Protein Electrophoresis, Immunofixation, and Immunodisplacement in Clinical Diagnosis. 2nd Edition,...

Protein Electrophoresis, Immunofixation, andImmunodisplacement in Clinical Diagnosis

A Compendium of Selected Cases Employingthe Procedures and Instruments of

Helena Laboratories, USA, and Sebia, France

by

Zia Uddin, Ph.D.

&

Christopher R. McCudden, Ph.D.

First Edition: September 2010Second Edition: May 2011

This compendium is dedicated to ourteachers, to whom we would like toexpress our heartfelt undying gratitudefor their relentless effort, sincerity,dedication, and virtuous contributiontowards 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 15Undermined Significance

Case #1 IgG-Lambda Monoclonal Gammopathy: Low Risk 18

Case #2 IgA-Kappa Monoclonal Gammopathy: 23Low Intermediate Risk

Case #3 IgA-Kappa Monoclonal Gammopathy: 28High 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 46Concomitant Serum Iron Deficiency

Case#7 Post Acute Phase Reaction: Monoclonal Band 51Superimposed 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) 57b) 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 73with Beta-Gamma Globulin Bridging

Case#12 Polyclonalgammopathy: Pancytopenia with Bone 78Marrow Containing <5% Plasma Cells

Section E: Hypogammaglobulinemia/Light Chain 83Multiple Myeloma

Case #13 Hypogammaglobulinemia Secondary to Chemotherapy 83for 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) 94Multiple Myeloma

Case#16 Light Chain (Lambda) Multiple Myeloma with normal 99Gammaglobulins

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)4

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 158with Splenomegaly

Section H: Unknown Cases 164

Case#29 Unknown Diagnosis: Please make the diagnosis 164on the basis of the information provided.

(iii)5

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, NCWarren, Michigan 48093, USA [email protected]@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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mailto:[email protected]


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 CenterSt. John Macomb-Oakland Hospital, Warren, Michigan

Jessica Poisson, MD, Resident PhysicianDepartment of Pathology, University of North Carolina,Chapel Hill, NC

Stephanie Mathews, MD, Assistant Professor,Department of Pathology, University of North Carolina,Chapel Hill, NC

Yuri Fedoriw, MD, Associate Director Core Laboratory & HematopathologyDepartment of Pathology, University of North Carolina,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, USAP.O. Box 752, Beaumont, Texas 77704-0752, USAe-mail: [email protected]: 409-842-3714www.helena.com

Helena Bioscience EuropeQueensway South, Team Valley Trading EstateGateshead, Tyne and Wear, NE11 OSD, United KingdomTel: 44 (0) 191 482 8440e-mail: [email protected]

Sebia, FranceParc Technologique Leonard de VinciCP 8010 LIsses-91008 EVRY Cedex-Francee-mail: [email protected]: 33(0) 1 69 80 80

Sebia, USA1705 Corporate DriveNorcross, GA 30093-2991Tel: 770-446-3707Fax: 700-446-5511http://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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http://www.sebia-usa.com/


http://www.helena.com/

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 fractionsTrack # 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 TrackCZE scan of IgG pretreatment serum CZE scan of IgA pretreatment serumCZE scan of IgM pretreatment serumCZE scan of Kappa pretreatment serumCZE 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 TrackCZE scan of IgG pretreatment serumCZE scan of IgA pretreatment serumCZE scan of IgM pretreatment serumCZE scan of Kappa pretreatment serumCZE 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.

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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.

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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.

17

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/dLHct 21 35-48 %WBC 18.6 4.0-11.00 Thou/cc mmCEA 64.4 0.0-3.4 ng/mLCA 19-9 102 Less than 37 u/mLIron 19 30-160 mcg/dLTIBC 417 205-380 mcg/dLSaturation 4.0 20-50%

Free Kappa Chains-Serum 2.40 0.33 – 1.94 mg/dLFree Lambda Chains-Serum 1.35 0.57-2.63 mg/dLFree Kappa/Lambda Ratio 1.03 0.26- 1.65

Urine Protein Electrophoresis:No evidence of monoclonal band.

18

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

22

had cardiac catheterization, successful PTCA of the circumflex artery with stent deployment.

Protein Electrophoresis-Serum: Immunoglobulins: Total Protein: 5.9 (6.4-8.3 g/dL) IgG: 600 (751-1560 mg/dL) Albumin: 3.1 (3.8-5.8 g/dL) IgA: 488 (82-453 mg/dL) Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 88 (46-304 mg/dL) Alpha-2 Globulin: 0.8 (0.4-0.9 g/dL) Beta Globulin: 0.9 (0.5-1.1 g/dL) Gamma Globulin: 0.9 (0.5-1.3 g/dL)

Note: Mini-monoclonal IgA-Kappa band identified from IF.


Free Kappa Chains-Serum 0.95 0.33-1.94 mg/dLFree Lambda Chains-Serum 0.81 0.57-2.63 mg/dLFree Kappa/Lambda Ratio 1.17 0.26-1.65

Urine Protein Electrophoresis:Negative for Bence Jones protein.

23

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

24

the IF does not demonstrate any abnormality in the region.

25

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


27

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.

Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 5.8 (6.4-8.3 g/dL) IgG: 1010 (751-1560 g/dL)Albumin: 2.8 (3.8-5.8 g/dL) IgA: 916 (82-453 mg/dL)Alpha-1 Globulin: 0.3 (0.1-0.2 g/dL) IgM: 99 (46-304 mg/dL)Alpha-2 Globulin: 0.8 (0.4-0.9 g/dL) Beta-Globulin: 1.1 (0.5-1.1 g/dL)Gamma Globulin: 0.8 (0.5-1.3 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/dLFree Lambda Chain-Serum 1.84 0.33-1.94 mg/dLFree Kappa/Lambda Ratio 2.03 0.26-1.65

Free Kappa Chain-Urine 56.2 0.14-2.42 mg/24 HrFree Lambda Chain-Urine 7.04 0.02-0.67 mg/24 HrFree Kappa/Lambda Ratio 7.98 2.04-10.37

Protein Electrophoresis-Urine:Non-selective proteinuria.

28

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).

29



32

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.

Protein Electrophoresis-Serum: Immunoglobulins:Total Protein: 6.5 (6.4-8.3 g/dL) IgG: 1220 (751-1560 mg/dL)Albumin: 3.7 (3.8-5.8 g/dL) IgA: 86 (82-453 mg/dL)Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 19 (46-304 mg/dL)Alpha-2 Globulin: 0.8 (0.4-0.8 g/dL)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/dLHct 25.0 35.0-48.0 %MCV 128 79-98 fLProtime 20.6 11.3-14.5 second(s)PTT 87.0 22.5-35.0 seconds(s)Iron 29 30-160 mcg/dLTIBC 297 205-380 mcg/dLSaturation 9.0 20.0-50.0 %Ferritin 338 8-120 ng/mL


33

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

37

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.

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

38

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.

39

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/mm3RBC 2.70 4.6-6.2 Mil/mm3Hemoglobin 8.4 13.5-18.0 g/dLHematocrit 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/LIron-Serum 90 45-160 ug/dLTIBC 109 205-380 ug/dL% Saturation 82.6 20-50Ferritin 10429 23-265 ng/mL

C-Reactive Protein 4.1 0-1.0 mg/dL

40

Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 5.1 (6.4-8.3 g/dL) IgG: 793 (751-1560 mg/dL)Albumin: 2.1 (3.8-5.8 g/dL) IgA: 185 (82-453 mg/dL)Alpha-1 Globulin: 0.3 (0.1-0.2 g/dL) IgM: 164 (46-304 mg/dL)Alpha-2 Globulin: 1.4 (0.4-0.9 g/dL)Beta Globulin: 0.7 (0.5-1.1 g/dL)Gamma Globulin: 0.6 (0.5-1.3 g/dL)

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.

41

Case #6: Acute Phase Reaction (Bacterial Meningitis) with Concomitant Serum Iron Deficiency

45

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.

Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 5.8 (6.4-8.3 g/dL) IgG: 1070 (751-1560 mg/dL)Albumin: 2.6 (3.8-5.8 g/dL) IgA: 357 (82-453 mg/dL)Alpha-1 Globulin: 0.3 (0.1-0.2 g/dL) IgM: 128 (46-304 mg/dL)Alpha-2 Globulin: 1.2 (0.4-0.9 g/dL) Beta Globulin: 0.9 (0.5-1.1 g/dL) Gamma Globulin: 0.8 (0.5-1.3 g/dL) 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/dLTIBC 308 205-380 mcg/dL% Saturation 3.0 20-50Ferritin 229 8-120 mcg/dLWBC 25.9 4.0-11.0 x 109/LWBC-CSF 3700 0-5 mm3

Protein-CSF 687 15-45 mg/dLGlucose-CSF Not Detected 50-70 mg/dL

46

Case #7: Post Acute Phase Reaction: Monoclonal Band Superimposed on C3 Band (Elevated Fibrinogen)

50

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.

Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 5.9 (6.4-8.3 g/dL) IgG: 1010 (751-1560 mg/dL)Albumin: 2.2 (3.8-5.8 g/dL) IgA: 475 (82-453 mg/dL)Alpha-1 Globulin: 0.3 (0.1-0.2 g/dL) IgM: 48 (46-304 mg/dL)Alpha-2 Globulin: 0.7 (0.4-0.9 g/dL) Beta Globulin: 1.9 (0.5-1.1 g/dL)Gamma Globulin: 0.8 (0.5-1.3 g/dL)

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).

Urine Protein Electrophoresis:No evidence of monoclonal band.

51

Another way to confirm the fibrinogen band is to perform IF using anti-fibrinogen.

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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 freelight 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

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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.

Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 6.7 (6.4-8.3 g/dL) IgG: 970 (751-1560 mg/dL)Albumin: 3.7 (3.8-5.8 g/dL) IgA: 994 (82-453 mg/dL)Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 29 (46-304 mg/dL)Alpha-2 Globulin: 0.9 (0.4-0.9 g/dL) Beta Globulin: 1.2 (0.5-1.1 g/dL)Gamma Globulin: 0.7 (0.5-1.3 g/dL)



Comment: No follow up data of the patient was available.

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Case #9 Double Gammopathy: a) IgM-Kappa (major band)b) IgG-Kappa (minor band)

61

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/dLFree Lambda Chains-Serum 2.30 0.57 – 2.63 mg/dLFree 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.

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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)

66

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/LPTT 94.7 22.5-35.0 second(s)BUN 58 8-20 mg/dLCalcium-serum 14.5 8.4-10.5 mg/dLCalcium-ionized 1.81 1.13-1.32 mmol/LPTH-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.

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Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 6.1 (6.4-8.3 g/dL) IgG: 877 (751-1560 mg/dL)Albumin: 3.3 (3.8-5.8 g/dL) IgA: 83 (82-453 mg/dL)Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 1610 (46-304 mg/dL) Alpha-2 Globulin: 0.5 (0.4-0.9 g/dL)Beta Globulin: 0.6 (0.5-1.1 g/dL) Gamma Globulin: 1.5 (0.5-1.3 g/dL)

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

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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.

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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).

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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 Kappa Chains-Serum 4.67 0.33-1.94 mg/dLFree Lambda Chain-Serum 8.47 0.57-2.63 mg/dLFree Kappa/Lambda Ratio 0.55 0.26-1.65Beta-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.


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*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.

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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).

Protein Electrophoresis-Serum Immunoglobulins: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)Alpha-2 Globulin: 1.2 (0.4-0.9 g/dL) Beta Globulin: 0.7 (0.5-1.1 g/dL) Gamma Globulin: 0.1 (0.5-1.3 g/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/LFree Lambda Chains-Serum 1.77 5.7-26.3 mg/LFree Kappa Chains-Urine (24 Hr) < 0.40 < 2.0 mg/dL

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Free Lambda Chains-Urine (24 Hr) < 0.40 < 2.0 mg/dLSerum IgA1 < 9.5 50-314 mg/dLSerum IgA2 < 0.3 9.7-156 mg/dLAnti-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.

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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.

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Case #14 Simultaneous Adenocarcinoma of Lung andLight 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

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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):Total Protein: 5.8 (6.4-8.3 g/dL) IgG: 540 (751-1560 mg/dL)Albumin: 3.3 (3.8-5.8 g/dL) IgA: 23 (82-453 mg/dL)Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: <13 (46- 304 mg/dL)Alpha-2 Globulin: 1.0 (0.4-0.9 g/dL)Beta Globulin: 1.0 (0.5-1.1 g/dL) Gamma Globulin: 0.3 (0.5-1.3 g/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/dLCreatinine 5.5 0.92 0.67-1.54 mg/dLFree Kappa Chains-Serum 2230.0 67.1 0.33-1.94 mg/dLFree Lambda Chains-Serum 0.37 0.54 0.57-2.63 mg/dLFree 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).

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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.

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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).

Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 6.5 (6.4-8.3 g/dL) IgG: 490 (751-1560 mg/dL)Albumin: 4.4 (3.8-5.8 g/dL) IgA: 78 (82-453 mg/dL)Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 51 (46-304 mg/dL)Alpha-2 Globulin: 0.7 (0.4-0.9 g/dL)Beta Globulin: 0.8 (0.5-1.1 g/dL) Gamma Globulin: 0.4 (0.5-1.3 g/dL) Note: No monoclonal band was detectedby SPE

Urine Protein Electrophoresis: Positive for free lambda chains.

Test Result Reference ValueCalcium-Serum 11.8 8.4-10.5 mg/dLPTH,intact 7.8 15-65 pg/mLVitamin D 25-hydroxy 13.0 >30.0 mg/dLFree Kappa Chains-Serum 0.7 0.33-1.94 mg/dLFree Lambda Chains-Serum 357.2 0.57-2.63 mg/dLFree Kappa/Lambda Ratio <0.01 0.26-1.65Protein-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.

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

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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.

Protein Electrophoresis-Serum Immunoglobulins:Total protein: 5.6 (6.4-8.3 g/dL) IgG: 688 (751-1560 mg/dL)Albumin: 2.8 (3.8-5.8 g/dL) IgA: 37 (82-453 mg/dL)Alpha-1 Globulin: 0.4 (0.1-0.2 g/dL) IgM: 38 (46-304 mg/dL)Alpha-2 Globulin: 1.0 (0.4-0.9 g/dL)Beta Globulin: 0.6 (0.5-1.1 g/dL) Gamma Globulin: 0.8 (0.5-1.3 g/dL) Note: Monoclonal protein restriction (cathodic to C3 on agarose gel) was identified as “Free Lambda Chains.”

Test Result Reference ValueFree Kappa Chains-Serum: 2.85 0.33-1.94 mg/dLFree Lambda Chains-Serum: 968.00 0.57-2.63 mg/dLFree 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.”

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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.

Protein Electrophoresis-Serum Immunoglobulins:Total protein: 6.0 (6.4-8.3 g/dL) IgG: 260 (751-1560 mg/dL)Albumin: 3.0 (3.8-5.8 g/dL) IgA: 26 (82-453 mg/dL)Alpha-1 Globulin: 0.3 (0.1-0.2 g/dL) IgM: 2380 (46-304 mg/dL)Alpha-2 Globulin: 0.7 (0.4-0.9 g/dL)Beta Globulin: 0.5 (0.5-1.1 g/dL) Gamma Globulin: 1.5 (0.5-1.3 g/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).

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WBC 7.2 4.5-11.0 x 109/LHgb 9.0 12.0-16.0 g/dLCreatinine 1.0 0.60-1.0 mg/dLCalcium 10.0 8.5-10.2 mg/dLAlkaline Phosphatse 194 38-126 U/LLD 189 338-610 U/LBeta-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.

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Case #18 IgG-Lambda Multiple Myeloma

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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.

Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 8.9 (6.4-8.3 g/dL) IgG: 5400 (751-1560 mg/dL)Albumin: 3.1 (3.8-5.8 g/dL) IgA: 64 (82-453 mg/dL)Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 18 (46-304 mg/dL)Alpha-2 Globulin: 0.6 (0.4-0.9 g/dL)Beta Globulin: 0.6 (0.5-1.1 g/dL) Gamma Globulin: 4.4 (0.5-1.3 g/dL)

Serum Immunofixation: IgG-Lambdamonoclonalgammopathy (approximately 4.1g/dL from densitometry of SPE).

Test Result Reference ValueFree Kappa Chains-Serum 8.97 0.33-1.94 mg/dLFree Lambda Chains-Serum 404.00 0.57-2.63 mg/dLFree Kappa/Lambda Ratio <0.02 0.26-1.65

CEA 3.6 <2.5 ng/mLCA 19-9 33 <37 U/mLSed 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.

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*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).

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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.

Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 6.8 (6.4-8.3 g/dL) IgG: 573 (751-1560 mg/dL)Albumin: 4.5 (3.8-5.8 g/dL) IgA: 36 (82-453 mg/dL)Alpha-1 Globulin: 0.3 (0.1-0.2 g/dL) IgM: <25 (46-304 mg/dL)Alpha-2 Globulin: 0.7 (0.4-0.9 g/dL)Beta Globulin: 0.7 (0.5-1.1 g/dL) Gamma Globulin: 0.6 (0.5-1.3 g/dL)

Immunofixation:IgD-Lambda monoclonal gammopathy was confirmed by immunofixation studies.

Test Result Reference ValueFree Kappa Chains-Serum 0.14 0.33-1.94 mg/dLFree Lambda Chains-Serum 35.1 0.57-2.63 mg/dLFree Kappa/Lambda Ratio 0.004 0.26-1.65

Kappa Light Chains-24 hr Urine <0.70 <0.70 mg/dLLambda Light Chains-24 hr Urine 5.60 <0.40 mg/dLFree 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.

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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 ImmunoglobulinsTotal Protein: 7.9 (6.4-8.3 g/dL) IgG: 555 (751-1560 mg/dL)Albumin: 4.7 (3.8-5.8 g/dL) IgA: 35 (82-453 mg/dL)Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 30 (46-304 mg/dL)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)Gamma Globulin: 1.2 (0.5-1.3 g/dL)

Serum Immunofixation: IgE-Lambda monoclonalgammopathy (approximately 1.0 g/dL from densitometry of SPE)Urine Protein Electrophoresis & Immunofixation: Free lambda chains were detected.


Free Kappa Chains-Serum 0.79 0.33-1.94 mg/dLFree Lambda Chains-Serum 8.1 0.57- 2.63 mg/dLFree Kappa/Lambda Ratio 0.099 0.26-1.65

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).

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Case #21 Light Chain (Lambda) Multiple Myeloma

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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.

Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 7.0 (6.4-8.3 g/dL) IgG: 490 (751-1560 mg/dL)

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.

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Albumin: 3.9 (3.8-5.8 g/dL) IgA: 648 (82-453 mg/dL)Alpha-1 Globulin: 0.3 (0.1-0.2 g/dL) IgM: 39 (46-304 mg/dL)Alpha-2 Globulin: 1.0 (0.4-0.9 g/dL) Beta Globulin: 1.0 (0.5-1.5 g/dL)Gamma Globulin: 0.8 (0.5-1.3 g/dL)

Note: Monoclonal gammopathy (lambda chains) confirmed by immunofixation studies.


Free Kappa Chains-Serum 0.25 0.33-1.94 mg/dLFree Lambda Chains-Serum 80.30 0.57-2.63 mg/dLFree Kappa/Lambda Ratio <0.01 0.26-1.65

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.

Protein Electrophoresis-Serum Immunoglobulins:Total Protein: 5.4 (6.4-8.3 g/dL) IgG: 381 (751-1560 mg/dL)Albumin: 2.9 (3.8-5.8 g/dL) IgA: 42 (82-453 mg/dL)Alpha-1 Globulin: 0.3 (0.1-0.2 g/dL) IgM: 12 (46-304 mg/dL)Alpha-2 Globulin: 0.8 (0.4-0.9 g/dL) Beta Globulin: 0.7 (0.5-1.5 g/dL) Gamma Globulin: 0.7 (0.5-1.3 g/dL)

Note: Monoclonal band (free lambda chains)was still present.

Urine Protein Electrophoresis & Immunofixation: Positive for free lambda chains.

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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.

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Note: 1.2 g/dL of IgG-Kappa monoclonal gammopathy was identified by SPE densitometry. Test Result Reference ValueCreatinine 1.35 0.60-1.00 mg/dLCalcium 10.2 8.5-10.2 mg/dLFree Kappa Chains-Serum 30.2 0.33-1.94 mg/dLFree Lambda Chains-Serum 1.75 0.57-2.63 mg/dLFree Kappa/Lambda Ratio 17.25 0.26-1.65

Iron 22 30-160 mcg/dLTIBC 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%

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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.

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Case #23 Autologous Stem Cell Transplantation

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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. Test Result Reference ValueCreatinine 1.67 0.70-1.30 mg/dLCalcium 9.8 8.5-10.2 mg/dLBeta-2 Microglobulin 3.40 0.70-1.80 mcg/mL Free Kappa Chains-Serum 2.06 0.33-1.94 mg/dLFree Lambda Chains-Serum 1.15 0.57-2.63 mg/dLFree Kappa/Lambda Ratio 1.79 0.26-1.65

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.

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Case #24 Waldenstrom’s Macroglobulinemia

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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.

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Protein Electrophoresis–Serum (2009): Immunoglobulins:Total Protein: 8.5 (6.4-8.3 g/dL) IgG: 1791 (751-1560 mg/dL)Albumin: 4.0 (3.8-5.8 g/dL) IgA: 402 (82-453 mg/dL)Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 1951 (46-304 mg/dL)Alpha-2 Globulin: 0.9 (0.4-0.9 g/dL)Beta Globulin: 1.1 (0.5-1.1 g/dL)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/dLFree Lambda Chains-Serum 3.83 2.51 0.57-2.63 mg/dLFree 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.

Protein Electrophoresis-Random Urine:Negative for Bence Jones protein.

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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

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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.

Protein Electrophoresis-Serum: Immunoglobulins:Total Protein: 7.9 (6.4-8.3 g/dL) IgG: 1970 (751-1560 mg/dL)Albumin: 3.6 (3.8-5.8 g/dL) IgA: 232 ( 82-453 mg/dL)Alpha-1 Globulin: 0.4 (0.1-0.2 g/dL) IgM: 68 (46-304 mg/dL)Alpha-2 Globulin: 1.2 (0.4-0.9 g/dL) Beta-Globulin: 1.2 (0.5-1.1 g/dL) Gamma globulin: 1.5 (0.5-1.3 g/dL)

Immunofixation-Serum: No evidence of monoclonal band.


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Case #26 Plasma Cell Leukemia

A 39 year-old woman presented to the emergency department with pneumonia and fever. At the time of admission, laboratory results revealed significant anemia (hemoglobin of 5.5 g/dL) and leukocytosis (WBC 45.4 x109 /L). A bone marrow biopsy was performed and a diagnosis of plasma cell leukemia was made. Bone

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marrow showed kappa restricted disease and CD38 and CD56 positivity. There was no evidence of lytic lesions by X-ray. The specimen included here is from a follow-up 6 months later.

Protein Electrophoresis-Serum: Immunoglobulins:Total Protein: 6.8 (6.4-8.3 g/dL) IgG: 1223 (751-1560 mg/dL)Albumin: 4.2 (3.8-5.8 g/dL) IgA: 11 (82-453 mg/dL)Alpha-1 Globulin: 0.4 (0.1-0.2 g/dL) IgM: <25 (46-304 mg/dL)Alpha-2 Globulin: 1.0 (0.4-0.9 g/dL) Beta-Globulin: 0.8 (0.5-1.1 g/dL) Gamma Globulin: 0.4 (0.5-1.3 g/dL)

Note:1. Serum immunofixation revealed a monoclonal component typed as IgG-

Kappa.

2. Urine immunofixation showed a large monoclonal component typed as free kappa light chains and a second (minor) monoclonal component typed as IgG-Kappa.

Test Result Reference ValueWBC 11.4 4.5-11.0 x109 /LHgb 10.8 12.0-16.0 g/dLCreatinine 2.03 0.60-1.00 mg/dLCalcium 11.5 8.5-10.2 mg/dLAlkaline phosphatase 93 38-126 U/LLD 1642 338-610 U/LFree Kappa Chains-Serum 2460 0.33-1.94 mg/dLFree Lambda Chains-Serum <0.04 0.57-2.63 mg/dLFree Kappa/Lambda Ratio >61809 0.26-1.65 Discussion: Plasma cell leukemia (PCL) is a clinical variant of plasma cell myeloma. Defined by the presence of >2x109 clonal plasma cells in peripheral blood (and/or >20% of the leukocyte differential), PCL develops in 2-5% of myeloma cases; PCL also occurs as a primary disorder. As evident in this case, plasma cell leukemia is commonly associated with light chains (or IgD). In contrast to this case, PCL typically does not express CD56. The prognosis of PCL is poor, with a median survival time of 7-11 months.

Protein Electrophoresis-Random Urine:Negative for Bence Jones protein.

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Case #27 Solitary Plasmacytoma of Bone:

A 71 year-old male presented to the emergency department after a femur fracture, and underwent a nailing of the left femur. Surgical Pathology Report: Reemed Bones Left Hip: CD138 immunohistochemical stain for plasma cells performed on three separate blocks showed a mild plasmacytosis involving bone marrow with a cell count of about 7%.

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Bone Marrow Biopsy: Normal to slightly mild hypercellular marrow with normal myeloid erythroid activity, elements of plasma cells were seen, but there was no evidence of tumor, lymphoma or granuloma throughout the biopsy material. Bone Marrow Aspirate: 6% plasma cells, some of the plasma cells had nucleoli, suggesting of myeloma cells. Flow Cytometry: Small population of CD56 positive monoclonal plasma cells (approximately 2% of the leukocytes) indicative of a plasma cell dyscrasia. No flow cytometric evidence of acute leukemia, or lymphoproliferative disorder.Cytogenetic Studies: No clonal abnormality was apparent. Normal male karyotype: 46,XY[14].MRI and X-ray: No evidence of bone lesion.

Protein Electrophoresis-Serum: Immunoglobulins-Serum:Total Protein: 5.0 (6.4-8.3 g/dL) IgG: 1020 (751-1560 mg/dL) Albumin: 2.4 (3.8-5.8 g/dL) IgA: 80 (82-453 mg/dL) Alpha-1 Globulin: 0.3 (0.1-0.2 g/dL) IgM: 30 (46-304 mg/dL) Alpha-2 Globulin: 0.8 (0.4-0.9 g/dL) Beta Globulin: 0.7 (0.5-1.1 g/dL)Gamma Globulin: 0.8 (0.5-1.3 g/dL)

Note: IgG-Kappa monoclonal band identified in serum from immunofixation studies.

Test Result Reference ValueFree Kappa Chains-Serum 8.31 0.33-1.94 mg/dLFree Lambda Chains-Serum 2.80 0.57-2.63 mg/dLFree Kappa/Lambda Ratio 2.96 0.26-1.15

Criteria for the diagnosis of solitary plasmacytoma of bone (Kyle RA et al. Br J Haem 2001; 115: 13-18)

Low concentration or no M-protein in serum and/or urine Single area of bone destruction due to clonal plasma cells Bone marrow not consistent with multiple myeloma Normal skeletal survey (and MRI of spine and pelvis if done) No related organ or tissue impairment (no end organ damage

other than a solitary bone lesion)

Protein Electrophoresis-Urine: No evidence of monoclonal band

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Section G: Rheumatic Diseases

Case #28 Felty’s Syndrome: Advanced Arthritis with Splenomegaly

This female patient was first examined in 1971 at the age of 25 years by a rheumatologist, and a diagnosis of rheumatoid arthritis was made. She was initially

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put on gold therapy, and subsequently showed microscopic hematuria without proteinuria. She was examined by a urologist, and urinalysis in his office showed some micropyuria but was negative for red blood cells. Endoscopic studies showed a severe chronic trigonitis that very well could be responsible for her cellular findings.

From 1977-97, she was examined by several physicians. She also had several surgical procedures performed on her (joint replacement surgery in 1988, multiple foot surgeries related to her arthritis, bilateral breast biopsies for benign disease, a total abdominal hysterectomy and bilateral salpingo-oophorectomy for endometriosis, and a tendon repair to her right hand). Her diagnoses in 1997 were rheumatoid arthritis, osteoporosis, Felty’s syndrome, chronic microhematuria and previous possible myasthenia gravis after using Penicillin.

A follow up examination in 2002 by a hematologist-oncologist indicated the presence of MGUS (IgG-kappa), leucopenia, pulmonary nodule from CT scan of the chest, and a decrease in her white blood count. The patient remained essentially asymptomatic (2002) and her physical examination was also noncontributory.

Hematological follow-up of the patient for her anemia, leukopenia, Felty’s Syndrome, MGUS was continued approximately on 6-9 months interval basis, and it was the opinion of the hematologist-oncologist that the patient remained hematologically stable without any significant changes. The SPE, serum quantitative immunoglobulins, and free kappa lambda chains in serum were monitored (2009-2010), but no significant change was noticed.

Date IgG Free Kappa Free Lambda Kappa/LambdaRatio

1/13/2009 1631 18.30 8.92 2.056/25/2009 1674 16.80 7.91 2.123/22/2010 1677 21.40 9.11 2.35Reference Value

694-1618 (mg/dL) 3.3-19.4(mg/L) 5.7-26.3 (mg/L) 0.26-1.65

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Protein Electrophoresis-Serum: Immunoglobulins-Serum:Total Protein: 7.4 (6.4-8.3 g/dL) IgG: 1677 (751-1560 mg/dL) Albumin: 4.2 (3.8-5.8 g/dL) IgA: 83 (82-453 mg/dL) Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 102 (46-304 mg/dL) Alpha-2 Globulin: 0.8 (0.4-0.9 g/dL) Beta Globulin: 0.8 (0.5-1.1 g/dL)Gamma Globulin: 1.4 (0.5-1.3 g/dL)

Discussion: During the past decade, rheumatologists at our institutions have noticed an increase in the rheumatoid arthritis (RA) patients, and this trend was also

observed at some other Americana and European medical centers. In general rheumatic diseases display polyclonal B-cell activation, high concentrations of autoimmune antibodies and polyclonal elevations of serum immunoglobulins. Occasionally, patients with rheumatoid arthritis were presented with a monoclonal band in serum, and thus a follow-up by a hematologist-oncologist was triggered,

thus contributing to the increase in health care costs of RA.b Hoffman et al, found elevated free light chains in rheumatic diseases as compared to blood donors, but in all these cases the kappa / lambda chains ratio in serum were normal, indicating

polyclonal synthesis.c In another study by Gottenberg et al, involving 50 patients with RA, 36% had elevated serum light chains but normal serum light chain ratios

except in three cases.d Even though in this case the light chains ratio in serum was slightly increased but constant over the monitoring period (15 months), thus indicating any active hematologic process.

a. Myasoedova E, Crowson CS, Maradit Kremers H, Therneau TM, Gabriel SE. Is the Incidence of Rheumatoid Arthritis Rising? Results from Olmsted County, Minnesota, 1955-2007. Arthritis Rheum 2010; 62: 1576-82.

b. Birnbaum H, Pike, C, Kaufman R, Maynchenko M, Kidolezi, Cifaldi M. Social cost of rheumatoid arthritis patients in U.S. Curr Med Res Opin 2010; 26: 77-90.

c. Hoffman U, Opperman M, Kuchler S., Ventur Y, Teuber W, Michels H, Welcker J, Landenberg PV, Helmke K. Free immunoglobulin light chains in opatients with rheumatic diseases. Z Rheumatol 2003; 62, Suppl.1: Pfr-40, p1051.

d. Gottenberg JE, Aucouturier F, Goetz J, Sordet C, Jhan I, Busson M, Cayuela J-M, Sibilia J, Mariette X. Serum immunoglobulin free light chain assessment in rheumatoid arthritis and primary Sjogren’s syndrome. Ann Rheum Dis 2007; 66: 23-27.

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Section H: Unknown Cases

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Case #29 Unknown Diagnosis: Please make the diagnosis on the basis of the information provided.

Diagnostic Possibilities: a) IgM Waldendtrom’s Macroglobulinemia, b) IgM-Related Disorder or IgM Monoclonal Gammopathy of Undetermined Significance, c) Plasma Cell Dyscrasia with a separate IgM Monoclonal Gammopathy, d) Probably Pre-Lymphoplasmacytic Lymphoma, e) Splenic Marginal Zone Lymphoma,a,b,c f) Large Cell Lymphoma, g) ?

64 years old male with symptoms of dizziness, loss of balance, fatigue, leg edema, and previous history of Hepatitis C (genotype 2b) was examined at the out-patient clinic, and was found to have severe hyponatremia. Patient was admitted to the hospital and the following abnormal laboratory results were noticed:

Hemoglobin: 10.2 (12-18 g/dL), Hematocrit: 30.4 (36.0-54.0%), RBC: 2.77 (4.0-5.5 M/uL), MCV: 109.7 (80-94 fL), MCH: 36.7 (27-31 pg), Platelet: 57 (130-400 Th/UL), Sodium: 103(135-145 mmol.L), Potassium: 3.0 (3.5-5.4 mmol/L, Chloride: 75 (98-109 mmol/L), CO2 Content: 19 (23-34 mmol/L), Protime: 22.2 (11.3-14.5 sec), APTT: 55.1 (22.5-35.0 sec), Fibrinogen: 62 (245-498 mg/dL), Aldosterone-serum: 2.5 (supine: 16.0 ng/dL or less) Renin Activity: 0.1 (supine: 0.2 -1.6 ng/ml/Hr).

Peripheral Blood Smear Review by the Pathologist:1) Hyporegenerative, macrocytic anemia 2) Platelets decreased 3) Neutrophils morphologically normal 4) Lymphopenia 1+ Toxicity

Bone-Marrow Aspirate: Microscopic Examination: 3-4% plasma cells. Non-Diagnostic. No evidence of tumor, lymphoma or granuloma.Flow Cytometry: Small population of monoclonal plasma cells (approximately 1% of the leucocytes) consistent with a plasma cell dyscrasia. There is no evidence of acute leukemia or lymphoproliferatiive disorder.


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Viscosity, Serum 6.09 1.10-1.8 cPACTH 14 7-69 pg/mLCortisol-Serum 1.2 4.0-20.0 ug/dL

Haptoglobin Less than 20 30-200 mg/dLSed Rate 60 0-20 mm/HrBeta-2 Microglobulin 2.7 1.1-2.4 mg/dLFree Kappa Chains-Serum 3.32 0.33-1.94 mg/dLFree Lambda Chains-Serum 2.54 0.57-2.63 mg/dLFree Kappa/Lambda Ratio 1.31 0.26-1.65

Protein Electrophoresis-Serum Immunoglobulins: *

Total protein: 8.8 (6.4-8.3 g/dL) IgG: 2385 (751-1560 mg/dL)Albumin: 2.6 (3.8-5.8 g/dL) IgA: 1075 (82-453 mg/dL)Alpha-1 Globulin: 0.2 (0.1-0.2 g/dL) IgM: 4220 (46-304 mg/dL)Alpha-2 Globulin: 0.6 (0.4-0.9 g/dL) Beta Globulin: 0.6 (0.5-1.1 g/dL) Gamma Globulin: 4.8 (0.5-1.3 g/dL)Serum Immunofixation: IgM-Kappa monoclonalgammopathy

In April 2003, the Consensus Panel Recommendations from the Second International Workshopd on Waldenstrom’s Macroglobulinemia (WM), held in Athens, Greece (September 2002) for the diagnosis of WM were:

IgM monoclonalgammopathy of any concentration Bone marrow infiltration by small lynphocytes showing plasmacytoid/plasma

cell differentiation Inter-trabecular pattern of bone marrow infiltration Surface IgM+, CD10-, CD19+, CD20-, CD22+, CD23+, CD25+,

CD27+,FMC7+,CD103-, CD138- immunotype (variations from this immunophenotypic profile can occur)

In view of the above stated criteria the diagnosis of IgM WM is ruled out in this patient. It is also obvious that this patient does not have IgM multiple myeloma (which is very rare, please see case # 17).

The Paneld also considered it to be inappropriate to define an IgM concentration to distinguish MGUS from WM, however it was also pointed out that IgM concentration rarely if ever exceeds 3g/dL in MGUS. This patient had IgM of 4.2 g/dL.

The Paneld proposed a term IgM-related disorders, in view of the fact that these patients may have symptoms (such as cryoglobulinemia, amyloidosis, or autoimmune phenomena such as peripheral neuroparthy, cold agglutinin disease.

*Assay performed after 1:5 dilution of serum with normal saline.

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etc.) attributable to the IgM monoclonal protein, but no overt evidence of lymphoma. This patient does not meet the criteria for the proposed IgM-related disorder.

In summary, patients will be considered to have WM, whenever there is unequivocal bone marrow infiltration by lymphoplasmacytic lymphoma, conversely patients without such evidence of infiltration will be considered to have MGUS as per the recommendations of Panel.d

a Novara F, Arcaini L, Merli M, et al. High-resolution genome-wide array comparative hybridization in splenic marginal zone B-cell lymphoma. Hum Pathol 2009; 40: 1628-37.

b De Renzo A, Perna F, Persico M, et al. Excellent prognosis and prevalence of HCV infection of primary hepatic and splenic non-Hodgkin’s lymphoma. Eur J Haematol 2008; 81(1): 51-7.

c Matutes E, Oscier D., Montalban C, et al. Splenic marginal zone lymphoma proposals for a revision of diagnostic, staging and therapeutic criteria. Leukemia 2008; 22(3): 487-95

d Owen RG, Treon SP, Al-Katib A, Fonseca R, Greipp PR, McMaster ML, Morra E, Pangalis GA, San Miguel JF, Branagan AR, Dimopoulos MA. Clinicopathological definition of Waldenstrom’s Macroglobulinemia: Consensus panel recommendations from the second international workshop on Waldenstrom’s Macrglobulinemia. Sem in Onc 2003; 30:110-115.

Additional 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

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Protein Electrophoresis, Immunofixation, and Immunodisplacement in Clinical Diagnosis. 2nd Edition,...

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