(04-06-2009 10:02A.M.)

Comparison of Agarose Gel Serum Protein Electrophoresis and Immunofixation with

Capillary Electrophoresis and Immunotyping

A Compendium of Selected Cases by

Zia Uddin, Ph.D., Consultant, Clinical Chemistry,

St. John Macomb-Oakland Hospital, Warren, Michigan 48093

This compendium is dedicated to the nuns, who painted houses in Detroit during the summer break,

and founded the St. John Hospital, Detroit, Michigan

Contents Introduction 1 Selected Case Studies:

Section A: Case # 1 Normal Person 8

Case # 2 Restricted band superimposed 11 on C3 complement band

Case # 3 Atypical shape of the Beta1-Beta2 14 region in the capillary zone

electrophoresis, and heavy staining of the beta1 region (transferrin) in agarose gel electrophoresis

Section B: Acute-Phase Reaction Pattern 17

Case # 1 Acute-phase reaction pattern with 17

monoclonal gammopathy

Section C: Double Gammopathy and Biclonal Gammopathy 20

Case # 1 Double gammopathy (IgG-Lambda 20 and IgM-Lambda)

Case # 2 Biclonal gammopathy (IgM-Kappa 23 and IgM-Lambda)

Case # 3 Biclonal gammopathy (IgM-Lambda 26 and IgG-Kappa)

Case # 4 Double Gammopathy (IgA1 – Kappa 29

and IgA2 – Kappa)

(i)

Section D: IgA Monoclonal Gammopathies 32

Case # 1 IgA monoclonal band masking the 33 beta globulin region

Case # 2 IgA monoclonal band cathodal to C3 36 complement

Section E: IgM Monoclonal Gammopathies 39

Case # 1 IgM monoclonal gammopathy in 39 multiple myeloma

Case # 2 IgM monoclonal gammopathy in 42 malignant lymphoma (IgM-Kappa)

Case # 3 IgM monoclonal gammopathy (MGUS) 45 without any clinical symptoms (IgM-Kappa)

Section F: IgG Monoclonal Gammopathies 48

Case # 1 IgG-Kappa multiple myeloma 48 Case # 2 IgG-Kappa monoclonal gammopathy 51

in prostate cancer patient with bone metastasis

Case # 3 IgG-Lambda monoclonal gammopathy 54 of undetermined significance (MGUS)

Case # 4 IgG-Lambda monoclonal gammopathy 57 of undetermined significance (MSUG)

Case # 5 IgG-Kappa monoclonal gammopathy 60 with persistent anemia and elevated serum ferritin

Case # 6 IgG-Kappa monoclonal gammopathy 63 without manifestation of myeloma from the bone marrow and clinical symptoms

Case # 7 IgG-Lambda monoclonal gammopathy 66 (MGUS)

(ii)

Section G: Hypogammaglobulinemia and 69

Hypergammaglobulinemia Case # 1 Polyclonalgammopathy 69 Case # 2 Hypogammaglobulinemia 72

Case # 3 Apparent hypogammaglobulinemia 75 with monoclonal gammopathy

Section H: Oligoclonal Bands in Serum Gammaglobulin 78

Region

Case # 1 Oligoclonal bands without any 78 clinical significance

Section I: Light Chain Multiple Myeloma 81 Case # 1 Lambda Chains Myeloma 81

Case # 2 Kappa Chains Myeloma 84

Section J: Cryoglobulins 87 Case # 1 Brouet Type I 87

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(04-06-2009 10:04A.M.)

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

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

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

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

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

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

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Track # 1 Serum Protein Electrophoresis-six fractions Track # 2 Immunoglobulin G Track # 3 Immunoglobulin A Track # 4 Immunoglobulin M Track # 5 Kappa Chains Track # 6 Lambda Chains Capillary Zone Electrophoresis: The proteins in serum were separated into six fractions using a liquid-based system (Sebia CAPILLARYS), without the use of a solid support medium i.e., agarose gel. In this method a fused silica capillary (into which a small volume of serum is aspirated) with strong negative charge on the interior of the capillary provided a large negative surface area. Under the conditions of electrophoresis (movement of protein molecules under charge) and endosmotic flow of cations toward the cathode, the proteins were separated into six fractions, and quantified by an ultraviolet detector using the absorbance of the amide band of the proteins at 200-215 nm. All the serum CZE scans wherever illustrated in this compendium are in green color. Immunotyping of Serum Proteins: The monoclonal protein present in serum was detected and identified by an antigen-antibody reaction in liquid phase followed by CZE using the Sebia CAPILLARYS. In this automated methodology the specific immunoglobulin and the antisera react to provide an antigen-antibody complex (pretreatment step). The pretreated samples (reference, IgG, IgA, IgM, kappa and lambda) were electrophoresed simultaneously in six capillaries. The antigen-antibody complexes migrated slowly than an unbound immunoglobulin, i.e., migrated anodically to albumin, or migrated as an increased baseline co-migrating with albumin, alpha-1 and/or alpha-2 globulin. This procedure is obverse of immunofixation (see above), as the monoclonal proteins were removed and thus not detected on the electropherogram after CZE. The interpretation (identification of the monoclonal band) was made by the “overlay” of the untreated reference electrophoretic curve on each of the five electrophoretic curves of the pretreated samples. All the serum CZE scans after the each pretreatment step (imunosubtraction process with anti-IgG, anti-IgA, anti-IgM, anti-kappa, and anti-lambda) wherever illustrated in this compendium are in red color.

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Selection of Patients for Case Studies: We performed serum protein electrophoresis on over 5000 patients over a period of nine months. All the sera having either abnormal band or atypical pattern were sequestered. The patient’s medical record was reviewed to obtain clinical information. For each case we have presented in this compendium, a brief medical history, prior diagnosis and the diagnosis after hospitalization (if applicable), and any other information that may be helpful in the understanding of the electrophoretic patterns. First the serum protein electrophoretic (agrose gel) scan, and serum immunofixation (agarose gel) picture were presented. We also presented a picture of the electrophoretic separation on the agarose gel. The five fractions of serum (albumin, alpha-1, alpha-2, beta, and gamma globulin) in gram/dL along with the quantified immunoglobulins were also provided. An enlarged version of the serum protein CZE scan was also presented separately for interpretative purposes. Finally, the serum protein electrophoresis pattern obtained from the automated CZE method (Sebia CAPILLARYS) was presented six different times on a separate page. One of these scan was used as a reference. On the remaining five scans the electrophoretic pattern after the antigen-antibody pretreatment reaction of the serum with each of the five immunoglobulins (IgG, IgA, IgM, kappa and lambda chains) was electronically superimposed for the indentification of monolclonal bands. In some cases the identification of the mini-monoclonal band was facilitated by the electronic magnification of the area under study of the electrophoretic pattern. Comments were made in some cases in order to explain the laboratory results otherwise the scans of protein electrophoresis, immunofixation and immunotyping were obvious to interpret.

Detection and Identification of Monoclonal Bands: The majority of the readers of this compendium are familiar with the interpretation of the serum protein electrophoresis and immunofixation patterns. The interpretation of the serum protein electrophoresis by CZE is virtually identical to the agarose gel serum protein electrophoresis, and thus needs no further comments. The detection of protein bands with restricted mobility by immunotyping needs careful evaluation of the five individual CZE scans of the pretreated samples ( anti-IgG, anti-IgA, anti-IgM, anti-kappa, and anti-lambda) in conjunction with CZE scan of the neat serum.

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In order to familiarize with the salient features of electrophoresis, immunofixation electrophoresis, immunotyping based on CZE either after immunosubtraction using antibodies coated to Sepharose beads (Beckman Coulter Paragon CZE 2000), or after immunosubtraction using liquid phase antigen-antibody reaction (Sebia CAPILLARYS), the reader is advised to review the first three chapters of the “Protein Electrophoresis in Clinical Diagnosis” by David F. Keren, MD , 2003 Edition (Arnold, a member of the Holder Headline Group, Great Britain), ISBN 0340 812133. The interpretation of the immunotyping patterns requires the visualization of the subtle changes in the shape of the electrophoretic scans after the antigen-antibody reaction. A large monoclonal band (M-protein greater than 100 mg/dL) can be accurately identified without much difficulty. It is the minimonoclonal bands (M-protein less than 25 mg/dL) that could not be identified easily due to not so obvious large changes in the shape of the electrophoretic curve, especially if the minimonoclonal band is either embedded with a polyclonal gamma globulin, transferrin, complement C3, or associated with an artifact (fibrinogen, contrast media, etc.). First, let us examine the symmetrical changes in the shape of the electrophoretic curve after the immunosubtraction step in a normal person. Please see the hypothetical figure No A.

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The difference between the two curves (green minus red) is plotted in violet color. This violet colored curve is plotted only to exhibit symmetrical polyclonal immunoreduction process in a normal person, and one will never notice it on the immunotyping scans of any of the five tracks (anti-IgG, anti-IgA, anti-IgM, anti-kappa, and anti-lambda). A smooth and symmetrical shaped curve after the immunoreduction step (red curve) indicates a polyclonal reduction of the immunoglobulins from the serum. This kind of smooth and symmetrical shaped curve (red color) is most commonly seen in a normal person after the liquid phase reaction with anti-IgG. Uniform immunoreduction in the green curve, Fig No. A, cannot be construed as a monoclonal band, as polyclonal immunosubtraction results in the reduction of the gamma globulin fraction in a symmetrical manner. In a normal person the serum IgA is lower than the serum IgG, therefore a very small difference is observed between the serum CZE scan and the serum CZE scan after the anti-IgA reaction. This immunoreduction after reaction with anti-IgA is primarily witnessed in the beta-globulin region. Similarly in a normal person the serum IgM is even lower than IgA, therefore again a very little reduction without change of the symmetry of the fraction is observed (virtually mirror image of each other).

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The situation in case of kappa and lambda chains is slightly different. Kappa chains are normally present in an approximate 2:1 ratio to lambda chains. Therefore in a normal person after liquid phase reaction with anti-kappa, one should expect 2/3 reduction in the gamma region. Obversely, in a normal person after liquid phase reaction with anti-lambda, one should expect 1/3 reduction in the gamma region. The magnitude of the reduction in the gamma globulin region for both the kappa and lambda chains is very low as compared to the IgG, and this is obviously due to the high concentration of serum IgG as compared to kappa and lambda chains in serum. Let us consider another hypothetical case with a protein restriction (monoclonal band) in the gamma globulin region (Fig No. B).

It is pointed out that in this hypothetical case of monoclonal band detected in the gamma globulin region from CZE (green color, Fig No. B), there is also a symmetrical shaped immunoreduction of the gamma globulin region after the reaction with anti-IgG (red curve, Fig No. B). The difference between the green and the red curve is illustrated by violet colored curve, thus indicating a sharp monoclonal

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peak. It is again pointed out that in actual practice the violet colored curve will never be depicted on the CZE scan after the immunosubtraction step.

Assignment of monoclonal bands: The first step is the visual examination of serum CZE scan to note the presence of, a) sharp peak, b) unsymmetrical band (from the alpha-2 to the farthest cathode end of gamma globulin region), c) distortion (similar to electrical noise of a signal) of the shape of any of the alpha-2, beta-1, beta-2, and gamma globulin bands, and d) decreased gamma globulin. A decreased gamma globulin band also suggests the electrophoretic migration of the possible restricted immunoglobulin towards the anode, therefore one might observe unsymmetrical bands for any of the alpha-2, beta-1, and beta-2 globulin bands. The location of the abnormal / unsymmetrical band on the CZE scan must be kept in mind while comparing it with the five pretreated (anti-IgG, anti-IgA, anti-IgM, anti-kappa, and anti-lambda) CZE scans. Generally speaking (except in a very rare case of heavy chain disease associated with gamma, alpha, or mu chains), any abnormal / unsymmetrical disappearance of the immunoglobulin fraction (IgG, IgA, IgM) in the liquid phase immunosubtraction step followed by CZE must be associated with a concomitant abnormal / unsymmetrical disappearance of either kappa or lambda chains or both. Also the position (electrophoretic mobility) of the abnormal / unsymmetrical heavy chain and the light chain must be the same on the CZE scan (after immunosubtraction step) in order to correctly assign the monoclonal band. There are rare cases in which abnormal / unsymmetrical disappearance of the light chain is observed after the immunosubtraction step without any concomitant subtraction of the IgG, IgA, and IgM. In these cases the laboratory must rule out the possibility of either IgD or IgE monoclonal gammopathy, and also the possibility of light chain gammopathy.

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Selected Case Studies: Section A: Case # 1 Normal Person Case # 2 Restricted band superimposed on C3 complement band Case # 3 Atypical shape of the Beta1-Beta2 region in the capillary zone electrophoresis,

and heavy staining of the beta1 region (transferrin) in agarose gel electrophoresis

Hint: The presence of fibrinogen in serum due to any reason, e.g. patient on

heparin therapy, improper processing of specimen, etc., can mimic the pattern of monoclonal gammopathy.

Case # 1 Normal Person

Serum of a 28 years old male employee, presented for annual physical examination. No abnormality observed in the laboratory tests and examination by the physician.

Total Protein: 7.5 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 4.5 g/dL (3.8-5.8 g/dL) IgG: 902 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 208 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 81 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.2 g/dL (0.5-1.3g/dL)

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Case # 2 Restricted band superimposed on C3 complement band 51 years male admitted with hypotension, acute renal failure,

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

Total Protein: 2.9 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 1.3 g/dL (3.8-5.8 g/dL) IgG: 786 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 208 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.4 g/dL (0.4-0.9 g/dL) IgM: 32 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.5 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.5 g/dL (0.5-1.3g/dL) Comments: The intense band detected in both the agarose gel and capillary zone electrophoresis (superimposed on the C3 complement band position) is an artifact, perhaps due to fibrinogen, as no monoclonal band was detected either by immunofixation or immunotyping. We did not confirm it by reacting the patient’s specimen with anti-fibrinogen. In view of the high intensity of this band, we believe that the patient sample was most likely plasma and not serum. A follow up inquiry confirmed that indeed it was plasma specimen.

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NOTE: The apparent increase in β-2 globulin was persistent as indicated by an arrow, perhaps due to fibrinogen. Similar bands in the β-2 globulin region as an artifact due to fibrinogen were reported in the CZE (Xavier Bossuyt, et at, Automated serum protein electrophoresis by Capillarys, Clin Chem Lab Med 2003; 41:704-710).

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Case # 3 Atypical shape of the Beta1-Beta2 region in the capillary zone

electrophoresis, and heavy staining of the beta1 region (transferrin) in agarose gel electrophoresis.

76 years old female with severe symptomatic anemia (hemoglobin of

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

Total Protein: 5.8 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.1 g/dL (3.8-5.8 g/dL) IgG: 701 mg/dL(751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 668 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 170 mg/dL ( 46-304 mg/dL) Beta Globulin: 1.2 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.6 g/dL (0.5-1.3g/dL) Comments: The serum protein electrophoresis by agarose gel indicated increased concentration of the beta1 globulin region (suggesting increased transferrin due to serum iron deficiency as the patient is severely anemic). Another possibility of increased transferrin may be double transferrin bands due to genetic reasons in some Afro-Americans. The third possibility is of embedded monoclonal band under the transferrin band. The serum protein electrophoresis by capillary zone electrophoresis indicated asymmetrical beta1-beta2 globulin region with a minor restriction on the cathode side of the C3 complement band. It is obvious from the study of Cases # 2- 4 that fibrinogen can be the possible cause of the atypical electrophoretic pattern. A monoclonal IgA band is known to migrate between alpha-2 globulin to any place in the gamma globulin region. Indeed immunofixation and immunotyping indicated the presence of monoclonal IgA-kappa in serum of this patient. Therefore it is imperative to perform immunotyping or immunofixation of serum, whenever a minor restriction band or atypical shape of the fractions are observed on electrophoresis in the area of beta 1-beta 2 globulin region.

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NOTE: IgA-Kappa monoclonal band after the immunoreduction step is indicated by an arrow.

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Section B: Acute-Phase Reaction Pattern The serum protein electrophoresis is a useful tool to detect acute

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

Case # 1 Acute-phase reaction pattern with monoclonal gammopathy 63 years old male with a known history of anemia, nephrotic

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

Total Protein: 4.3 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 0.9 g/dL (3.8-5.8 g/dL) IgG: 924 mg/dL(751-1560 mg/dL) Alpha-1 Globulin: 0.3 g/dL (0.1-0.2 g/dL) IgA: 280 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 1.0 g/dL (0.4-0.9 g/dL) IgM: 401 mg/dL ( 46-304 mg/dL) Beta Globulin: 1.0 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.1 g/dL (0.5-1.3g/dL) Comments: The serum electrophoretic pattern by both the agarose gel and the capillary zone electrophoresis depicts features of acute-phase reaction pattern. In view of the fact that gamma globulin concentration though not increased but greater than albumin in serum, this pattern suggested active, chronic inflammation.

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NOTE: The difference between the red and green curve for the IgM and Kappa chain is not symmetrical (indicated by an arrow), thus suggesting monoclonal band.

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

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

Double gammopathy: If the light chains identified by either immunofixation or immunotyping are the same, i.e. both are either “kappa” or “Lambda” then it is called “double gammopathy” as these light chains originate from a same clone. The presence of the heavy chain can be the same or different in either the double or biclonal gammopathy. It is pointed out that the clinical course and treatment is the same for biclonal and double gammopathies.

Case # 1 Double gammopathy (IgG-Lambda and IgM-Lambda) 63 years old female under chemotherapy for a previously known case

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

Total Protein: 6.4 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.2 g/dL (3.8-5.8 g/dL) IgG: 1940 mg/dL(751-1560 mg/dL) Alpha-1 Globulin: 0.1 g/dL (0.1-0.2 g/dL) IgA: 165 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.6 g/dL (0.4-0.9 g/dL) IgM: 904 mg/dL ( 46-304 mg/dL) Beta Globulin: 1.6 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.9 g/dL (0.5-1.3g/dL)

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IgG IgM

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Case # 2 Biclonal gammopathy (IgM-Kappa and IgM-Lambda)

61 years old male admitted for urinary tract infection with renal dysfunction, and was discharged with the diagnosis of monoclonal gammopathy of undetermined significance (MGUS).

Total Protein: 8.4 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.9 g/dL (3.8-5.8 g/dL) IgG: 693 mg/dL(751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 132 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 3670 mg/dL ( 46-304 mg/dL) Beta Globulin: 1.8 g/dL (0.5-1.1 g/dL) Gamma Globulin: 2.8 g/dL (0.5-1.3g/dL)

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IgM-Kappa IgM-Lambda

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

lymphoproliferative disorder (Waldenstrom’s macroglobulinemia). Total Protein: 7.0 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.9 g/dL (3.8-5.8 g/dL) IgG: 1120 mg/dL(751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 52 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.8 g/dL (0.4-0.9 g/dL) IgM: 745 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.7 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.4 g/dL (0.5-1.3g/dL)

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Case # 4 Double Gammopathy (IgA1 – Kappa and IgA2 – Kappa)

55 years old unconscious female admitted to the hospital for liver cirrhosis, asthma, hypertension, etc. Her kidney function was normal. Serum creatinine was 0.6 mg/dL (0.7 – 1.5 mg/dL), and the calculated

glomerular filtration rate (GFR) was 105 (> 60 ml/min/1.73 m2 ). Total Protein: 6.3 g/dL Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 2.9 g/dL (3.8 – 5.8 g/dL) IgG: 1300 mg/dL (751 –1560 mg/dL) Alpha-1 Globulin: 0.1 g/dL (0.1 – 02 g/dL) IgA: 1640 mg/dL (82 – 453 mg/dL) Alpha-2 Globulin: 0.6 g/dL (0.4 – 0.9 g/dL) IgM: 105 mg/dL (46 - 304 mg/dL) Beta Globulin: 1.4 g/dL (0.5 – 1.1 g/dL) Free κ 37.6 mg//L (3.3 – 19.4 mg/L) Gamma Globulin: 1.3 g/dL (0.5 – 1.3 g/dL) Free λ 20.7 mg/L (5.7 – 26.3 mg/L) Free κ /λ 1.82 (0.26 – 1.65) Comments: Serum protein electrophoresis (Sebia, HYDRASIS) showed a dark staining for C3 band, and another monoclonal band adjacent to the C3 band (cathode side). According to Feinstein et al (Nature 212: 1496-1498, 1966), IgA in

human beings is composed of two antigenically different subclasses IgA1 and IgA2,

and approximately 80-90% is IgA1 in serum (Gray et al., Exp. Med. 128: 1233-1236, 1969).

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Section D: IgA Monoclonal Gammopathies

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

Upon visual examination of the electrophoretic pattern, if any distortion of the beta globulin region is detected, it is advised to perform immunotyping or immunofixation.

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Case # 1 IgA monoclonal band masking the beta globulin region 67 years old male admitted to hospital and was diagnosed with anemia, acute renal failure, urinary tract infection, congestive heart failure, coronary artery disease, and gastrointestinal hemorrhage.

Positive for Bence Jones protein in urine. Total Protein: 6.8 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.1 g/dL (3.8-5.8 g/dL) IgG: 441 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 2410 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 43 mg/dL (46-304 mg/dL) Beta Globulin: 2.4 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.4 g/dL (0.5-1.3g/dL)

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Case # 2 IgA monoclonal band cathodal to C3 comlement

65 years old male with multiple medical problems (chronic renal failure, hypertension, coronary artery disease, non-ST elevation myocardial infarction, anemia, osteomyelitis, diabetes mellitus, GI bleed, hepatitis C) was admitted because of the shortness of breath and lower lobe pneumonia.

Total Protein: 5.6 g/dL (6.4-8.3 g/dL) Immunoglobulins Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 2.6 g/dL (3.8-5.8 g/dL) IgG: 1240 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.3 g/dL (0.1-0.2 g/dL) IgA: 549 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.8 g/dL (0.4-0.9 g/dL) IgM: 72 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.7 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.2 g/dL (0.5-1.3g/dL)

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Section E: IgM Monoclonal Gammopathies:

IgM monoclonal gammopathies when detected are generally associated with either plasma cell proliferations (IgM myeloma) or a low-grade B-cell lymphoplasmacytic disorder (Waldenstrom’s macroglobulinemia). In both the diseases the bone marrow is involved, however one major characteristic of the Waldenstrom’s macrglobulinemia is the absence of lytic skeletal lesions.

Case # 1 IgM monoclonal gammopathy in multiple myeloma

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

Total Protein: 7.1 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.9 g/dL (3.8-5.8 g/dL) IgG: 695 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 128 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.6 g/dL (0.4-0.9 g/dL) IgM: 1770 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.5 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.9 g/dL (0.5-1.3g/dL)

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Case # 2 IgM monoclonal gammopathy in malignant lymphoma (IgM-Kappa)

60 years old male with a past medical history of malignant lymphoma was examined after first round of chemotherapy and radiation for bone marrow and flow cytometric tests.

Total Protein: 6.7 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 4.2 g/dL (3.8-5.8 g/dL) IgG: 922 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.1 g/dL (0.1-0.2 g/dL) IgA: 195 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 373 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.8 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.9 g/dL (0.5-1.3g/dL)

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

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

Total Protein: 7.1 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 4.1 g/dL (3.8-5.8 g/dL) IgG: 1240 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 253 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.8 g/dL (0.4-0.9 g/dL) IgM: 355 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.1 g/dL (0.5-1.3g/dL)

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Section F: IgG Monoclonal Gammopathies

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

Case # 1 IgG-Kappa multiple myeloma

73 years old female with a past medical history of multiple myeloma was examined during her treatment and laboratory tests were performed.

Total Protein: 7.2 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 4.1 g/dL (3.8-5.8 g/dL) IgG: 1440 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 202 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.9 g/dL (0.4-0.9 g/dL) IgM: 22 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.8 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.2 g/dL (0.5-1.3g/dL)

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Case # 2 IgG-Kappa monoclonal gammopathy in prostate cancer patient with bone metastasis

79 years old male with known prostate cancer was admitted with severe anemia (hemoglobin 6.1), renal failure, and hypertension.

Total Protein: 6.8 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.8 g/dL (3.8-5.8 g/dL) IgG: 1160 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 158 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 1.0 g/dL (0.4-0.9 g/dL) IgM: 16 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.1 g/dL (0.5-1.3g/dL)

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Case # 3 IgG-Lambda monoclonal gammopathy of undetermined significance (MGUS)

85 years old female, known case of dementia was admitted to the hospital for multiple disorders (septicemia, renal failure, anemia, respiratory failure, etc.).

Total Protein: 6.2 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.2 g/dL (3.8-5.8 g/dL) IgG: 1200 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.3 g/dL (0.1-0.2 g/dL) IgA: 181 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.9 g/dL (0.4-0.9 g/dL) IgM: 59 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.8 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.0 g/dL (0.5-1.3g/dL)

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Case # 4 IgG-Lambda monoclonal gammopathy of undetermined significance (MSUG)

48 years old male was admitted to the hospital for a viral illness, and was placed on acylclovir but with no relief of his hyperpyrexia. After the antiviral therapy the patient was administered antibiotics, and the pulmonary symptoms were resolved. Several serological tests were performed during hospitalization, but all of them were negative.

Total Protein: 5.9 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 2.6 g/dL (3.8-5.8 g/dL) IgG: 1150 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.5 g/dL (0.1-0.2 g/dL) IgA: 202 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 1.0 g/dL (0.4-0.9 g/dL) IgM: 77 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.0 g/dL (0.5-1.3g/dL)

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Case # 5 IgG-Kappa monoclonal gammopathy with persistent anemia

and elevated serum ferritin 61 years old female with persistent anemia, weakness, multiple falling episodes, refractory lumbosacral pain, hypothyroidism, hypertension, pelvic ramus fracture in 2003 and sacral fracture in 2003 was examined by an oncologist, but no morphologic cause for the patient’s anemia was demonstrated. The mini-monoclonal band detected (indicated by the shaded area) at the most cathode part of the electrophoretic pattern was very faint in both the agarose and the capillary zone electrophoresis (approximately 0.08 gram/dL).

Total Protein: 5.0 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 2.4 g/dL (3.8-5.8 g/dL) IgG: 794 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.3 g/dL (0.1-0.2 g/dL) IgA: 146 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 1.1 g/dL (0.4-0.9 g/dL) IgM: 53 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.7 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.6 g/dL (0.5-1.3g/dL)

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Case # 6 IgG-Kappa monoclonal gammopathy without manifestation of

myeloma from the bone marrow and clinical symptoms 56 years old male with IgG-Kappa monoclonal gammopathy was examined for bone marrow aspirate, however the bone marrow features and other hematopathology assays did not meet the criteria of multiple myeloma.

Total Protein: 7.9 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 4.8 g/dL (3.8-5.8 g/dL) IgG: 1890 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.1 g/dL (0.1-0.2 g/dL) IgA: 149 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.6 g/dL (0.4-0.9 g/dL) IgM: 26 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.8 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.6 g/dL (0.5-1.3g/dL)

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Case # 7 IgG-Lambda monoclonal gammopathy (MGUS)

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

Total Protein: 6.4 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.5 g/dL (3.8-5.8 g/dL) IgG: 1270 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 307 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.8 g/dL (0.4-0.9 g/dL) IgM: 77 mg/dL ( 46-304 mg/dL) Beta Globulin: 1.0 g/dL (0.5-1.1 g/dL) Gamma Globulin: 1.0 g/dL (0.5-1.3g/dL)

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Section G Hypogammaglobulinemia and Hypergammaglobulinemia

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

Case # 1 Polyclonalgammopathy 68 years old female admitted for rectal bleeding, anemia

and infection Total Protein: 6.5 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.2 g/dL (3.8-5.8 g/dL) IgG: 2090 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.1 g/dL (0.1-0.2 g/dL) IgA: 446 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.5 g/dL (0.4-0.9 g/dL) IgM: 119 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.6 g/dL (0.5-1.1 g/dL) Gamma Globulin: 2.1 g/dL (0.5-1.3g/dL)

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Case # 2 Hypogammaglobulinemia

84 years old female with several previously known clinical conditions was admitted to the hospital for acerbating weakness and chronic obstructive pulmonary disease.

Total Protein: 4.6 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 2.3 g/dL (3.8-5.8 g/dL) IgG: 426 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.3 g/dL (0.1-0.2 g/dL) IgA: 40 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 1.0 g/dL (0.4-0.9 g/dL) IgM: 16 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.6 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.4 g/dL (0.5-1.3g/dL)

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Case # 3 Apparent Hypogammaglobulinemia with monoclonalgammopathy

65 years old female previously diagnosed for multiple myeloma was checked for clinical response for chemotherapy, and serum protein electrophoresis along with quantitative immunoglobulins assay was performed on out-patient basis.

Total Protein: 7.1 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 4.4 g/dL (3.8-5.8 g/dL) IgG: 1170 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 34 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 45 mg/dL ( 46-304 mg/dL) Beta Globulin: 1.5 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.2 g/dL (0.5-1.3g/dL) Comment: In this patient though the gammaglobulin region appears to be low, it is an

aberration as the monoclonal gammaglobulin band (IgG-Kappa) migrates with C3 complement. In this manner the concentration of C3 complement and the total beta1-beta2 region appears to be increased, which in fact is not.

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Section H: Oligoclonal Bands in Serum Gammaglobulin Region

The term “oligo” means few (more than one). The gammaglobulin region of the serum protein electrophoresis very rarely exhibits oligoclonal bands of different intensities spaced at equal distance or crowded very close to each other. Sometimes one band is very prominent as compared to the other bands. In general there is polyclonal increase in the serum gammaglobulins, and the concomitant presence of oligoclonal bands are observed in few clinical conditions, e.g. chronic infection, autoimmune diseases and less frequently in lymphoproliferative processes. In this situation immunotyping and/or immunofixation is helpful in the interpretation of these bands. In cases the clinical condition of the patient requires the diagnosis of light chain multiple myeloma, it is suggested to perform urine protein electrophoresis and immunofixation or alternatively the assay of free kappa and lambda chains in serum.

Case # 1 Oligoclonal bands without any clinical significance

79 years old female with known peripheral neuropathy was evaluated to rule out lumbar radiculopathy (EMG was negative for radiculopathy). She was having some gait dysfunction and parathesia in her feet. The bone X-ray and CT scan were negative. A few laboratory tests were slightly abnormal (BUN, creatinine, PTT, WBC, and Vitamin B12), but were not diagnostic of any settled clinical condition.

Total Protein: 6.3 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.5 g/dL (3.8-5.8 g/dL) IgG: 763 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 107 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 1.1 g/dL (0.4-0.9 g/dL) IgM: 59 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.6 g/dL (0.5-1.3g/dL) Comments: The oligoclonal bands (indicated by dashed lines) were detected in both the agarose gel and capillary zone electrophoresis, however, they were not present on the immunofixation plate and the immunotyping scan. The laboratory report was signed off (without any prior access to the clinical and other laboratory data), a) oligoclonal bands present perhaps due to immune complex formation, and b) Suggest urine protein electrophoresis to rule out Bence Jones proteinuria.

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Section I: Light Chain Multiple Myeloma Case #1 Lambda Chains Myeloma

72 years old male (previously known case of light chain multiple myeloma for more than three years), was admitted to the hospital due to renal failure, fever, and urinary tract infection. In the most recent serum specimen, there was no evidence of a monoclonal band from agarose gel electrophoresis. The shape of the C3 complement band was not symmetrical, but diffuse and slanted, and this triggered serum immunofixation studies. The initial laboratory interpretation provided to the physician was: " Acute inflammation pattern, possibility of monoclonal band." "Final interpretation to follow after immunofixation studies." Serum immunofixation studies indicated a monoclonal band due to free lmbda chains, and no precipitation band was detected for any of the heavy chains (IgG, IgA, IgM, IgD, and IgE). The immunofixation results using the anti-IgD and anti-IgE are not displayed on next page.

Total Protein: 5.8 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 2.9 g/dL (3.8-5.8 g/dL) IgG: 522 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.4 g/dL (0.1-0.2 g/dL) IgA: 29 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 1.2 g/dL (0.4-0.9 g/dL) IgM: 6 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.4 g/dL (0.5-1.3g/dL)

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Case #2 Kappa Chains Myeloma 69 years old male brought to the emergency room of the hospital with abdominal pain, ileus, fever, anemia, and weakness in lower extremities. Patient was admitted and oncologist was consulted. Serum protein electrophoresis (agarose gel) indicated hypogammaglobulinemia and a very faint monoclonal band (identified as free kappa chains from immunofixation). There was no evidence of monoclonal heavy chains (lgG, lgA, lgM, lgD, and lgE) from immunofixation studies. Bone marrow examination showed 42% plasma cells. Total Protein: 5.8 g/dL (6.4-8.3 g/dL) Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 3.7 g/dL (3.8-5.8 g/dL) IgG: 234 mg/dL (751-1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 23 mg/dL (82-453 mg/dL) Alpha-2 Globulin: 1.1 g/dL (0.4-0.9 g/dL) IgM: 5 mg/dL ( 46-304 mg/dL) Beta Globulin: 0.6 g/dL (0.5-1.1 g/dL) Gamma Globulin: 0.3 g/dL (0.5-1.3g/dL) Free Kappa = 901 mg/L (3.3 – 19.4 mg/L) Free Lambda = 15.0 mg/L (5.7 – 26.3 mg/L) Kappa/Lambda Ratio = 60 (0.3 – 1.2)

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Section I: Case #2

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Section J: Cryoglobulins Agarose gel serum protein electrophoresis is not the procedure for the detection of the cryoglobulins, however in some cases due to the employment of cooler temperatures, one might observe a precipitate at the application point / abnormal electrophoretic pattern. This formation of the precipitate or abnormal pattern triggers further investigation, e.g., serum immunofixation. It is the observation of several lanes with precipitate upon imuunofixation (and repeat immunofixation upon dilution of the serum and also replacement of one of the antisera with buffer or saline), that alerts the laboratorian about the possibility of cryoglobulins. It is emphasized that the confirmation of cryoglobulins in serum requires establishment of the cryocrit, and immunofixation of the washed cryoglobulin. The assay for Hepatitis C virus antibody, rheumatoid factor and complement C4 for other diagnostic reasons are also recommended. Conversely the CZE and the immunotyping by the Sebia System is performed at 35.5o C, thus the cryoglobulins remain dissolved and do not precipitate, thus the presence of cryoglobulins are eluded. Quantitative analysis for IgG, IgA, igM, Free κ, Free λ were performed at 37o C, therefore accurate results were obtained. Case # 1 Brouet Type I

68 year old male, with a known history of several clinical disorders, e.g., hypentension, anemia, weakness in the legs, deep venous thrombosis, etc. was admitted to the hospital. Negative for Hepatitis C.

Total Protein: 7.7 g/dL Immunoglobulins: Agarose gel electrophoresis: Beckman Coulter IMMAGE: Albumin: 4.2 g/dL (3.8 – 5.8 g/dL) IgG: 1480 mg/dL (751 –1560 mg/dL) Alpha-1 Globulin: 0.2 g/dL (0.1 – 02 g/dL) IgA: 160 mg/dL (82 – 453 mg/dL) Alpha-2 Globulin: 0.8 g/dL (0.4 – 0.9 g/dL) IgM: 943 mg/dL (46 - 304 mg/dL) Beta Globulin: 1.2 g/dL (0.5 – 1.1 g/dL) Free κ 56.9 mg//L (3.3 – 19.4 mg/L) Gamma Globulin: 1.4 g/dL (0.5 – 1.3 g/dL) Free λ 31.3 mg/L (5.7 – 26.3 mg/L) Free κ /λ 1.82 (0.26 – 1.65)

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Comments: The serum protein electrophoresis by agarose gel electrophoresis (HYDRASYS) indicated three restrictions. Serum immunofixation indicated precipitate lanes in all five sectors (serum protein electrophoresis, IgG, IgA, IgM, kappa, and lambda). Repeat serum immunofixation after 1:5 dilution of the serum again indicated the precipitate lanes in all the five areas. The CZE at 35.5o C indicated no such phenomenon and the elctrophoretic pattern indicated a monoclonal band in the gamma globulin region. Immunotyping indicated a distinct IgM-Kappa monoclonal band.

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