proteins by the use of monospecific antisera prior to performing protein
electrophoresis.
Monoclonal proteins present in serum were detected and identified by an antigen-
antibody reaction in liquid phase followed by CZE using either Sebia’s
CAPILLARYS or Helena’s V8. In this automated methodology, the specific
immunoglobulin and the anti-sera bind together forming an antigen-antibody
complex (pretreatment step). The reference (untreated) and the pretreated samples
(anti-IgG, anti-IgA, anti-IgM, anti-kappa and anti-lambda) were electrophoresed
simultaneously. The antigen-antibody complexes migrate slower than unbound
immunoglobulin, i.e. appear before albumin or as an increased baseline co-
migrating with albumin, alpha-1 and / or alpha-2 globulin. This procedure is
essentially the reverse of immunofixation (see above), as in immunosubstration
immunoglobulins are removed rather than visualized. The interpretation
(identification of monoclonal bands) was made by the “overlay” of the untreated
reference curve on each of the electrophoretic curves (IgG, IgA, IgM, kappa and
lambda) All the subtracted serum CZE scans in this compendium are shown in red.
Selection of Patients for Case Studies: We performed SPE on >4000 patients
over a period of one year at St. John Macomb-Oakland Hospital, Warren, MI, and
the University of North Carolina McLendon Clinical Laboratories, Chapel Hill, NC.
All the sera having either abnormal or atypical pattern were sequestered. The
patient’s medical record was reviewed to obtain the clinical information, and in most
cases the admitting or primary physician of the patient was also consulted. In cases
of lymphoproliferative diseases, we also consulted the hematologist, pathologist,
and oncologist associated with the patient’s diagnosis and treatment.
University of North Carolina Samples: Sera were salvaged from patient samples
after completion of physician-ordered electrophoresis testing. By necessity, these
samples were stored refrigerated for 1-2 days while testing was done before being
frozen for transport and testing by the Helena methods. Samples were transported
on dry ice. The reader should note that because of the storage times, it is probable
that there was degradation of some proteins (e.g. C3). It is also probable that there
was differential overall storage times/thawing times when analyzing the samples
using different methods and different institutions.
St. John Health System Samples: Sera for SPE were collected at eight hospitals
and > 125 drawing stations affiliated with the system. Here again by necessity,
these samples were stored refrigerated for 1-2 days while testing was done before
being frozen for transport and testing at the University of North Carolina McLendon
Clinical Laboratories, and Helena Laboratories. We anticipate similar possibilities of
degradation of samples as mentioned above for the specimens of the University of
North Carolina.

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We therefore reiterate that this was not designed as a method comparison
study, but an educational endeavor and readers should not focus on minute
differences between traces, but the main point of each case. Specific method
comparison studies are likely to appear in the literature in the future.
Presentation of Clinical Cases and the Electrophoretic Data: For each case
presented in this compendium, we have presented a brief medical history, prior
diagnoses (if available), diagnosis at the time of the admission to the hospital (if
applicable), and any other information that might be helpful in the diagnosis of the
disease and the understanding of the electrophoretic patterns. The electrophoretic
data and other laboratory results for each case are presented as follows (except in
few cases where this protocol is deviated, e.g., Case #7, Case #13, Case #20,
Case #22, Case #23, and Case #26).
Immediately after the case history for each patient, we have provided the
concentration (g/dL) of the total protein, albumin, alpha-1 globulin, alpha-2 globulin,
beta-globulin and gamma globulin obtained from the manual agarose gel SPE
procedure (Sebia, HYDRASYS), and the quantitative values (mg/dL) of IgG, IgA,
and IgM (Beckman Coulter Image). In situations, where urine protein
electrophoresis and immunofixation were performed by the manual agarose gel
method (Sebia, HYDRASYS), the final interpretation was provided.
In cases, where it was felt to be informative for diagnostic purposes, we have
provided additional laboratory data, e.g. results of free kappa, free lambda in serum
and their ratio. In cases, where we had the opportunity to discuss the treatment of
the patient with the oncologist and hematologist, we have briefly provided their
conclusions.
We have presented on the top half of the first page for each case the
agarose gel SPE scan (Sebia, HYDRASYS), and on top of the scan we have
embossed the agarose gel IF picture (Sebia, HYDRASYS). Besides the IF picture
we have also presented (left side corner of the first page for each case) the picture
of the separation (SPE) by the agarose gel procedure (Sebia, HYDRASYS).
On the bottom half of the first page, we have presented the agarose gel SPE
scan (Helena, SPIFE 3000), and on top this scan we have embossed the agarose
gel IF picture (Helena, SPIFE 3000). Besides the IF picture, we have also
presented the picture of the separation (SPE) by the agarose gel procedure
(Helena, SPIFE 3000) on the left side of the bottom half of first page for each case.
On the top half of the second page for each case, we have presented the
enlarged SPE scan obtained from CZE (Sebia, CAPILLARYS).
On the bottom half of the second page for each case, we have presented the
enlarged SPE scan obtained from CZE (Helena, V8).

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On the third page for each case we have presented the following six scans
obtained from the immunotyping (Sebia, CAPILLARYS) of the serum proteins:
SPE scan (CZE)-Reference Track
CZE scan of IgG pretreatment serum
CZE scan of IgA pretreatment serum
CZE scan of IgM pretreatment serum
CZE scan of Kappa pretreatment serum
CZE scan of Lambda pretreatment serum
Note: The SPE scan (CZE) of Reference Track (green color) is superimposed on
the remaining five CZE scans of pretreatment sera (red color).
On the fourth page, we have presented the following six scans obtained from
the immunodisplacement (Helena, V8) of the serum proteins:
SPE scan (CZE)-Reference Track
CZE scan of IgG pretreatment serum
CZE scan of IgA pretreatment serum
CZE scan of IgM pretreatment serum
CZE scan of Kappa pretreatment serum
CZE scan of Lambda pretreatment serum
Note: The SPE scan (CZE) of Reference Track (green color) is superimposed on
the remaining five CZE scans of pretreatment sera (red color).
Detection and Identification of Monoclonal Bands: Adopted from our first
compendium: http://www.scribd.com/doc/14114106
The majority of the readers of this compendium are familiar with the
interpretation of the serum protein electrophoresis and immunofixation patterns
(agarose gel). The interpretation of the serum protein electrophoresis by CZE is
virtually identical to scanning densitometry used in 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.
In order to familiarize the naïve reader 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

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phase antigen-antibody reaction (Sebia CAPILLARYS), we recommend a review of
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
thesubtle 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 mini-monoclonal band (M-
protein less than 25 mg/dL) that may not be identified easily due to subtle changes
in the shape of the electrophoretic curve, especially if the mini-monoclonal 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 A.
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 is not actually
shown 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

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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 typically healthy 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).
The situation in the 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. Conversely, 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 significantly lower 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 restricted protein
(monoclonal band) in the gamma globulin region (figure B).

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Please note that in this hypothetical case of monoclonal band detected in the
gamma globulin region from CZE (green color, figure B), there is also a symmetrical
shaped immunoreduction of the gamma globulin region after the reaction with anti-
IgG (red curve, figure B). The difference between the green and the red curve is
illustrated by violet colored curve, thus indicating a sharp monoclonal 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.

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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. Adopted from: http://www.scribd.com/doc/14114106

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Section A: Monoclonal Gammopathy of Undetermined Significance
The term monoclonal gammopathy of undetermined significance (MGUS)
was first coined in 1978 by Kyle,a and the assignment of a restriction in the serum
protein electrophoresis to MGUS has to meet the following recent criteria.b-c
i) M-protein in serum 3.0 g/dL or less
ii) No evidence of B-cell proliferative disorder (multiple myeloma, AL
amyloidosis, Waldenstrom’s macroglobulinemia)
iii) Bone marrow clonal plasma cells <10% and low level of plasma cell
infiltration in a trephine biopsy (if done)
iv) No M-protein or only small amounts of monoclonal light chain in the
urine
v) Absence of lytic bone lesions, anemia, hypercalcemia or renal
insufficiency related to the M-protein
MGUS is also called pre-malignant plasma cell disease in view of the fact
that there is a risk of progression in some individuals to multiple myeloma, but in
vast majority of cases of MGUS a lymphoproliferative malignancy will never
develop,d and the patient dies of other causes. The reason for this discrepancy in
the progression of MGUS to MM is based on the “Risk Stratification Model” of
Rajkumar et al.d This model is based on three characteristics or risk factors, a)
serum M protein concentration, b) IgG isotype, and c) Free Light Chains ratio in
serum, and the four levels of the risk of progression of MGUS to MM are
summarized in Table 1.
In summary, a MGUS patient with all the three abnormal risk factors
[abnormal serum light chain ratio, non-IgG MGUS and a high serum M-protein level
(> 1.5 g/dL)] shall have a 58% absolute risk of developing MM after 20 years of
follow-up; as compared to a MGUS patient in whom all the risk factors are normal,
there is only 5% risk of progression to MM in the same 20 years.

16
Table 1
Risk stratification model
incorporating all three
predictive factors:
Absolute risk of progression
to MM at 20 years, %
Absolute risk of
progression to MM at 20
years accounting for death
as competing risk, %
Low Risk [serum M protein
<1.5g/dL, IgG subtype, normal
FLCs ratio (0.26-1.65)]
5 2
Low-intermediate risk (any
one factor abnormal)
21 10
High-intermediate risk (any
two risk factors abnormal)
37 18
High risk factor (all three
factors abnormal)
58 27
MGUS has prevalence of 3.2% in the Caucasian population 50 years of age or
older,e and this prevalence increases two- to threefold in African-Americansf but
interestingly the prevalence of MGUS is substantially lower in Asians.g In our study
of 2307 patients with abnormal SPE results at St. John Health System, we found 21
patients at low risk for progression to MM, 15 patients at low-intermediate risk for
progression to MM, and 2 patients at high intermediate risk for progression to MM.
Serum protein electrophoresis is requested for several other clinical reasons
besides B-cell dyscrasia by the physicians in many fields of clinical practice
(neurologists, dermatologists, rheumatologists, etc.). Therefore it is imperative for
the laboratory to provide a correct interpretation and also guide physicians in the
future course of action. The laboratory is advised to alert the physician with the risk
of the progression of the MGUS to MM, and the patients with the highest risk must
be periodically monitored and also their records must be kept for future reference.
In this regard readily accessible electronic medical records are imperative.

17
a. Kyle, RA. Monoclonal gammopathy of undetermined significance. Natural
history in 241 cases. Am J Med 1978; 64: 814-826.
b. Bradwell, AR. Serum Free Light Chain Analysis, Fifth Edition, 2008. The
Binding Site Ltd. PO Box 11712, Birmingham, B14 4ZB, UK, Chapter 19,
pp159.
c. International Myeloma Working Group, Criteria for the classification of
monoclonal gammopathies, multiple myeloma and related disorders: a report
of the International Myeloma Working Group. Br J Heaematol. 2003; 121:
749-757.
d. Rajkumar SV, Kyle RA, Therneau TM, Melton III LJ, Bradwell AR, Clark RJ
et al. Serum free light chain ratio is an independent risk factor for
progression in monoclonal gammopathy of undetermined significance. Blood
2005; 106: 812- 817.
e. Kyle RA, Therneau TM, Rajkumar SV, Larson DR, Plevak MF, Offord JR et
al. Prevalence of monoclonal gammopathy of undetermined significance. N
Eng J Med 2006; 354: 1362-1369
f. Landgren O, Gridley G, Turesson I, Caporaso NE, Goldin LR, Baris D et al.
Risk of monoclonal gammopathy of undetermined significance (MGUS) and
subsequent multiple myeloma among African American and white veterans
in the United States. Blood 2006; 107: 904-906.
g. Landgren O, and Weiss BM. Patterns of monoclonal gammopathy of
undetermined significance and multiple myeloma in various ethnic/racial
groups: support of genetic factors in pathogenesis. Leukemia 2009; 23:
1691-1697.

18
Case #1 IgG-Lambda Monoclonal Gammopathy: Low Risk
A 79 year-old female from the nursing home presented to the emergency
department with right facial droop, severe Alzheimer’s dementia, hypertension,
atrial fibrilation, stool positive for blood, low hemoglobin, and low hemotocrit. Upon
admission, endoscopy and colonscopy were performed which indicated mass in her
colon with distal lesions in the duodenum; pathology reports indicated an infiltrating
adenocarcinoma of the colon. After consent with family members, a stent was
deployed for the colon cancer with radiation therapy employed to keep the stent
open and prevent any further GI bleeding.
Protein Electrophoresis-Serum Immunoglobulins:
Total Protein: 5.9 (6.4-8.3 g/dL) IgG: 1160 (751-1560 mg/dL)
Albumin: 2.9 (3.8-5.8 g/dL) IgA: 573 (82-453 mg/dL)
Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 147 (46-304 mg/dL)
Alpha-2 Globulin: 0.8 (0.4-0.9 g/dL)
Beta-Globulin: 1.0 (0.5-1.1 g/dL)
Gamma Globulin: 1.0 (0.5-1.3 g/dL)
Note: Monoclonal band identified as IgG-
Lambda.
Test Result Reference Value
RBC 3.1 4.0-5.5 Mil/mm3
Hgb 7.0 12.0-16.0 g/dL
Hct 21 35-48 %
WBC 18.6 4.0-11.00 Thou/cc mm
CEA 64.4 0.0-3.4 ng/mL
CA 19-9 102 Less than 37 u/mL
Iron 19 30-160 mcg/dL
TIBC 417 205-380 mcg/dL
Saturation 4.0 20-50%
Free Kappa Chains-Serum 2.40 0.33 – 1.94 mg/dL
Free Lambda Chains-Serum 1.35 0.57-2.63 mg/dL
Free Kappa/Lambda Ratio 1.03 0.26- 1.65
Urine Protein Electrophoresis:
No evidence of monoclonal band.

23
Case #2 IgA-Kappa Monoclonal Gammopathy: Low Intermediate Risk
A 65 year-old male was admitted to hospital with complaints of chest pain radiating
to the left and shortness of breath with a diagnosis of unstable angina. The patient
had cardiac catheterization, successful PTCA of the circumflex artery with stent
deployment.
Protein Electrophoresis-Serum: Immunoglobulins:
Beta Globulin: 0.9 (0.5-1.1 g/dL)
Note: Mini-monoclonal IgA-Kappa band
identified from IF.
Free Kappa Chains-Serum 0.95 0.33-1.94 mg/dL
Free Kappa/Lambda Ratio 1.17 0.26-1.65
Negative for Bence Jones protein.

24
Note: There is a faint restriction (possibility of a mini-monoclonal band) in the
Kappa region (cathode side). This is most likely an artifact, as the SPE scan below
the IF does not demonstrate any abnormality in the region.

26
*There is an unexpected lack of reduction in the kappa and lambda channels,
cause unknown.

27
cause unknown.

28
Case #3 IgA-Kappa Monoclonal Gammopathy: High-Intermediate Risk
A 63 year-old female fell at home in bathroom and the X-ray obtained in the
emergency department of the hospital showed right femoral hip fracture. The
patient underwent a right hip hemiarthroplasty and then sent for physical therapy
and rehabilitation to an extended care facility.
Total Protein: 5.8 (6.4-8.3 g/dL) IgG: 1010 (751-1560 g/dL)
Note: Monoclonal band superimposed on the C3 band in the Beta-Globulin region,
and Identified as IgA-Kappa from IF studies.
Free Kappa Chain-Serum 3.75 0.33-1.94 mg/dL
Free Lambda Chain-Serum 1.84 0.33-1.94 mg/dL
Free Kappa Chain-Urine 56.2 0.14-2.42 mg/24 Hr
Free Lambda Chain-Urine 7.04 0.02-0.67 mg/24 Hr
Protein Electrophoresis-Urine:
Non-selective proteinuria.

29
Note: The SPE scans (HYDRASYS and SPIFE 3000) exhibit asymmetrical shape
of the gamma globulin band, suggesting a possibility of an embedded monoclonal
band, however neither the IF nor the immunosubtraction studies (page 31, 32)
definitively delineated an additional monoclonal band (it may represent an
additional very low concentration of IgG-Kappa).

31
cause unknown.

32
cause unknown.

33
Case #4 IgG-Kappa Monoclonal Gammopathy: Low Risk
A 69 year-old female presented at the physician office with pleuritic left-sided chest
pain and dyspnea on minimal exertion. The patient was admitted after the chest CT
indicated the presence of bilateral pulmonary emboli, and the ultrasound of venous
duplex lower extremities indicated findings positive for left lower extremity deep
venous thrombosis.
Bone Marrow Aspirate: Normocellular with maturating granulocytic hyperplasia,
dysplastic erythropoieses, and adequate megakaryocytes. A small population of
monoclonal plasma cells (6%) was noted. Flow cytometry also indicated small
population of monoclonal plasma cells (0.4% of leukocytes) consistent with a
plasma cell dyscrasia, and no evidence of acute leukemia or lymphoproliferative
disorder.
Peripheral Blood Smear: Macrocytic anemia and reticulocyte response.
Beta-Globulin: 1.0 (0.5-1.1 g/dL) Urine Protein Electrophoresis:
Gamma Globulin: 0.8 (0.5-1.3 g/dL) No evidence of monoclonal band.
Note: Protein restriction in SPE was identified as IgG-Kappa from immunofixation.
RBC 1.88 4.0-5.5 Mil/mm3
Hgb 8.6 12.0-16.0 g/dL
Hct 25.0 35.0-48.0 %
MCV 128 79-98 fL
Protime 20.6 11.3-14.5 second(s)
PTT 87.0 22.5-35.0 seconds(s)
TIBC 297 205-380 mcg/dL
Saturation 9.0 20.0-50.0 %
Ferritin 338 8-120 ng/mL

38
Section B: Acute Phase Reaction Pattern
A frequently observed SPE pattern in hospitalized patients is the Acute
Phase Reaction (APR). This is not unexpected in lieu of patients admitted for acute
episodes of tissue damage (infection, tissue injury, tumor necrosis) with or without
inflammation. The proteins that are associated with APR are a group derived from
hepatocytes. A classical SPE pattern of APR depicts, a) low albumin, b) increased
alpha-1 globulin, c) increased alpha-2 globulin, and d) decreased transferrin.a
Sometimes a faint band in the gamma globulin region (either in the mid gamma
globulin region or at the tail end of the gamma globulin region) is also detected in
the agarose gel SPE. This band in most cases is due to C-reactive protein and
perhaps can be misconstrued as a mini-monoclonal gammopathy. It is emphasized
here that this C-reactive protein band is never detected in SPE by the CZE system,
because of the differing migration compared with agarose gels. In patients with
concomitant serum iron deficiency (Case #6), the serum transferrin band (beta-1
globulin) is increasedb
and this causes deviation from the classical APR pattern
described above. The APR pattern varies qualitatively in shape as a function of time
(e.g. in sub-acute phase some of the regions may appear normal), and also by the
presence of other proteins (e.g. α-1 acid glycoprotein, α-1 antitrypsin, haptoglobin,
fibrinogen, etc.) the SPE pattern may have an atypical shape.
Fibrinogen (soluble precursor of fibrin) is a glycoprotein encoded by the
genes-FGA, FGB, FGG-clustered on chromosomes 4q,c
and synthesized in
hepatocytes from three homologous polypeptide chains, Aα, Bβ, and γ. These
chains are assembled to form a 340-kDa hexamer held together by 29 disulfide
bonds. Approximately 72-96 hours after an APR episode, the peak concentration of
fibrinogen is achieved in plasma, after which it gradually decreases in response to
clinical intervention (e.g. antibiotic therapy).
We noticed an interesting patient of APR (Case # 7) in which the serum had a
presumptive monoclonal band superimposed on the C-3 band (beta-2 globulin).
Immunofixation studies with anti-sera to IgG, IgA, IgM, IgD, IgE, free and total kappa,
and lambda chains ruled out monoclonal band. We assayed fibrinogen in a plasma
specimen collected 24 hours after the admission (SPE electropherogram #1,
HYDRASYS-Sebia), and found 886 mg/dL of fibrinogen (reference interval: 250-498
mg/dL). This patient had a para-spinal abcess and was treated with IV vancomycin
upon admission to the hospital. We concluded that the monoclonal band in SPE was
indeed due to elevated fibrinogen, as four weeks later (SPE electropherogram #2,
HYDRASYS-Sebia), there was no detectable monoclonal band.

39
Interpreters should be aware of potential fibrinogen artifacts and ensure that they
use immunofixation to confirm apparent SPE abnormalities. Interpreters should also
be cautioned that fibrinogen has been reported to cross-react with IgA (Snyder et al.
Clinical Chemical Acta 2006; 368: 192-194).
a. David F Karen, Protein Electrophoresis in Clinical Diagnosis, London,
England: Arnold 2003: pp 124
b. Jollif C. Agarose gel electrophoresis in the acute and chronic inflammatory
states. Clin Immunnol Newsl 1991; 11: 132-135.
c. Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features
of fibrinogen and fibrin. Ann NY Acad Sci 2001; 936: 11-30.

40
Case #5: Acute Phase Reaction without Serum Iron Deficiency
A 61 year-old male came to the emergency department complaining of excruciating
mid-back pain lasting for 3-4 weeks. The patient had blisters on his lower back, and
had an infected hematoma with cellulitis on his right dorsal foot. The foot abscess
was surgically drained in the emergency department and the patient was put on an
intravenous drip of vancomycin. X-rays indicated that a previous spinal fusion (see
below) was stable, but showed a T10 compression fracture.
Note: The blood drawn at the time of arrival in the emergency department was used
for all the electrophoretic, immunochemistry, and some laboratory tests presented
here.
Past Medical History: The patient had a history of pulmonary embolism, rheumatoid
arthritis, hypertension, gout, deep venous thrombosis, arteriosclerotic coronary
artery disease, and myelodysplastic syndrome with recent diagnosis of acute
myelogenous leukemia status post-induction chemotherapy.
Past Surgical History: Spinal fusion L4-L5 in 2009, left knee arthroscopy, right
rotator cuff repair, and bilateral carpal tunnel release.
Peripheral Blood Smear (2010): Monocytosis. The interpretative report
recommended a bone marrow biopsy to evaluate for a process such as chronic
myelomonocytic leukemia.
WBC 59.5 4.0-11.0 Th/mm3
RBC 2.70 4.6-6.2 Mil/mm3
Hemoglobin 8.4 13.5-18.0 g/dL
Hematocrit 24.8 38.0-54.0%
Neutrophils 19.0 36-66%
Lymphocytes 10.0 24-44%
Monocytes 67.0 0-10%
LDH 447 0-240 U/L
Iron-Serum 90 45-160 ug/dL
TIBC 109 205-380 ug/dL
% Saturation 82.6 20-50
Ferritin 10429 23-265 ng/mL
C-Reactive Protein 4.1 0-1.0 mg/dL

41
Note: No evidence of monoclonal band in serum and urine from immunofixation
studies.
Comment: Please compare the serum electrophoretic patterns of this case with
Case #6, in which the transferrin band is increased due to serum iron deficiency.

46
Case #6: Acute Phase Reaction (Bacterial Meningitis) with
Concomitant Serum Iron Deficiency
A 69 year-old female was brought to the emergency department appearing quite ill
with shortness of breath, ear pain and mental status changes (positive Kernig
sign). The patient was intubated upon admission for respiratory failure as well as
mental status changes and airway protection, and was treated with multiple
antibiotics. A lumbar puncture was performed, revealing pustular CSF. The CSF
culture was positive for Haemophilus influenzae. A blood culture was positive for
Haemophilus influenzae and Streptococcus pneumoniae and a urine culture was
positive for Klebsiella pneumoniae. A CSF culture was negative for acid-fast bacilli
(AFB) growth after six weeks.
Note: Apparent increase in transferrin, and no evidence of monoclonal band by IF.
Urine Protein Electrophoresis: No evidence of monoclonal band.
C-Reactive Protein 5.5 0-1.0 mg/dL
Serum-Iron 10 30-160 mcg/dL
TIBC 308 205-380 mcg/dL
% Saturation 3.0 20-50
Ferritin 229 8-120 mcg/dL
WBC 25.9 4.0-11.0 x 109
/L
WBC-CSF 3700 0-5 mm3
Protein-CSF 687 15-45 mg/dL
Glucose-CSF Not Detected 50-70 mg/dL

51
Case #7: Post Acute Phase Reaction: Monoclonal Band
Superimposed on C3 Band (Elevated Fibrinogen)
A 61 year-old female with lower back pain was admitted to the hospital and a spinal
abscess was found. The patient was treated with antibiotics and discharged after
full recovery from the spinal infection.
Past Medical History: Insulin dependent diabetes, urinary tract infection,
hypertension, coronary artery disease, renal failure (required frequent dialysis),
history of respiratory failure, anemia, cataract, C-section, hysterectomy.
MRI showed abnormal signal in T6 and T7 with destruction of the intervertebral disc
and residual paraspinal soft tissue abnormalities suggestive of abscess with
paraspinal extension.
Note: Restricted band showed no evidence of monoclonality.
Plasma Fibrinogen 886 250-498 mg/dL
(2nd day after admission to hospital)
Plasma Fibrinogen 156 “ “
(4th week after admission to hospital)
Comment: SPE four weeks after the initiation of antibiotic therapy, showed no
monoclonal band, therefore confirming the disappearance of elevated fibrinogen
restriction (see page 52-54).
Another way to confirm the fibrinogen band is to perform IF using anti-fibrinogen.
No evidence of monoclonal band.

56
Section C: Double and Biclonal Gammopathy
In about 3-5% of monoclonal gammopathy cases, patients produce more
than one monoclonal protein, which is defined as biclonal gammopathy or double
gammopathy. Double gammopathy is designated when the light chain (either kappa
or lambda) is the same in both bands. Conversely, biclonal gammopathy is
designated when the light chain is different in both the bands. In either double or
biclonal cases, the heavy chains can be the same or different. This is because B-
lymphocytes always produce the same light chain, but can express more than one
heavy chain at a time. According to Weinstein et al, the various heavy chain
combinations in the double or biclonal bands are prevalent in the following
decreasing frequency: IgG-IgM IgG-IgA IgG-IgG
(probably different subclasses) IgA-IgM.a
We found three years ago a rare case
of double gammopathy (IgA1-kappa and IgA2-kappa) that involved the two
subclasses of IgA.b
In rare cases, there are two separate and distinct bands
between the alpha-2 globulin and gamma globulin region. These double bands are
also composed of (similar to most monoclonal bands in SPE) a heavy chain and a
light chain, except in a few cases when one of the double band represents a free
light chain when produced in very excess. According to Kyle et al, from the
physician’s perspective there is no difference between double and biclonal
gammopathy, and approximately 97-99 % are MGUS.c
In spite of the rare
encounter of double and biclonal gammopathy, the risk of their stratification to MMd
is also similar to MGUS (personal communication, Prof. JA Katzman, Mayo Clinic,
Rochester, MN). It is emphasized that in biclonal gammopathy MM, the free
kappa/lambda ratio may be normal. However, it is likely that both the free light
chains are elevated in biclonal gammopathy MM, and in different amounts.e
a Weinstein S, Jain A, Bjagavan NV, Scorrolini AG. Biclonal IgA and IgM
gammopathy in lymphocytic lymphoma. Clin Chem 1984; 30: 17110-1712.
b Uddin Z. Comparison of Agarose Gel Serum Protein Electrophoresis and
Immunofixation with Capillary Electrophoresis and Immunotyping.
http://www.scribd.com/doc/14114106, pp29
c Kyle RA, Robinson RA, Katzman JA. The clinical aspects of biclonal
gammopathies. Review of 57 cases. Am J Med 1981; 71: 999-1008.
d Rajkumar SV, Kyle RA, Therneau TM, Melton LJ 3rd
, Bradwell AR,
Clark RJ, Larson DR, Plevak MF, Dispenzieri A, Katzman JA. Serum free
light chain ratio is an independent risk factor for progression in
monoclonal gammopathy of undetermined significance. Blood 2005; 106:
812-817.
e Ramasamy I. Letter to the Editor: Serum Free Light Chain Analysis in
B-cell Dyscrasia. Am Clin Lab Sci 2007; 37: 291-94

57
Case #8 Double Gammopathy: a) IgA-Lambda (major band)
b) IgG-Lambda (minor band)
A 72-year-old nursing home resident complained persistent abdominal pain. The
patient was first examined by her family physician who determined that the patient
was afebrile and did not have chest pain or shortness of breath. The woman was
subsequently referred to other specialists, who requested several laboratory tests
including serum protein electrophoresis. No other abnormality was found in
peripheral blood smear or bone marrow examination.
Comment: No follow up data of the patient was available.

62
Case #9 Double Gammopathy: a) IgM-Kappa (major band)
b) IgG-Kappa (minor band)
A 67 year-old male was admitted to hospital for urosepsis and hemorrhagic cystitis
with chronic renal failure. The patient’s hemoglobin dropped from 10.8 to 7.0 over
the past two months prior to hospitalization prompting a blood transfusion. There
was no evidence of active bleeding. Patient had a cystoscopy and bladder biopsy,
which were negative.
Past medical history: Hypothyroidism, hypertension, chronic renal failure and
dementia.
TEST RESULT REFERENCE VALUES
Free Kappa Chains-Serum 9.42 0.33 – 1.94 mg/dL
Free Lambda Chains-Serum 2.30 0.57 – 2.63 mg/dL
Free Kappa/Lambda Ratio 4.09 0.26 – 1.65
Comment: The patient was treated for the urinary tract infection and discharged
back to the nursing home. No further treatment details were available after
discharge to the nursing home.

67
Case #10 IgM-Kappa and IgM-Lambda Biclonal Gammopathy
A 79 year-old male was admitted to the hospital, where his EKG showed a right
bundle branch block with atrial flutter. He was placed on telemetry and was found to
be quite bradycardic, where his rate dropped into the 20s while he was sleeping. A
pacemaker was inserted stabilizing his clinical condition. His comprehensive
workup indicated serum calcium of 14.5 mg/dL (Reference Value: 8.4-10.5 mg/dL)
and also elevated urinary total protein of 399 mg/24 hr (Reference Value: 0-150
mg/24Hr). The patient had a parathyroid hormone concentration of 387 pg/mL
(Reference Value: 15-65 pg/mL) prompting a CT scan of neck to screen for a tumor
as the source for the hyperparathyroidism; the CT did not show evidence of soft
tissue tumor. Chest X-ray showed no pneumothorax or pneumoperitoneum. Ultra-
sound showed kidneys within normal limits. X-ray of the spine cervical indicated no
evidence of traumatic spondylolisthesis.
Past Medical History: Hypertension, chronic kidney failure, coronary artery disease,
myocardial infarction, hyperlipedemia, benign prostatic hypertrophy, hernia,
cataract and carotid stents.
Platelet 80 150-450 x109/L
PTT 94.7 22.5-35.0 second(s)
BUN 58 8-20 mg/dL
Calcium-serum 14.5 8.4-10.5 mg/dL
Calcium-ionized 1.81 1.13-1.32 mmol/L
PTH-intact 387 15-65 pg/mL
Peripheral Smear: Normocytic / normochromic anemia and thrombocytopenia.
Bone Marrow Aspirate: Cellularity: Normal. Trabeculae: Normal. Other Findings: A
majority of the cells were small lymphoid cells with a scattering of mast cells,
plasma cells, a few hematopoietic cells. Iron: Absent. Less than 5% plasma cells
were present.
No evidence of AL amyloidosis, Waldenstrom’s macroglobulinemia,
lymphoproliferative disorders, plasmacytoma or related conditions.

68
Comments: No further treatment data was available and the patient expired in the
nursing home.
Urine Protein Electrophoresis and IF:
Both free kappa and lambda chains
were detected
chains were detected in urine
but not IgM.

72
Note: The reader will note the inferiority of immunosubtraction to immunofixation in
this case. Although, both bands are difficult to identify by immunosubtraction, there
would be no clinical significance to missing the second band.

73
Section D: Polyclonal Gammopathy
Case #11 Polyclonal Gammopathy: Increased IgG, IGA, and IGM with
Beta-Gamma Globulin Bridging
A 67 year-old female living in a nursing home presented to the emergency
department with altered mental status changes, diarrhea, and lethargy. She was
diagnosed with a urinary tract infection (UTI) for which she received intravenous
vancomycin. As a result of the antibiotic, her serum creatinine increased and she
developed interstitial nephritis. She was also found to have neutropenia, anemia,
and thrombocytopenia concerning for an underlying bone marrow disease versus
the antibiotic use. There was no evidence of intracranial mass or hemorrhage from
CT of brain. X-ray of the chest and abdomen were non-specific. The family
members of the patient decided against a bone marrow biopsy.
Discussion: The patient recovered from urinary tract infection and was returned to
the nursing home. The most probable cause for the beta gamma bridge is the
polyclonal increase in IgA that migrates in this region, and there is also increase in
IgG and IgM (though borderline) levels that completes the ‘bridge’ and is indicative
of broad increase in gamma globulin. In general, a polyclonal increase in gamma
globulin relates to immune response to enteric antigens (UTI in this case).

78
Case #12 Polyclonalgammopathy: Pancytopenia with Bone Marrow
Containing <5% Plasma Cells
A 54 year-old female was admitted to the hospital with complaints of upper quadrant
pain, fatigue, lower abdomen pain, and was found to be pancytopenic. CT scan of
the abdomen and pelvis were essentially normal. X-Ray of the hip showed no
acutely displaced bone fractures and without hip joint space narrowing.
Peripheral Blood: Normochromic, normocytic anemia with minimal polychromosia,
leucopenia and thrombopenia.
Bone Marrow Aspirate: Essentially normocellular marrow with normoblastic
erythropoieses, mature granulopoieses, and adequate megakaryopoieses. Flow
cytometric evidence of a small population of CD56 positive monoclonal plasma cells
(approximately 0.7% of the leukocytes), indicative of a plasma cell dyscrasia. There
was no flow cytometric evidence of acute leukemia or lymphoproliferative disorder.
Cytogenetic studies performed on bone marrow showed a normal karyotype.
Free Lambda Chain-Serum 8.47 0.57-2.63 mg/dL
Beta-2 Microglobulin: 1.8 1.1-2.4 mg/L
Comment: Patient was followed by the family physician and we had no access to
her laboratory data.

82
*There should be a reduction in the polyclonal IgG to baseline. This is shown to
exemplify what happens if the sample is not appropriately diluted to account for
high immunoglobulin concentrations.

83
Section E: Hypogammaglobulinemia/Light Chain Multiple Myeloma
Hypogammaglobulinemia in adults is observed in several clinical conditions (e.g.,
light chain disease) and also in iatrogenic situations (e.g., chemotherapy, with or
without autologus stem cell transplantation). If the other serum protein fractions are
in normal range but the gamma globulin is decreased, the laboratories investigate
the reason by performing additional tests (e.g., urine protein electrophoresis, serum
immunofixation and serum free kappa and lambda chains). We have selected the
following cases in order to illustrate this point.
Case #13 Hypogammaglobulinemia Secondary to Chemotherapy for
Non-Hodgkin’s Lymphoma: Anaphylactic Reaction in Response
to Intravenous Immunoglobulin Infusion.
A 58 year-old female was admitted to the hospital with an acute deep venous
thrombosis involving her left lower extremity. CT scan of her abdomen confirmed
the presence of significant splenomegaly with generalized intra-abdominal
lymphadenopathy. Surgical pathology (left axillary lymph node) studies along with
flow cytometry, and bone marrow examination led to a diagnosis of follicular center
cell lymphoma. The patient was successfully treated with chemotherapy.
Five years later the patient was again admitted to the hospital, this time with sepsis.
A hepatobiliary imino-diacetic acid (HIDA) scan did not show definite gallbladder
activity. Severe hypogammaglobulinemia was detected and the patient had an
anaphylactic reaction in response to intravenous immunoglobulin infusion
(Gammaguard).
Total Protein: 4.1 (6.4-8.3 g/dL) IgG: <23 (751-1560 mg/dL)
Albumin: 1.7 (3.8-5.8 g/dL) IgA: < 7 (82-453 mg/dL)
Alpha-1 Globulin: 0.4 (0.1-0.2 g/dL) IgM: < 4 (46-304 mg/dL)
Serum Immunofixation: IgG, IgA, IgM, IgD, IgE, Kappa and Lambda chains (free &
total) were not detected.
Urine Immunofixation: No monoclonal band was detected.
Tests Result Reference Value
Free Kappa Chains-Serum < 0.06 3.3-19.4 mg/L
Free Lambda Chains-Serum 1.77 5.7-26.3 mg/L
Free Kappa Chains-Urine (24 Hr) < 0.40 < 2.0 mg/dL

84
Free Lambda Chains-Urine (24 Hr) < 0.40 < 2.0 mg/dL
Serum IgA1 < 9.5 50-314 mg/dL
Serum IgA2 < 0.3 9.7-156 mg/dL
Anti-IgA Antibody, IgG (Serum) 18 <100 Units (Negative)
It is recognized in the literature that some patients after successful chemotherapy
for lymphoma, (e.g. with rituximab alone or in combination with an alkylating agent
and purine analog), exhibit profound hypogammaglobulinemia,a-e The same
phenomenon was also observed in cases of chemotherapy treatment along with
stem cell transplantation.
Most of the patients who exhibited immunodeficiency after successful
chemotherapy for lymphoma, may be given intravenous immunoglobulin treatments
resulting in complete resolution of the immunodeficiency and without any
complications, e.g., transfusion reaction.
No clinical or theoretical reason has been proven scientifically for the observation of
profound hypogammaglobulinemia 5-7 years after cessation of the chemotherapy
for lymphoma, except for the possible relationship to the genotype FCGR3-158V/F
polymorphism.b
Selective serum IgA deficiency is currently recognized as the most frequent
immunodeficiency in humans, who are otherwise considered to be healthy;
however, on rare occasions it is considered of concern when these individuals
require blood or blood products. Anaphylactoid antigenic sites on IgA are
responsible for anaphylactic transfusion reactions in IgA deficient patients.
Approximately ten (10) days after the transfusion reaction, anti-IgA antibodies (IgG
class) are positive in the serum of the patient. In this patient (see above) the anti-
IgA antibodies (IgG class) were negative, as expected since no immunoglobulin
therapy was instituted ten days prior to the drawing of the blood for this test. There
is a possibility, however, that IgE anti-IgA sensitizing antibody could have been
involved.
a. Profound hypogammaglobulinemia 7 years after treatment for indolent
lymphoma, Walker, AR et al., Cancer Invest 2008, 26(4): 431-3.
b. FCGR3A-158V/F polymorphism may correlate with the levels of
Immunoglobulin in patients with non-Hodgkin’s lymphoma after rituximab
treatment as an adjuvant to autologus stem cell transplantation. Nisho M, et
al., Eur J Heamatol 2009, 82(2): 143-7.
c. Delayed redistribution of CD27, CD40 and CD80 positive B cells and the
impaired in vitro immunoglobulin production in patients with non-Hodgkin
lymphoma after rituximab treatment as adjuvant to autologus stem cell
transplantation. Nisho, M. et al., Br. J. Haematol 2007, 137(4): 349-54.

85
d. Immune recovery in children with malignancy after cessation of
Chemotherapy. Mustafa, MM. et al., J Pediatr Hematol Oncol 1998, 20(5):
451-7.
e. Immunoglobulin subclass levels in patients with non-Hodgkin lymphoma.
Biggar, RJ et al., Int J Cancer 2009, 124(1): 2616-20.

89
Case #14 Simultaneous Adenocarcinoma of Lung and
Light Chain (Kappa) Multiple Myeloma
A 77 year-old male presented to the emergency department with excruciating pain
in his left arm, neck and shoulder, and was admitted to the hospital as a result of
the MRI finding of a destructive lesion at the cervical vertebral # 6. Bone biopsy
performed of the cervical lesion indicated malignancy. In a palliative effort radiation
therapy was delivered to the cervical spine utilizing CT for 3D treatment planning
and DVH construction. The total dose to the area was 3500 cGy. Patient
experienced discomfort with swallowing, and no further radiation treatment was
rendered.
Chest X-Ray: Abnormality in the right upper lobe. Chest CT: Two-centimeter mass
against the left chest wall and a 5-centimeter mass in the right inferior hilum.
Surgical Pathology: Left chest wall mass: Plasmacytoma, consistent with
involvement with multiple myeloma. Lung Biopsy: Moderately differentiated
adenocarcinoma. Bone Marrow Biopsy: 41% plasma cells, consistent with bone
marrow involvement. Flow Cytometry: CD56 positive monoclonal kappa positive
plasma cell population (approximately 15% of the leukocytes) indicative of plasma
cell dyscrasia.
Protein Electrophoresis-Serum (4/16/09) Immunoglobulins (4/16/09):
Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: <13 (46- 304 mg/dL)
Note: IF (HYDRASYS and SPIFE 3000) of serum indicated
a faint “Free Kappa Chains” restriction, but there was no
evidence of “Free Kappa Chains” band from immunotyping
(CAPILLARYS) and immunodisplacement (V8).
Test 1/21/09 4/16/09 Reference Value
BUN 65 24 7-25 mg/dL
Creatinine 5.5 0.92 0.67-1.54 mg/dL
Free Kappa Chains-Serum 2230.0 67.1 0.33-1.94 mg/dL
Free Lambda Chains-Serum 0.37 0.54 0.57-2.63 mg/dL
Free Kappa/Lambda Ratio 6027.03 124.26 0.26-1.65
Treatment: Alternating treatment every week for multiple myeloma and lung cancer
restored normal renal function; chest X-rays did not show any worsening of his right
lung adenocarcinoma.
Protein Electrophoresis-Random
Urine: Positive for Bence Jones
protein (Free Kappa Chains).

94
Case #15 Hypogammaglobulinemia and Light Chain (Lambda)
Multiple Myeloma
A 58 year-old male presented to the emergency department after onset of left arm
pain, right knee pain, double vision, and severe headache. The patient was found to
have a fractured left humerus.
Chest X-Ray: Soft tissue lesion associated with the left ninth rib. Thoracic CT:
Multiple lytic lesions including the left transverse process of T6, ribs, left scapula,
left humerus, right AC joint, right clavicle and right glenohumeral joint.
Bone Marrow Biopsy and Aspirate: 31% plasma cells; trilineage differentiation and
adequate iron stores. Flow Cytometry-Bone Marrow Aspirate:
Increased CD56 positive monoclonal plasma cells (approximately 32% of the
leukocytes) indicative of plasma cell dyscrasia.
Bone Biopsy Right Femur: Portions of the bone with sheets of plasma cells within
the bone marrow. Consistent with multiple myeloma (immunoperoxidase stain
positive for CD 138).
Note: No monoclonal band was detected
by SPE
Positive for free lambda chains.
Calcium-Serum 11.8 8.4-10.5 mg/dL
PTH,intact 7.8 15-65 pg/mL
Vitamin D 25-hydroxy 13.0 >30.0 mg/dL
Free Kappa/Lambda Ratio <0.01 0.26-1.65
Protein-24 hour urine 7336 0-150 mg/dL
Discussion: Treatment of light chain multiple myeloma was rendered for six months,
and according to the oncologist there was improvement in his clinical condition but
the patient expired due to other medical reasons. Additional information was not
available.
No monoclonal band (Free Lambda Chains) was detected in serum from CZE (V8
and CAPPILLARYS), but both the serum IF procedures (HYDRASYS and SPIFE
3000) were positive for free Lambda chains.

98
Note: As with the earlier case (#14), it is more difficult to identify low concentration
monoclonal light chains by immunosubtraction.

99
Case #16 Light Chain (Lambda) Multiple Myeloma
with normal Gammaglobulins
A 72 year-old male with a recent history of mental changes, confusion, trouble
ambulating, and diffuse body pain came to the emergency department. A workup
revealed anemia, GI bleeding, tachycardia, and dyspnea. Prior to hospitalization
the patient had severe back pain, and also underwent kyphoplasty procedures to
multiple areas of his spine. CT-Scan of the head was unremarkable. X-Ray of the
abdomen showed lytic lesions on the ileus. Chest X-Ray showed an old rib fracture
and basilar patchy infiltrate. EKG showed left bundle branch block. The patient also
had metabolic alkolosis, hypernatremia, hypercalcemia, and thrombocytopenia.
During hospitalization he continued to have sepsis and eventually died due to renal
failure. Esophageal biopsy: No evidence of specific inflammation, dysplasia, or
malignancy.
Bone Marrow Biopsy and Aspirate: Plasmacytic dyscrasia (46% replacement of
hematopoietic tissue by plasma cells). Trilineage differentiation, and normal male
karyotype determined from cytogenetic studies. The population of monoclonal
plasma cells demonstrated partial (~ 2.5% of the leukocytes) CD56 positivity by flow
cytometry, indicative of plasma cell dyscrasia.
Total protein: 5.6 (6.4-8.3 g/dL) IgG: 688 (751-1560 mg/dL)
Note: Monoclonal protein restriction
(cathodic to C3 on agarose gel) was
identified as “Free Lambda Chains.”
Free Kappa Chains-Serum: 2.85 0.33-1.94 mg/dL
Free Lambda Chains-Serum: 968.00 0.57-2.63 mg/dL
Free Kappa/Lambda Ratio: <0.01 0.26-1.65
Comment: (1) Please compare this with another case of “Light Chain Multiple
Myeloma” (Case #15) where the gamma globulin was decreased and no
monoclonal band was detected from SPE (V8 and CAPILLARYS). (2) The free
lambda chains in the serum are quantified as 968.0 mg/dl. It should be noted that
this concentration is however highly overestimated because the entire gamma-
globulin region has a quantity of only 800.0 mg/dl. This means that the
nephelometric FLC concentration overestimates the ‘true’ FLC concentration with a
factor of 6 to 10. In a recent publication it is demonstrated that serum FLC are in
fact consistently overestimated using immunophelometry (ref de Kat Angelino et al.
2010)
Protein Electrophoresis &
Immunofixation-Urine:
Monoclonal band identified
as “Free Lambda Chains.”

104
Section F: Multiple Myeloma and other B-Cell Dyscrasia
Case #17 IgM-Lambda Multiple Myeloma
A 65 year-old female was brought to the emergency department from a nursing
home after she developed severe pain in her left hip, shortness of breath, dizziness,
and altered mental status.
Past Medical History: Non-insulin dependent diabetes, hypertension, anemia,
osteopenia, hearing loss and right ear surgery.
CT scan of the brain was normal. CT scan of the chest was consistent with the
bilateral lower lobe pneumonia. The pelvic CT images were indicative of pathologic
fracture involving the left hip, and a small amount of non-specific pelvic fluid. A bone
scan showed abnormal uptake in the left hip. The patient underwent a closed
reduction and intermedullary nailing of the peritrochanteric fracture of the left femur.
She had a complicated postoperative course including respiratory failure,
subsequent intubation. She had vent-dependent respiratory failure with subsequent
tracheostomy placed in the ICU.
Bone marrow biopsy revealed a plasma cell neoplasm consistent with multiple
myeloma. Marrow had 80-90% plasma cells with CD138 staining. Flow cytometry
showed around 40% plasma cells, with expression of CD45.
The legal guardian of the patient was advised about her extremely poor medical
condition, and that treatment of her underlying hematological neoplasm would not
be of any benefit to her in terms of her quality of life. She was transferred to a
hospice, where she died.
Total protein: 6.0 (6.4-8.3 g/dL) IgG: 260 (751-1560 mg/dL)
Serum Immunofixation: Monoclonal IgM-Lambda; concentration of 1.3 g/dL by
scanning densitometry. In addition to the monoclonal IgM-Lambda band, another
restriction due to “Free Lambda Chains” was also detected by both methods
(HYDRASYS and SPIFE 3000).

105
WBC 7.2 4.5-11.0 x 109/L
Hgb 9.0 12.0-16.0 g/dL
Creatinine 1.0 0.60-1.0 mg/dL
Calcium 10.0 8.5-10.2 mg/dL
Alkaline Phosphatse 194 38-126 U/L
LD 189 338-610 U/L
Beta-2 Microglobulin 3.8 1.1-2.4 mg/L
Discussion: IgM-producing multiple myeloma (MM) is a rare occurrence accounting
for only 0.5-1% of MM cases1. Laboratory findings often show overlap of
Waldenstrom’s macroglobulinemia and MM, which can make diagnosis difficult.
Recent reports indicate unique immunophenotypes for IgM MM where they usually
express CD38, CD138, and MUM1/IRF4, but are negative for CD20, CD56, and
CD117. A common genetic translocation in IgM MM is t(11;14), although other
deletions and rearrangements can occur2. Prognosis for this disease is generally
worse (<36 months) than other MM variants.
1. Willenbacher E, Erdel M, Strasser U, Gastl G, Schmidt S, Gunsilius E,
Willenbacher W. IgM myeloma: more on a rare entity. Br J Haematol. 2008
Sep; 143(1): 146-8.
2. Feyler S, O’Connor SJ, Rawstron AC, Subash C, Ross FM, Pratt G, Drayson
MT, Ashcroft J, Cook G, Owen RG. IgM myeloma: a rare entity characterized
by a CD20-CD56-CD117- immunotype and the t(11;14). Br J Haematol. 2008
Mar; 140(5): 547-51.

110
Case #18 IgG-Lambda Multiple Myeloma
A 78 year-old male was admitted for pneumonia, COPD exacerbation, rapid arterial
fibrillation. No evidence of hypercalcemia, and renal failure.
Past-History: Adenocarcinoma of the colon, hypertension, diabetes mellitus Type II,
and dementia.
Peripheral Blood Smear: Red blood cell, white blood cell, and platelet morphology
were within normal limits.
Bone Marrow Aspirate: The hematopoietic tissue was mildly hyper-cellular.
Megakaryopoiesis was adequate with adequate maturation. There was an infiltrate
of atypical enlarged plasma cells. CD138 immunohistochemical stain showed 75%
plasma cells. Flow cytometry indicated a large population of monoclonal plasma
cells (approximately 7.8% of the leukocytes) consistent with plasma cell dyscrasia.
Cytogenetic studies indicated normal male karyotype.
Serum Immunofixation: IgG-Lambda
monoclonalgammopathy (approximately
4.1g/dL from densitometry of SPE).
CEA 3.6 <2.5 ng/mL
CA 19-9 33 <37 U/mL
Sed Rate 91 0-15 mm/Hr
Comment: No information about the treatment of the patient was available.
Protein Electrophoresis-Urine:
Monoclonal band akin to serum
detected in urine but no free
lambda chains were detected.

114
*Note lack of complete reduction of IgG and Lambda channels. This is shown to
demonstrate the need to dilute specimens with very high concentration monoclonal
proteins (compare with Capillarys traces on page 113).

115
Case #19: IgD-Lambda Multiple Myeloma
A 58 year-old man presented to the emergency department after recurrent severe
back pain. X-rays revealed numerous lytic lesions and compression fractures of the
spine. Laboratory results were notable for hypercalcemia. A bone marrow biopsy
showed hypercellularity and 80% involvement by plasma cells based on CD138
staining. FISH testing did not demonstrate evidence of any high risk abnormalities.
Alpha-1 Globulin: 0.3 (0.1-0.2 g/dL) IgM: <25 (46-304 mg/dL)
Immunofixation:
IgD-Lambda monoclonal gammopathy
was confirmed by immunofixation studies.
Kappa Light Chains-24 hr Urine <0.70 <0.70 mg/dL
Lambda Light Chains-24 hr Urine 5.60 <0.40 mg/dL
Free Kappa/Lambda Ratio-24 hr Urine <0.12 0.7-6.2
Discussion: IgD multiple myeloma is a relatively rare type of plasma cell dyscrasia.
IgD accounts for ~1% of all cases of multiple myeloma and is associated with a
poor prognosis (median survival is 12-17 months). Occurring more commonly in
men between 55-60 years of age, the clinical features of IgD MM are bone pain,
fatigue, and weight loss. Typically, patients with IgD MM have relatively low
concentration monoclonal proteins. These features are reflected in this case, where
the patient presented with bone pain and the SPE revealed
hypogammaglobulinemia with a monoclonal protein concentration <0.3 g/dL. The
presence of associated kappa light chains is also common in this type of MM. This
patient was treated with chemotherapy and eventually underwent successful
autologous stem cell transplant.

120
Case #20 IgE-Lambda Multiple Myeloma
A 71 year-old female presented with low back pain, and arthralgia of the knees and
shoulders. No abnormalities concerning heart, lung, and abdomen were apparent
upon physical examination. There was no evidence of hepatosplenomegaly. No
pathological lymph nodes were palpable. Skeletal X-ray showed diffuse osteopenia
(patient was diagnosed with osteoporosis in 2008), and multiple lytic lesions were
detected in the skull, spine, and both femurs. Bone marrow aspirate indicated 25%
plasma cells; and flow cytometry and genetic studies were not performed. No
abnormalities of the skin were observed, and also there was no indication of allergy.
Protein Electrophoresis-Serum Immunoglobulins
Alpha-2 Globulin: 1.0 (0.4-0.9 g/dL) IgD: 2 (0-14 mg/dL)
Beta-Globulin: 0.8 (0.5-1.1 g/dL) IgE: 3,790,000 (<25 kU/L)
Serum Immunofixation:
IgE-Lambda monoclonalgammopathy (approximately 1.0 g/dL from
densitometry of SPE)
Urine Protein Electrophoresis & Immunofixation:
Free lambda chains were detected.
Free Lambda Chains-Serum 8.1 0.57- 2.63 mg/dL
Treatment: Patient was treated with melphalan, prednisone and thalidomide.
Since it is a very recent case of diagnosis and treatment, therefore no data was
available about the remission status of IgE-Lambda MM.
Special Note: The CZE electrophoretic and immunotyping (immunodisplacement)
scans are not provided for this case. Reason: Due to the lack of detection channels
for IgE in the CZE systems (Sebia & Helena), the interpretation can be misleading
as only the lambda chains monoclonality might be exhibited.
Acknowledgement:
We are highly indebted to Dr. Joannes F.M. Jacobs, Ph.D., MD (Laboratory Medical
Immunology, Radbound University Nijmegen Medical Center, Geert Grooteplein 10,
6525 GA Nijmegen, The Netherlands) for providing us the case history and the
laboratory data (Clin Chem 2010; 56:1368).

122
Case #21 Light Chain (Lambda) Multiple Myeloma
A 49 year-old female was brought to the emergency department with severe back
pain and shortness of breath. Chest CT showed a large right posterolateral chest
wall mass involving the right back musculature and destroying the tenth rib.
Additionally, there were multiple lytic lesions of the ribs and the thoracic and lumber
spine. CT guided biopsy of the chest wall mass revealed a poorly differentiated,
pankeratin-negative, non-epithelial infiltrating neoplasm confirmed with CD 138
immunostain to be a plasmacytoma.
Past Medical History: Hydrocephalus
Past Surgical History: VP shunt, cholecystectomy, and tonsillectomy
Past CT, X-ray &
Ultrasound Studies:
Bone Marrow Biopsy and Aspirate: Three days after CT guided biopsy of the chest
wall mass (see above):
Cellularity: Hypercellular for patient’s age, on account of extensive replacement
of normal hematopoitic tissue by plasma cells, including many abnormal and
dysplastic forms, estimated 60%. This was confirmed by CD138 immunostain of
plasma cells.
Trabeculae: Adequate
Lynphocytes & Plasma Cells: Marked plasmacytosis, estimated at 33% on
smear, but closer to 60% on CD138 staining of the biopsy specimen. Marked
dysplasia of plasmacytes was present, including multinuclear form.
Lymphocytes were not increased and showed no dysplasia.
Flow cytometry: CD56 positive monoclonal plasma cell population
(approximately 12% of the leukocytes) indicative of a plasma cell dyscrasia.
Final Diagnosis: Plasmacytosis consistent with multiple myeloma.
No lesion detected upon ultrasound images of
abdomen and kidney post cholecystectomy.
Mammography on several occasions negative for a
dominant suspicious, mass. CT of head manifested
normal pressure hydrocephalus.

123
Note: Monoclonal gammopathy (lambda
chains) confirmed by immunofixation
studies.
Comments: The patient underwent radiation and chemotherapy treatment, and the
SPE and quantitative immunoglobulins were as follows two months after the start of
the treatment.
Note: Monoclonal band (free lambda chains)
was still present.
Urine Protein Electrophoresis &
Immunofixation: Positive for free
lambda chains.

128
Case #22 Smoldering Myeloma vs Symptomatic Myeloma
A 84 year-old woman presented to the emergency department with recurrent back
pain. Imaging and laboratory studies showed multiple spinal compression fractures,
anemia, and osteopenia. A bone marrow biopsy showed 40% cellularity, 17%
plasma cells on aspirate, and 10% plasma cells by CD138 immunohistochemistry;
cells were monoclonal kappa light chain restricted.
Note: 1.2 g/dL of IgG-Kappa monoclonal gammopathy was identified by SPE
densitometry.
TIBC 315 205-380 mcg/dL
% iron saturation 7 20-50
Discussion: Multiple myeloma is broadly classified as symptomatic and
asymptomatic (also known as smoldering or indolent myeloma)1. This classification
essentially represents the clinical spectrum, as asymptomatic cases progress to
symptomatic disease at a rate of 10% per year.2 The key difference between
classifications is the presence of symptoms represented by the “CRAB” mnemonic
(hypercalcemia, renal failure, anemia, bone lesions). In symptomatic disease, there
is no threshold for the quantity of the monoclonal protein or the % of plasma cells in
bone marrow (plasma cells typically exceed 10%). In asymptomatic disease, the
classification requires an M-protein in serum >3.0 g/dL and/or >=10% clonal plasma
cells in bone marrow without evidence of end organ damage (i.e. no CRAB
findings). At first glance, the present case appears to meet the criteria for
symptomatic myeloma (anemia, fractures, 1.2g/dL monoclonal protein, and 17%

129
plasma cells). However, the patient’s anemia and fractures may be due to iron
deficiency and osteoporosis. At this snapshot in time, the patient is presumed to
have smoldering myeloma pending response to treatment for anemia and
osteoporosis.
1. WHO classification of tumors of haematopoietic and lymphoid tissues. 4th
edition. Edited by SH Swerdlow et al. WHO Press, 2008.
2. Dispenzieri A et al., Immunoglobulin Free Light Chain Ratio is an
Independent Risk Factor for prognosis of Smoldering (asymptomatic)
Multiple Myeloma. Blood, 15 January 2008, III: 785-789.

134
Case #23 Autologous Stem Cell Transplantation
A 67 year-old man with a history of stage I multiple myeloma returned for a routine
follow-up. He was diagnosed with multiple myeloma 2-years prior and received
autologous stem cell transplant (SCT) 1-year prior to this visit. At the time of
diagnosis (two years ago) he had 1.8 g/dL IgG-kappa monoclonal protein in his
serum.
Urine Protein Electrophoresis & Immunofixation:
Mild glomerular proteinuria; no monoclonal band
was evident from urine immunofixation.
Beta-2 Microglobulin 3.40 0.70-1.80 mcg/mL
Discussion:
Small abnormal protein bands (APBs) are frequently observed after high dose
chemotherapy and stem cell transplantation for multiple myeloma. It is proposed
that the bands appear as a result of transient dysregulation of B-cells as the
immune system recovers from therapy. The emergence of these bands can make
treatment monitoring somewhat challenging, as it can be difficult to differentiate the
original disease clone from newly emerging monoclonal bands. It is reported that
the presence of APBs after hematopoietic cell transplantation is a positive
prognostic indicator of overall survival. a, b
a Hall, SL, Tate J, Gill D, Mollee P., Significance of Abnormal Protein Bands in
Patients with Multiple Myeloma following Autologous Stem Cell
Transplantation. Clin Biochem Rev. 2009 Aug;30(3):113-8.
b Zent CS, Wilson CS, Tricot G, Jagannath S, Siegel D, Desikan, KR, Munshi
N, Bracy D, Barlogie B, Butch AW., Oligoclonal bands and lg isotype
switching in multiple myeloma treated with high-dose therapy and
hematopoietic cell transplantation. Blood. 1998 May 1;91(9):3518-23.

137
Case #24 Waldenstrom’s Macroglobulinemia
A 60 year-old male complained of severe back pain in 1999, and since then
underwent extensive examination including visits to a cardiologist, rheumatologist,
hematopathologist, neurologist, oncologist, and neurosurgeon.
Medical History: Hypercholesterolemia, hypertension, and chronic back pain
Surgical History: Laminectomy at L3, L4, and L5 in 1999. In 2001 underwent a
fusion to L2, L3, L4, L5 vertebrae.
In May 2007, a work up ensued to determine etiology of an elevated sed rate of
81(normal: 0-15 mm/hr), and increased C-reactive protein (8.7; normal: 0-1.0
mg/dL). According to a rheumatologist, there was no medical history to suggest a
connective-tissue disease such as rheumatoid arthritis, lupus, or temporal arteritis.
Electrophysiologic evidence suggested L5 (and possibly L4) lumbar radiculopathy
with acute and chronic denervation / reinvernation. There was evidence suggestive
of, but not diagnostic of a left distal polyneuropathy. MRI revealed multiple
extradural defects in the iliac bone. CT of the pelvis revealed 2.4 cm bony
destructive lesion in the right iliac bone. A bone scan revealed both right and left
sacroilial joint, with increased uptake on the right, findings were felt to be non-
specific.
Bone Marrow Biopsy, Aspirate & Peripheral Blood (2007): Hypercellular marrow for
age with trilineage hematopoietic maturation and focal lymphoplasmacytic infiltrate
along with small population of lambda positive monoclonal B-cells that lacked CD5
and CD10 expression, consistent with low level marrow involvement by
lymphoplasmacytic lymphoma.
Surgical Pathology (right ileum biopsy, 2007): Scant portions of fibrous tissue with
small lymphoid cells.
Flow Cytometry (bone marrow, 2007): Small population of lambda positive
monoclonal B-cells (approximately 2% of the leukocytes) that lacked CD5 and
CD10 expression. These results were suggestive of a B-cell lymphoproliferative
disorder, but not diagnostic without other confirmatory findings since small
monoclonal B-cell populations can occasionally be seen in reactive and auto
immune processes.
Chromosome Analysis (bone marrow, 2007): Karyotype: 45,X,-Y[7]/46,XY[11]
The patient was diagnosed asymptomatic Waldenstrom’s macroglobulinanemia
(without hyperviscosity, constitutional, or B-cells), and did not receive any treatment
at diagnosis.

138
Protein Electrophoresis–Serum (2009): Immunoglobulins:
Gamma Globulin: 2.3 (0.5-1.3 g/dl)
Serum Immunofixation: IgM-Lambda
monoclonal gammopathy. IgM-Lambda
monoclonal band was approximately,
1.1 g/dL from densitometry of SPE.
Test 6/20/2007 4/27/2009 Reference Value
Free Kappa Chains-Serum 3.12 2.57 0.33-1.94 mg/dL
Free Lambda Chains-Serum 3.83 2.51 0.57-2.63 mg/dL
Free Kappa / Lambda Ratio 0.81 1.02 0.26-1.65
Discussion: The patient consulted two other physicians, who also advised him that
his chronic back pain was not related to Waldenstrom’s macroglobulinemia. It was
the opinion of his hematologist/oncologist that he required no treatment for his
Waldenstrom’s macroglonulinemia, as he has remained asymptomatic since
diagnosis.
Reference: Ansell SM, Kyle RA et al. Diagnosis and Management of Waldenstrom’s
Macroglobulinemia: Mayo Stratification of Macroglobulinemia and Risk Adapted
Therapy (mSMART) Guidelines. Mayo Clinic Proc. September 2010; 85(9): 824-833
Protein Electrophoresis-Random Urine:

143
Case #25 Hodgkin’s Lymphoma
A 45 year-old female admitted to hospital through emergency department (ED) with
chief complaint of swelling and pain on both sides of the neck. No neck abscess or
thyroid mass was detected. Initial EKG revealed sinus tachycardia, in an otherwise
normal EKG. Abnormal laboratory results (ED specimen): WBC = 18.6 (4.0 – 11.0
thou/cu mm), serum iron = 19 (30 – 160 mcg/dL),TIBC = 170 (205 – 380 mcg/dL),
iron saturation = 11.2 (20.0-50.0%), LDH = 757 (2-240 u/L), Protime = 15.8 (11.3 –
14.5 seconds), PTT = 44.5 (22.5 - 35.0 seconds). She was found to have extensive
bulky mediastinal and bilateral hilar neck lymphadenopathy from CT. A hemato-
oncologist and a general surgeon were consulted. Right side cervical lymph node
biopsy was done followed by bone marrow aspirate and biopsy of right posterior
iliac spine.
Peripheral Blood: Normochromic, normocytic anemia and leukocytosis
Cervical Lymph Node Biopsy: Nodular sclerosing Hodgkin’s disease.
Bone Marrow Biopsy & Aspirate: Cytogenetic studies performed on the bone
marrow aspirate showed a normal karyotype. Flow cytometry did not demonstrate
any evidence of monoclonality. The fragmented marrow showed a focal area of
fibrosis without any distinct Reed-Sternberg cells or variants were identified,
however the CD30 stain showed focal staining. The T-cells appeared phenotypically
normal and have a CD4:CD8 ratio of 2.4. The B-cells were polytypic and had a
kappa:lambda ratio of 1.5. These morphologic features, along with the
immunohistochemical stains were suggestive of involvement by Hodgkin’s disease.
Albumin: 3.6 (3.8-5.8 g/dL) IgA: 232 ( 82-453 mg/dL)
Gamma globulin: 1.5 (0.5-1.3 g/dL)
Immunofixation-Serum: No evidence of monoclonal band.

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