SPEP UPEP Interp

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AN ATLAS OF PROTEIN ELECTROPHORESIS Serum, Urine, Cerebrospinal Fluid Donald R. Hoffman, Ph.D. Department of Pathology and Laboratory Medicine East Carolina University Brody School of Medicine Greenville North Carolina

Copyright © 2002, 2006, Donald R. Hoffman

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This book is dedicated to the memory of Doctor Seymour Bakerman. Dr. Bakerman was the founding Chairman of the Department of Pathology at East Carolina University School of Medicine. He was an outstanding teacher, who received numerous awards from his students, both for the quality of his teaching and for his genuine concern for them as people. In all respects Dr. Bakerman was a true "Mensch." His knowledge of clinical chemistry was unparalleled and his ability to transmit this knowledge unrivaled. This work was begun at his urging and with his support and assistance.

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Introduction Although there are a number of books available that discuss and give examples of protein electrophoresis, there is not a comprehensive series of patterns published. This book contains scans of electrophoresis gels from a variety of diseases, both common and rare, along with some discussion of the abnormalities and some of the important features of the cases. All of the patterns and cases are actual cases from Pitt County Memorial Hospital and East Carolina University School of Medicine, but in a few of the examples some general information or data has been added to complete the case. Electrophoreses were run on Beckman Paragon, Paragon II or HRE gels and scanned on Beckman Densitometers. The scans are shown, since we emphasize the use of quantitative as well as qualitative data. We feel it is important to look at both the original stained gel and densitometric scan, as well as any other relevant laboratory data, before interpreting electrophoresis patterns. When appropriate, immunoelectrophoresis or immunofixation electrophoresis patterns are shown. The choice between these techniques was based primarily upon the date the test was run. Our laboratory has now switched completely to immunofixation electrophoresis. I would like to thank Dr. Seymour Bakerman, late Chairman of the Department of Pathology at East Carolina University, and Dr. William Castellani of the Department of Clinical Pathology and Diagnostic Medicine for their many helpful discussions. The results shown in this book were produced by the staff of Special Chemistry at Pitt County Memorial Hospital including Felix Seay, Anita Parks, Sandra Paul, Debbie Johnson and Linda Braddy and at the ECU School of Medicine Clinic Laboratories by James Fennell. These individuals have all made this work possible. I would also like to thank Drs. R. Page Hudson, L. Stanley Harris, and Carl Bentzel for helping to review and correct the original manuscript and for their helpful suggestions.

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TABLE OF CONTENTS SERUM ELECTROPHORESIS......................................1 High Resolution Serum Electrophoresis.................1 Normal Serum Pattern..................................2 Aged Serum............................................3 Normal Plasma.........................................4 Ligand Binding........................................5 Polyclonal Elevation of Gamma.........................6 Sepsis................................................7 Infection with Immune Complexes.......................9 Infection with Oligoclonal Response..................10 Monoclonal Gammopathy of Undetermined Significance...11 Hypogammaglobulinemia................................14 Agammaglobulinemia...................................15 Dysgammaglobulinemia.................................18 Bisalbuminemia.......................................19 Alpha-1-Antitrypsin Deficiency.......................21 Alpha-1-Antitrypsin Deficiency with Cirrhosis........22 Inflammation.........................................23 Rheumatoid Disease...................................24 Progressive Systemic Sclerosis.......................25 Biliary Tract Obstruction............................26 Hepatitis............................................27 Severe Alcoholism....................................28 Hepatic Cirrhosis - early............................29 Hepatic Cirrhosis - Advanced.........................30 Cirrhosis with Ascites...............................31 Liver Failure - Juvenile.............................33 Protein Loss - GI or Vascular........................34 Short Bowel Syndrome.................................35 Nephrotic Syndrome...................................36 Nephrotic Syndrome with Inflammation.................37 Nephrotic Syndrome with Renal Failure................38 Nephrotic Syndrome with Immune Deficiency............39 Renal Failure with Pneumonia.........................40 Banding Pattern......................................41 HIV-Related Complex and Uremia.......................43 Other Serum Patterns.................................44 URINE ELECTROPHORESIS.....................................48 Normal Urine.........................................49 Nonselective Proteinuria.............................50 Hematuria into Hypotonic Urine.......................52 Myoglobinuria........................................54 Partially Selective Proteinuria - Inflammation.......55 Nonselective Benign Proteinuria......................56 Glomerular Proteinuria...............................57 Renal Tubular Disease................................58 Tubular Proteinuria with Inflammation................59 Overflow Proteinuria - Inflammation..................60 Overflow Proteinuria with Inflammation...............61

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Urine from a Septic Patient..........................62 Urine from a Septic Patient..........................63 Nephrotic Syndrome...................................64 Nephrotic Syndrome...................................65 Other Urine Patterns.................................66 CEREBROSPINAL FLUID ELECTROPHORESIS.......................67 Normal Cerebrospinal Fluid...........................68 Cerebrovascular Accident.............................70 Central Nervous System Infection.....................71 Bacterial Meningitis.................................72 Meningitis...........................................73 Fungal Meningitis....................................74 Human Immunodeficiency Virus Infection...............75 AIDS - Cryptococcal Meningitis.......................76 Systemic Lupus with CNS Involvement..................77 Central Nervous System Lupus.........................78 Meningioma...........................................80 Malignant Brain Tumor................................81 Ependymoma with Froin's Syndrome.....................82 Central Nervous System Lymphoma......................83 Pseudo-Tumor of Brain................................84 Multiple Sclerosis...................................85 Neuromyelitis Optica.................................89 Single Gamma Band in CSF.............................90 Chronic and Relapsing Inflammatory Polyneuropathy....91 Guillain-Barre Syndrome..............................92 CSF with Large Beta Spike............................93 Benign Monoclonal Gammopathy with Neuropathy.........94 Paraprotein Polyneuropathy...........................95 Paraprotein Polyneuropathy...........................96 Malignant Paraprotein Polyneuropathy.................98 Isoelectric Focusing of CSF.........................100 Other CSF Patterns..................................101 OTHER FLUIDS.............................................103 Pleural Fluid.......................................104 Pleural Fluid - Infection...........................105 Synovial Fluids.....................................107 PARAPROTEINS (M-COMPONENTS)..............................108 Paraproteins........................................109 Waldenstrom's Macroglobulinemia.....................113 Waldenstrom's Macroglobulinemia.....................115 B Cell Lymphoma.....................................117 Chronic Lymphocytic Leukemia........................118 IgM Paraproteinemia.................................119 Secondary Malignancy................................120 Secondary malignancy................................121 Carcinoma of Lung...................................122 Thymoma with Paraproteinemia........................124 IgG Cryoglobulinemia................................126

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Type II Cryoglobulinemia............................127 Monoclonal Cryogelglobulin..........................129 Primary Amyloidosis (AL)............................130 Peritoneal Fluid - Amyloidosis......................134 Solitary Plasmacytoma...............................136 Oligoclonal Gammopathy..............................137 Oligoclonal Gammopathy - Infection..................139 IgG-Lambda Myeloma..................................141 IgG-Kappa Myeloma...................................142 IgG-Lambda Myeloma - Beta Spike.....................143 IgG-Lambda Myeloma - Broad Peak.....................144 IgG-Kappa Myeloma - Defective Protein...............145 IgG-Kappa Myeloma with Bence-Jones Protein..........146 IgG-Lambda Myeloma with Three Urine Spikes..........147 Free Light Chain in Serum...........................148 Multiple Myeloma with Renal Failure.................150 Nephrosis with MGUS.................................154 Monoclonal Gammopathy of Undetermined Significance..155 Smoldering or Asymptomatic Myeloma..................156 Trace Immunoglobulin Spike..........................157 Nephrotic Syndrome with Monoclonal Gammopathy.......158 IgA-Kappa Myeloma...................................159 IgA-Kappa Paraprotein...............................160 IgA-Lambda Paraprotein with Two Bands...............162 IgA-Lambda Myeloma with Diabetic Renal Disease......163 IgD-Kappa Myeloma...................................165 IgD-Kappa Myeloma...................................166 IgD-Lambda Myeloma..................................168 Kappa Light Chain Disease...........................170 Plasma Cell Leukemia with Lambda Myeloma............171 Multiple Myeloma with CNS Involvement...............172 Immunoglobulin Fragments in Urine...................176 Kappa Bence-Jones Protein and IgG in Urine..........177 Multiple Spikes in Urine - Myeloma..................178 Biclonal Gammopathy.................................179 Triclonal Gammopathy - Myeloma......................181 Hepatitis in Myeloma................................183 Gamma Heavy Chain Disease...........................184 Additional Illustrated M-component Cases............187 Sebia Electrophoresis...............................197 REFERENCES...............................................198

High Resolution Serum Electrophoresis

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% g/dlAlbumin 46.1 3.00 Alpha-1 6.0 0.39 Alpha-2 10.1 0.66 Beta 13.2 0.86 Gamma 24.6 1.60 Total Protein 6.50

This pattern shows a typical serum separated on a high resolution electrophoresis gel. The vertical marks define the five standard fractions from left to right, albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin. Two peaks may be visible in the alpha-2 region and two to four in the beta region. A faint prealbumin peak, which moves faster than albumin, is sometimes visible in serum. Although there are hundreds of proteins found in serum, only a limited number usually are visible in electrophoretic patterns. The prealbumin peak consists of the acidic transport proteins. The albumin peak contains almost entirely albumin. The alpha-1 peak is about 90% alpha-1-anti-trypsin. The alpha-2 peak consists of haptoglobin and alpha-2-macroglobulin. The beta peak consists of transferrin, the left hand peak; C3 complement, the right hand peak; beta-2-lipoprotein, which makes a sharp peak; hemopexin and immunoglobulins. The gamma peak is composed of immunoglobulins and also contains lysozyme and C reactive protein. In most cases only IgG affects the level of the gamma peak. Occasionally other protein constituents are found in serum patterns, for example fibrinogen or hemoglobin. The locations of these will be identified on the appropriate patterns.

Normal Serum Pattern

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This is a typical serum electrophoretic pattern. The C3 complement peak is labeled to compare with the pattern on the next page. The first peak visible in the beta region on this pattern is beta-lipoprotein. It should be noted that many of the alpha and beta globulins have allotypes, which differ in electrophoretic mobility. Normal Ranges g/dl Albumin 3.6 - 5.2 Alpha-1 0.15 - 0.40 Alpha-2 0.5 - 1.0 Beta 0.6 - 1.2 Gamma 0.5 - 1.6 Total protein 6.2 - 8.3 Note that albumin and total protein are usually lower in hospitalized patients. Because of dilution with intravenous fluids, the lower limit of normal for albumin should be 3.0g/dl for inpatients.

Aged Serum

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This pattern was produced by electrophoresis of old serum, actually a commercial normal control serum. The C3 peak in the beta region is no longer visible, because of the natural cleavage of C3. In addition many of the other bands have broadened and become less distinct. Whenever possible, fresh serum should be used in high resolution electrophoresis.

Normal Plasma

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This is a representative pattern of normal human plasma. The fibrinogen peak is labeled and is integrated into the beta region. Serum should always be used for electrophoresis, not plasma. A fibrinogen peak is seen in specimens from patients with coagulation defects, those treated with anti-coagulants, and some patients with paraproteins that inhibit coagulation. Fibrinogen can be identified by the use of thrombin to convert it to insoluble fibrin or by immunochemical tests.

Ligand Binding % g/dl

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Albumin 34.2 2.26 Alpha-1 6.9 0.46 Alpha-2 9.0 0.59 Beta 15.4 1.02 Gamma 34.5 2.28 Total Protein 6.6

Various molecules including drugs, metabolites like bilirubin and proteins like hemoglobin can bind to serum proteins causing alterations in the shape and mobility of peaks. This pattern shows a serum from a patient with advanced liver disease with ligand binding generating a large prealbumin peak and causing some broadening and displacement of the albumin and other peaks. Another common cause of anodal broadening of the albumin peak is the presence of heparin, used as an anti-coagulant. In serum from patients with hemolytic anemia or other hemolysis some haptoglobin-hemoglobin complex may be seen. Rarely immunoglobulins may either complex or specifically bind to other serum proteins generating peaks of abnormal mobility.

Polyclonal Elevation of Gamma

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% g/dlAlbumin 49.6 3.87 Alpha-1 3.1 0.24 Alpha-2 8.7 0.68 Beta 13.1 1.02 Gamma 25.4 1.98 Total Protein 7.8 The gamma globulin peak is elevated in the serum of persons with repeated exposure to antigens and/or infections. This type of pattern is seen typically in patients with chronic asthma, severe rheumatoid disease in the inactive phase, and episodes of severe or repeated infection. It is also seen in the later convalescence of hepatitis. It is important to distinguish this pattern from other polyclonal elevations of gamma in which the beta and gamma regions show prominent bridging and from active disease patterns which show elevations of alpha and/or beta globulin acute phase proteins. Polyclonal elevations are not uncommon in relatively healthy individuals, especially those from lower socioeconomic stations.

Another case

Sepsis

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This pattern was generated by serum from a patient suffering an extended systemic bacterial infection. There is a large polyclonal elevation of gamma globulins and a significantly decreased serum albumin. The acute phase proteins are not visibly elevated. The patient's poor nutritional status is reflected in the low serum albumin. Many of the acute phase reactants are also nutrition sensitive and are primarily synthesized in the liver. The older literature commonly states that the concentration of serum albumin decreases as the concentrations of globulins increase as part of the homeostatic control of plasma protein concentration. However improved management of critically ill and chronic disease patients in recent years has shown that nutrition is a very important factor in the serum albumin level. Immunochemically measured prealbumin is a very useful parameter for assessing current nutritional status, since it decreases much faster than albumin in a state of negative nitrogen balance.

Another case

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Infection with Immune Complexes

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This electrophoresis pattern was produced by the serum of a patient with severe pneumonia, anemia and confusion. The gamma region shows a significant polyclonal increase and has an irregular banding pattern superimposed. Immunofixation electrophoresis shows class and light chain heterogeneity of the bands. A small amount of cryoglobulin containing both kappa and lambda chains is present. The bands represent soluble immune complexes in the serum. Immune complexes are commonly seen in the active phase of viral hepatitis. In addition, the serum albumin is low, reflecting the poor nutritional status of the patient. Both the alpha-1 and alpha-2 regions show increases, reflecting increased synthesis of alpha-1-antitrypsin and haptoglobin acute phase proteins. The C3 complement is not elevated, since it is being consumed in the immune complexes. This case of pneumonia is a bacterial infection and the blood contained significantly increased numbers of mature and immature neutrophils.

Infection with Oligoclonal Response

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IgG = 2450 mg/dl IgA = 453 mg/dl IgM = 363 mg/dl This serum was obtained from a patient with Hemophilus influenzae pneumonia. There is a polyclonal increase in the gamma region reflected in IgG, IgA and IgM. In addition a clear banding pattern is visible as marked by the arrows on the scan. These bands were typed by immunofixation electrophoresis and found to consist of IgG2-lambda. Antibodies of the IgG2 subclass are commonly produced against carbohydrate antigens. Individuals with IgG2 deficiencies have poor responses to H. influenzae vaccine and have increased incidence of infection with encapsulated bacteria. This pattern also shows the decreased albumin typical of the nutritional status of the severely ill patient and some increases in acute phase reactants.

Monoclonal Gammopathy of Undetermined Significance

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SERUM

IgG = 571 mg/dl IgA = 110 mg/dl IgM = 216 mg/dl % mg/dlAlbumin 72.6 122 Alpha-1 7.2 12 Alpha-2 3.5 6 Beta 3.5 6 Gamma 13.2 22 Total Protein 168

URINE

These patterns show the serum and urine from a woman in her 70's who was admitted to the hospital after suffering a myocardial infarction. A small peak is visible in the gamma region in the urine. Typing by immunofixation electrophoresis showed it to be free kappa chains, Bence-Jones protein. Immunofixation electrophoresis of the serum revealed a small IgM-kappa paraprotein peak in the beginning of the gamma region. Small amounts of monoclonal paraproteins are found in many

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sera. The incidence increases with age. In Europe most investigators refer to this condition as benign monoclonal gammopathy, but in the United States it is referred to as monoclonal gammopathy of undetermined significance. Studies in the older literature indicated that there was little chance of progression to malignant diseases such as multiple myeloma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, AL amyloidosis etc. More recent studies by Kyle at the Mayo Clinic, where he has carefully followed cases for many years, have indicated that 19% of cases of MGUS developed a malignant paraproteinemia within 10 years. An additional 39% of the group died from unrelated causes during the 10 year follow-up. Bence-Jones proteins are uncommon in MGUS being found in only a few percent of cases. The presence of Bence-Jones protein or increasing levels of serum protein requires careful surveillance of the patient. Present recommendations are that cases of MGUS be monitored with periodic electrophoresis, at first repeated in 4-6 weeks to see if paraprotein levels are increasing, and approximately every 6 months if levels are constant or decreasing. The presence of any discrete bone lesions, anemia, neuropathy or renal compromise requires more extensive evaluation. A bone marrow biopsy should be performed at the time of initial evaluation and repeated at a later stage, if needed. Additional information may be obtained by measuring serum beta-2-microglobulin levels and by immunohistochemical studies of bone marrow and peripheral blood lymphocytes. Patients with MGUS should not be given chemotherapy unless they develop malignant disease.

Hypogammaglobulinemia

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IgG = 255mg/dl IgA = 10mg/dl IgM = 22mg/dl This serum was obtained from an adult who had experienced several severe infections during the preceding year. He had not had any unusual infections prior to this time. The gamma region is low at 0.2g/dl and all three immunoglobulins are well below normal limits. IgG subclasses showed low levels of IgG1, IgG2 and IgG3. This supports a diagnosis of common variable immunodeficiency.

Agammaglobulinemia

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IgG = 75 mg/dl IgA = 0 mg/dl IgM = 0 mg/dl Infantile X-linked agammaglobulinemia or Bruton's disease is a genetic defect that prevents the production of almost all immunoglobulins. Typically the patients have low levels of IgG, less than 150 mg/dl, and undetectable amounts of the other immunoglobulins. Without replacement therapy affected individuals suffer repeated severe bacterial infections including pneumonias, meningitis and osteomyelitis. Pneumocystis carinii pneumonia and viral infections like cytomegalovirus also occur. At present the patients are managed by infusions of intravenous gamma globulins and early use of antibiotic therapy. The availability of i.v. gamma globulin has greatly decreased the morbidity from when intramuscular gamma globulin was the only available choice. It is only practical to replace IgG, since it has a half life of about 23 days in the circulation. IgA and IgM have half lives of 5.8 and 5.1 days respectively, making replacement impractical. Many individuals with IgA deficiencies experience anaphylactic reactions to immunoglobulin or plasma preparations due to the presence of class or allotype specific antibodies against IgA. It is important to use an IgA free preparation for these individuals. The currently available preparations of intravenous gamma globulin are controlled to guarantee that significant antibody titers against an extensive list of common organisms are present in each lot, unlike the older intramuscular preparations which were not as standardized. Other cases

Dysgammaglobulinemia

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% g/dlAlbumin 52.4 3.3 Alpha-1 6.3 0.4 Alpha-2 14.3 0.9 Beta 12.7 0.8 Gamma 14.3 0.9 Total Protein 6.3 IgG = 9 mg/dl IgA = 56 mg/dl IgM = 1680 mg/dl As can be seen in this pattern, dysgammaglobulinemias are not always apparent from serum protein electrophoresis. This serum was obtained from a teenage boy who had immunoglobulin deficiency with hyper-IgM, which is sometimes seen as a variant of infantile X-linked agammaglobulinemia. In this case there was no family history of repeated or extensive infection or early childhood death. The maternal uncles were healthy. The presence of significant IgA also suggests that this individual's disease is not genetic agammaglobulinemia. This patient has been followed for 8 years on gamma globulin therapy with no evidence that the immune deficiency is transient. The IgM is polyclonal and the patient produces IgM antibodies to antigenic stimulation.

Bisalbuminemia

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% g/dlAlbumin 32.1 2.05 Alpha-1 6.1 0.39 Alpha-2 6.8 0.44 Beta 13.4 0.86 Gamma 41.5 2.66 Total Protein 6.4 Total Protein 48mg/dl These patterns show an uncommon genetic condition in which two variants of albumin are synthesized. One is the normal sequence and the other has at least one amino acid residue different. There are at least 13 types recognized. On the gel two albumin bands are visible, the faster one showing up as a shoulder on the main peak in the scan. The urine shows the same albumin pattern. Two other genetic variants of albumin synthesis include a form that readily forms dimers, giving a broad albumin peak, and analbuminea, the absence of serum albumin. This patient also has hepatic cirrhosis, which will be discussed later.

SERUM

URINE

Another case

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Alpha-1-Antitrypsin Deficiency

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% g/dlAlbumin 62.2 4.7 Alpha-1 0 0 Alpha-2 12.3 0.9 Beta 9.0 0.7 Gamma 16.4 1.2 Total Protein 7.6

IgG = 1370 mg/dl IgA = 464 mg/dl IgM = 79 mg/dl AAT = 0 mg/dl This pattern was found among routine orders for serum protein electrophoresis. There was no alpha-1-antitrypsin band visible on the gel. Alpha-1-antitrypsin was not detectable immunochemically. There are 5 common alleles of AAT and over 25 rare forms. Although some have atypical mobility in standard high resolution protein electrophoresis, many require special techniques like acid starch gel electrophoresis for detection. The very low and absent phenotypes Pi null and Pi Z are associated with development of neonatal cryptogenic hepatitis or childhood cirrhosis in 5-20% of affected individuals. Pi Z phenotype individuals are at high risk to develop a basilar emphysema in adulthood. Occasionally adult onset liver disease appears, but usually careful review finds a history of neonatal jaundice. The development of emphysema is greatly aggravated by smoking and high degrees of air pollution. The normal mean level for AAT is 135mg/dl. The Pi Z phenotype usually has levels of 10-20% of normal and is found in about 1 in 1500 northern European caucasians. The Pi - or null phenotype is rare. Individuals with levels below 40% of normal are at increased risk of disease. Other types of neonatal hepatitis and idiopathic respiratory distress syndrome of infancy may show low levels of AAT because of consumption. Phenotyping and family studies can readily distinguish these from genetic deficiencies.

Alpha-1-Antitrypsin Deficiency with Cirrhosis

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% g/dlAlbumin 31.1 1.97 Alpha-1 0.6 0.04 Alpha-2 6.6 0.42 Beta 11.4 0.72 Gamma 50.2 3.16 Total Protein 6.3 Alpha-1-Antitrypsin = 9 mg/dl This serum was obtained from an adult who was admitted to the hospital with severe cirrhosis, which had progressed to ascites. The patient gave a history of long standing cirrhosis and did not consume alcohol. He was a moderate cigarette smoker and evaluation showed a mild emphysema. Other medical history included a previous splenectomy for pancytopenia. The AAT was Pi Z type.

The electrophoretic pattern shows a low serum albumin, due to loss to ascites and decreased production due to liver destruction. The alpha-1 band is absent. There is a massive polyclonal gammopathy with total fusion of the beta and gamma regions. Both IgG and IgA are significantly elevated. Another case

Inflammation

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Increases in the acute phase reactive proteins are seen as a response to inflammation. The traditional characterization of patterns as acute, subacute and chronic is of limited value to the clinician, and is not always accurate. This pattern shows an increase in alpha-1-antitrypsin, haptoglobin and C3 complement, as well as some polyclonal elevation of gamma globulins. Following surgery C reactive protein increases in 6-8 hours reaching a maximum at 48-72 hours, followed closely by alpha-1-acid glycoprotein. Alpha-1-antitrypsin, haptoglobin and fibrinogen levels increase at 24 hours. In the next few days prealbumin, albumin, alpha-lipoprotein and transferrin may decrease, because of decreased nutrition. C3 complement and ceruloplasmin may increase during the subacute phase. In myocardial infarction C-reactive protein, alpha-1-acid glycoprotein, alpha-1-antitrypsin, haptoglobin and fibrinogen rapidly increase, peaking at about 5 days and returning to normal in about a month. The nutritionally sensitive proteins and IgG reach minimum levels in 5 days and return to normal. Ceruloplasmin and C3 reach a maximum during the second week. In infectious disease of bacterial origin alpha-1-acid glycoprotein, alpha-1-antitrypsin, haptoglobin and C-reactive protein may reach very high levels. In most viral diseases there is much less increase in C-reactive protein and alpha-1-acid glycoprotein. The convalescent phase of infectious diseases may be accompanied by a rise in immunoglobulins. This is most marked in hepatitis.

Rheumatoid Disease

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IgG = 1660 mg/dl IgA = 769 mg/dl IgM = 65 mg/dl Severe polyarticular rheumatoid arthritis and systemic lupus erythematosus may show a variety of electrophoretic changes. This pattern illustrates a moderate polyclonal gammopathy with elevation of IgA and IgG. This may be more marked in severe SLE. Acute phase reactants are of some value in assessing the activity of the disease, especially elevations of haptoglobin and both increases and decreases of C3. The C3 peak may decline or disappear in cases with large scale immune complex deposition, especially in SLE with severe renal involvement. Alpha-1-antitrypsin does not change as much as in infectious disease. Immune complex patterns (see page 8) may be seen during periods of disease activity. Serial measurements of total hemolytic complement, C3, C4 and B can be of use in following the disease activity of selected patients.

Progressive Systemic Sclerosis

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IgG = 1780 mg/dl IgA = 310 mg/dl IgM = 68 mg/dl This pattern is typical of severe rheumatoid disease in the active phase. The alpha-2 globulin and beta globulins are elevated reflecting acute and subacute inflammation. There is some polyclonal elevation of gamma, with an irregular banding pattern caused by the presence of immune complexes. The alpha-2 band is much broader than normal.

Biliary Tract Obstruction

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Cholesterol = 1262 mg/dl LDL = 1229 mg/dl alkaline phosphatase = 1400mU Blood urea nitrogen = 6 mg/dl Urine protein = less than 6mg Bilirubin = 13mg/dl This pattern was observed from a plasma sample obtained from a 31 month old female with choledocal cysts causing bile duct obstruction. A prominent fibrinogen peak is present in the gamma. The specimen was bright yellow from the elevated bilirubin. The sharp peak in the beta region is the greatly increased beta-lipoprotein. This is reflected in the extremely high serum cholesterol and directly measured low density lipoprotein. The triglyceride level was normal. Liver enzymes including alkaline phosphatase, SGOT and amylase were all elevated. The alpha-2 region shows an increase in both major components. This patient had no evidence of renal disease.

Hepatitis % g/dlAlbumin 20.1 1.9 Alpha-1 4.7 0.4 Alpha-2 7.0 0.7 Beta 7.2 0.7 Gamma 61.0 5.7 Total Protein 9.3 IgG = 5530 mg/dl

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IgA = 209 mg/dl IgM = 520 mg/dl C3 = 106mg/dl C4 = 7mg/dl B = 32mg/dl This specimen was obtained from a patient with a severe case hepatitis A, who developed serious complications including

ofbacterial meningitis and liver failure. Viral hepatitis causes very potent stimulation of the immune response, even in tients with partial acquired immune deficiency from malignant matopoietic disease. In the early stages of the disease, there a significant increase in specific and total IgM. The IgM tibodies decrease over the course of the disease. This patient ill has a relatively high IgM. An IgG response follows the M, very high levels may be reached as in this case. Much of e IgG is not specific for the infectious organism and the

ts of immune complexes. till normal, the C4 is f the high rate of consumption.

m is d s f the acute phase proteins are e large amount of liver damage.

a paheisanstIgthresponse is polyclonal as is shown by the broad gamma peak and kappa-lambda ratio of 1.8. In the later stages of disease a gnificant IgA response is usually observed. si

The irregular appearance of the gamma zone in this serum is due to the presence of rge a la mouAlthough C3 complement and B are s

n y d d b e o

n

significa tl epresse ecaus The albu in low an ome olower t e cted be se ofhan xpe cau th

Severe Alcoholism % g/dlAlbumin 44.3 4.4 Alpha-1 2.1 0.2 Alpha-2 5.2 0.5 Beta 9.1 0.9 Gamma 39.4 3.9

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Total Protein 10.0 Many chronic alcoholics develop severe liver disease. Tearliest and still reversible phase is the deposition of fate hepatocytes. About 30% of heavy drinkers de

he in

velop alcoholic patitis, characterized by random necrosis of hepatocytes, flammation and hyaline changes. Protracted hepatitis leads to rrhosis in many cases. Alcoholic cirrhosis starts out as a cronodular pattern, where fibrous bands bridge the nodules and generative areas are seen. In the end stages the liver may pear grossly macronodular and consist primarily of fibrous ssue.

This patient was a severe alcoholic who did not show any gns of cirrhosis. re for a number of derate infections ome elevations of ver enzymes and bi coholic hepatitis. the time this specimen was obtained the patient had normal

no ificant medical complaints. The clonal increase in gamma. t shows good nutritional albumin. The sample was tal protein reflects the summer day.

thheincimireapti si He had received medical camo and had in the past shown s

lirubin consistent with alliAtserum chemistries and signonly abnormality visib is a le polyUnlike many alcoholics, this patienstatus, r fl ed in t 4.4 g/e ect he dl collected in August and the high toshort-term dehydration from the hot

Hepatic Cirrhosis - Early % g/dlAlbumin 45.8 3.16 Alpha-1 6.5 0.45 pha-2 11.3 0.78

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AlBeta 17.9 1.24 mma 18.5 1.28

tal Protein 6.9

This pattern shows the relatively early stages of hepatic rrhosis. There is an increase in the acute phase reacting oteins in the alpha-1 and beta. The only other visible normality is the bridging between the beta and gamma zones. is reflects the production of IgM or IgA at increased rates. cirrhosis IgA levels are usually high. Serum chemistries owed a moderate liver disease consistent with alcoholism.

Ga To ciprabThInsh

Hepatic Cirrhosis - Advanced % g/dlAlbumin 31.2 2.0 Alpha-1 3.3 0.2 Alpha-2 7.0 0.5 Beta 9.9 0.6 Gamma 48.5 3.2 tal Protein 6.5

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To IgG = 3270 mg/dl

IgA = 883 mg/dl IgM = 232 mg/dl

This pattern is typical of advanced alcoholic cirrhosis. e albumin and many of the proteins in the alpha and beta gions are present in subnormal levels, because of decreased nthesis, due to the large amount of liver damage. The beta gion is totally fused with the gamma region and the serum IgA vel is high. There is a massive polyclonal increase in IgG.

The polyclonal gammopathy in cirrhosis is thought to result

reticuloendothelial damage and shunting of antigens into the systemic circulation, where they are processed in lymph nodes leading to a more vigorous response.

Thresyrele from a combination of immunoregulatory abnormalities,

Cirrhosis with Ascites

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SERUM

% mg/dlAlbumin 1.8 2 Alpha-1 7.9 10 Alpha-2 2.5 3 Beta 4.1 5 Gamma 83.8 110 Total Protein 131 Protein/24hrs 1.1g

URINE

These are the serum and urine patterns from an alcoholic patient with end stage cirrhosis admitted to the hospital with ascites. The serum pattern is even more extreme than the previous figure, having a lower albumin and higher polyclonal gammopathy. The urine pattern shows that the extremely low albumin level is not due to loss through the kidneys, but is the result of not being synthesized. Most of the urine protein is in the gamma region. Immunoelectrophoretic studies showed the presence of significant amounts of both kappa and lambda chains, and of fragments of immunoglobulin. The large excretion of protein reflects overflow and hypercatabolism of the immunoglobulin.

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Bone marrow biopsies on similar patients have shown an extreme degree of plasmacytosis, as much as 45% of marrow white cells. If a severe alcoholic patient is suspected of having a monoclonal gammopathy, it is essential to perform complete immunohistochemical studies on the marrow. Typical severe cirrhosis patients will show plasma cells positive for both gamma and alpha chains, and the kappa-lambda staining ratio will be between 1 and 2.

Liver Failure - Juvenile

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IgG = 862 mg/dl IgA = 157 mg/dl IgM = 104 mg/dl A 1.5 year old boy was admitted to the hospital with liver failure. An extensive work-up did not reveal no the cause. The pattern shown here has a very low serum albumin and is otherwise normal. Immunoglobulins are normal for age. Urine protein excretion was normal.

Protein Loss - GI or Vascular

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% g/dlAlbumin 61.5 2.2.9 Alpha-1 9.2 0.3 Alpha-2 14.6 0.7 Beta 8.7 0.4 Gamma 7.9 0.4 Total Protein 4.9

This pattern with a relatively low total protein and decreases in all fractions including alpha-2 is typical of protein loss through the gastrointestinal system or vascular loss. In this case the immunoglobulin levels are also decreased to subnormal levels. In renal protein loss the very large proteins alpha-2-macroglobulin and IgM are usually normal or increased. Severe protein losing syndromes may also present clinically as acquired immune deficiency.

Short Bowel Syndrome

35


IgG = 1390 mg/dl IgA = 322 mg/dl IgM = 70 mg/dl Kappa/lambda = 1.83 This pattern was obtained from the serum of a patient with the short bowel syndrome and consequent severe malabsorption. The very low albumin reflects the poor nutritional state. Immunoglobulins are normal as are the alpha globulins. The beta-lipoprotein peak is decreased. A prominent fibrinogen peak is seen in the gamma region, consistent with the clotting defect observed.

Nephrotic Syndrome

36

% g/dlAlbumin 31.4 2.4 Alpha-1 5.9 0.5 Alpha-2 28.9 2.2 Beta 19.6 1.5 Gamma 14.2 1.1 Total Protein 7.6 IgG = 1260 mg/dl IgA = 948 mg/dl IgM = 185 mg/dl This pattern illustrates the nephrotic syndrome occurring in a severe alcoholic patient. The albumin is low and there is an elevation of the alpha-2 peak, specifically alpha-2-macroglobulin. There is also a prominent beta-2-lipoprotein peak. These findings are typical of nephrotic syndrome. There is a significant amount of beta-gamma bridging and a very high serum IgA, typical of post-necrotic cirrhosis. Immunoelectrophoresis did not show the presence of paraprotein. Urine protein was 12g/day.

Another case

Nephrotic Syndrome with Inflammation

37


IgG = 459 mg/dl IgA = 181 mg/dl IgM = 51 mg/dl This pattern shows a decreased total protein with decreased serum albumin and gamma. The alpha-1 peak is increased and the alpha-2 peak is slightly increased, primarily in the alpha-2-macroglobulin. Immunoglobulin G was slightly decreased. Blood counts were consistent with infection. Qualitative urine protein was 4+.

Nephrotic Syndrome with Renal failure

38


This serum was obtained from a patient who had a long history of nephrotic syndrome, which had progressed to renal failure. The total protein, albumin and gamma globulins are all significantly decreased. The alpha-2 macroglobulin and beta-lipoprotein are increased. The alpha-1 shows an absolute increase and some of the beta globulins show relative increases, because of acute phase responses probably due to the uremia.

Nephrotic Syndrome with Immune Deficiency

39


Cholesterol = 658 mg/dl % g/dlAlbumin 33.1 1.36 Alpha-1 6.9 0.28 Alpha-2 25.5 1.05 Beta 31.0 1.27 Gamma 3.7 0.15 Total Protein 4.1

IgG = 89 mg/dl IgA = 62 mg/dl IgM = 73 mg/dl Cholesterol = 1104 mg/dl These two patterns both illustrate nephrotic syndrome with functional hypogammaglobulinemia. Both sera have relative elevations of alpha-2-macroglobulin, and extreme elevations of beta-lipoprotein as shown by the serum cholesterol. All other fractions are significantly decreased. The IgG levels are insufficient to be protective. Because of their larger size there is less loss of IgA and little, if any loss of IgM. In nephrotic syndrome it is rare for molecules larger than 200,000 molecular weight to pass through the kidney. The increased beta-lipoprotein reflects a greatly increased risk of atherosclerotic heart disease.

Renal Failure with Pneumonia

40


Patients with nephrotic type renal failure have an increased susceptibility to severe infection. This specimen was obtained when a patient with renal failure was hospitalized with severe bacterial pneumonia. The total protein is low with relative elevations of alpha-1, alpha-2 and beta-lipoproteins. The gamma region shows a significantly higher level than the two patients' sera on the previous page. A clear banding pattern is visible in the gamma corresponding to immune complexes and/or a restricted immune response.

Banding Pattern % g/dl

41

Albumin 31.3 3.1 Alpha-1 2.0 0.2 Alpha-2 5.4 0.5 Beta 7.0 0.7 Gamma 54.4 5.3 Total Protein 9.8

IgG = 6490 mg/dl IgA = 234 mg/dl IgM = 183 mg/dl This pattern was obtained from the serum of a 27 year old male admitted to the critical care unit for treatment of an overdose of alcohol and doxepin. There is a large increase in the gamma region and a very large increase in IgG, without increased IgA or IgM. A clear banding pattern was visible on top of the polyclonal increase in gamma. Three of the bands were identified as IgG-kappa by immunofixation electrophoresis, including the small band following the beta-gamma division mark. These results are suggestive of an occult hematologic malignancy or possibly an autoimmune disease. Further laboratory results included a negative hepatitis panel, negative toxoplasmosis and cytomegalovirus, positive RPR and FTA-ABS for syphilis and positive human immunodeficiency virus by ELISA and western blot. Polyclonal and oligoclonal gammopathies are not unusual in HIV infections. Another case

HIV-Related Complex and Uremia

43


SERUM

% mg/dlAlbumin 35.3 417 Alpha-1 10.2 120 Alpha-2 10.9 129 Beta 16.6 196 Gamma 27.0 319 Total Protein 1180 Protein/24hrs 28.3g Human immunodeficiency virus infections cause a large variety of abnormalities. This individual has HIV related complex, which has not yet developed into acquired immune deficiency syndrome. The patient has severe uremia and nephrosis, losing 28 grams of protein per day. The serum protein is low and the albumin extremely low. Bridging of the beta and gamma regions is present. The beta-lipoprotein peak is prominent, but the alpha fractions are normal. The urine reflects the serum pattern with the exception of alpha-2-macroglobulin and beta-lipoprotein, which do not pass through the kidney. The abnormalities observed are the result of renal disease in this case.

URINE

Other Serum Patterns

Alpha-1 Anti-Trypsin PiMS Heterozygote

Cryofibrinogenemia

44

Haptoglobin-Hemoglobin Complex

Human Immunodeficiency Virus Gammopathy

Iron Deficiency with Low Transferrin

45

Sickle Cell Crisis

Sprue with Malnutrition

46

48

URINE ELECTROPHORESIS

49

Authorities disagree as to the upper limit of protein in ivi cally values of 100 to 300

we use 150mg/day. Normal 2/3 plasma proteins and 1/3 tract. This is most readily retic maps. The plasma proteins 0) proteins that can pass ompletely resorbed by the

y larger proteins can also be y concentrated normal urine

ntains a small amount of albumin and a trace of transferrin, no her discrete peaks are usually visible.

There are several relatively benign conditions, which cause ni cant increases of plasma proteins in the urine. These lu pregnancy, postural hematuria, exercise hematuria and its riant march hematuria. The patterns in these conditions pically mirror serum electrophoretic patterns.

e

mmopathies. Patients with renal tubular disease may also have protein excretions within the normal range. Urine

ctrophoresis should be performed whenever it may be of inical value to identify the proteins in urine in order to aluate the nature or extent of renal damage.

Normal Urine urine from healthy ind duals. Typi

le, mg/day are used. As a general ruurinary proteins consist of about proteins derived from the urinary seen in two-dimensional electrophoare the low molecular weight (<40,00hrough the glomerulus and are not cttubules. Small amounts of slightlound. A typical pattern of highlfcoot sig finc deivaty Trace amounts of protein in urine are important in the monitoring of some disease states. The early stages of diabetic renal disease are monitored by following the excretion of serum albumin. The upper limit of normal for urinary microalbumin is 20-30 mg/day. Urinary microalbumin is typically measured by using low level immunodiffusion plates. The presence of any amount of urinary free light chain (Bence-Jones protein) can bmportant in the evaluation of patients with monoclonal iga

eleclev

50

Nonselective Proteinuria % mg/dlAlbumin 49.3 123 Alpha-1 11.0 28 Alpha-2 14.3 36 Beta 10.9 27 Gamma 14.5 36 Total Protein 250 This pattern shows a nonselective type of proteinuria. The inary electrophoresis pattern is almost identical o the serum ttern, except for the absence in urine of the largest plasma oteins like beta-lipoprotein. Nonselective patterns suggest vere damage to some of the glomeruli. The total amount of oteinuria reflects the degree of damage and fraction of damaged omeruli. Nonsel en in benign oteinurias and e kidney.

Urine

ur tpaprseprgl ective patterns may also be sepr in inflammatory conditions of th

52

c Urine Hematuria into Hypotoni % mg/dlAlbumin 4.4 1.0 Alpha-1 1.4 0.3 Alpha-2 3.5 0.8 Beta 79.7 17.5 Gamma 11.0 2.4 Total Protein 22.0

URINE

Urine may vary in osmolarity. When it is hypotonic, red cells will lyse in it. This sample was obtained at the limit of detectable color in the urine, about 0.3% red blood cells by volume. The large beta spike is hemoglobin A. The presence of heme in urine can also result from myoglobin. To distinguish myoglobin from hemoglobin, the urine should be dialyzed before electrophoresis. The myoglobin is more basic than hemoglobin A, but can be confused with hemoglobin C or E. In these cases isoelectric focusing is the method of choice. The salt precipitation method for separating hemoglobin and myogreliable an

lobin is d should be replaced by electrophoretic or

immunochemical techniques.

un

Another case

53

Myoglobinuria URINE

% mg/dlAlbumin 44.2 51 Alpha-1 18.0 21 Alpha-2 12.1 14 Beta 17.5 20 Gamma 8.3 10 Total Protein 115 This uect

rine specimen was obtained from a victim of near-fatal

iable

el rocution two days following the accident. There are two peaks in the beta; the first is transferrin and the second slightly larger peak is myoglobin. The myoglobin peak is slightly more basic than hemoglobin A, but has the same mobility as hemoglobin C or E at pH 8.6. Isoelectric focusing is the best method to distinguish myoglobin from abnormal hemoglobins. The salt precipitation method for identifying myoglobin is unreland should be abandoned in favor of electrophoresis or immunoassay.

54

55

Partially Selective Proteinuria with Inflammation % mg/dlAlbumin 58.7 106 Alpha-1 14.3 26

Alpha-2 13.7 25 Beta 7.8 14 Gamma 5.6 10 tal Protein 180 To

This specimen illustrates a primarily glomerular pattern ofproteinuria with a large albumin peak and a transferrin peak ithe beta. There is a prominent alpha-1 peak from overflow ofacute phase proteins. The gamma globulins are underrepresentin the urine relative to the plasma levels. This pattern issul

n the ed the

RINEU

re t of a lesser degree of glomerular permeability than thepattern illustrating nonselective proteinuria.

56

Nonselective Benign Proteinuria

% mg/dlAlbumin 42.0 21 Alpha-1 9.6 5 Alpha-2 9.8 5 Beta 10.0 5 Gamma 28.7 14 Total Protein 50 otein/24hrs 0.9g Pr

This urine electrophoresis pattern is almost identical to the patient's serum pattern. It is typical of conditions in which small amounts of blood enter the urine. In this case red cells were also seen in the initial specimen. This pattern comesfrom a case of march hematuria, an episodic hematuria first described in military trainees. In this disease hematuria typically follows prolonged or strenuous activity. Related conditions include other variants of exercise proteinuria and postural proteinuria.

Glomerular Proteinuria

57

% mg/dlAlbumin 7pha-1

4.9 124 7.9 13

pha-2 3.6 6 ta 10.3 17

5

otein/24hrs 3.2g

r type proteinuria xcretions over several grams per day. Occasionally protein peak is found with mobility identical or

use of the renal disease may be unsuspected paraprotein disease AL amyloidosis.

AlAlBeGamma 3.3 tal Protein 165

URINE

ToPr This urine specimen is classic illustration of glomerular proteinuria. There is a large albumin peak with a prominent transferrin peak in the beta. The alpha-1 peak is increased relative to the serum percentage. The alpha-2 peak is significantly lower than in the serum and very little gamma globulin is passed into the urine. Immunoelectrophoresis or immunofixation electrophoresis

formed on all cases of glomerulashould be perwith protein eBence-Jonesa

similar to transferrin. If Bence-Jones protein is found, the caor

58

Renal Tubular Disease % mg/dlAlbumin 28.2 28 Alpha-1 30.0 30 Alpha-2 25.1 25 ta 16.1 16 Be

Gamma 0.7 0.7

tal Protein 100

e antibiotics, pyelonephritis, Fanconi syndrome, se, sarcoid, cystinosis, tubular acidosis and a her conditions. Tubular damage also occurs as part

other renal diseases including myeloma kidney. Tubular sfunction is manifested by the loss of the ability to resorb

oteins that can be used for quantitative assessment of tubular nction are lysozyme and beta-2-microglobulin. In tubular

the urine, but there is no specific test available for its measurement and its function is unknown. Workers exposed to heavy metals and patients on long-term or high dose aminoglycoside therapy should have urine electrophoresis, urine lysozyme and/or urine beta-2-microglobulin periodically checked. The typical tubular pattern shows a relatively small albumin peak, with a large alpha-2 peak consisting of alpha-2-microglobulin. There is usually a broad undefined alpha-1 peak. The small peak in the beta runs after transferrin and is beta-2-microglobulin. There is usually an undefined smear in the gamma region. Protein excretion levels in tubular proteinuria are much lower than in other types of proteinuria.

URINE

To

Renal tubular disease can be caused by heavy metal exposure,

aminoglycosidWilson's diseariety of otva

ofdysmall protein molecules of about 15,000 molecular weight. Two prfuproteinuria a large amount of alpha-2-microglobulin is found in

59

Tubular Proteinuria with Inflammation

but a similar pattern some cases of pyelonephritis.

other case

Total P ei 52 mg/rot n = dl The electrophoresis of this urine gives a broad smear with some faint bands visible on top. The albumin is shaded in the figure. The increased alpha-2 of the tubular disease pattern is present, along with a massive increase in alpha-1. This is overflow of the acute phase protein into the urine. Most of the urinary proteins are unresolved in the electrophoresis, suggesting that they may be partially metabolized. This urine

from a patient with pneumonia, was obtained can be seen in An

URINE

60

Overflow Proteinuria - Inflammation % /dl mgAlbumin 28.7 13 Alpha-1 15.2 7

5 Alpha-2 11.1 Beta 21 mma 2 11

.5 10 3.6

tal Protein 45

d the proportionally large ak result from iron deficiency anemia. The in this patient results from bacterial sepsis.

Another case

Ga To Much of the urinary protein visible in the scan of this specimen results from overflow of acute phase proteins into the urine. These are primarily alpha-1 and alpha-2 globulins. In high resolution electrophoresis the alpha-2 region may show

The increased beta anthree peaks. transferrin peflammation in

URINE

61

Overflow Proteinuria with Inflammation % mg/dlAlbumin 26.8 27 Alpha-1 25.5 26 Alpha-2 31.8 32 Beta 9.9 10 Gamma 5.9 6 Total Protein 102 This urine specimen was tained from a female patient hospitalized with an inflammatory lyn d

d

URINE

obpo europathy. The acute phase proteins in the alpha-1 analpha-2 regions are prominent. Moderate amounts of albumin antransferrin are also present. A small spike is present in the gamma corresponding to a serum paraprotein. Another case

62

Urine from a Septic Patient % mg/dlAlbumin 26.2 9.4 Alpha-1 24.0 8.6 Alpha-2 10.4 3.7 Beta 18.6 6.7 Gamma 20.9 7.5 Total Protein 36 This urine was collected om a patient suffering from cterial cellulitis and psis. There is a large opo

ing n

frbasepr rtion of alpha-1 acute phase proteins as well as some alpha-2 microglobulins. A bandpattern is seen in the gamma region, caused by partial digestioof immune complexes. Both kappa and lambda light chains are found throughout the pattern. Urine patterns similar to this usually indicate severe infection. ANOTHER CASE

URINE

Urine from a Septic Patient U

63

% mg/dlAlbumin 20.8 25 Alpha-1 4.7 6 Alpha-2 11.9 14 Beta 8.3 10 Gamma 54.2 65 tal Protein 120 To

ion ded

he ified as immunoglobulin. Heavy

gamma is due to digestion products of the immunoglobulins and immune complexes present in the plasma. The large gamma spike may be a fragment of C3.

RINE

This urine was obtained from a patient with bacterial sepsis. The serum showed a polyclonal elevation of gamma and some acute phase reaction. The urine shows the presence of serum proteins with inflammatory proteins. The gamma region is greatlyincreased and shows a prominent banding pattern. There is a clear spike at the beginning of the gamma region. Immunofixatelectrophoresis showed that the smaller gamma bands corresponto kappa chains, and also two weaker lambda chain bands. Trger gamma peak was not identla

chains gave only diffuse staining. The banding pattern in the

64

Nephrotic Syndrome

% mg/dlAlbumin 51.1 118 Alpha-1 8.6 20 Alpha-2 4.9 11 Beta 7.7 18 Gamma 27.8 64 Total Protein = 230mg/dl Nephrotic syndrome usually begins as a glomerular type proteinuria and becomes less selective as it progresses. The specimen illustrated here shows a relatively non-selective proteinuria with gamma globulin excreted proportionally to the plasma level. The alpha-2 and beta globulins are underrepresented relative to the plasma. The serum electrophoresis pattern for this patient showed low total protein d albumin, with elevated alpha-2-macroglobulin a d beta-poprotein similar to the case illustrated for nephrotic ndrome serum.

URINE

an nlisy

65

Nephrotic Syndrome

%

mg/dlA .2 382

.6 9 lbumin 67lpha-1 1lpha-2 7.8 44 eta 8.3

otal Protein = 569 mg/dl This urine from a woman with nephrotic syndrome shows a primarily glomerular pattern of proteinuria, with some excretion of gamma globulin. There are only small amounts of acute phase proteins present. The alpha-1 is particularly low and indicates alpha-1-antitrypsin deficiency, which can be verified by measuring the serum level. The proteinuria is relatively massive. It should be mentioned that there is not a regular correlation between the amount of proteinuria and the size permeability of the glomeruli.

URINE

AAB 47 Gamma 15.1 86

T

66

THER URINES

abetic Nephrosis

O Di

Urine Beta Spike

67

Cerebrospinal Fluid Electrophoresis

and Neurological Diseases

Normal Cerebrospinal Fluid

68

% mg/dlPrealbumin 7.4 2 Albumin 65.0 14 Alpha-1 3.5 0.4 Alpha-2 5.4 0.8 Beta 13.8 2 amma 5.0 0.8 G SF protein 18 C gG/albumin 0.08I

CEREBROSPINAL FLUID

Normal Values mg/dlPrealbumin 0.3 - 2.7 Albumin 8.2 - 33 Alpha-1 0.3 - 3.0 Alpha-2 0.6 - 3.6 Beta 1.5 - 7.6 Gamma 0.3 - 4.5 CSF protein 15 - 45

G/albumin ratio less than 0.28

G synthesis index = IgG(CSF)/Albumin(CSF)

Ig Ig less than 0.85

IgG(serum)/Albumin(serum) mean = 0.5

Cerebrospinal fluid patterns are readily distinguishable om serum patterns. The prealbumin peak is significantly larger CSF. When the prealbumin is less than 2% of CSF protein, ere is probably a large amount of plasma protein leakage across

the blood-brain barrier. There are a number of proteins in CSF not found in serum. An easily visible peak in the beta-2 region is the carbohydrate deficient "CSF-specific" transferrin. A faint peak in the far beta is the non-immunoglobulin gamma trace protein. Faint banding is sometimes observed in normal CSF. The large proteins alpha-2-macroglobulin and beta-lipoprotein should not normally be visible in CSF. There are usually much lower proportions of alpha globulins in CSF than in serum. When CSF electrophoresis is performed, the albumin and IgG

frinth

69

ould be measured immunochemically. In addition, if possible, rum electropho ould be rformed at the are found in th serum and CS mic condition

Ma ysicians do not cooperate in this sar ro al

s lammatory s s, myelin basic protein should

evels have relatively low inflammatory CNS disease. round 0.7 and the IgG synthesis definite oligoclonal bands has iagnosis of multiple sclerosis.

itation and interpretation, umin on CSF and serum, CSF myelin basic

otein, IgG/albumin and IgG synthesis index.

shse resis and serum IgG and albumin shpe same time. If any abnormalities

F, they are the result of a systeboand not CNS specific. ny phregard, so it is neces y to p vide less than optim

es of suspected infevaluations such ca . In cin ses aconditions or demyelinating disea ealso be m as d by im oassaye ure mun . Abnormalities of CSF protein lsensitivities and specificities for

aThe IgG/albumin ratio has valuesindex around 0.8. The finding of

- he da sensitivity of 79 94% in t Whenever possible CSF workups should include electrophoresis of CSF and serum with quantmunochemical IgG and albim

pr

Cerebrovascular Accident

70

% mg/dlPrealbumin 8.3 2.7 Albumin 41.4 13.2 Alpha-1 4.3 1.4 Alpha-2 2.9 0.9 Beta 39.2 12.5 Gamma 3.9 1.2

CEREBROSPINAL FLUID

CSF protein 32 G/albumin 0.27 Ig

This specimen was obtained from an elderly woman after she suffered a stroke. All of the fractions are normal except the beta, which contains a large spike. The CSF had a clear red color, and after concentration was deep red. Immunoelectrophoresis was performed and the spike was neither immunoglobulin, nor transferrin. From the color it was entifieid

CVA were no longer present. There were only a few intact red d as hemoglobin. Most of the plasma proteins from the

cells in the CSF.

Central Nervous System Infection % mg/dl

71

Prealbumin 4.8 3.9 Albumin 44.4 36.0 Alpha-1 3.9 3.2 Alpha-2 10.6 8.6 Beta 17.0 13.8 Gamma 19.3 15.6 CSF protein 81 IgG/albumin 0.68 IgG Synthesis index 1.36

of CSF a strong

lyclonal immunoglobulin response is present with significantly evated indices. The proteins are of both plasma and CNS igin, since albumin is not synthesized in the CNS compartment.

CEREBROSPINAL FLUID

This pattern was obtained from a CSF specimen that appeared bloody. The 4.8% prealbumin, however, suggests that there is little, if any dilution by blood. There is a polyclonal elevation of the beta and gamma regions and the alpha-1 and -2 acute phase proteins are visible. The total protein, globulin fractions, IgG, IgG/albumin ratio and IgG synthesis index are allelevated. The clinical diagnosis was sepsis with meningitis. Most cases of bacterial meningitis lead to elevations oteins, many time as high as 1000mg/dl. Usually pr

poelor

Bacterial Meningitis

72

% mg/dlPrealbumin 0.7 4 Albumin 66.1 400 Alpha-1 4.3 26 Alpha-2 4.8 29 Beta 12.1 73 Gamma 12.0 73 CSF protein 605

CEREBROSPINAL FLUID

IgG/albumin 0.22 Large increases in CSF protein are found in bacterial meningitis. The relative amount of prealbumin is usually decreased, since the much of the protein enters by leakage from plasma. The relative proportion of gamma and IgG/albumin may be either normal or increased. The gamma region usually has a polyclonal appearance, although immune complex banding may be visible.

Meningitis % mg/dlPrealbumin 2.6 4.5 CEREBROSPINAL FLUID

73

Albumin 64.9 111.0 Alpha-1 3.2 5.5 Alpha-2 7.2 12.3 Beta 12.4 21.2 Gamma 9.8 16.8 CSF protein 171

0IgG/albumin .25 elin basic protei <1 My n

is as

e

This pattern was obtained on CSF from a patient with sepsand meningitis in which no organism could be identified. It wthought to be of viral etiology. The total CSF protein is markedly increased, with a pattern reminiscent of serum in the alpha and beta regions. Although the IgG is significantly elevated at 29 mg/dl, the indices are normal. Myelin basic protein was not detected. No banding pattern was visible in thgamma. The increased protein in this CSF is mainly due to increased permeability of the blood-brain barrier.

Fungal Meningitis

74


CEREBROSPINAL FLUID

CSF protein 139 IgG/albumin = 0.66 IgG synthesis index = 0.83 An 11 year old girl was hospitalized with sepsis and meningitis. Multiple cultures grew Candida albicans. She had no known predisposing condition like hematologic malignancy or immune deficiency. Cerebrospinal fluid contained high levels of protein and all fractions. The IgG level was 39 mg/dl and the IgG/albumin ratio was elevated, but the synthesis index was borderline. The gamma region showed some irregularity, but an oligoclonal pattern was not observed. This pattern is typical of fungal meningitis, many cases of tuberculosis meningitis and some viral meningitis. Typically in these conditions CSF proteins are elevated much less than in cterial meningitis. ba

75

Human Immunodeficiency Virus Infection % mg/dlPrealbumin 4.1 1.1 Albumin 40.1 10.4

Alpha-1 3.6 0.9Alpha-2 6.7 1.7 Beta 21.6 5.6 Gamma 24.0 6.2

CEREBROSPINAL FLUID

CSF protein 26 IgG/albumin 0.38 This is cerebrospinal fluid from a young man with human immunodeficiency virus-1 infection and lymphadenopathy. The total protein and all fractions are within normal limits. An oligoclonal banding pattern is easily visible in the gamma zonRecently a number of research groups in the United States and Europe have reported studies of CSF abnormalities in HIV infection. About 65% of HIV infected individuals have oligoclonal bands and about 60% have an elevated IgG synthesis index. There is not a correlation with stage of disease nor with length of infection. Virtually all patients had demonstrable antibodies to HIV-1 in CSF. The bands in CSF were many times different than those in serum, indicating synthesis in the CNS compartment.

Oligoclonal CSF responses against viruses are found in high

e.

equency in other CNS infections including Herpes fr encephalitis, bacute sclerosing panencephalitis, kuru and infectious viral cephalitides.

suen

76

AIDS - Cryptococcal Meningitis % mg/dlPrealbumin 6.0 1.4 Albumin 23.8 5.7 Alpha-1 10.9 2.6 Alpha-2 11.4 2.7 Beta 21.3 5.1 Gamma 26.6 6.4 CSF protein 24 IgG/albumin 1.83 This specimen had a normal CSF total protein level and all of the fractions were within or below normal limits except gamma globulins. The immunochemical IgG was 11 mg/dl. The patient habeen previously diagnosed with acquired immune deficiency syndrome and was admitted with symptoms of meningitis. Cells

CEREBROSPINAL FLUID

d

of Cryptococcus were seen in CSF and the cryptococcal polysaccharide agglutination d beta regi

test was positive. In this specimen the alpha-1 ons show significant relative elevations, indicating

the presence of acute phase reactants. The IgG is elevated and the gamma region shows a clear oligoclonal banding pattern. It is not known in this case whether the bands are antibody against Cryptococcus

an

or against human immunodeficiency virus.

77

Systemic Lupus with CNS Involvement % mg/dlPrealbumin 1.6 1 Albumin 50.8 27 Alpha-1 6.5 4 Alpha-2 7.4 4 Beta 14.5 8 Gamma 19.2 10 CSF protein 54 IgG/albumin 0.56 Serum C3 = 42 mg/dl Systemic lupus erythematosus is a disease of protean manifestations. This specimen was obtained from a child with SLE presenting as severe renal disease, typical skin rash and arthritis. A spinal tap was performed because of behavioral changes and recent onset of neurologic dysfunction. The CSF protein was slightly elevated, with an IgG of 14 mg/dl. Although the IgG/albumin ratio was elevated in CSF, the IgG synthesis index was marginal because of the polyclonal gammopathy in the serum and renal disease. At the time of the tap the disease was active by clinical and laboratory criteria, and the serum C3 was significantly depressed. An irregular appearance was clearly visible in the CSF gamma region suggesting the presence of immune complexes in the CSF. Oligoclonal bands were not visible in the CSF electrophoresis gel.

CEREBROSPINAL FLUID

Central Nervous System Lupus

78

% mg/dlPrealbumin 3.6 0.6 Albumin 44.2 7.1 Alpha-1 9.2 1.5 Alpha-2 7.5 1.2 Beta 19.3 3.1 Gamma 16.3 2.6 CSF protein 16.0 IgG/albumin 0.41 This patient with active ste

F

CEREBROSPINAL FLUID

sy mic lupus erythematosus had recently reported experiencingsevere headaches. His wife noticed personality changes. The CShad a normal protein level with an elevated IgG/albumin. Threefaint bands were visible on top of a diffuse gamma region, corresponding to immune complexes. The acute phase reactive protein, alpha-1-antitrypsin was also elevated. Another case

80

Meningioma % mg/dlPrealbumin 4.8 1.2 Albumin 56.2 14.1 Alpha-1 1.1 0.3 Alpha-2 2.1 0.5 Beta 17.4 4.4 Gamma 18.5 4.6 CSF protein 25 IgG/albumin 0.50 Myelin basic protein <1 Cerebrospinal fluid was obtained from a middle aged woman with a large benign brain tumor and a slowly progressing neurological deficit of one leg, suggestive of a demyelinating disease. Brain plaques were observed on magnetic resonance studies. The tumor was removed and found to be a meningioma. The neurological deficits showed some improvement, but full function was not restored. The CSF shows normal protein levels and the concentrations of all fractions are normal. CSF IgG is slightly elevated at 7 /dl and the Igmg

regions show a polyclonal pattern and no immunoglobulin bands are G/albumin ratio is elevated. The beta and gamma

visible. Myelin basic protein was not detected.

REBROSPINAL FLUID CE

Malignant Brain Tumor % mg/dlPrealbumin 1.9 2.8 Albumin 57.2 84.1

81

Alpha-1 2.8 4.1 Alpha-2 6.8 10.0 Beta 18.4 27.1 Gamma 12.9 19.0 CSF protein 147 IgG/albumin 0.35 Cerebrospinal fluid was obtained from a woman with a malignant brain tumor. The total protein, albumin, alpha, beta and gamma globulins were all elevated. No oligoclonal bands were observed and the IgG indices were slightly elevated. The relatively low prealbumin level suggests that much of the increased protein is of plasma origin, due to disturbance of the blood-brain barrier by the tumor. The increased indices suggest that there is also an inflammatory component in the CNS.

CEREBROSPINAL FLUID

82

Ependymoma with Froin's Syndrome

%

mg/dlPrealbumin 0 Albumin 59.7 1594 Alpha-1 3.3 88 Alpha-2 8.0 214 Beta 15.5 414 Gamma 13.4 358 CSF protein 2670 IgG/albumin 0.24 This specimen illustrate unusual conditio referre

s n d

as Froin's syndrome. The tient had a small ependymoma cated between L2 and L4 in the spinal cord. The tumor caused ockage of the CSF circulation. When tapped there was a very gh

gels

antopaloblhi pressure and the CSF was thick and bright yellow. The protein content was extreme, but ratios were generally within normal ranges. There was a relative decrease in prealbumin, but alpha-2-macroglobulin and beta-lipoprotein were not present. Additional faint bands were visible in the beta, but there were no visible bands in the gamma. The CSF became thicker on frire geration, but did not gel.

Froin's syndrome consists of subarachnoid blockage of the CSF circulation leading to extreme elevations in CSF protein, xanthochromia, high lumbar pressure and frequently CSF thatat 4 degrees C.

CEREBROSPINAL FLUID

Central Nervous System Lymphoma

83

% mg/dlPrealbumin 2.0 10.4 Albumin 58.0 301.0

G/albumin 0.07

ob f n a, toms ystem lymphoma. The diagnosis nation of the CSF. The total levated, with a relative decrease te phase proteins in the alpha t 21 mg/dl, but the indices were in the gamma region. The protein be the result of increased . CSF beta-2-microglobulin when

beta-2-microglobulin can be a useful ocytic malignancies of the CNS.

Alpha-1 5.9 30. 6 Alpha-2 12.1 62.8 Beta 16.9 87.7 Gamma 5.0 25.9 CSF protein 519

CEREBROSPINAL FLUID

Ig This specimen was tained rom a patient with a

who developed sympestablished gnosis lympdia of homconsistent with central nervous s

r d b ogi amiwas confi me y cytol c exprotein a d fracti wern all ons e ein prealbumin and evidence of acuand beta. CSF IgG was elevated anormal. No banding was visible increase in this case appears to

abilityblood-brain barrier permeelevated relative to serumst for monitoring lymphte

Pseudo-Tumor of Brain % mg/dl

84

Prealbumin 3.6 1.8 Albumin 63.6 31.2 Alpha-1 3.7 1.8

2.8 Alpha-2 5.8 Beta 15.6 mma 7.7

7.6 3.8

F protein 49

G/albumin 0.10

m a patient whose admitting n." A final diagnosis was not The pattern is included here ng pattern was observed in the herwise relatively normal.

the CSF of patients with multiple sclerosis and similar seases, HIV infection, slow virus CNS infections, some cephalitis, TB meningitis, some fungal meningitis and urosyphilis. Bands are found with lesser frequency in CSF from tients with chronic and relapsi

ic n is, rome, and cca ly bands are seen in CSF from CNS

conditions of the CNS and neurologic disease. In s the presence and amount of ty. although not totally remission have somewhat lower goclonal antibodies than those

CEREBROSPINAL FLUID

Ga CS Ig This specimen was obtained frodiagnosis was "pseudo-tumor of brai

available when this was written.because a faint oligoclonal bandi

n is otgamma region. The patter

Oligoclonal immunoglobulin bands are found in high frequency indiennepa ng inflammatory

Guillain-Barre syndpolyneuropathies, opt euritother neuropathies. O sionaltumor patien other lammatts, inf oryrarely in CSF from subjects without

n inf ry ionchronic a d lammato conditbands is el d to di se actr ate sea ivireflective of status. Patients in incidence and lesser amounts of oliwith active disease.

85

Multiple Sclerosis % mg/dlPrealbumin 14.3 2 Albumin 41.2 16 Alpha-1 2.3 1 Alpha-2 3.3 1 Beta 6.8 3 Gamma 42.2 17 CSF protein 40 IgG/albumin = 1.05 % mg/dlPrealbumin 4.7 2 Albumin 46.2 22 Alpha-1 3.3 2 Alpha-2 5.4 3 Beta 11.2 5 Gamma 29.2 14 CSF protein 48 IgG/albumin = 0.95

Electrophoresis patterns are illustrated from 6 cases of ltiple sclerosis. All the examples shown have normal to derately elevated total protein, elevated IgG, oligoclonal

banding and elevated IgG/albumin. The myelin basic protein was also elevated in each specimen. The most consistent feature of multiple sclerosis CSF is oligoclonal banding, even in remission. IgG levels are not consistently elevated and IgG/albumin is only elevated in about 80% of cases. The clinical diagnosis of this disease can be very difficult, and the laboratory can provide significant help to the neurologist.

CEREBROSPINAL FLUID

CEREBROSPINAL FLUID

mumo

86

% mg/dlPrealbumin 4.1 1 Albumin 49.5 18 Alpha-1 1.5 1 Alpha-2 3.6 1 Beta 10.8 4 Gamma 30.5 11 CSF protein 36 IgG/albumin = 0.61 % mg/dlPrealbumin 5.4 1.4 Albumin 50.1 13.0 Alpha-1 2.0 0.5 Alpha-2 4.6 1.2 Beta 16.1 4.2 Gamma 21.8 5.7

F protein 26

G/albumin = 0.57

%

CS Ig

mg/dlPrealbumin 3.1 2.9 Albumin 57.6 53.6 Alpha-1 4.0 3.7 Alpha-2 8.7 8.1 Beta 14.6 13.6 Gamma 12.0 11.2 CSF protein 93 IgG/albumin = 0.50

CEREBROSPINAL FLUID

CEREBROSPINAL FLUID

CEREBROSPINAL FLUID

87

% mg/dlPrealbumin 2.4 1.1 Albumin 51.9 23.9 Alpha-1 2.0 0.9

Alpha-2 3.0 1.4Beta 14.0 6.4 Gamma 26.7 12.3

CEREBROSPINAL FLUID

CSF protein 46 IgG/albumin = 0.70

Neuromyelitis Optica

CEREBROSPINAL FLUID

% mg/dlPrealbumin 6.1 2 Albumin 57.0 19 Alpha-1 3.7 1 Alpha-2 7.2 2 Beta 16.9 6 Gamma 9.0 3 CSF protein 33 IgG/albumin 0.14 Neuromyelitis optica or Devic's disease is thought to be a variant of multiple sclerosis. The usual presentation is eye pain followed by vision loss in one or both eyes. Spinal cord involvement may be present; it usually appears after the eye symptoms. Spinal involvement presents as back pain with neurologic deficits in the legs and plantar responses. It mimispinal chord compression, because significant edema may be present. The involved optic nerves show demyelinationpic

cs

and

erns in

tis optica are similar to those in multiple sclerosis.

ty al multiple sclerosis type plaques. Many times symptoms can be reversed by treatment with high dose corticosteroids. This 32 year old woman presented with symptoms of frontal headache followed by recent onset of blindness. The cerebrospinal fluid electrophoresis pattern showed the presenceof 3 bands in the gamma region. CSF electrophoretic patturomyeline

89

Single Gamma Band in CSF CEREBROSPINAL FLUID

% mg/dlPrealbumin 3.8 2 Albumin 53.4 25 Alpha-1 2.3 1 Alpha-2 5.7 3 Beta 14.5 7 Gamma 20.2 9 CSF protein 47 IgG/albumin = 0.5 Occasionally a single band is observed in the gamma region of a cerebrospinal fluid electrophoresis. Statistically this finding is of much less significance than oligoclonal banding. The single band may be a variant of oligoclonal banding, but confirmation is necessary from clinical and other laboratory data. The band may also be a paraprotein, in which case the same protein should be found in the serum and/or in the urine. Immunoelectrophoresis or immunofixation electrophoresis on nceco ntrated CSF can be performed, when paraproteinemia is

suspected. Monoclonal bands of immunoglobulin are rarely foundin CSF in cases of chronic lymphocytic leukemia and immunoblastilymphoma. A large fraction of single gamma bands in CSF are noof any diagnostic significance. The pattern illustrated here is from a patient with mild multiple sclerosis. The IgG/albumin is elevated as was myelin sic protein.

90

c t

ba

91

Chronic and Relapsing Inflammatory Polyneuropathy % mg/dlPrealbumin 2.0 3.0 Albumin 66.3 44.5 Alpha-1 3.2 4.8 Alpha-2 8.6 12.8 Beta 19.3 28.8 Gamma 24.4 36.4

CEREBROSPINAL FLUID

CSF protein 149 IgG/albumin = 0.55 The chronic and relapsing inflammatory polyneuropathy syndromes (CRIPS) are a group of diverse polyneuropathies. Theusually but not always have motor involvement with proximal and distal weakness. The spinal fluid proteins are always elevated and nerve conduction velocities are slowed. The course of the disease is progressive or recurrent. The disease is usually responsive to corticosteroids, plasma exchange and/or immunosuppression.

y

The typical electrophoretic pattern shows an elevated total otein and albumin. Alpha and beta globulins are variable. The mma may be elevated in a polyclonal or oligoclonal pattern. G/albumin and IgG synthesis index are not consistently evated.

prgaIgel

Guillain-Barre Syndrome

92


CEREBROSPINAL FLUID

CSF protein 101 IgG/albumin = 0.20 Guillain-Barre syndrome is acute inflammatory neuropathy that follows a viral or mycoplasmal infection, surgery, neoplasmor immunization. About 40% of cases have no obvious precedent.The syndrome usually starts with back or leg pain and paresthesia, which progresses to weakness, which then becomes the primary symptom. The disease can rapidly progress to paralysis d a

an utonomic involvement can occur. A cluster of cases occurred following the abortive swine flu vaccination program in the U.S. The typical electrophoretic pattern in Guillain-Barre syndrome shows an elevation of total protein and albumin. The gamma fraction and alpha and beta globulins usually remain in approximately normal proportions to albumin. An oligoclonal banding pattern is found in some, but not most cases. There is no evidence for much IgG synthesis in the CNS compartment in thiscondition. Similar patterns are seen in transverse myelitis.

CSF with Large Beta Spike CEREBROSPINAL FLUID % mg/dl

93

Prealbumin 3.7 1.3 Albumin 47.1 16.5 Alpha-1 1.6 0.6 Alpha-2 1.8 0.6 Beta 34.1 11.9 Gamma 11.6 4.1 CSF protein 35

G/albumin = 0.10

elin basic protein <1

clear and colorless ter concentration. The beta peak was not immunoglobulin when

mobility as transferrin; however, there was insufficient specimen volume to confirm this identification.

Ig My This CSF specimen was obtained from a patient with a diagnosis of cerebellar ataxia. The bands marked in the gamma e artefactual stain dots. The specimen was

araftested on an immunochemical analyzer. The spike has the same

Benign Monoclonal Gammopathy with Neuropathy

94


CEREBROSPINAL FLUID

CSF protein 41 IgG/albumin = 0.21 This patient was referred to a neurologist with a mild

ngle oclo eak was found in the CSF resis as free lambda

rine showed the presence of s protein of identical mobility

dyscrasia syndromes is usually Histologically there is egeneration. It is somewhat patients. In almost all cases

e l lambda, either free or associated with G, mber of studies of these cases have shown e h oglobulin deposited on the affected nerves, pecially in the sheaths. The IgM type of polyneuropathy is owly progressive and shows severe reduction of conduction loc s to react with a carbohydrate

polyneuropathy. A si monand was identified by unoel

nal prophoimm ect

chains. Examination of serum and usmall amo nt f lambd ence-Ju s o a B oneto the CSF peak. Polyneuropathy of plasma cell

.of a mixed motor and sensory typeusually demyelination and axonal dre d younger mo common in males an

th ight chain type isIg IgA or IgM. A nu

omogeneous immunthesslve ity. The antibody seemdeterminant on central and peripheral myelin-associated glycoprotein in half of the cases. The remaining cases show antibodies reacting with other glycolipids and glycoproteins of the peripheral nerve. The IgG type antibodies are associated with osteosclerotic type myeloma react with other myelin antigens. Cases of polyneuropathy with paraproteinemia have recently been found in which the immunoglobulin reacts with thenerve gangliosides.

95

Paraprotein Polyneuropathy

%

mg/dlAlbumin 56.6 187 pha-1 7.1 23 pha-2 5.2 17 ta 5.7 19 mma 25.4 84

tal Protein 330

This scan illustrates a urine electrophoresis from a patient o presented w of identical bility was found in the serum and urine and was identified by

opho s as lambda.

AlAlBeGa To

ith polyneuropathy. A moderate spikewhmoimmunofixation electr resi

IgG-

URINE

96

neuropathy Paraprotein Poly % mg/dlPrealbumin 3.9 1.9 Albumin 58.0 27.8

2.4 Alpha-1 4.9 pha-2 10.0 Al 4.8 ta 14.1 6.8 mma 9.1 4.4

F protein 48

G/albumin = 0.19

%

BeGa CS Ig IgG synthesis index = 0.34 g/dlAlbumin 43.9 2.99 pha-1 6.0 0.41 pha-2 13.8 0.94 ta 13.5 0.92 mma 22.8 1.55

Serum electrophoresis showed a one distinct and two faint bands labeled 1 was identified as an IgM-aint bands, 2, were IgG by

Band 1, the IgM-lambda rebrospinal fluid. The urine verflow Proteinuria with

fla

CEREBROSPINAL FLUID

AlAlBeGa Total Protein 6.8

SERUM

IgG = 1 mg/dl 620 IgA = 377 mg/dl IgM = 38 mg/dl This patient presented

opathy.with progressive polyneurpolyclonal gammopathy withpresent. The distinct bandlambda paraprotein and the fimmunofixation electrophoresis.paraprotein was also found in ceom this patient is shown as Ofr

In mmation.

Malignant Paraprotein Polyneuropathy % g/dlAlbumin 56.5 1.8 Alpha-1 8.8 0.3 Alpha-2 13.4 0.4 Beta 10.6 0.3 Gamma 10.7 0.3

SERUM

98

Total Protein 3.2 IgG = 410 mg/dl


Beta-2-microglobulin = 23mg/l % mg/dlAlbumin trace Alpha-1 Alpha-2 Beta Gamma 100 1400 Total Protein 1450 Protein/24hrs = 14.5 g Continued on next page.

URINE

% mg/dl

99

Prealbumin 3.7 bumin 63.2 31.0

1.8

pha-1 3.6 1.8 Alpha-2 5.8 2.8 Beta 15.0 7.3 Gamma 8.7 4.3 CSF protein 49 IgG/albumin = 0.10 IgG synthesis index = 0.44 Beta-2-microglobulin = 2.3mg/l This elderly man had been followed by a neurologist for approximately two years. During that time he developed a progressive polyneuropathy, culminating in an inability to walk. When admitted to the hospital he had significant anemia and renal impairment. He was given contrast medium for a CNS CAT scan, developed rapidly progressive renal failure and expired in a few days. The urine electrophoretic pattern shows a huge double spike

t 14 g/day and a small amount of albumin. The ified as kappa Bence-Jones protein by

immunoelectrophoresis. The serum electrophoresis showed suppression of normal immunoglobulins and a trace of kappa chain in the far gamma. The CSF electrophoresis appeared normal except

Serum beta-2-microglobulin was highly elevated and the CSF level was at the upper limit of normal. It is unusual for a kappa chain plasma cell dyscrasia to lead to polyneuropathy, the overwhelming majority of cases reported in the literature have lambda light chains. Neurological symptoms can result from deposition of light chains or fragments in nerves, which may appear as typical amyloid deposits or as the more recently described non-amyloid light chain deposition disease. Immunofluorescent or immunoperoxidase staining of a biopsy specimen provides a definitive diagnosis.

CEREBROSPINAL FLUID

AlAl

amounting to abouspikes were ident

for a trace band at the end of the gamma, which was found to be kappa light chain.

100

Isoelectric Focusing of CSF Isoelectric focusing (IEF) is electrophoresis in a pH gradient. Most proteins form sharp bands at their isoelectric points in this method. Silver stains are significantly more sensitive than the usual organic dyes used for detecting proteins, although silver stains are much less reliable for quantitation. Unconcentrated cerebrospinal fluid can be studied by the combination of IEF and silver staining, yielding information not apparent in high resolution protein electrophoresis. The gel illustrated shows CSF from patients with six different conditions. The sample in track 1 comes from a patient with viral encephalitis. The protein, albumin and IgG are within the normal range. The IEF pattern shows a polyclonal synthesis of immunoglobulin. The second pattern shows oligoclonal bands from a patient with multiple sclerosis. Two band patterns are visible; one at the cathode and one about a quarter of the way toward the anode. This sample had an IgG of 10mg/dl and IgG-albumin ratio of 0.46. The sample in track 3 shows the presence of hemoglobin A near the middle of the pattern, this may indicate a cerebrovascular accident, red cell leakage into the CNS or a traumatic tap. The fourth track shows a normal CSF pattern. The

anode are prealbumin. The sample in track 5 is from a patient with a metastatic carcinoma in the

tral nervous system. The albumin was 34mg/dl and IgG 5mg/dl. he pattern shows polyclonal immunoglobulins with a C-reactive otein band nearest the cathode. Some serum proteins not rmally visible in CSF can be seen anodal to the position of moglobin. Track 6 illustrates HIV infection. The CSF protein s 49mg/dl with an albumin of 23mg/dl and an IgG of 14mg/dl. veral clonal patterns are visible in the immunoglobulin region.

Viral encephalitis, 2-Multiple sclerosis oligoclonal Hemoglobin A stroke, 4-Normal CSF, 5-Polyclonal carcinoma with reactive protein, 6-Human immunodeficiency virus

three sharp bands at the

cen TprnohewaSe 1-3-C-

101

HER CEREBROSPINAL FLUIDS

F Cervical Myelitis

OT CS

CSF CNS Sarcoid

102

F CNS Toxoplasmosis CS

CSF Transverse Myelitis

103

OTHER FLUIDS

104

Pleural Fluid - Transudate

%

g/dlAlbumin 61.0 0.30 Alpha-1 6.5 0.03 Alpha-2 6.8 0.03 Beta 13.0 0.06 Gamma 12.8 0.06 Total Protein 0.50 This pattern shows a sample of pleural fluid with a relatively low protein concentration. The electrophoretic pattern is similar to that of serum without the alpha-2 macroglobulin and beta-lipoprotein. Since the fluid has a low LDH and a low total protein, it is a transudate.

other case An

PLEURAL FLUID

Pleural Fluid - Infection

105

%

g/dlAAlbumin 46.0 0.9 lpha-1 10.4 0.2 Alpha-2 8.8 0.2 Beta 15.2 0.3 Gamma 19.6 0.4 Total Protein 1.9 This pleural fluid was obtained from a patient with an infection of unknown origin. The protein concentration is about one-third of plasma protein concentration. The fluid LDH level was 101 and the serum LDH was 350, giving a ratio of 0.29. The electrophoretic pattern is similar to that of plasma, except for the significant elevation of alpha-1 globulin. A prominent fibrinogen peak is present in the gamma, along with an irregular elevation of gamma globulin probably representing an early immune response. This fluid is a transudate with a protein level below 3g/dl and an LDH ratio below 0.6.

PLEURAL FLUID

SYNOVIAL FLUIDS Synovial Fluid Exudate

Synovial Fluid Gout

107

108

PARAPROTEINS

M-COMPONENTS

109

Paraproteins

Paraproteins or M-components are abnormal plasma proteins, most always immunoglobulins. Most paraproteins are monoclonal,

myeloma proteins, Bence-Jones proteins, cryoglobulins and Waldenstrom's macroglobulin. Paraproteins are produced by abnormal proliferations of cells of the B lymphocyte lineage, either lymphocytes or plasma cells. The proliferation may be benign or malignant, transient or permanent. Immunoglobulin paraproteins are identified by either immunoelectrophoresis or immunofixation electrophoresis. Immunochemically measured immunoglobulin levels and kappa-lambda ratios can provide additional evidence. As can be seen in many of the following patterns, the interpretation is not always simple. In the present cost cutting atmosphere several investigators have suggested replacing immunoelectrophoresis and immunofixation electrophoresis with immunoglobulin levels and kappa-lambda ratios. This system is available on a computerized system, which even generates interpretations. A large fraction of the cases we see are atypical, and we find that immunofixation electrophoresis is the most appropriate test to characterize these. It is not necessary to repeat the complete series of tests on patients with monoclonal gammopathies in following the course of the disease and response to therapy. Repeating the electrophoresis and/or appropriate immunoglobulin levels are sufficient. Paraprotein identification needs only to be repeated in the rare case in which a new paraprotein appears. Repeating urine electrophoresis is very valuable for evaluating the amount and nature of renal disease in monoclonal gammopathy and is the method of choice for quantitating Bence-Jones protein. Urine electrophoresis results should be given as protein excretion per 24 hours in order to account for differences in fluid intake and output. The levels of paraprotein generally reflect the relative tumor mass in a given patient, although synthesis rates vary widely among different patients. Serial measurement of serum paraprotein levels and/or urinary Bence-Jones protein allow the hematologist to assess the effectiveness of therapy and to recognize relapse at an early stage. Serum beta-2-microglobulin levels are also of value in following some patients. A discussion of monoclonal gammopathy of undetermined significance (MGUS) or benign monoclonal gammopathy (BMG) can be found in the first section of this book. Multiple myeloma is a malignant proliferation of plasma cells in the bone marrow. The disease exhibits a wide spectrum and many cases are difficult to classify as either myeloma or

alalthough some are oligoclonal. Examples of paraproteins are

110

US. Most hematologists use some type of score chart. An MGexample of one from the Southwestern Oncology Group follows. DIAGNOSIS OF MULTIPLE MYELOMA Major Criteria I Bone marrow over 30% plasma cells (monoclonal)

noclonal)

2000mg/dl IgA, 1000mg/dl IgD

g/day

II Biopsy pr n plasm toma ove acy (mo III Over 3000mg/dl monoclonal IgG, or IgE Be over 1000mIV nce-Jones protein

Minor Criteria

e marrow 10-30% ma Bon onoclonal plasma cells

Any monoclonal paraprotein present b c Discrete lytic bone lesions d Suppression of normal immunoglobulins, IgM < 50mg/dl, IgA < 100mg/dl, IgG < 600mg/dl Diagnosis of Multiple Myeloma Any 2 major criteria I + b, c, or d II + a, b, c, or d III + a IV + a a + b + c a + b + d is Th has now been replaced in most academic institutions with

current WHO score chart, shown below.

the

Diagnosis of Multiple Myeloma (World Health Organization) Major Criteria Minor CriteriaI Bone marrow over 30% plasma cells a 10-30% plasma cells II Biopsy proven plasmacytoma b Monoclonal protein

present III Monoclonal over 3.5g% IgG or 2g% IgA c Lytic bone lesions

d Reduced normal immunoglobulins IgM<50mg/dl, IgA<100mg/dl, IgG<600mg/dl

of one major and one minor ree minor criteria which must include (a) and (b).

ptomatic and have progressive disease or ll moldering or indolent myeloma.

LUSTRATIONS rum Immunoelectrophoresis M-components

Bence-Jones protein over 1g/day

e diagnosis requires a minimum Th

criterion or the pTh atient must be sym

wi be classified as s ILSe

Urine Immunoelectrophoresis M-components

111

eloma Untreated Natural Course My

112

114

Waldenst inemia rom's Macroglobul % g/dlAlbumin 47.3 4.1 Alpha-1 3.1 0.3 Alpha-2 6.0 0.5 Beta 13.0 1.1 Gamma 30.5 2.7 Total Protein 8.7

IgG = 471 mg/dl IgA = 32 mg/dl IgM = 4490 mg/dl This pattern illustrates Waldenstrom's macroglobulinemia. This disease is defined by finding a large IgM spike in the serumand a proliferation of monoclonal cells ranging from B lymphocytes to almost fully differentiated plasma cells in the bone marrow. The cells are typically intermediate between lymphocytes and plasma cells. Macroglobulinemia is less aggressive than multiple myeloma and behaves like a low grade lymphoma. Rarely discrete bone lesions are present, then the disease behaves like multiple myeloma and is treated as such. The IgM proteins typically are found in the beta or gamma region.his one is at the junction

and is integrated into the gamma.

kappa. Kappa Bence-Jones protein was present in the urine. Although most patients with macroglobulinemia have significant Bence-Jones proteinuria, renal disease is uncommon and when present less severe than in myeloma. In many cases the dominant symptoms are from increase in plasma viscosity from the large amount of macroglobulin. This is clinically most apparent by imaging the eye grounds, but can be measured in the laboratory. Hyperviscosity syndrome is managed by plasma removal. Many of the IgM paraproteins are cryoglobulins (precipitate in the cold), either as single paraproteins or as weak antibodies against other immunoglobulins (mixed cryoglobulins). Rarely they are pyroglobulins, which precipitate on heating. Cryoglobulins can be difficult to type and may require isolation. Mixed cryoglobulins cannot be accurately measured by routine immunochemical methods. The presence of cryoglobulins can also cause problems in routine electrophoresis, when they do not remain soluble. This is especially true in high resolution techniques, where cooling is

TThe IgG and IgA exhibit suppression. The IgM was typed as IgM-

115

quired. Some IgM paraproteins have antibody activity. There e a group of IgM- e a typical cold glutinin disease, ariable region quence. Other pa tting factors,

es a ther autoantigens.

rear kappa paraproteins which causag which mainly have the same v

raproteins may react with closeglycoproteins on nerv nd o % g/dlAlbumin 50.7 3.9 Alpha-1 3.0 0.2 Alpha-2 6.9 0.5 Beta 10.7 0.8 mmaGa 28.9 2.2

Total Protein IgG = 789 mg/dl


a paraprotein in the gamma times, but not always somewhat ias are Waldenstrom's

This sc shows a gM-la an n I mbdregion. IgM paraproteins are manybroad. About 11% of paraproteinemmacroglobulinemia. Another case

Waldenstrom's Macroglobulinemia

116

% g/dlAlbumin 30.2 3.2 Alpha-1 1.9 0.2 Alpha-2 5.1 0.5 Beta 7.1 0.7 Gamma 55.7 5.9 talTo Protein 10.6

IgG = 981 mg/dl IgA = 122 mg/dl

IgM = 4600 mg/dl % mg/dlAlbumin 15.3 10 Alpha-1 7.8 5 Alpha-2 4.8 3 Beta 10.7 7 Gamma 61.3 40 Total Protein 65

d at time of diagnosis of a case . The paraprotein was IgM- elevated at 6.4 centistokes. arge number of small to the cells in a small cell sis pattern contained three

ike ion. The largest was identified as lambda nce immunofixation electrophoresis. The other o s ied as mu Fc fragments and Fab fragments.

ERUMS

URINE

Protein/24hrs 770 Thes s imens w obtae pec ere ineof Waldenstrom's macroglobulinemialambda and the serum viscosity wasThe bone marrow biopsy showed a llymphocytes, similar in appearancemph lectrophorely oma. The urine e

sp s in the gamma regBe -Jones protein by tw pikes were identif

B Cell Lymphoma

118



IgM = 125 mg/dl This serum was obtained from a patient with a B cell non-Hodgkins lymphoma. A small paraprotein spike is visible in the gamma. It was typed as IgG-lambda. Some B cell lymphomas produce Bence-Jones protein. Cases of non-Hodgkins lymphoma have been reported in which defective heavy chains were synthesized (heavy chain disease) or half immunoglobulin molecules secreted. Paraproteinemia is not typical of B-cell lymphomas. Occasionally oligoclonal immunoglobulins are produced by

lymphomas and cases have been described where the tumor produces polyclonal immunoglobulins.

119

Chronic Lymphocytic Leukemia



IgM = 140 mg/dl % g/dlAlbumin 55.1 3.5 Alpha-1 5.1 0.3 Alpha-2 10.2 0.7 Beta 22.6 1.5

6.9 Gamma 0.4 tal Protein 6.4 To

IgG = 684 mg/dl


that al

ulin ppression in CLL have a less favorable prognosis.

These patterns illustrate two cases of chronic lymphocytic leukemia with paraproteins. The first with IgG-kappa and the second with IgM-lambda. The first pattern also shows an acute phase reaction. The most common form of CLL is of B cell origin and in many cases the cells make monoclonal immunoglobulin, usually in small amounts. Some investigators have suggested patients whose sera show either the presence of monoclonmunoglobulin, polyclonal gammopathy, or immunoglobim

su

120

IgM Paraproteinemia

%

g/dlAlbumin 59.8 4.1 Alpha-1 3.4 0.2 Alpha-2 6.2 0.4 Beta 10.7 0.7 Gamma 19.9 1.4 Total Protein 6.8 IgG = 757 mg/dl IgA = 42 mg/dl IgM = 1600 mg/dl This serum pattern was found as part of the evaluation of a woman being worked up for iron deficiency anemia. Clinically she had only slight hepatosplenomegaly. The spike in the gamma region was identified as an IgM-kappa paraprotein. This probably represents an early stage of Waldenstrom's macroglobulinemia. A significant fraction of patients with malignant monoclonal gammopathies have atypical presentations. These diseases should be included in the differential diagnoses of anemia and peripheral polyneuropathy in older individuals. Serum and urine electrophoresis can provide valuable screening tools when ordered at an appropriate point in the diagnostic evaluation.

Secondary Malignancy

% g/dlAlbumin 46.8 3.1 Alpha-1 3.7 0.3 Alpha-2 7.4 0.6 Beta 10.7 0.7 Gamma 29.3 2.0 Total Protein 6.7 IgG = 2400 mg/dl IgA = 129 mg/dl IgM = 137 mg/dl Another case This patient had been treated several years previously ldens

for trom's macroglobulinemia. She had no symptoms and her

developed recurrent bleeding into the gastrointestinal system. Her serum developed a series of four bands in the gamma region. The relative proportions of the bands changed with time. Typing showed the bands to consist entirely of IgG1-kappa. The patient's urine contained about 100mg/dl of serum proteins with no Bence-Jones protein. IgG2 was undetectable in serum and only traces of IgG3 were found. After eighteen months the bleeding became uncontrollable and the patient expired.

Waserum showed no detectable paraprotein for five years. She then

121

Secondary Malignancy

122


IgG = 1740 mg/dl IgA = 204 mg/dl IgM = 81 mg/dl This patient had been treated for non-Hodgkins lymphoma with chemotherapy four years before this sample was obtained. There were no electrophoretic abnormalities at the time of his initiadiagnosis. The lymphoma was in remission for three years. The specimen shown here contained three spikes in the gamma region each of which typed as IgG-lambda.

l

Carcinoma of the Lung

123

% g/dlAlbumin 62.9 4.0

SERUM

Alpha-1 4.1 0.3 Alpha-2 11.4 0.7 Beta 10.3 0.7 Gamma 11.3 0.7 talTo Protein 6.4


IgM = 128 mg/dl URINE % mg/dlAlbumin 47.1 68 Alpha-1 17.5 25 Alpha-2 18.3 27 Beta 8.8 13 Gamma 8.3 12

145 Total Protein Patients with various types of carcinomas occasionally produce small amounts of paraproteins. The serum from this man with lung cancer showed two small spikes identified by immunofixation electrophoresis as IgG-kappa. The urine contains a clearly visible spike identified as free kappa chain. The urine also shows the presence of significant amounts of inflammatory proteins in the alpha-1 and 2 regions.

125

Thymoma with Paraproteinemia

%

g/dlAlbumin 56.9 3.9 Alpha-1 3.8 0.3 Alpha-2 15.6 1.1 Beta 7.9 0.6 Gamma 15.7 1.1 Total Protein 6.9 IgG = 1360 mg/dl

IgA = 264 mg/dl IgM = 539 mg/dl This patient was an older woman who had previously had a malignant thymoma removed surgically. The tumor had recurred andshe was undergoing radiation therapy. The electrophoretic pattern showed three small spikes, which were identified as IgM-kappa, IgG-kappa and IgG-lambda by immunofixation. One contemporary theory of the origin of paraproteins is that they are caused by an imbalance of T cell function. This has been proposed as the mechanism of paraprotein production in immunodeficiency diseases, allogeneic and syngeneic bone marrow transplantation, human immunodeficiency virus infection, and the increased incidence of small paraprotein spikes with age. It is well known that thymomas can cause severe alterations in immune function such as the hypogammaglobulinemia known as Good's syndrome. This case may illustrate a functional imbalance in CD4 and CD8 function leading to an oligoclonal stimulation of B cells.

SERUM

127

IgG Cryoglobulinemia % g/dlAlbumin 27.5 2.3 Alpha-1 1.0 0.1 Alpha-2 3.1 0.3 Beta 9.8 0.8 mma 58.6 4.9 Ga

Total Protein 8.3

IgG = 5060 mg/dl IgA = 1090 mg/dl IgM = 280 mg/dl C3 = 19 mg/dl C4 = 18 mg/dl

osplenomegaly and proteinuria. A bone marrow ma cells in sheets. A broad irregular peak gamma region of serum electrophoresis,

with weaker bands extending to the beta. All of the immunoglobulins were elevated, the IgG and IgA remarkably. C3 complement was very low, but other complement components were

s of urine showed a non-selective oglobulin precipitated at room temperature

and the centrifuged cryocrit was 12% at 4 degrees. Cryoglobulin was isolated and identified as IgG1-kappa. Many IgG cryoglobulins are IgG3 subclass. The patient was treated by extensive plasmapheresis and was discharged. He did not report for follow-up care and expired shortly thereafter.

This patient presented with anemia, thrombocytopenia, lymphopenia, hepatbiopsy showed 80% plaswas visible in the far

normal. Electrophoresiproteinuria. Some cry

128

Type II Cryoglobulinemia

%

g/dlAlbumin 44.4 2Alpha-1 2.4 0.1

.8

Alpha-2 11.1 0.7 Beta 16.6 1.1 Gamma 25.4 1.6

.4

IgG = 1700 mg/dl IgA = 319 mg/dl IgM = 104 mg/dl %

Total Protein 6

g/dlAlbumin 61.0 4.3 Alpha-1 8.1 0.6 Alpha-2 9.3 0.7 Beta 6.9 0.5 Gamma 14.7 1.0 Total Protein 7.0 Another case

Both of these sera contain IgM-kappa paraproteins with eumatoid factor like activity against IgG and IgA. Significant ounts of cryoprecipitate were found in both sera. munofixation electrophoresis studies on the precipitate using ssociating buffers may be necessary to properly work-up mixed yoglobulins. In both of these cases the IgG and IgA contained th kappa and lambda, while the IgM from the precipitate ntained only kappa. Because of the overwhelming size of the noclonal IgM molecules, the complexes appear as restricted raprotein spikes. The pattern of the second serum shows the esence of both free IgM-kappa and IgM-kappa-IgG/IgA complexes two distinct spikes.

Type II cryoglobulinemia is associated with mphoproliferative disease, chronic infectious and autoimmune eumatoid diseases. Cases without these underlying causes are lled essential cryoglobulinemia. Typical presentations are

rhamImdicrbocomopapras lyrhca

129

rpura, polyarthralgias, and cutaneous vasculitis. Liver sease is found in many cases and about 55% develop renal sease. Development of renal disease is a poor prognostic dicator. Essential cryoglobulinemia and lymphoproliferative sease associate teins. eumatoid diseas gG or IgA.

The presence of cryoprecipitate causes interference in anal , m extremely high

nd kept warm, then WBC histogram usually shows an descending to baseline. Manual

f our no bject to this interference.

pudidiindi d cases usually show IgM paraproRh e associated cases may be IgM, I automated hematology yzerswhite counts. If the cimen

any times giving apparatus are spe a

normal counts are obtained. The y xis gra ly elevated -a slope dual

counts, o c se, are t su

130

Monoclonal Cryogelglobulin % g/dlAlbumin 40.3 3.9 Alpha-1 3.2 0.3 Alpha-2 10.8 1.0 Beta 9.1 0.9 Gamma 36.6 3.5 Total Protein 9.6 IgG = 538 mg/dl IgA = 1784 mg/dl* IgM = 20 mg/dl * Value in error because of cryoprecipitation. This serum specimen was obtained from a patient with multiple myeloma. Both whole blood and serum formed a gel, when the specimens cooled to below 35‘C. This pattern was from the initial serum specimen and may not reflect the total amount of paraprotein present because of partial cryoprecipitation. It was not possible to get an immunochemical IgA measurement that was consistent with the electrophoretic pattern. The serum viscosity was 7.9 centistokes at 38‘ with complete gelling between 32 and 35‘. After plasma exchange performed in a room heated above 38‘, the serum viscosity was 1.8cS at 33‘ and 3.4cS at 22‘. This is an extreme amount of temperature dependence of the viscosity and shows that the gelling occurs by a continuous increase in viscosity. The gelling does not take place even at 4‘, when the paraprotein concentration is sufficiently low. This monoclonal cryoglobulin is an IgA-kappa protein, but similar properties have been reported with IgM and rarely IgG paraproteins.

SERUM

Primary Amyloidosis (AL)

131


SERUM

Alpha-1 5.7 0.2 Alpha-2 32.6 1.1 Beta 21.1 0.7

15.0 0.5

Gamma talTo Protein


IgM = 91 mg/dl See track 106

132

% mg/dlAlbumin 50.9 1069Alpha-1 5.4 113

an was

evi AL ylo l ction rapidly deteriorated th

ma

. The amount of Bence-Jones protein and IgG

diagnosis. Two courses of myeloma chemotherapy had no significant effect on either paraprotein levels or the course of the disease. Secondary amyloidosis is a common finding in multiple myeloma. There is not a clear distinction between primary amyloidosis and multiple myeloma in many cases. Primary AL amyloidosis is usually associated with lambda light chains with kappa chains in a quarter of the cases and no free light chain in about 20%. Primary amyloidosis is usually unresponsive to chemotherapy. This patient has survived over two years despite an albumin excretion of over 15g/day.

URINE

Alpha-2 13.0 273 Beta 10.1 212 Gamma 20.5 431 Total Protein 2100

Protein/24hrs = 30 g This 36 year old mdiagnosed five years pr ously with primary

idosis. His renaamfunto e stage shown here. The serum contained an extremely low level of albumin with only a marginal improvement with intravenous albumin solution. There was a large alpha-2-macroglobulin peak and a prominent sharp beta-lipoprotein peak, as are typical of nephrosis. The small peak in the beta was identified as free lambda chains and the larger peak in the gamwas 600 mg/dl of IgG-lambda paraprotein. The urine electrophoresis has the appearance of a serum pattern. There is a relatively small lambda Bence-Jones protein peak in the beta (actually 1.2g/day) and an intact paraprotein peak in the gamma 5.5 g/dayof

paraprotein had increased significantly from the time of

133

es Additional casAL-Amyloidosis Serum

AL-Amyloidosis Urine

134

AL-Amyloidosis IgG Serum

AL Amyloidosis IgG Urine

135

Peritoneal Fluid - Amyloidosis %

mg/dlAlbumin 71.2 46 Alpha-1 4.1 3 Alpha-2 7.6 5 Beta 9.4 6 Gamma 7.7 5 Total Protein 65 Peritoneal and pleural fluids may be either exudates or transudates. This fluid is a transudate. A trace of prealbumin is visible, along with the alpha-2 inflammatory proteins, transferrin, C3 complement and fibrinogen. No abnormal proteins are visible. Exudates and transudates are usually distinguished by protein or lactic dehydrogenase levels. Pleural exudates have protein over 3g/dl 90% of the time and a ratio of fluid LDH to serum LDH over 0.6, while transudates have lower levels. Peritoneal fluid exudates usually have protein over 2g/dl (some authorities use 2.5). Some authors recommend the use of protein ratios rather than absolute levels; pleural fluids with protein concentrations above 50% of plasma are considered to be exudates.

PERITONEAL FLUID

In some cases detailed composition studies are necessary to distinguish exudates from transudates.

136

Solitary Plasmacytoma

137

% g/dlAlbumin 37.6 3.0 Alpha-1 3.2 0.3 Alpha-2 16.3 1.3 Beta 13.4 1.1 Gamma 29.6 2.4

SERUM

Total Protein IgG = 2330 mg/dl IgA = 364 mg/dl IgM = 69 mg/dl % mg/dlAlbumin 46.4 11.1 Alpha-1 5.4 1.3 Alpha-2 13.8 3.3 Beta 5.8 1.4 Gamma 28.6 6.9

URINE

Total Protein 24 This patient was hospitalized because of a mass on his chest wall. Serum electrophoresis showed an elevated alpha-2, probablyan acute phase response, and a small spike superimposed on the polyclonal gamma. The spike was typed by immunofixation electrophoresis as IgG-kappa. The urine contained a relatively small amount of protein reflecting the serum. IgG-kappa paraprotein and a small amount of kappa Bence-Jones protein wfound by immunofixation. In general solitary plasmacytomas nthesiz

ere

e little or no detectable paraprotein. A fraction ogress to multiple myeloma, but most can be removed surgically. ome are polyclonal.

sypr S

Oligoclonal Gammopathy

138


IgG = 3610 mg/dl IgA = 774 mg/dl IgM = 48 mg/dl This pattern was found on a routine protein electrophoresis der

e

e

or ed on a patient admitted to the psychiatric unit for depression. Both IgG and IgA are elevated. Two heavy bands arvisible as well as three faint bands in the gamma region. Immunofixation electrophoresis identified the strong bands as IgG-kappa and the weak bands as IgG-lambda. Oligoclonal responses are occasionally seen following antigenic stimulus likinfection. They are usually transient. In this case the cause unknown. is

139

other case An Oligoclonal Banding Serum

Oligoclonal Banding Urine

Oligoclonal Gammopathy - Infection

140


SERUM

IgG = 1240 mg/dl IgA = 414 mg/dl IgM = 56 mg/dl

% mg/dl

141

Albumin 17.8 pha-1 29.0 9.9

This patient was hospitalized for fever of unknown origin. aluation revealed acute tubular necrosis and endocarditis. rum electrophoresis showed a low albumin with three bands sible in the gamma. Typing by immunofixation electrophoresis

G-ka Ur lectrophoresis gave a s inflammation. The gamma nt kappa spikes. This case esponse to infection.

6.0

URINE

AlAlpha-2 13.0 4.4Beta 11.0 3.7 Gamma 29.3 10.0

Total Protein 34 EvSeviidentified them as Ig ppa. pattern suggesting tub r dise

ine ee andula a

region of the urine contained faie oli l i e rillustrat s goclona mmun

IgG-Lambda Myeloma

142


SERUM

Alpha-1 4.8 0.5 Alpha-2 8.1 0.8 Beta 6.8 0.7 Gamma 53.4 5.4 tal

To Protein 10.1 IgG = 7000 mg/dl IgA = 11 mg/dl

IgM = 19 mg/dl % mg/dlAlbumin 31.2 29 Alpha-1 16.9 16 Alpha-2 13.8 13 Beta 12.3 11

5.9 Gamma 2 24 tal Protein 92 To

Protein/24hrs 1000

n ak in the beta of about 40 mg/day. The patient presented with cterial meningitis and profound anemia. This patient has shown poor response to chemotherapy. The serum pattern is an

synthesis by multiple myeloma. About 53% of myeloma cases produce IgG paraproteins.

URINE

These are typical serum and urine patterns from case of IgGmultiple myeloma at time of diagnosis. The serum contained an IgG-lambda paraprotein at 7 g/dl and the urine contained serum proteins, with an inflammatory pattern, an intact paraprotein ak of about 0.2 g/day and a small lambda Bence-Jones proteipe

pebaa excellent example of the suppression of normal immunoglobulin

IgG-Kappa Myeloma

143

% g/dlAlbumin 38.0 4.0 Alpha-1 3.1 0.3 Alpha-2 11.6 1.2 Beta 9.5 1.0 Gamma 37.7 4.0 Total Protein 10.6 IgG = 5200 mg/dl IgA = 69 mg/dl IgM = 42 mg/dl % mg/dlAlbumin 39.8 159 Alpha-1 6.2 25 Alpha-2 8.2 33 Beta 11.4 46 Gamma 34.4 138 Total Protein 400 This patient also is typical of multiple myeloma at diagnosis. The dominant symptoms in this case were renal with a nephrotic syndrome. There is a large IgG-kappa paraprotein peak in the gamma region of the serum as well as an increased alpha-2. The urinary protein pattern is non-selective with moderate amounts of both IgG-kappa paraprotein and kappa Bence-Jones protein.

URINESERUM

URINE

144

IgG-Lambda Myeloma Beta Spike % g/dlAlbumin 39.8 3.2 Alpha-1 2.7 0.2 Alpha-2 12.8 1.0 Beta 43.7 3.5

0.1 Gamma 1.0 Total Protein 8.0


nd d

This serum specimen was obtained from a patient with IgG-lambda myeloma after a successful course of chemotherapy. Theparaprotein spike is in the beta, not gamma. Typically IgG2 aIgG4 proteins are less basic than IgG1 and IgG3. Many IgG2 anIgG4 paraproteins migrate in the beta. Before therapy the IgG paraprotein concentration was 7000 mg/dl.

145

IgG-Lambda Myeloma Broad Peak % g/dlAlbumin 45.3 2.8 Alpha-1 4.9 0.3 Alpha-2 15.8 1.0 Beta 11.1 0.7 mmaGa 22.8 1.4

Total Protein 6.1


a r el ad paraprotein spike in the apr typed as IgG-lambda. Broad spikes

i abo of ases and in many IgA, IgM, and IgD to charge heterogeneity of the hydrate portion.

IgG = 1540 mg/dl

This serum shows elativ

i . y bro

gamma reg on The par oteinare seen n ut 10% IgG cparaprote ns The bre h is i . adt duemolecules, many times in the carbo

IgG-Kappa Myeloma Defective Protein

146

% g/dlAlbumin 35.1 3.2 Alpha-1 2.9 0.3 Alpha-2 8.3 0.8 Beta 14.3 1.3 Gamma 39.4 3.6

SERUM

URINE

Total Protein IgG = 4200 mg/dl **


% mg/dlAlbumin 9.3 30 Alpha-1 3.1 10 Alpha-2 2.8 9 Beta 6.1 20 Gamma 78.7 258 Total Protein 327 otein/24hrs = 3.4 g Pr

These patterns illustrate an IgG-kappa paraprotein in the serum, with the intact paraprotein excreted selectively in the urine. There is very little albumin and other proteins in the urine with the 2.7 g of IgG. The serum paraprotein appears to be defective, either half molecules and/or missing part of the heavy chain. The serum IgG measured by rate nephelometry was 838 mg/dl. When measured by radial immunodiffusion, there was a dense ring of 725 mg/dl and an atypical faint ring at 4200 mg/dl. The larger number was in agreement with the integration of the electrophoresis scan.

IgG-Kappa Myeloma With Bence-Jones Protein

147

%

g/dlAlbumin 35.5 3.0 Alpha-1 2.2 0.2 Alpha-2 4.8 0.4 Beta 7.8 0.7 Gamma 49.7 4.2 Total Protein 8.5 IgG = 4400 mg/dl IgA = 46 mg/dl IgM = 69 mg/dl

% mg/dl Albumin 20.0 21 Alpha-1 2.0 2 Alpha-2 5.5 6 Beta 56.2 58 Gamma 16.0 17 Total Protein 104 This serum has a large IgG-kappa paraprotein spike in the gamma. The urine shows albumin and two spikes. The smaller spike in the gamma is IgG-kappa and the larger spike in the beta is kappa Bence-Jones protein.

SERUM

URINE

IgG-Lambda Myeloma with Three Urine Spikes

148

% g/dlAlbumin 38.2 3.6 Alpha-1 1.4 0.1 Alpha-2 5.4 0.5 Beta 54.0 5.0 Gamma 1.3 0.1 Total Protein 9.3 IgG = 5380 mg/dl IgA = 16 mg/dl IgM = 24 mg/dl % mg/dlAlbumin 49.1 73 Alpha-1 2.3 3 Alpha-2 26.4 39 Beta 21.0 31 Gamma 1.0 2 Total Protein 148

The serum contains a large IgG-lambda paraprotein spike ich

SERUM

URINE

wh migrates at the beginning of the beta region. There are three spikes visible in the urine. The smallest corresponds to the serum paraprotein. The two spikes in the alpha-2 region and between the alpha-2 and beta are lambda Bence-Jones protein.

149

Free Light Chain in Serum % g/dlAlbumin 52.9 3.3 Alpha-1 5.1 0.3 Alpha-2 10.2 0.6 Beta 23.7 1.5 Gamma 8.1 0.5 Total Protein 6.2

IgM = 51 mg/dl

other case

IgG = 1450 mg/dl

IgA = 101 mg/dl An

ERUMS

150

% mg/dlAlbumin 5.9 pha-1 1.8 2

This patient has multiple myeloma with only a small amount intact IgG-kappa paraprotein in the serum. mun

8

URINE

AlAlpha-2 2.8 4 Beta 80.1 106 Gamma 9.3 12 Total Protein 132

Protein hr = 4.3 g/24 s ofIm oelectrophoresis showed a double "gull wing" arc for the kappa, but only a single paraprotein arc for the IgG. Urine contained almost pure kappa Bence-Jones protein with a small amount of albumin. Free light chains usually form dimers of about 46,000 molecular weight. These can pass through the glomerulus. Occasionally tetramers are made which can accumulate in serum. The dimers are linked by disulfide bonds.

151

Multiple Myeloma with Renal Failure

%

g/dlAlbumin 27.2 2.6 Alpha-1 1.4 0.1 Alpha-2 5.0 0.5 Beta 3.3 0.3 Gamma 63.0 6.1 Total Protein 9.7 IgG = 6670 mg/dl IgA = 34 mg/dl IgM = 15 mg/dl

SERUM

% mg/dlAl 4.8 31

6.1 40

152

bumin pha-1 pha-2 5.0 33

AlAlBeta 11.5 75 Gamma 72.6 476 Total Protein 655 Protein/24hrs 491mg

er nistration of

intravenous contrast fluid. He was noted at the time of arrival to have a Streptococcal infection. He was started on peritoneal dialysis and he remained anuric for almost 4 weeks. The urine

URINE

This patient was transferred from a community hospital aftdeveloping total renal failure following the admi

153

ecimen shown here was obtained four weeks after the serum

ppa chains. Several investigators have reported that highly pear be extremely nephrotoxic. The urine

serum proteins and a small The kappa Bence-Jones protein y.

spsample. The serum shows a low albumin level with two paraprotein spikes in the gamma region. The large spike was identified as an G-kappa paraprotein and the small very basic spike was free Ig

kabasic light chains ap to specimen showed small unts amo of amount of intact IgG paraprotein.

d ble of /daformed a ou spike 0.35g % g/dlAlbumin 34.0 2.7 Alpha-1 2.1 0.2 pha 0.5 Al -2 6.4

Beta 4.2 0.3 mma 4.2

tal

Ga 53.3

Protein 7.8 To % mg/dlAlbumin 43.4 87 Alpha-1 2.1 4 Alpha-2 4.9 10 Beta 4.6 9 Gamma 44.9 90 tal Protein 200 To

Protein/24hrs 16grams

The second serum specimen was obtained two months after the initial specimen, approximately one month after initiation of chemotherapy. The paraprotein level had decreased over one-third, and has continued to decrease with further chemotherapy. The patient is maintained on eight liters of peritoneal dialysis

ERUMS

PERITONEAL DIALYSIS FLUID

154

ily at home. A sample of the dialysis fluid at the time of the

e normal range. Urine electrophoresis showed almost pure xcre .

dasecond serum specimen is shown above. At this time the dialysis was removing 16g/day of protein, 7.2g of paraprotein. Renal nction had improved significantly and output volume was within fu

thBence-Jones protein e tion Another case Myeloma with Nephrosis Serum

Myeloma with Nephrosis Urine

155

nal pa hy of Undetermined Significance Nephrosis with Monoclo Gammo t % g/dlAlbumin 43.9 2.7 Alpha-1 6.7 0.4 Alpha-2 20.2 1.3

Beta 7.4 0.5mma 21 1.4 Ga .8

tal Protein 6.2

IgG = 1310 mg/dl

%

To IgA = 174 mg/dl IgM = 90 mg/dl

mg/dlAlbumin 57.9 181 Alpha-1 5.7 18 Alpha-2 10.9 34 Beta 6.9 22 Gamma 18.7 58 Total Protein 312 These specimens are from a patient with nephrosis and renal failure. The serum showed a low albumin, elevated alpha-2 and a small IgG-lambda paraprotein spike in the gamma. The urine pattern was identical to the serum except for the alpha-2-macroglobulin and beta-lipoprotein. Specimens obtained two years later, just prior to the patient's death from renal failure, showed no change in the amount of paraprotein in the serum.

ERUMS

URINE

156

Monoclonal Gammopathy of Undetermined Significance

%

g/dlAlbumin 50.1 3.6 Alpha-1 2.5 0.2 Alpha-2 10.3 0.7 Beta 7.5 0.5 Gamma 29.6 2.1 Total Protein 7.2 IgG = 2020 mg/dl IgA = 90 mg/dl IgM = 97 mg/dl % mg/dlAlbumin 57.3 76 pha-1 3.7 5 Al

Alpha-2 6.3 8 Beta 6.5 9 Gamma 26.2 35 Total Protein 132 The serum from this patient has a broad IgG-kappa paraprotein peak in the gamma at a total of 2 g/dl. The kappa-lambda ration was significantly elevated at 5.7. The urine electrophoresis reflects the serum pattern with intact IgG, but no free light chain detected.

SERUM

URINE

Smoldering or Asymptomatic Myeloma

157


IgG = 3870 mg/dl IgA = 219 mg/dl IgM = 94 mg/dl This serum shows two spikes in the gamma region, both of which are IgG-lambda. There is a group of about 5-7% of myeloma patients who fulfill the diagnostic criteria for multiple myeloma, but have a form of the disease of much lower malignancy. These are called asymptomatic myeloma by Alexanian and smoldering myeloma by Kyle. Typically these patients have between 2 and 5 g/dl of serum monoclonal protein, 10% or more marrow plasmacytosis and few symptoms. About 10% of the group has 1 or 2 discrete bone lesions. When followed serially, paraprotein levels tend to remain stable or only increase slowly. The presence of severe anemia or further bone lesions or a rise in serum paraprotein over 5 g/dl indicates the disease has become malignant. Alexanian recently reported that 35 patients with this variant of myeloma had a mean survival of 105 months and that the 25 patients without any bone lesions survived a mean of 125 months. Chemotherapy was deferred in all these cases until evidence of malignancy was present. Patients with the typical malignant form of myeloma have life expectancies of 6-9 months without therapy and about 36 months with appropriate therapy. About 1% of all myeloma cases do not have detectable paraprotein in either serum or urine. Some of these tumors produce paraprotein, but do not secrete it; and others do not produce any paraprotein. Such cases are generally malignant.

158

bulin Spike Trace Immunoglo % g/dlAlbumin 61.7 3.9 Alpha-1 4.5 0.3 pha-2 7.6 0.5 Al

Beta 13.9 0.9 mma 12.4 0.8

tal Protein 6.4

IgG = 1090 mg/dl

.

Ga To IgA = 190 mg/dl IgM = 37 mg/dl This is an example of a small paraprotein spike. The spike is at the very far end of the gamma region and types as IgG-ppa

ka . The serum kappa/lambda ratio is slightly elevated at 2.3 This peak is found in the area where C reactive protein migrates. Occasionally a highly elevated CRP will give a spike in the far gamma. This can be verified by immunochemical quantitation of CRP.

Nephrotic Syndrome with Monoclonal Gammopathy

159

% g/dlAlbumin 33.7 1.9 Alpha-1 5.0 0.3 Alpha-2 13.9 0.8 Beta 11.2 0.6 Gamma 36.2 2.1 talTo Protein

IgG = 1650 mg/dl IgA = 160 mg/dl IgM = 101 mg/dl % mg/dlAlbumin 57.5 361 Alpha-1 6.7 42 pha-2 8.2 51 Al

Beta 22.2 139 mma 5.4 34

tal Protein 627

These patterns illustrate nephrotic syndrome with a monoclonal gammopathy. The serum has low albumin and a broad IgG-lambda paraprotein peak between the beta and gamma. The kappa/lambda ratio is abnormal at 0.47. A prominent beta-lipoprotein spike is visible. The urine reflects the serum pattern except for the macro-proteins and contains intact IgG-lambda paraprotein with no Bence-Jones protein.

RUMSE

URINE

Ga To

IgA-Kappa Myeloma

160

% g/dlAlbumin 28.3 3.4 Alpha-1 2.1 0.3 Alpha-2 2.8 0.3 Beta 54.7 6.6 Gamma 12.0 1.4 tal

To Protein 12.0 IgG = 300 mg/dl IgA = 10000 mg/dl IgM = 25 mg/dl A few patients present with enormous paraprotein levels at

e ha en ase with 17 g/dl of tro is e shows a huge spike in the e i gamma. Both spikes typed as IgA-

o po and ge variants are seen with IgA atively normal serum albumin. ts that albumin decreases to ulin in myeloma. As therapy and this has turned out to be a loss, myeloma patients can n levels. The decrease in

t ease in the status of the patient, e.g. tritional, rather than paraprotein level. Approximately 23% of eloma cases make IgA paraproteins.

time of diagnosis. W ve separaprotein. The elec phores

sma

one cabov

beta with a ll spik n thekappa. B th lymers charproteins. T patien as a his t h relMuch of the older literature repor

ate or rea lobcompens f the inc sed gpatient management have improved,fallacy. In the absence of renal

al albumimaintain relatively normbumin reflec s a decral

numy

IgA-Kappa Paraprotein % g/dl

161

Albumin 35.1 2.1 AlAlpha-2 pha-1 3.2 0.2

12.6 0.8 Beta 42.4 2.7 Gamma 6.7 0.4 Total Protein 6.3 IgG = 433 mg/dl IgA = 2830 mg/dl IgM = 16 mg/dl

% mg/dl

162

Albumin 56.1 690 Alpha-1 1.6 20

n IgA-kappa paraprotein which makes a sharp ike e cannot identify the type of paraproteins om tion or peak shape. The urine shows two ge beta spikes of intact IgA-kappa paraprotein and two small mma spikes of kappa Bence-Jones protein.

Alpha-2 10.0 123 Beta 25.5 314 Gamma 6.9 85

Total Pro ei 1230 t n Protein/24hrs 15.5g This scan shows asp in the beta. On

either the migrafrlarga

163

IgA-Lambda Paraprotein with Two Bands % g/dlAlbumin 46.9 3.5 Alpha-1 3.5 0.3 Alpha-2 6.9 0.5 Beta 10.3 0.8 Gamma 32.4 2.4 Total Protein 7.5 IgG = 1120 mg/dl IgA = 1900 mg/dl IgM = 139 mg/dl A broad spike is seen in the scan of this serum electrophoresis. On the original gel there are two equal closely spaced bands visible at the location of the spike. In immunofixation electrophoresis both are IgA-lambda paraprotein.

164

IgA-Lambda Myeloma with Diabetic Renal Disease % g/dlAlbumin 44.1 2.6 Alpha-1 5.3 0.3 Alpha-2 10.0 0.6 Beta 27.4 1.6 Gamma 13.2 0.8 Total Protein 5.8

SERUM


% mg/dlAl 46.2 455

6.7 66 bumin pha-1

pertension was admitted to the hospital with a suspected brain mor. On radiologic examination discrete bone lesions were und in the skull. A plasmacytoma was removed from the skull. e serum electrophoresis shows a low albumin with a double spike the beta, which types as IgA-lambda. A faint spike at the

a re was d as free lambda chains.

is similar to the serum, but with a i in mma is unusual for IgA to pass readily

A level was borderline for IgA calization of the disease to a gest that the myeloma was at a rom a solitary plasmacytoma, dvanced nephrotic syndrome bly the result of long-standing

abetes and poorly controlled hypertension.

165

AlAlpha-2 7.2 71 Beta 22.1 218 Gamma 17.8 175 Total Protein 985

This patient with a long previous history of diabetes and hytufoThinbeginning of the gamm gion

u ne

type

The ri pattern verylarger sp ke the ga . Itthrough t e ney. T serumh kid he Igmyeloma. The IgA level and the losingle tumor mass in the skull sugrelatively early stage evolving fwhich is not consistent with the aen. The renal damage was probase

di

URINE

IgD-Kappa Myeloma

166

% g/dlAlbumin 55.3 3.3 pha-1 4.6 0.3

is pattern was obtained with serum from a ye ultiple myeloma. Two paraprotein spikes re visible in the gamma region. One typed as free kappa chain d the other as IgD-kappa. Significance suppression of IgG, IgA

produce IgD paraproteins. IgE paraproteins are produced by less than 0.1% of all patients.

AlAlpha-2 9.2 0.5 Beta 12.7 0.8 Gamma 18.2 1.1 Total Protein 6.0


IgD = 480 mg/dl This electrophores

ar old man with m77weanand IgM is also present. From 1-2% of all myeloma patients

167

IgD-Kappa Myeloma


.3

IgG = 344 mg/dl IgA = 21 mg/dl IgM = 34 mg/dl IgD = 2380 mg/dl

Alpha-1 2.8 0.2 Alpha-2 7.5 0.6 Beta 42.6 3.3 Gamma 3 .4 0 tal Protein 7.8

To % mg/dlAlbumin 2.1 7 Alpha-1 1.9 6 Alpha-2 1.4 4 Beta 92.9 292 Gamma 1.7 5 Total Protein 314 Protein/24hrs = 5.9 g These patterns were obtained at time of diagnosis of multiple myeloma in a 43 year old man. A broad spike was visiblein the beta region of the serum, along with profound suppression normal immunoglobulin produc

tion. The spike was typed as IgD-ppa. The urine contained a large broad spike of the same bility as the serum paraprotein, but which did not contain any munochemically measurable IgD, and was identified as 5.9 g of

present in the urine, and no measurable immunoglobulin. At time of diagnosis serum beta-2-microglobulin was 15.2 mg/l. The patient was profoundly anemic at presentation.

SERUM

URINE

ofkamoimKappa Bence-Jones protein. There was only a trace of albumin

168

The patient received several courses of chemotherapy over two and a half years. IgD levels were reduced to about 500 mg/dl. Bence-Jones protein excretion could not be brought below 1 g/day and beta-2-microglobulin remained above 5 mg/l. This contrasts with many other myeloma patients, who can experience almost complete remission, with undetectable paraprotein levels following chemotherapy.

169

IgD-Lambda Myeloma % g/dlAlbumin 54.8 3.9 Alpha-1 4.8 0.3 Alpha-2 9.0 0.6 Beta 13.5 1.0 Gamma 17.9 1.3 Total Protein IgG = 622 mg/dl IgA = 26 mg/dl IgM = 16 mg/dl IgD = 960 mg/dl

% mg/dlAlbumin pha-1

2.2 2 2.1 2

Alpha-2 2.4 3 Beta 85.4 92 Gamma 7.9 9 Total Protein 108 These specimens show a serum with two spikes; the gamma spike typing as IgD-lambda and the beta spike as free lambda chains. The urine shows a single beta spike of free lambda chains with traces of serum proteins present. The patient had renal disease, anemia, 21% plasma cells in the bone marrow biopsy and a serum beta-2-microglobulin of 19.7 mg/l.

SERUM

URINE

Al

Kappa Light Chain Disease

171


SERUM

Alpha-1 15.0 0.8 Alpha-2 12.2 0.7 Beta 9.3 0.5 Gamma 12.3 0.7 Total Protein 5.4


IgM = 186 mg/dl URINE % mg/dlAlbumin 13.9 9 Alpha-1 12.8 8 Alpha-2 15.0 10 Beta 51.3 32 Gamma 7.0 4 tal Protein 63 To

The serum pattern from this patient was normal, except for a low albumin level and elevated alpha-1. The urine showed a large kappa Bence-Jones protein spike, with a moderate amount of other serum proteins. This can be contrasted with the more selective patterns seen in the two previous urines shown. Light chain disease without intact immunoglobulin paraprotein production is found in 21% of all myeloma patients. About 85% of myeloma patients produce Bence-Jones protein.

172

Plasma Cell Leukemia with Lambda Myeloma % g/dlAlbumin 45.0 2.6

SERUM

Alpha-1 6.9 0.4 Alpha-2 8.7 0.5 Beta 32.1 0.4 Gamma 7.4 0.4

Total Protein


IgM = 15 mg/dl % mg/dlAlbumin 6.4 48 Alpha-1 1.7 13 Alpha-2 2.3 17 Beta 85.0 646 Gamma 4.5 34 Total Protein 760

URINE

This patient presented with anemia, renal failure, oteinuria and plasma cell leukemia. The serum had suppression

typed as free lambda chains. The serum albumin and gamma globulins were low. The urine showed a very large lambda Bence-Jones protein peak with a small amount of albumin. Plasma cell leukemia is an unusual finding seen in only a few percent of multiple myeloma cases.

prof normal immunoglobulins with a large spike in the beta, which

Multiple Myeloma with CNS Involvement % g/dlAlbumin 22.0 1.9 Alpha-1 3.9 0.3 Alpha-2 8.8 0.8 Beta 6.5 0.6 mma 5.2 Ga 58.8

Total Protein 8.8


IgM = 25 mg/dl

RUMSE

173

174

% mg/dlAlbumin 6.6 6.8 Alpha-1 4.4 4.5 Alpha-2 5.6 5.8 Beta 5.5 5.7 Gamma 78.0 80.3 Total Protein 103 Protein/24hrs 3832mg This 78 year old man was admitted to the hospital with a major complaint of altered mental status. Within hours after admission he became comatose, but recovered consciousness following treatment. Laboratory studies on admission showed a serum calcium of 10.0mg/dl, serum albumin of 1.9g/dl and red blood cell count of 1.7 x 1012/l. The patient was given packed red cells and it was possible to normalize his serum calcium. Serum electrophoresis showed a large spike which was identified by immunofixation electrophoresis as an IgG-kappa paraprotein. Urine electrophoresis showed moderate amounts of serum proteins with two large closely spaced spikes in the gamma region. The first was identified as 2.1g of kappa Bence-Jones protein per day and the second as intact IgG-kappa paraprotein. Other measures of renal function were also abnormal. A spinal tap had been performed because of the mental changes and coma. Only a few red cells were noted and no plasma cells were seen. The total CSF protein was 68mg/dl of which 43.7% was a large spike in the gamma region, identified by immunofixation as IgG-kappa paraprotein. The amounts of albumin and prealbumin were normal, indicating that the blood-brain barrier is intact. No significant free light chain was found in the CSF. The relatively large amount of intact IgG paraprotein with normal ounts of other CSF proteins suggests possible central nervous stem growth of the myeloma. No CNS tumor masses were visible magnetic resonance imaging.

URINE

amsyby

175

% mg/dlPrealbumin 3.8 2.6 Albumin 29.6 20.1 Alpha-1 5.3 3.6 pha-2 6.4 4.3 Al

Beta 11.2 7.6 mma 43.7 29.7 Ga

CSF rotein p 68

G/a

G synthesis index 0.66

ID CEREBROSPINAL FLU

lbumin 2.1 Ig

Ig

Another case

176

177

Immunoglobulin Fragments in Urine URINE

% mg/dlAlbumin 44.9 42 Alpha-1 4.6 4 Alpha-2 6.5 6 Beta 12.3 12 Gamma 31.8 30 Total Protein 94 Protein/24hrs = 0.6 g This pattern shows a urine from a patient with IgG-kappa myeloma. Four spikes are visible in the gamma region. The largest one contains kappa chains. The other three precipitate with anti-immunoglobulin and anti-gamma chain. The spikes represent proteolytic fragments of the IgG-kappa paraprotein, that are small enough to pass through the glomerulus. The glomeruli are not normal as can be seen from the albumin excretion of 275 mg/day.

178

Kappa Bence-Jones and IgG in Urine % mg/dlAlbumin 7.2 8 Alpha-1 2.0 3 Alpha-2 2.0 3

3.0 4 Beta Gamma 84.5 95

112

RINEU

Total Protein Protein/24hrs = 3.4 g This urine contains a small albumin peak, a very large kappaBence-Jones protein peak and a small intact IgG-kappa paraproteinpeak. The Bence-Jones protein excretion is 2.9 g/day. The intact protein in the urine is due to an overflow type of proteinuria, because of the large amount in the serum. The relatively low albumin excretion of 240 mg/day out of a total protein of 3400 mg/day indicates relatively early renal disease.

179

ikes in Urine - Myeloma %

Multiple Sp

mg/dlAlbumin 21.9 3 Alpha-1 5.2 1 Alpha-2 6.5 1 Beta 8.2 1 Gamma 58.1 8 Total Protein 14 Protein/24hrs = 1.0 g is urine shows a more advanced renal disease than the previous pattern, although total protein excretion is lower. Albumin makes up a larger fraction of the total protein and various alpha and beta globulins are visible. Three spikes are visible in the gamma region. The largest spike is intact IgG-lambda paraprotein. The medium spike is free lambda chains and the smallest spike is the Fc fragment of IgG. Renal disease in multiple myeloma is a function of the particular light chain and not solely of the amount of Bence-Jones protein excreted. It should be noted that multiple myeloma patients who do not produce Bence-Jones protein may develop renal disease, and occasionally a rare patient who does not make any paraprotein develops renal disease. Secondary amyloidosis is common in myeloma. Although many patients with Waldenstrom's macroglobulinemia produce excess light chain, severe renal disease is very rare in macro

URINE

Th

globulinemia.

180

Biclonal Gammopathy %

g/dlAlbumin 39.6 2.2 Alpha-1 5.0 0.3 Alpha-2 14.6 0.8 Beta 26.1 1.4 Gamma 14.7 0.8 Total Protein 5.5 IgG = 1800 mg/dl IgA = 56 mg/dl IgM = 44 mg/dl This serum specimen was submitted from an 85 year old lady with a mass on the fifth lumbar vertebra causing a lytic lesion and spinal chord compression. Two prominent spikes were visible in the beta and gamma. The spikes were both identified as IgG-kappa by immunofixation electrophoresis. The beta spike was IgG2 subclass and the gamma spike IgG3. A bone marrow biopsy showed 13% atypical plasma cells not growing in sheets. Serum chemistries were normal except for low albumin, calcium, ionized calcium and phosphorus, with an elevated lactic dehydrogenase. There was only a trace of protein in the patient's urine with a few red cells; no significant Bence-Jones protein was detected. About 2% of paraproteins are biclonal. All possible combinations of classes and subclasses have been reported. Some cases of biclonal gammopathy have identical variable regions in both immunoglobulins, suggesting that both immunoglobulins are derived from the same clone undergoing a class switch. In other cases both immunoglobulins have differences in both the heavy and light chain variable regions, suggesting that they are produced by two independent clones.

182

Triclonal Gammopathy - Myeloma % g/dlAlbumin 18.0 1.6 Alpha-1 2.5 0.2 Alpha-2 5.4 0.5 Beta 71.1 6.3 Gamma 3.0 0.3 Total Protein 8.8 IgG = 6020 mg/dl IgA = 24 mg/dl IgM = 32 mg/dl % g/dlAlbumin 53.2 3.4 Alpha-1 3.0 0.2 Alpha-2 8.7 0.6 Beta 21.7 1.4 Gamma 13.5 0.9 Total Protein 6.3 IgG = 1700 mg/dl IgA = 112 mg/dl IgM = 84 mg/dl This patient was admitted to the critical care unit with profound anemia, hypercalcemia and general deterioration. On admission the red blood cell count was 2.9x106, hemoglobin was 7.8 g/dl and hematocrit 22.9%. The erythrocyte sedimentation rate was over 150 mm/hr and serum chemistries gave a blood urea nitrogen of 37 mg/dl, potassium of 2.5 meq/l, chloride of 83 meq/l, CO2 content of 38 meq/l and creatinine of 2.0 mg/dl. The serum calcium reached a high of 16.5 mg/dl with 1.4 g/dl of serum albumin. Serum phosphorus was low at 2.0 mg/dl, uric acid was high at 11.3 mg/dl, and serum beta-2-microglobulin was high at 13.2 mg/l. The serum pattern showed severe hypoalbuminemia and a huge paraprotein spike in the beta with some small trace spikes

PRE-CHEMOTHERAPY

POST-CHEMOTHERAPY

183

ped as IgG-lambda. The ble on the post-chemotherapy

pattern. The left hand gamma spike typed as IgG-kappa and the right one as IgG-lambda. Bone marrow biopsy showed almost a total replacement by sheets of plasma cells.

%

in the gamma. The beta spike was tysmaller spikes are more readily visi

mg/dlAlbumin 26.8 8 Alpha-1 8.7 3 Alpha-2 10.2 3 Beta 52.0 16 Gamma 2.3 1 Total Protein 30 Protein/24hrs = 1.16 g This patient's urine at time of diagnosis is shown above. There is a large albumin peak and a large beta spike of 600 mg/day, which types as intact IgG-lambda. No Bence-Jones protein was found. This patient required about 6 weeks of hospitalization and was discharged in remission. The post-chemotherapy pattern shown was obtained after a year of

It is reported that 2% of myeloma patients produce oligoclonal immunoglobulin. Biclonal myeloma accounts for about 90% of these. Cases with three or more clones are rare.

URINE

outpatient follow-up.

184

Hepatitis in Myeloma % g/dlAlbumin 38.0 2.7 Alpha-1 4.0 0.3 Alpha-2 6.1 0.4 Beta 28.3 2.0 Gamma 23.7 1.7 Total Protein 7.1 Patients with multiple myeloma are highly susceptible to

Despite the partial immune typically show the rises in IgM,

followed by IgG and then IgA levels typical of viral hepatitis. This serum was obtained from a patient in almost total long term remission from myeloma. This specimen shows the total fusion of

tion of IgA and some ility to bacterial

infections is traditionally reported in multiple myeloma patients, this is at least as much a result of poor overall status as it is of immune suppression. In recent years the improved management of myeloma patients has greatly decreased the incidence of severe bacterial infections.

viral hepatitis of all types.suppression, myeloma patients

the beta and gamma regions caused by producpolyclonal rise in IgG. Although susceptib

185

Gamma Heavy Chain Disease % g/dlAlbumin 63.8 4.0 Alpha-1 2.9 0.2 Alpha-2 9.6 0.6 Beta 12.5 0.8 Gamma 11.2 0.7 Total Protein IgG = 719 mg/dl IgA = 66 mg/dl IgM = 37 mg/dl A man in his mid-thirties presented with a moderate sized stomach tumor. It was removed and determined to be a monoclonal plasmacytoma. The serum showed only a trace spike which typed as gamma, with no light chains. Immunoglobulin subtyping showed it to be a defective gamma-3 chain. Urine contained only 16 mg/dl of albumin. Samples of the tumor were subjected to exhaustive immunohistochemical studies. The cells contained gamma, with no light chain, Leu-12 (pan-B cell) and Leu-17 (plasma cell). This tumor was unusual in that it expressed the C3DR (Epstein-Barr virus receptor) along with Leu-12. A weak expression of Leu-3ab, the T-helper/inducer antigen was also seen. Tumor cells were found in the regional lymph nodes, possibly in the spleen, but not in the liver. Another case

Serum Electrophoresios

Serum Scan

Serum Immunofixation

Urine Electrophoresis

186

Urine Immunofixation

Purified Gamma Chain

187

188

OTHER M-COMPONENT PATTERNS

Biclonal M-components IgG and IgA

Bence-Jones Protein and Kappa Ladder – Urine

189

Bone Marrow Transplant with Oligoclonal Gammopathy

190

Monoclonal Cold Agglutinin Disease

Incomplete IgA M-component

Biclonal M-components with Deposition Disease

IgA M-component Broad spike

IgA M-component Small Spike

191

IgA M-component CSF from Previous Patient

IgA M-component Very Small

192

IgA M-component in Alpha-2 Serum

IgA M-component in alpha-2 Urine

193

IgA-Lambda M-component with Hemolysis

IgG-Lambda Myeloma

194

195

IgG-Lambda and Free Lambda M-components

IgM Anti-IgA M-component

196

Kappa in Alpha-2 Region Urine

Papular Mucinosis IgM M-component

198

Sebia Electrophoresis

Immunofixation Urine in Track 14

199

UREFERENCES U 1. Alexanian R., Barlogie B., and Dixon D.: Prognosis of asymptomatic multiple myeloma. Arch. Intern. Med. 1988; 148:1963-1965. 2. Bakerman S.: UABC's of Interpretive Laboratory Data U. 2nd edition, Interpretive Laboratory Data Inc., Greenville NC, 1984. 3. Bakerman S.: UClinical Chemistry Notes U. Greenville NC, 1986. 4. Brady R.O., and Quarles R.H.: Myelin-associated glycoprotein in demyelinating diseases. ISI Atlas of Science, Immunology, 1988; 1:11-14. 5. Buxbaum J.N., Chuba J.V., Hellman G.C., Solomon A., and Gallo G.R.: Monoclonal immunoglobulin deposition disease: Light chain and light and heavy chain deposition diseases and their relation to light chain amyloidosis. Ann. Int. Med. 1990; 112:455-464. 6. Carstens K.S., Sepulveda-Pacheco A.M., and Romfh P.C.: UIntroduction to High Resolution Protein Electrophoresis and Associated Techniques U. Helena Laboratories, Austin TX, 1986. 7. Fauchier P., and Catalan F.: UInterpretive Guide to Clinical Electrophoresis. Important Normal and Abnormal Patterns U. Helena Laboratories, Beaumont TX, 1988. 8. Feiner H.D.: Pathology of Dysproteinemia: Light chain amyloidosis, non-amyloid immunoglobulin deposition disease, cryoglobulinemia syndromes and macroglobulinemia of Waldenstrom. Human Pathol. 1988; 19:1255-1272. 9. Gandara D.R. and Mackenzie M.R.: Differential diagnosis of monoclonal gammopathy. Med. Clin. North Amer. 1988; 72:1155-1167. 10. Grimaldi L.M.E., Castagna A., Lazzarin A., and Roos R.P.: Oligoclonal IgG bands in cerebrospinal fluid and serum during asymptomatic human immunodeficiency virus infection. Ann. Neurol. 1988; 24:277-279. 11. Keren D. F.: UHigh-Resolution Electrophoresis and Immunofixation U. Butterworths, Boston, 1987. 12. Killingsworth L.M.: UHigh Resolution Protein Electrophoresis: A Clinical Overview with Case Studies U. Helena Laboratories, Austin Tx, 1985.

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13. Kostulas V.K., Link H., and Lefvert A.: Oligoclonal IgG bands in cerebrospinal fluid: principles for demonstration and interpretation based on findings in 1,114 neurological patients. Arch. Neurol. 1987; 44:1041-1044. 14. Radl J., Hijmans W., and Van Camp B., eds.: UMonoclonal Gammapathies: Clinical Significance and Basic Mechanisms U. Eurage, Rijswijk, 1985. 15. Radl J., and Van Camp B., eds.: UMonoclonal Gammapathies II: Clinical Significance and Basic Mechanisms U. Eurage, Rijswijk, 1989. 16. Raine C.S., ed.: UAdvances in Neuroimmunology U. Annals N.Y. Acad. Sci., 1988; 540. 17. Ritzmann S.E., ed.: UProtein Abnormalities Volume 1 Physiology of Immunoglobulins: Diagnostic and Clinical Aspects U. Alan R. Liss, Inc., New York, 1982. 18. Ritzmann S.E., ed.: UProtein Abnormalities Volume 2 Pathology of Immunoglobulins: Diagnostic and Clinical Aspects U. Alan R. Liss, Inc., New York, 1982. 19. Ritzmann S.E., and Killingsworth L.M., eds.: UProtein Abnormalities Volume 3 Proteins in Body Fluids, Amino Acids and Tumor Markers: Diagnostic and Clinical Aspects U. Alan R. Liss, Inc., New York, 1983.

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Additional Cases

HTUAlpha-1 Anti-Trypsin PiMS Heterozygote UTH

HTUAlpha-1 Anti-Trypsin Deficiency PiZZ with Cirrhosis UTH

HTUAL-Amyloidosis Serum UTH

HTUAL-Amyloidosis Urine UTH

HTUAL-Amyloidosis IgG Serum UTH

HTUAL-Amyloidosis IgG Urine UTH

HTUBiclonal M-components IgG and IgA UTH

HTUBisalbuminemia UTH

HTUBisalbuminemia enlarged UTH

HTUBence-Jones Protein and Kappa Ladder - Urine UTH

HTUBone Marrow Transplant with Oligoclonal Gammopathy UTH

HTUMonoclonal Cold agglutinin Disease UTH

HTUCommon Variable Hypogammaglobulinemia UTH

HTUCryofibrinogenemia UTH

HTUCSF Cervical Myelitis UTH

HTUCSF Oligoclonal Bands Immunofixation UTH

HTUCSF Oligoclonal Bands Scan UTH

HTUCSF CNS Myeloma UTH

HTUCSF CNS Sarcoid UTH

HTUCSF CNS Systemic Lupus Erythematosus UTH

HTUCSF CNS Toxoplasmosis Encephalitis UTH

202

HTUCSF Transverse Myelitis UTH

HTUDiabetic Nephrosis UTH

HTUIncomplete IgA M-component UTH

HTUBiclonal M-components with Deposition Disease UTH

Gamma Heavy Chain Disease

HTUSerum Electrophoresis UTH

HTUSerum Scan UTH

HTUSerum Immunofixation UTH

HTUUrine Electrophoresis UTH

HTUUrine Immunofixation UTH

HTUPurified Gamma Chains UTH

HTUHaptoglobin-Hemoglobin Complex UTH

HTUHuman Immunodeficiency Virus Gammopathy UTH

HTUIgA M-component Broad Spike UTH

HTUIgA M-component small serum UTH

HTUIgA M-component CSf from previous UTH

HTUIgA M-component very small UTH

HTUIgA M-component in Alpha-2 Serum UTH

HTUIgA M-component in Alpha-2 Urine UTH

HTUIgA-Lambda M-component with Hemolysis UT H

HTUIgG-Lambda Myeloma UT H

HTUIgG-lambda and Free Lambda M-components UTH

203

HTUIgM Anti-IgA M-component UTH

HTUImmunodeficiency Case 1 UTH

HTUImmunodeficiency Case 2 UTH

HTUImmunodeficiency with Hyper IgM UTH

HTUImmunodeficiency Secondary UTH

HTUIron Deficiency with Low Transferrin UTH

HTUKappa in Alpha-2 region Urine UTH

HTUKappa Chains in Serum UTH

HTUMyeloma with Nephrosis Serum UTH

HTUMyeloma with Nephrosis Urine UTH

HTUMyeloma Untreated Natural Course UTH

HTUNephrosis UTH

HTUOligoclonal Banding Carcinoma UTH

HTUOligoclonal Banding Serum UTH

HTUOligoclonal Banding Urine UTH

HTUPapular Mucinosis IgM M-component UTH

HTUPleural Effusion UTH

HTUPleural Fluid Transudate UTH

HTUPolyclonal Increase in Gamma UTH

HTUSecondary Malignancy UTH

HTUSickle Cell Crisis UTH

HTUSerum Immunoelectrophoresis M-components UTH

204

HTUSprue with Malnutrition UTH

HTUStaphylococcal Sepsis UTH

HTUSynovial Fluid Exudate UTH

HTUSynovial Fluid Gout UTH

HTUType 2 Mixed Cryoglobulinemia UTH

HTUUrine Immunoelectrophoresis M-components UTH

HTUUrine Beta Spike UTH

HTUUrine with Hemoglobin UTH

HTUUrine Inflammatory Proteinuria UTH

HTUUrine Sepsis UTH

HTUUrine Spike and Ladder UTH

HTUUrine Tubular and Inflammatory Proteinuria UTH

HTUWaldenstrom's Macroglobulinemia UTH

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