SPEP UPEP Interp

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
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.
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.
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 4
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 5
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
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein
% 46.1 6.0 10.1 13.2 24.6
g/dl 3.00 0.39 0.66 0.86 1.60 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
% 62.8 3.6 9.4 14.7 9.5
g/dl 4.46 0.26 0.67 1.04 0.67 7.1
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 Albumin Alpha-1 Alpha-2 Beta Gamma Total protein g/dl 3.6 - 5.2 0.15 - 0.40 0.5 - 1.0 0.6 - 1.2 0.5 - 1.6 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
% 63.9 3.5 10.4 12.8 9.4
g/dl 3.96 0.22 0.64 0.79 0.58 6.2
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
% 60.8 2.3 9.3 16.6 11.0
g/dl 4.6 0.2 0.7 1.2 0.8 7.5
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
% 34.2 6.9 9.0 15.4 34.5
g/dl 2.26 0.46 0.59 1.02 2.28 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
% 49.6 3.1 8.7 13.1 25.4
g/dl 3.87 0.24 0.68 1.02 1.98 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
% 23.0 4.5 10.4 11.6 50.5
g/dl 1.68 0.33 0.76 0.85 3.69 7.3
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
Infection with Immune Complexes
% 26.4 7.0 16.7 9.1 40.7
g/dl 1.95 0.52 1.24 0.67 3.01 7.4
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 alpha1-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
% 34.5 6.7 13.1 15.5 30.2
g/dl 2.38 0.46 0.90 1.07 2.08 6.9
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.
10
Monoclonal Gammopathy of Undetermined Significance
% 60.9 7.3 11.1 10.1 10.6
g/dl 3.41 0.41 0.62 0.57 0.59 5.6
SERUM
% 72.6 7.2 3.5 3.5 13.2
mg/dl 122 12 6 6 22 168
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 IgMkappa paraprotein peak in the beginning of the gamma region. Small amounts of monoclonal paraproteins are found in many 11
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.
12
13
Hypogammaglobulinemia
% 61.2 4.1 19.0 10.2 3.6
g/dl 3.68 0.23 1.14 0.61 0.20 5.9
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.
14
Agammaglobulinemia
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG = 75 IgA = 0 IgM = 0
% 66.0 6.0 18.0 7.9 2.0
g/dl 3.96 0.36 1.08 0.47 0.12 6.0
mg/dl mg/dl 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
15
16
17
Dysgammaglobulinemia
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG = 9 IgA = 56 IgM = 1680
% 52.4 6.3 14.3 12.7 14.3
g/dl 3.3 0.4 0.9 0.8 0.9 6.3
mg/dl mg/dl 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.
18
Bisalbuminemia % 32.1 6.1 6.8 13.4 41.5 g/dl 2.05 0.39 0.44 0.86 2.66 6.4 SERUM
URINE
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.
19
Another case
20
Alpha-1-Antitrypsin Deficiency
% 62.2 0 12.3 9.0 16.4
g/dl 4.7 0 0.9 0.7 1.2 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.
21
Alpha-1-Antitrypsin Deficiency with Cirrhosis
% 31.1 0.6 6.6 11.4 50.2
g/dl 1.97 0.04 0.42 0.72 3.16 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
22
Inflammation
% 37.3 5.9 12.3 15.5 28.9
g/dl 3.17 0.50 1.05 1.32 2.46 8.5
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-1acid 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. 23
Rheumatoid Disease
% 46.2 4.5 16.7 11.3 21.3
g/dl 3.8 0.4 1.4 0.9 1.7 8.2
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-1antitrypsin 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.
24
Progressive Systemic Sclerosis
% 45.9 3.3 14.6 16.2 20.0
g/dl 3.9 0.3 1.2 1.4 1.7 8.5
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.
25
Biliary Tract Obstruction % 37.0 6.5 20.6 16.6 19.2 g/dl 2.26 0.40 1.26 1.01 1.17 6.1
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.
26
Hepatitis
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG IgA IgM C3 C4 B
% 20.1 4.7 7.0 7.2 61.0
g/dl 1.9 0.4 0.7 0.7 5.7 9.3
= 5530 mg/dl = 209 mg/dl = 520 mg/dl = 106mg/dl = 7mg/dl = 32mg/dl
This specimen was obtained from a patient with a severe case of hepatitis A, who developed serious complications including bacterial meningitis and liver failure. Viral hepatitis causes a very potent stimulation of the immune response, even in patients with partial acquired immune deficiency from malignant hematopoietic disease. In the early stages of the disease, there is a significant increase in specific and total IgM. The IgM antibodies decrease over the course of the disease. This patient still has a relatively high IgM. An IgG response follows the IgM, very high levels may be reached as in this case. Much of the IgG is not specific for the infectious organism and the response is polyclonal as is shown by the broad gamma peak and kappa-lambda ratio of 1.8. In the later stages of disease a significant IgA response is usually observed. The irregular appearance of the gamma zone in this serum is due to the presence of large amounts of immune complexes. Although C3 complement and B are still normal, the C4 is significantly depressed because of the high rate of consumption. The albumin is low and some of the acute phase proteins are lower than expected because of the large amount of liver damage.
27
Severe Alcoholism
% 44.3 2.1 5.2 9.1 39.4
g/dl 4.4 0.2 0.5 0.9 3.9 10.0
Many chronic alcoholics develop severe liver disease. The earliest and still reversible phase is the deposition of fat in the hepatocytes. About 30% of heavy drinkers develop alcoholic hepatitis, characterized by random necrosis of hepatocytes, inflammation and hyaline changes. Protracted hepatitis leads to cirrhosis in many cases. Alcoholic cirrhosis starts out as a micronodular pattern, where fibrous bands bridge the nodules and regenerative areas are seen. In the end stages the liver may appear grossly macronodular and consist primarily of fibrous tissue. This patient was a severe alcoholic who did not show any signs of cirrhosis. He had received medical care for a number of moderate infections and had in the past shown some elevations of liver enzymes and bilirubin consistent with alcoholic hepatitis. At the time this specimen was obtained the patient had normal serum chemistries and no significant medical complaints. The only abnormality visible is a polyclonal increase in gamma. Unlike many alcoholics, this patient shows good nutritional status, reflected in the 4.4 g/dl albumin. The sample was collected in August and the high total protein reflects the short-term dehydration from the hot summer day.
28
Hepatic Cirrhosis - Early
% 45.8 6.5 11.3 17.9 18.5
g/dl 3.16 0.45 0.78 1.24 1.28 6.9
This pattern shows the relatively early stages of hepatic cirrhosis. There is an increase in the acute phase reacting proteins in the alpha-1 and beta. The only other visible abnormality is the bridging between the beta and gamma zones. This reflects the production of IgM or IgA at increased rates. In cirrhosis IgA levels are usually high. Serum chemistries showed a moderate liver disease consistent with alcoholism.
29
Hepatic Cirrhosis - Advanced
% 31.2 3.3 7.0 9.9 48.5
g/dl 2.0 0.2 0.5 0.6 3.2 6.5
This pattern is typical of advanced alcoholic cirrhosis. The albumin and many of the proteins in the alpha and beta regions are present in subnormal levels, because of decreased synthesis, due to the large amount of liver damage. The beta region is totally fused with the gamma region and the serum IgA level is high. There is a massive polyclonal increase in IgG. The polyclonal gammopathy in cirrhosis is thought to result from a combination of immunoregulatory abnormalities, reticuloendothelial damage and shunting of antigens into the systemic circulation, where they are processed in lymph nodes leading to a more vigorous response.
30
Cirrhosis with Ascites
% 17.7 2.1 6.1 10.9 63.2
g/dl 1.49 0.18 0.51 0.92 5.31 8.4
SERUM
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein Protein/24hrs
% 1.8 7.9 2.5 4.1 83.8
mg/dl 2 10 3 5 110 131 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. 31
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.
32
Liver Failure - Juvenile
% 37.1 9.4 19.5 13.9 20.1
g/dl 1.4 0.3 0.7 0.5 0.7 3.7
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.
33
Protein Loss - GI or Vascular
% 61.5 9.2 14.6 8.7 7.9
g/dl 2.2.9 0.3 0.7 0.4 0.4 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.
34
Short Bowel Syndrome
% 32.1 5.7 12.9 27.2 22.2
g/dl 1.7 0.3 0.7 1.5 1.2 5.4
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 betalipoprotein peak is decreased. A prominent fibrinogen peak is seen in the gamma region, consistent with the clotting defect observed.
35
Nephrotic Syndrome
% 31.4 5.9 28.9 19.6 14.2
g/dl 2.4 0.5 2.2 1.5 1.1 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
36
Nephrotic Syndrome with Inflammation
% 38.1 10.2 25.0 15.8 10.9
g/dl 1.90 0.51 1.25 0.79 0.54 5.0
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-2macroglobulin. Immunoglobulin G was slightly decreased. Blood counts were consistent with infection. Qualitative urine protein was 4+.
37
Nephrotic Syndrome with Renal failure
% 30.3 12.1 30.3 19.7 7.6
g/dl 1.45 0.58 1.45 0.95 0.36 4.8
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 betalipoprotein 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.
38
Nephrotic Syndrome with Immune Deficiency % 42.4 5.1 25.6 24.2 2.7 g/dl 1.7 0.2 1.1 1.0 0.1 4.1
Cholesterol = 658 mg/dl
% 33.1 6.9 25.5 31.0 3.7
g/dl 1.36 0.28 1.05 1.27 0.15 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 betalipoprotein reflects a greatly increased risk of atherosclerotic heart disease.
39
Renal Failure with Pneumonia
% 38.9 9.2 21.9 17.5 12.6
g/dl 1.59 0.38 0.90 0.72 0.52 4.1
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.
40
Banding Pattern
% 31.3 2.0 5.4 7.0 54.4
g/dl 3.1 0.2 0.5 0.7 5.3 9.8
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 41
42
HIV-Related Complex and Uremia
% 24.0 5.2 15.1 32.0 23.7
g/dl 1.01 0.22 0.63 1.34 1.00 4.2
SERUM
% 35.3 10.2 10.9 16.6 27.0
mg/dl 417 120 129 196 319 1180 28.3g
URINE
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 betalipoprotein 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.
43
Other Serum Patterns Alpha-1 Anti-Trypsin PiMS Heterozygote
Cryofibrinogenemia
Haptoglobin-Hemoglobin Complex 44
Human Immunodeficiency Virus Gammopathy
Iron Deficiency with Low Transferrin 45
Sickle Cell Crisis
Sprue with Malnutrition 46
47
URINE ELECTROPHORESIS
48
Normal Urine Authorities disagree as to the upper limit of protein in urine from healthy individuals. Typically values of 100 to 300 mg/day are used. As a general rule, we use 150mg/day. Normal urinary proteins consist of about 2/3 plasma proteins and 1/3 proteins derived from the urinary tract. This is most readily seen in two-dimensional electrophoretic maps. The plasma proteins are the low molecular weight (<40,000) proteins that can pass through the glomerulus and are not completely resorbed by the tubules. Small amounts of slightly larger proteins can also be found. A typical pattern of highly concentrated normal urine contains a small amount of albumin and a trace of transferrin, no other discrete peaks are usually visible. There are several relatively benign conditions, which cause significant increases of plasma proteins in the urine. These include pregnancy, postural hematuria, exercise hematuria and its variant march hematuria. The patterns in these conditions typically mirror serum electrophoretic patterns. 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 be important in the evaluation of patients with monoclonal gammopathies. Patients with renal tubular disease may also have protein excretions within the normal range. Urine electrophoresis should be performed whenever it may be of clinical value to identify the proteins in urine in order to evaluate the nature or extent of renal damage.
49
Nonselective Proteinuria % 49.3 11.0 14.3 10.9 14.5 mg/dl 123 28 36 27 36 250
Urine
This pattern shows a nonselective type of proteinuria. The urinary electrophoresis pattern is almost identical to the serum pattern, except for the absence in urine of the largest plasma proteins like beta-lipoprotein. Nonselective patterns suggest severe damage to some of the glomeruli. The total amount of proteinuria reflects the degree of damage and fraction of damaged glomeruli. Nonselective patterns may also be seen in benign proteinurias and in inflammatory conditions of the kidney.
50
51
Hematuria into Hypotonic Urine
% 4.4 1.4 3.5 79.7 11.0
mg/dl 1.0 0.3 0.8 17.5 2.4 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 myoglobin is unreliable and should be replaced by electrophoretic or immunochemical techniques.
52
Another case
53
Myoglobinuria
% 44.2 18.0 12.1 17.5 8.3
mg/dl 51 21 14 20 10 115
URINE
This urine specimen was obtained from a victim of near-fatal electrocution 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 unreliable and should be abandoned in favor of electrophoresis or immunoassay.
54
Partially Selective Proteinuria with Inflammation
% 58.7 14.3 13.7 7.8 5.6
mg/dl 106 26 25 14 10 180
URINE
This specimen illustrates a primarily glomerular pattern of proteinuria with a large albumin peak and a transferrin peak in the beta. There is a prominent alpha-1 peak from overflow of the acute phase proteins. The gamma globulins are underrepresented in the urine relative to the plasma levels. This pattern is the result of a lesser degree of glomerular permeability than the pattern illustrating nonselective proteinuria.
55
Nonselective Benign Proteinuria
% 42.0 9.6 9.8 10.0 28.7
mg/dl 21 5 5 5 14 50 0.9g
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 comes from 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.
56
Glomerular Proteinuria
% 74.9 7.9 3.6 10.3 3.3
mg/dl 124 13 6 17 5 165 3.2g
URINE
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 should be performed on all cases of glomerular type proteinuria with protein excretions over several grams per day. Occasionally a Bence-Jones protein peak is found with mobility identical or similar to transferrin. If Bence-Jones protein is found, the cause of the renal disease may be unsuspected paraprotein disease or AL amyloidosis.
57
Renal Tubular Disease
% 28.2 30.0 25.1 16.1 0.7
mg/dl 28 30 25 16 0.7 100
URINE
Renal tubular disease can be caused by heavy metal exposure, aminoglycoside antibiotics, pyelonephritis, Fanconi syndrome, Wilson's disease, sarcoid, cystinosis, tubular acidosis and a variety of other conditions. Tubular damage also occurs as part of other renal diseases including myeloma kidney. Tubular dysfunction is manifested by the loss of the ability to resorb small protein molecules of about 15,000 molecular weight. Two proteins that can be used for quantitative assessment of tubular function are lysozyme and beta-2-microglobulin. In tubular proteinuria a large amount of alpha-2-microglobulin is found in 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-2microglobulin. There is usually a broad undefined alpha-1 peak. The small peak in the beta runs after transferrin and is beta-2microglobulin. 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.
58
Tubular Proteinuria with Inflammation
URINE
Total Protein = 52 mg/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 was obtained from a patient with pneumonia, but a similar pattern can be seen in some cases of pyelonephritis. Another case
59
Overflow Proteinuria - Inflammation
% 28.7 15.2 11.1 21.5 23.6
mg/dl 13 7 5 10 11 45
URINE
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 three peaks. The increased beta and the proportionally large transferrin peak result from iron deficiency anemia. The inflammation in this patient results from bacterial sepsis. Another case
60
Overflow Proteinuria with Inflammation
% 26.8 25.5 31.8 9.9 5.9
mg/dl 27 26 32 10 6 102
URINE
This urine specimen was obtained from a female patient hospitalized with an inflammatory polyneuropathy. The acute phase proteins in the alpha-1 and alpha-2 regions are prominent. Moderate amounts of albumin and transferrin are also present. A small spike is present in the gamma corresponding to a serum paraprotein. Another case
61
Urine from a Septic Patient
URINE % 26.2 24.0 10.4 18.6 20.9 mg/dl 9.4 8.6 3.7 6.7 7.5 36
Albumin Alpha-1 Alpha-2 Beta Gamma
Total Protein
This urine was collected from a patient suffering from bacterial cellulitis and sepsis. There is a large proportion of alpha-1 acute phase proteins as well as some alpha-2 microglobulins. A banding pattern is seen in the gamma region, caused by partial digestion of immune complexes. Both kappa and lambda light chains are found throughout the pattern. Urine patterns similar to this usually indicate severe infection. ANOTHER CASE
62
Urine from a Septic Patient
% 20.8 4.7 11.9 8.3 54.2
mg/dl 25 6 14 10 65 120
URINE
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 greatly increased and shows a prominent banding pattern. There is a clear spike at the beginning of the gamma region. Immunofixation electrophoresis showed that the smaller gamma bands corresponded to kappa chains, and also two weaker lambda chain bands. The larger gamma peak was not identified as immunoglobulin. Heavy chains gave only diffuse staining. The banding pattern in the 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.
63
Nephrotic Syndrome
% 51.1 8.6 4.9 7.7 27.8 = 230mg/dl
mg/dl 118 20 11 18 64
URINE
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 and albumin, with elevated alpha-2-macroglobulin and betalipoprotein similar to the case illustrated for nephrotic syndrome serum.
64
Nephrotic Syndrome
% 67.2 1.6 7.8 8.3 15.1
mg/dl 382 9 44 47 86
URINE
Total 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.
65
OTHER URINES Diabetic Nephrosis
Urine Beta Spike
66
Cerebrospinal Fluid Electrophoresis and Neurological Diseases
67
Normal Cerebrospinal Fluid
Prealbumin Albumin Alpha-1 Alpha-2 Beta Gamma CSF protein IgG/albumin
% 7.4 65.0 3.5 5.4 13.8 5.0
mg/dl 2 14 0.4 0.8 2 0.8 18 0.08
CEREBROSPINAL FLUID
Prealbumin Albumin Alpha-1 Alpha-2 Beta Gamma CSF protein
Normal Values mg/dl 0.3 - 2.7 8.2 - 33 0.3 - 3.0 0.6 - 3.6 1.5 - 7.6 0.3 - 4.5 15 - 45
IgG/albumin ratio less than 0.28 IgG synthesis index = IgG(CSF)/Albumin(CSF) IgG(serum)/Albumin(serum) less than 0.85 mean = 0.5
Cerebrospinal fluid patterns are readily distinguishable from serum patterns. The prealbumin peak is significantly larger in CSF. When the prealbumin is less than 2% of CSF protein, there 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 68
should be measured immunochemically. In addition, if possible, serum electrophoresis and serum IgG and albumin should be performed at the same time. If any abnormalities are found in both serum and CSF, they are the result of a systemic condition and not CNS specific. Many physicians do not cooperate in this regard, so it is necessary to provide less than optimal evaluations in such cases. In cases of suspected inflammatory conditions or demyelinating diseases, myelin basic protein should also be measured by immunoassay. Abnormalities of CSF protein levels have relatively low sensitivities and specificities for inflammatory CNS disease. The IgG/albumin ratio has values around 0.7 and the IgG synthesis index around 0.8. The finding of definite oligoclonal bands has a sensitivity of 79-94% in the diagnosis of multiple sclerosis. Whenever possible CSF workups should include electrophoresis of CSF and serum with quantitation and interpretation, immunochemical IgG and albumin on CSF and serum, CSF myelin basic protein, IgG/albumin and IgG synthesis index.
69
Cerebrovascular Accident
% 8.3 41.4 4.3 2.9 39.2 3.9
mg/dl 2.7 13.2 1.4 0.9 12.5 1.2 32 0.27
CEREBROSPINAL FLUID
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 identified as hemoglobin. Most of the plasma proteins from the CVA were no longer present. There were only a few intact red cells in the CSF.
70
Central Nervous System Infection % 4.8 44.4 3.9 10.6 17.0 19.3 mg/dl 3.9 36.0 3.2 8.6 13.8 15.6 81 0.68 1.36
CEREBROSPINAL FLUID
IgG Synthesis index
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 all elevated. The clinical diagnosis was sepsis with meningitis. Most cases of bacterial meningitis lead to elevations of CSF proteins, many time as high as 1000mg/dl. Usually a strong polyclonal immunoglobulin response is present with significantly elevated indices. The proteins are of both plasma and CNS origin, since albumin is not synthesized in the CNS compartment.
71
Bacterial Meningitis
% 0.7 66.1 4.3 4.8 12.1 12.0
mg/dl 4 400 26 29 73 73 605 0.22
CEREBROSPINAL FLUID
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.
72
Meningitis % 2.6 64.9 3.2 7.2 12.4 9.8 mg/dl 4.5 111.0 5.5 12.3 21.2 16.8 171 0.25
CEREBROSPINAL FLUID
Myelin basic protein <1
This pattern was obtained on CSF from a patient with sepsis and meningitis in which no organism could be identified. It was thought 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 the gamma. The increased protein in this CSF is mainly due to increased permeability of the blood-brain barrier.
73
Fungal Meningitis
% 4.5 49.5 6.0 6.7 14.2 19.1
mg/dl 6.2 68.8 8.3 9.3 19.7 26.6 139
CEREBROSPINAL FLUID
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 bacterial meningitis.
74
Human Immunodeficiency Virus Infection
% 4.1 40.1 3.6 6.7 21.6 24.0
mg/dl 1.1 10.4 0.9 1.7 5.6 6.2 26 0.38
CEREBROSPINAL FLUID
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 zone. Recently 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 frequency in other CNS infections including Herpes encephalitis, subacute sclerosing panencephalitis, kuru and infectious viral encephalitides.
75
AIDS - Cryptococcal Meningitis
% 6.0 23.8 10.9 11.4 21.3 26.6
mg/dl 1.4 5.7 2.6 2.7 5.1 6.4 24 1.83
CEREBROSPINAL FLUID
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 had been previously diagnosed with acquired immune deficiency syndrome and was admitted with symptoms of meningitis. Cells of Cryptococcus were seen in CSF and the cryptococcal polysaccharide agglutination test was positive. In this specimen the alpha-1 and beta regions 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 or against human immunodeficiency virus.
76
Systemic Lupus with CNS Involvement
% 1.6 50.8 6.5 7.4 14.5 19.2
mg/dl 1 27 4 4 8 10 54 0.56
CEREBROSPINAL FLUID
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.
77
Central Nervous System Lupus
% 3.6 44.2 9.2 7.5 19.3 16.3
mg/dl 0.6 7.1 1.5 1.2 3.1 2.6 16.0 0.41
CEREBROSPINAL FLUID
This patient with active systemic lupus erythematosus had recently reported experiencing severe headaches. His wife noticed personality changes. The CSF had a normal protein level with an elevated IgG/albumin. Three faint 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
78
79
Meningioma
% 4.8 56.2 1.1 2.1 17.4 18.5
mg/dl 1.2 14.1 0.3 0.5 4.4 4.6 25 0.50
CEREBROSPINAL FLUID
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 mg/dl and the IgG/albumin ratio is elevated. The beta and gamma regions show a polyclonal pattern and no immunoglobulin bands are visible. Myelin basic protein was not detected.
80
Malignant Brain Tumor
% 1.9 57.2 2.8 6.8 18.4 12.9
mg/dl 2.8 84.1 4.1 10.0 27.1 19.0 147 0.35
CEREBROSPINAL FLUID
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.
81
Ependymoma with Froin's Syndrome
% 0 59.7 3.3 8.0 15.5 13.4
mg/dl 1594 88 214 414 358 2670 0.24
CEREBROSPINAL FLUID
This specimen illustrates an unusual condition referred to as Froin's syndrome. The patient had a small ependymoma located between L2 and L4 in the spinal cord. The tumor caused blockage of the CSF circulation. When tapped there was a very high 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 refrigeration, 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 that gels at 4 degrees C.
82
Central Nervous System Lymphoma
% 2.0 58.0 5.9 12.1 16.9 5.0
mg/dl 10.4 301.0 30.6 62.8 87.7 25.9 519 0.07
CEREBROSPINAL FLUID
This specimen was obtained from a patient with an established diagnosis of lymphoma, who developed symptoms consistent with central nervous system lymphoma. The diagnosis was confirmed by cytologic examination of the CSF. The total protein and all fractions were elevated, with a relative decrease in prealbumin and evidence of acute phase proteins in the alpha and beta. CSF IgG was elevated at 21 mg/dl, but the indices were normal. No banding was visible in the gamma region. The protein increase in this case appears to be the result of increased blood-brain barrier permeability. CSF beta-2-microglobulin when elevated relative to serum beta-2-microglobulin can be a useful test for monitoring lymphocytic malignancies of the CNS.
83
Pseudo-Tumor of Brain
% 3.6 63.6 3.7 5.8 15.6 7.7
mg/dl 1.8 31.2 1.8 2.8 7.6 3.8 49 0.10
CEREBROSPINAL FLUID
This specimen was obtained from a patient whose admitting diagnosis was "pseudo-tumor of brain." A final diagnosis was not available when this was written. The pattern is included here because a faint oligoclonal banding pattern was observed in the gamma region. The pattern is otherwise relatively normal. Oligoclonal immunoglobulin bands are found in high frequency in the CSF of patients with multiple sclerosis and similar diseases, HIV infection, slow virus CNS infections, some encephalitis, TB meningitis, some fungal meningitis and neurosyphilis. Bands are found with lesser frequency in CSF from patients with chronic and relapsing inflammatory polyneuropathies, optic neuritis, Guillain-Barre syndrome, and other neuropathies. Occasionally bands are seen in CSF from CNS tumor patients, other inflammatory conditions of the CNS and rarely in CSF from subjects without neurologic disease. In chronic and inflammatory conditions the presence and amount of bands is related to disease activity. although not totally reflective of status. Patients in remission have somewhat lower incidence and lesser amounts of oligoclonal antibodies than those with active disease.
84
Multiple Sclerosis
% 14.3 41.2 2.3 3.3 6.8 42.2
mg/dl 2 16 1 1 3 17 40
CEREBROSPINAL FLUID
= 1.05
% 4.7 46.2 3.3 5.4 11.2 29.2
mg/dl 2 22 2 3 5 14 48
CEREBROSPINAL FLUID
= 0.95
Electrophoresis patterns are illustrated from 6 cases of multiple sclerosis. All the examples shown have normal to moderately 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.
85
% 4.1 49.5 1.5 3.6 10.8 30.5
mg/dl 1 18 1 1 4 11 36
CEREBROSPINAL FLUID
= 0.61
% 5.4 50.1 2.0 4.6 16.1 21.8
mg/dl 1.4 13.0 0.5 1.2 4.2 5.7 26
CEREBROSPINAL FLUID
= 0.57
CEREBROSPINAL FLUID Prealbumin Albumin Alpha-1 Alpha-2 Beta Gamma CSF protein IgG/albumin = 0.50 % 3.1 57.6 4.0 8.7 14.6 12.0 mg/dl 2.9 53.6 3.7 8.1 13.6 11.2 93
86
% 2.4 51.9 2.0 3.0 14.0 26.7
mg/dl 1.1 23.9 0.9 1.4 6.4 12.3 46
CEREBROSPINAL FLUID
= 0.70
87
88
Neuromyelitis Optica
% 6.1 57.0 3.7 7.2 16.9 9.0
mg/dl 2 19 1 2 6 3 33 0.14
CEREBROSPINAL FLUID
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 mimics spinal chord compression, because significant edema may be present. The involved optic nerves show demyelination and typical 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 presence of 3 bands in the gamma region. CSF electrophoretic patterns in neuromyelitis optica are similar to those in multiple sclerosis.
89
Single Gamma Band in CSF
% 3.8 53.4 2.3 5.7 14.5 20.2
mg/dl 2 25 1 3 7 9 47
CEREBROSPINAL FLUID
= 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 concentrated CSF can be performed, when paraproteinemia is suspected. Monoclonal bands of immunoglobulin are rarely found in CSF in cases of chronic lymphocytic leukemia and immunoblastic lymphoma. A large fraction of single gamma bands in CSF are not of any diagnostic significance. The pattern illustrated here is from a patient with mild multiple sclerosis. The IgG/albumin is elevated as was myelin basic protein.
90
Chronic and Relapsing Inflammatory Polyneuropathy
% 2.0 66.3 3.2 8.6 19.3 24.4
mg/dl 3.0 44.5 4.8 12.8 28.8 36.4 149
CEREBROSPINAL FLUID
= 0.55
The chronic and relapsing inflammatory polyneuropathy syndromes (CRIPS) are a group of diverse polyneuropathies. They usually 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. The typical electrophoretic pattern shows an elevated total protein and albumin. Alpha and beta globulins are variable. The gamma may be elevated in a polyclonal or oligoclonal pattern. IgG/albumin and IgG synthesis index are not consistently elevated.
91
Guillain-Barre Syndrome
% 3.1 65.6 4.0 5.7 10.5 11.1
mg/dl 3.1 66.3 4.0 5.8 10.6 11.2 101
CEREBROSPINAL FLUID
= 0.20
Guillain-Barre syndrome is acute inflammatory neuropathy that follows a viral or mycoplasmal infection, surgery, neoplasm or 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 and autonomic 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 this condition. Similar patterns are seen in transverse myelitis.
92
CSF with Large Beta Spike
% 3.7 47.1 1.6 1.8 34.1 11.6
mg/dl 1.3 16.5 0.6 0.6 11.9 4.1 35
CEREBROSPINAL FLUID
= 0.10 <1
Myelin basic protein
This CSF specimen was obtained from a patient with a diagnosis of cerebellar ataxia. The bands marked in the gamma are artefactual stain dots. The specimen was clear and colorless after concentration. The beta peak was not immunoglobulin when tested on an immunochemical analyzer. The spike has the same mobility as transferrin; however, there was insufficient specimen volume to confirm this identification.
93
Benign Monoclonal Gammopathy with Neuropathy
% 4.9 76.2 1.3 2.0 10.4 5.2
mg/dl 2.0 31.2 0.5 0.8 4.3 2.1 41
CEREBROSPINAL FLUID
= 0.21
This patient was referred to a neurologist with a mild polyneuropathy. A single monoclonal peak was found in the CSF and was identified by immunoelectrophoresis as free lambda chains. Examination of serum and urine showed the presence of small amounts of lambda Bence-Jones protein of identical mobility to the CSF peak. Polyneuropathy of plasma cell dyscrasia syndromes is usually of a mixed motor and sensory type. Histologically there is usually demyelination and axonal degeneration. It is somewhat more common in males and younger patients. In almost all cases the light chain type is lambda, either free or associated with IgG, IgA or IgM. A number of studies of these cases have shown the homogeneous immunoglobulin deposited on the affected nerves, especially in the sheaths. The IgM type of polyneuropathy is slowly progressive and shows severe reduction of conduction velocity. The antibody seems to react with a carbohydrate determinant 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 the nerve gangliosides.
94
Paraprotein Polyneuropathy
% 56.6 7.1 5.2 5.7 25.4
mg/dl 187 23 17 19 84 330
URINE
This scan illustrates a urine electrophoresis from a patient who presented with polyneuropathy. A moderate spike of identical mobility was found in the serum and urine and was identified by immunofixation electrophoresis as IgG-lambda.
95
Paraprotein Polyneuropathy
% 3.9 58.0 4.9 10.0 14.1 9.1
mg/dl 1.9 27.8 2.4 4.8 6.8 4.4 48
CEREBROSPINAL FLUID
= 0.19
IgG synthesis index = 0.34
% 43.9 6.0 13.8 13.5 22.8
g/dl 2.99 0.41 0.94 0.92 1.55 6.8
SERUM
Total Protein IgG = 1620 mg/dl IgA = 377 mg/dl IgM = 38 mg/dl
This patient presented with progressive polyneuropathy. Serum electrophoresis showed a polyclonal gammopathy with one distinct and two faint bands present. The distinct band labeled 1 was identified as an IgMlambda paraprotein and the faint bands, 2, were IgG by immunofixation electrophoresis. Band 1, the IgM-lambda paraprotein was also found in cerebrospinal fluid. The urine from this patient is shown as Overflow Proteinuria with Inflammation.
96
97
Malignant Paraprotein Polyneuropathy
% 56.5 8.8 13.4 10.6 10.7
g/dl 1.8 0.3 0.4 0.3 0.3 3.2
SERUM
IgG = 410 mg/dl IgA = 31 mg/dl IgM = 61 mg/dl Beta-2-microglobulin = 23mg/l
% Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein Protein/24hrs
mg/dl trace
URINE
100 = 14.5 g
1400 1450
Continued on next page.
98
% 3.7 63.2 3.6 5.8 15.0 8.7
mg/dl 1.8 31.0 1.8 2.8 7.3 4.3 49
CEREBROSPINAL FLUID
= 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 amounting to about 14 g/day and a small amount of albumin. The spikes were identified 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 for a trace band at the end of the gamma, which was found to be kappa light chain. 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.
99
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 IgGalbumin 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 three sharp bands at the anode are prealbumin. The sample in track 5 is from a patient with a metastatic carcinoma in the central nervous system. The albumin was 34mg/dl and IgG 5mg/dl. The pattern shows polyclonal immunoglobulins with a C-reactive protein band nearest the cathode. Some serum proteins not normally visible in CSF can be seen anodal to the position of hemoglobin. Track 6 illustrates HIV infection. The CSF protein was 49mg/dl with an albumin of 23mg/dl and an IgG of 14mg/dl. Several clonal patterns are visible in the immunoglobulin region. 1-Viral encephalitis, 2-Multiple sclerosis oligoclonal 3-Hemoglobin A stroke, 4-Normal CSF, 5-Polyclonal carcinoma with C-reactive protein, 6-Human immunodeficiency virus
100
OTHER CEREBROSPINAL FLUIDS CSF Cervical Myelitis
CSF CNS Sarcoid
101
CSF CNS Toxoplasmosis
CSF Transverse Myelitis
102
OTHER FLUIDS
103
Pleural Fluid - Transudate PLEURAL FLUID % 61.0 6.5 6.8 13.0 12.8 g/dl 0.30 0.03 0.03 0.06 0.06 0.50
Total Protein
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. Another case
104
Pleural Fluid - Infection
% 46.0 10.4 8.8 15.2 19.6
g/dl 0.9 0.2 0.2 0.3 0.4 1.9
PLEURAL FLUID
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.
105
106
SYNOVIAL FLUIDS Synovial Fluid Exudate
Synovial Fluid Gout
107
PARAPROTEINS M-COMPONENTS
108
Paraproteins Paraproteins or M-components are abnormal plasma proteins, almost always immunoglobulins. Most paraproteins are monoclonal, although some are oligoclonal. Examples of paraproteins are 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 BenceJones 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 109
MGUS. Most hematologists use some type of score chart. An example of one from the Southwestern Oncology Group follows. DIAGNOSIS OF MULTIPLE MYELOMA Major Criteria I Bone marrow over 30% plasma cells (monoclonal) II Biopsy proven plasmacytoma (monoclonal) III Over 3000mg/dl monoclonal IgG, 2000mg/dl IgA, 1000mg/dl IgD or IgE IV Bence-Jones protein over 1000mg/day Minor Criteria a Bone marrow 10-30% monoclonal plasma cells b Any monoclonal paraprotein present 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 This has now been replaced in most academic institutions with the current WHO score chart, shown below.
110
Diagnosis of Multiple Myeloma (World Health Organization)

Major Criteria I Bone marrow over 30% plasma cells II Biopsy proven plasmacytoma III Minor Criteria a 10-30% plasma cells b Monoclonal protein present Monoclonal over 3.5g% IgG or 2g% IgA c Lytic bone lesions Bence-Jones protein over 1g/day d Reduced normal immunoglobulins IgM<50mg/dl, IgA<100mg/dl, IgG<600mg/dl
The diagnosis requires a minimum of one major and one minor criterion or three minor criteria which must include (a) and (b). The patient must be symptomatic and have progressive disease or will be classified as smoldering or indolent myeloma. ILLUSTRATIONS Serum Immunoelectrophoresis M-components
Urine Immunoelectrophoresis M-components
111
Myeloma Untreated Natural Course
112
113
Waldenstrom's Macroglobulinemia
% 47.3 3.1 6.0 13.0 30.5
g/dl 4.1 0.3 0.5 1.1 2.7 8.7
This pattern illustrates Waldenstrom's macroglobulinemia. This disease is defined by finding a large IgM spike in the serum and 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. This one is at the junction and is integrated into the gamma. The IgG and IgA exhibit suppression. The IgM was typed as IgMkappa. 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 114
required. Some IgM paraproteins have antibody activity. There are a group of IgM-kappa paraproteins which cause a typical cold agglutinin disease, which mainly have the same variable region sequence. Other paraproteins may react with clotting factors, glycoproteins on nerves and other autoantigens.
% 50.7 3.0 6.9 10.7 28.9
g/dl 3.9 0.2 0.5 0.8 2.2
This scan shows an IgM-lambda paraprotein in the gamma region. IgM paraproteins are many times, but not always somewhat broad. About 11% of paraproteinemias are Waldenstrom's macroglobulinemia. Another case
115
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG = IgA = IgM =
% 30.2 1.9 5.1 7.1 55.7
g/dl 3.2 0.2 0.5 0.7 5.9 10.6
SERUM
981 mg/dl 122 mg/dl 4600 mg/dl
% 15.3 7.8 4.8 10.7 61.3
mg/dl 10 5 3 7 40 65 770
URINE
These specimens were obtained at time of diagnosis of a case of Waldenstrom's macroglobulinemia. The paraprotein was IgMlambda and the serum viscosity was elevated at 6.4 centistokes. The bone marrow biopsy showed a large number of small lymphocytes, similar in appearance to the cells in a small cell lymphoma. The urine electrophoresis pattern contained three spikes in the gamma region. The largest was identified as lambda Bence-Jones protein by immunofixation electrophoresis. The other two spikes were identified as mu Fc fragments and Fab fragments.
116
117
B Cell Lymphoma
% 64.8 4.3 9.3 13.2 8.5
g/dl 4.1 0.3 0.6 0.8 0.5 6.4
This serum was obtained from a patient with a B cell nonHodgkins 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.
118
Chronic Lymphocytic Leukemia % 47.7 6.6 19.1 14.1 12.4 g/dl 3.0 0.4 1.2 0.9 0.8 6.3
% 55.1 5.1 10.2 22.6 6.9
g/dl 3.5 0.3 0.7 1.5 0.4 6.4
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 that patients whose sera show either the presence of monoclonal immunoglobulin, polyclonal gammopathy, or immunoglobulin suppression in CLL have a less favorable prognosis.
119
IgM Paraproteinemia
% 59.8 3.4 6.2 10.7 19.9
g/dl 4.1 0.2 0.4 0.7 1.4 6.8
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 120
% 46.8 3.7 7.4 10.7 29.3
g/dl 3.1 0.3 0.6 0.7 2.0 6.7
IgG = 2400 mg/dl IgA = 129 mg/dl IgM = 137 mg/dl Another case This patient had been treated several years previously for Waldenstrom's macroglobulinemia. She had no symptoms and her serum showed no detectable paraprotein for five years. She then 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.
121
Secondary Malignancy
% 53.8 2.2 10.3 13.4 20.3
g/dl 4.1 0.2 0.8 1.0 1.5 7.6
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 initial diagnosis. 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.
122
Carcinoma of the Lung % 62.9 4.1 11.4 10.3 11.3 g/dl 4.0 0.3 0.7 0.7 0.7 6.4 SERUM
% 47.1 17.5 18.3 8.8 8.3
mg/dl 68 25 27 13 12 145
URINE
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.
123
124
Thymoma with Paraproteinemia
SERUM Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG = 1360 mg/dl IgA = 264 mg/dl IgM = 539 mg/dl % 56.9 3.8 15.6 7.9 15.7 g/dl 3.9 0.3 1.1 0.6 1.1 6.9
This patient was an older woman who had previously had a malignant thymoma removed surgically. The tumor had recurred and she was undergoing radiation therapy. The electrophoretic pattern showed three small spikes, which were identified as IgMkappa, 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.
125
126
IgG Cryoglobulinemia
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG IgA IgM C3 C4
% 27.5 1.0 3.1 9.8 58.6
g/dl 2.3 0.1 0.3 0.8 4.9 8.3
= 5060 mg/dl = 1090 mg/dl = 280 mg/dl = 19 mg/dl = 18 mg/dl
This patient presented with anemia, thrombocytopenia, lymphopenia, hepatosplenomegaly and proteinuria. A bone marrow biopsy showed 80% plasma cells in sheets. A broad irregular peak was visible in the far 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 normal. Electrophoresis of urine showed a non-selective proteinuria. Some cryoglobulin 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.
127
Type II Cryoglobulinemia Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG = 1700 mg/dl IgA = 319 mg/dl IgM = 104 mg/dl % 44.4 2.4 11.1 16.6 25.4 g/dl 2.8 0.1 0.7 1.1 1.6 6.4
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein Another case
% 61.0 8.1 9.3 6.9 14.7
g/dl 4.3 0.6 0.7 0.5 1.0 7.0
Both of these sera contain IgM-kappa paraproteins with rheumatoid factor like activity against IgG and IgA. Significant amounts of cryoprecipitate were found in both sera. Immunofixation electrophoresis studies on the precipitate using dissociating buffers may be necessary to properly work-up mixed cryoglobulins. In both of these cases the IgG and IgA contained both kappa and lambda, while the IgM from the precipitate contained only kappa. Because of the overwhelming size of the monoclonal IgM molecules, the complexes appear as restricted paraprotein spikes. The pattern of the second serum shows the presence of both free IgM-kappa and IgM-kappa-IgG/IgA complexes as two distinct spikes. Type II cryoglobulinemia is associated with lymphoproliferative disease, chronic infectious and autoimmune rheumatoid diseases. Cases without these underlying causes are called essential cryoglobulinemia. Typical presentations are 128
purpura, polyarthralgias, and cutaneous vasculitis. Liver disease is found in many cases and about 55% develop renal disease. Development of renal disease is a poor prognostic indicator. Essential cryoglobulinemia and lymphoproliferative disease associated cases usually show IgM paraproteins. Rheumatoid disease associated cases may be IgM, IgG or IgA. The presence of cryoprecipitate causes interference in automated hematology analyzers, many times giving extremely high white counts. If the specimen and apparatus are kept warm, then normal counts are obtained. The WBC histogram usually shows an elevated y-axis slope gradually descending to baseline. Manual counts, of course, are not subject to this interference.
129
Monoclonal Cryogelglobulin
SERUM Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG = 538 mg/dl IgA = 1784 mg/dl* IgM = 20 mg/dl * Value in error because of cryoprecipitation. % 40.3 3.2 10.8 9.1 36.6 g/dl 3.9 0.3 1.0 0.9 3.5 9.6
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 35C. 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.
130
Primary Amyloidosis (AL)
% 25.6 5.7 32.6 21.1 15.0
g/dl 0.9 0.2 1.1 0.7 0.5
SERUM
IgG = 676 mg/dl IgA = 31 mg/dl IgM = 91 mg/dl See track 106
131
% 50.9 5.4 13.0 10.1 20.5 = 30 g
mg/dl 1069 113 273 212 431 2100
URINE
This 36 year old man was diagnosed five years previously with primary AL amyloidosis. His renal function rapidly deteriorated to the 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-2macroglobulin 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 gamma was 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 of 5.5 g/day. The amount of Bence-Jones protein and IgG paraprotein had increased significantly from the time of 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.
132
Additional cases AL-Amyloidosis Serum
AL-Amyloidosis Urine
133
AL-Amyloidosis IgG Serum
AL Amyloidosis IgG Urine
134
Peritoneal Fluid - Amyloidosis % 71.2 4.1 7.6 9.4 7.7 mg/dl 46 3 5 6 5 65 PERITONEAL FLUID
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. 135
In some cases detailed composition studies are necessary to distinguish exudates from transudates.
136
Solitary Plasmacytoma % 37.6 3.2 16.3 13.4 29.6 g/dl 3.0 0.3 1.3 1.1 2.4 SERUM
% 46.4 5.4 13.8 5.8 28.6
mg/dl 11.1 1.3 3.3 1.4 6.9 24
URINE
This patient was hospitalized because of a mass on his chest wall. Serum electrophoresis showed an elevated alpha-2, probably an 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 were found by immunofixation. In general solitary plasmacytomas synthesize little or no detectable paraprotein. A fraction progress to multiple myeloma, but most can be removed surgically. Some are polyclonal. 137
Oligoclonal Gammopathy % 38.9 3.2 6.1 15.4 36.4 g/dl 3.3 0.3 0.5 1.3 3.1 8.5
This pattern was found on a routine protein electrophoresis ordered on a patient admitted to the psychiatric unit for depression. Both IgG and IgA are elevated. Two heavy bands are visible 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 like infection. They are usually transient. In this case the cause is unknown.
138
Another case Oligoclonal Banding Serum
Oligoclonal Banding Urine
139
Oligoclonal Gammopathy - Infection
% 39.0 7.3 16.2 16.5 21.1
g/dl 2.0 0.4 0.8 0.9 1.1 5.2
SERUM
140
% 17.8 29.0 13.0 11.0 29.3
mg/dl 6.0 9.9 4.4 3.7 10.0 34
URINE
This patient was hospitalized for fever of unknown origin. Evaluation revealed acute tubular necrosis and endocarditis. Serum electrophoresis showed a low albumin with three bands visible in the gamma. Typing by immunofixation electrophoresis identified them as IgG-kappa. Urine electrophoresis gave a pattern suggesting tubular disease and inflammation. The gamma region of the urine contained faint kappa spikes. This case illustrates oligoclonal immune response to infection.
141
IgG-Lambda Myeloma % 26.8 4.8 8.1 6.8 53.4 g/dl 2.7 0.5 0.8 0.7 5.4 10.1 SERUM
% 31.2 16.9 13.8 12.3 25.9
mg/dl 29 16 13 11 24 92 1000
URINE
These are typical serum and urine patterns from case of IgG multiple 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 peak of about 0.2 g/day and a small lambda Bence-Jones protein peak in the beta of about 40 mg/day. The patient presented with bacterial meningitis and profound anemia. This patient has shown a poor response to chemotherapy. The serum pattern is an excellent example of the suppression of normal immunoglobulin synthesis by multiple myeloma. About 53% of myeloma cases produce IgG paraproteins. 142
IgG-Kappa Myeloma % 38.0 3.1 11.6 9.5 37.7 g/dl 4.0 0.3 1.2 1.0 4.0 10.6 URINESERUM
% 39.8 6.2 8.2 11.4 34.4
mg/dl 159 25 33 46 138 400
URINE
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.
143
IgG-Lambda Myeloma Beta Spike
% 39.8 2.7 12.8 43.7 1.0
g/dl 3.2 0.2 1.0 3.5 0.1 8.0
This serum specimen was obtained from a patient with IgGlambda myeloma after a successful course of chemotherapy. The paraprotein spike is in the beta, not gamma. Typically IgG2 and IgG4 proteins are less basic than IgG1 and IgG3. Many IgG2 and IgG4 paraproteins migrate in the beta. Before therapy the IgG paraprotein concentration was 7000 mg/dl.
144
IgG-Lambda Myeloma Broad Peak
% 45.3 4.9 15.8 11.1 22.8
g/dl 2.8 0.3 1.0 0.7 1.4 6.1
This serum shows a relatively broad paraprotein spike in the gamma region. The paraprotein typed as IgG-lambda. Broad spikes are seen in about 10% of IgG cases and in many IgA, IgM, and IgD paraproteins. The breadth is due to charge heterogeneity of the molecules, many times in the carbohydrate portion.
145
IgG-Kappa Myeloma Defective Protein % 35.1 2.9 8.3 14.3 39.4 g/dl 3.2 0.3 0.8 1.3 3.6 SERUM
IgG = 4200 mg/dl ** IgA = 25 mg/dl IgM = 37 mg/dl
% 9.3 3.1 2.8 6.1 78.7 = 3.4 g
mg/dl 30 10 9 20 258 327
URINE
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.
146
IgG-Kappa Myeloma With Bence-Jones Protein
% 35.5 2.2 4.8 7.8 49.7
g/dl 3.0 0.2 0.4 0.7 4.2 8.5
SERUM
% 20.0 2.0 5.5 56.2 16.0
mg/dl 21 2 6 58 17 104
URINE
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.
147
IgG-Lambda Myeloma with Three Urine Spikes % 38.2 1.4 5.4 54.0 1.3 g/dl 3.6 0.1 0.5 5.0 0.1 9.3 SERUM
% 49.1 2.3 26.4 21.0 1.0
mg/dl 73 3 39 31 2 148
URINE
The serum contains a large IgG-lambda paraprotein spike which 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.
148
Free Light Chain in Serum
% 52.9 5.1 10.2 23.7 8.1
g/dl 3.3 0.3 0.6 1.5 0.5 6.2
SERUM
IgG = 1450 mg/dl IgA = 101 mg/dl IgM = 51 mg/dl Another case
149
% 5.9 1.8 2.8 80.1 9.3 = 4.3 g
mg/dl 8 2 4 106 12 132
URINE
This patient has multiple myeloma with only a small amount of intact IgG-kappa paraprotein in the serum. Immunoelectrophoresis 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.
150
Multiple Myeloma with Renal Failure % 27.2 1.4 5.0 3.3 63.0 g/dl 2.6 0.1 0.5 0.3 6.1 9.7 SERUM
151
% 4.8 6.1 5.0 11.5 72.6 491mg
mg/dl 31 40 33 75 476 655
URINE
This patient was transferred from a community hospital after developing total renal failure following the administration 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 152
specimen shown here was obtained four weeks after the serum sample. The serum shows a low albumin level with two paraprotein spikes in the gamma region. The large spike was identified as an IgG-kappa paraprotein and the small very basic spike was free kappa chains. Several investigators have reported that highly basic light chains appear to be extremely nephrotoxic. The urine specimen showed small amounts of serum proteins and a small amount of intact IgG paraprotein. The kappa Bence-Jones protein formed a double spike of 0.35g/day. % 34.0 2.1 6.4 4.2 53.3 g/dl 2.7 0.2 0.5 0.3 4.2 7.8 SERUM
% 43.4 2.1 4.9 4.6 44.9
mg/dl 87 4 10 9 90 200 16grams
PERITONEAL DIALYSIS FLUID
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 onethird, and has continued to decrease with further chemotherapy. The patient is maintained on eight liters of peritoneal dialysis 153
daily at home. A sample of the dialysis fluid at the time of the second serum specimen is shown above. At this time the dialysis was removing 16g/day of protein, 7.2g of paraprotein. Renal function had improved significantly and output volume was within the normal range. Urine electrophoresis showed almost pure Bence-Jones protein excretion. Another case Myeloma with Nephrosis Serum
Myeloma with Nephrosis Urine
154
Nephrosis with Monoclonal Gammopathy of Undetermined Significance
% 43.9 6.7 20.2 7.4 21.8
g/dl 2.7 0.4 1.3 0.5 1.4 6.2
SERUM
% 57.9 5.7 10.9 6.9 18.7
mg/dl 181 18 34 22 58 312
URINE
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-2macroglobulin 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.
155
Monoclonal Gammopathy of Undetermined Significance
% 50.1 2.5 10.3 7.5 29.6
g/dl 3.6 0.2 0.7 0.5 2.1 7.2
SERUM
% 57.3 3.7 6.3 6.5 26.2
mg/dl 76 5 8 9 35 132
URINE
The serum from this patient has a broad IgG-kappa paraprotein peak in the gamma at a total of 2 g/dl. The kappalambda ration was significantly elevated at 5.7. The urine electrophoresis reflects the serum pattern with intact IgG, but no free light chain detected.
156
Smoldering or Asymptomatic Myeloma
% 42.5 2.7 5.7 8.6 40.5
g/dl 3.6 0.2 0.5 0.7 3.4 8.5
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.
157
Trace Immunoglobulin Spike
% 61.7 4.5 7.6 13.9 12.4
g/dl 3.9 0.3 0.5 0.9 0.8 6.4
This is an example of a small paraprotein spike. The spike is at the very far end of the gamma region and types as IgGkappa. 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.
158
Nephrotic Syndrome with Monoclonal Gammopathy
% 33.7 5.0 13.9 11.2 36.2
g/dl 1.9 0.3 0.8 0.6 2.1
SERUM
% 57.5 6.7 8.2 22.2 5.4
mg/dl 361 42 51 139 34 627
URINE
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 betalipoprotein spike is visible. The urine reflects the serum pattern except for the macro-proteins and contains intact IgGlambda paraprotein with no Bence-Jones protein.
159
IgA-Kappa Myeloma
% 28.3 2.1 2.8 54.7 12.0
g/dl 3.4 0.3 0.3 6.6 1.4 12.0
A few patients present with enormous paraprotein levels at time of diagnosis. We have seen one case with 17 g/dl of paraprotein. The electrophoresis above shows a huge spike in the beta with a small spike in the gamma. Both spikes typed as IgAkappa. Both polymers and charge variants are seen with IgA proteins. This patient has a relatively normal serum albumin. Much of the older literature reports that albumin decreases to compensate for the increased globulin in myeloma. As therapy and patient management have improved, this has turned out to be a fallacy. In the absence of renal loss, myeloma patients can maintain relatively normal albumin levels. The decrease in albumin reflects a decrease in the status of the patient, e.g. nutritional, rather than paraprotein level. Approximately 23% of myeloma cases make IgA paraproteins.
160
IgA-Kappa Paraprotein
% 35.1 3.2 12.6 42.4 6.7
g/dl 2.1 0.2 0.8 2.7 0.4 6.3
161
% 56.1 1.6 10.0 25.5 6.9
mg/dl 690 20 123 314 85 1230 15.5g
This scan shows an IgA-kappa paraprotein which makes a sharp spike in the beta. One cannot identify the type of paraproteins from either the migration or peak shape. The urine shows two large beta spikes of intact IgA-kappa paraprotein and two small gamma spikes of kappa Bence-Jones protein.
162
IgA-Lambda Paraprotein with Two Bands
% 46.9 3.5 6.9 10.3 32.4
g/dl 3.5 0.3 0.5 0.8 2.4 7.5
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.
163
IgA-Lambda Myeloma with Diabetic Renal Disease % 44.1 5.3 10.0 27.4 13.2 g/dl 2.6 0.3 0.6 1.6 0.8 5.8 SERUM
164
% 46.2 6.7 7.2 22.1 17.8
mg/dl 455 66 71 218 175 985
URINE
This patient with a long previous history of diabetes and hypertension was admitted to the hospital with a suspected brain tumor. On radiologic examination discrete bone lesions were found in the skull. A plasmacytoma was removed from the skull. The serum electrophoresis shows a low albumin with a double spike in the beta, which types as IgA-lambda. A faint spike at the beginning of the gamma region was typed as free lambda chains. The urine pattern is very similar to the serum, but with a larger spike in the gamma. It is unusual for IgA to pass readily through the kidney. The serum IgA level was borderline for IgA myeloma. The IgA level and the localization of the disease to a single tumor mass in the skull suggest that the myeloma was at a relatively early stage evolving from a solitary plasmacytoma, which is not consistent with the advanced nephrotic syndrome seen. The renal damage was probably the result of long-standing diabetes and poorly controlled hypertension.
165
IgD-Kappa Myeloma % 55.3 4.6 9.2 12.7 18.2 g/dl 3.3 0.3 0.5 0.8 1.1 6.0
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG IgA IgM IgD
= 455 mg/dl = 20 mg/dl = 19 mg/dl = 480 mg/dl
This electrophoresis pattern was obtained with serum from a 77 year old man with multiple myeloma. Two paraprotein spikes were visible in the gamma region. One typed as free kappa chain and the other as IgD-kappa. Significance suppression of IgG, IgA and IgM is also present. From 1-2% of all myeloma patients produce IgD paraproteins. IgE paraproteins are produced by less than 0.1% of all patients.
166
IgD-Kappa Myeloma % 43.6 2.8 7.5 42.6 3.4 g/dl 3.4 0.2 0.6 3.3 0.3 7.8 mg/dl mg/dl mg/dl mg/dl SERUM
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG IgA IgM IgD = 344 = 21 = 34 = 2380
% 2.1 1.9 1.4 92.9 1.7 = 5.9 g
mg/dl 7 6 4 292 5 314
URINE
These patterns were obtained at time of diagnosis of multiple myeloma in a 43 year old man. A broad spike was visible in the beta region of the serum, along with profound suppression of normal immunoglobulin production. The spike was typed as IgDkappa. The urine contained a large broad spike of the same mobility as the serum paraprotein, but which did not contain any immunochemically measurable IgD, and was identified as 5.9 g of Kappa Bence-Jones protein. There was only a trace of albumin 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. 167
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.
168
IgD-Lambda Myeloma
Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein IgG IgA IgM IgD
% 54.8 4.8 9.0 13.5 17.9
g/dl 3.9 0.3 0.6 1.0 1.3
SERUM
= 622 mg/dl = 26 mg/dl = 16 mg/dl = 960 mg/dl
% 2.2 2.1 2.4 85.4 7.9
mg/dl 2 2 3 92 9 108
URINE
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.
169
170
Kappa Light Chain Disease % 51.2 15.0 12.2 9.3 12.3 g/dl 2.8 0.8 0.7 0.5 0.7 5.4 SERUM
% 13.9 12.8 15.0 51.3 7.0
mg/dl 9 8 10 32 4 63
URINE
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.
171
Plasma Cell Leukemia with Lambda Myeloma % 45.0 6.9 8.7 32.1 7.4 g/dl 2.6 0.4 0.5 0.4 0.4 SERUM
% 6.4 1.7 2.3 85.0 4.5
mg/dl 48 13 17 646 34 760
URINE
This patient presented with anemia, renal failure, proteinuria and plasma cell leukemia. The serum had suppression of normal immunoglobulins with a large spike in the beta, which typed as free lambda chains. The serum albumin and gamma globulins were low. The urine showed a very large lambda BenceJones 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.
172
Multiple Myeloma with CNS Involvement
SERUM
% 22.0 3.9 8.8 6.5 58.8
g/dl 1.9 0.3 0.8 0.6 5.2 8.8
173
URINE % 6.6 4.4 5.6 5.5 78.0 mg/dl 6.8 4.5 5.8 5.7 80.3 103 3832mg
Total Protein Protein/24hrs
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 IgGkappa 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 amounts of other CSF proteins suggests possible central nervous system growth of the myeloma. No CNS tumor masses were visible by magnetic resonance imaging.
174
% 3.8 29.6 5.3 6.4 11.2 43.7
mg/dl 2.6 20.1 3.6 4.3 7.6 29.7 68
CEREBROSPINAL FLUID
2.1 0.66
IgG synthesis index
175
Another case
176
Immunoglobulin Fragments in Urine
% 44.9 4.6 6.5 12.3 31.8 = 0.6 g
mg/dl 42 4 6 12 30 94
URINE
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.
177
Kappa Bence-Jones and IgG in Urine
% 7.2 2.0 2.0 3.0 84.5 = 3.4 g
mg/dl 8 3 3 4 95 112
URINE
This urine contains a small albumin peak, a very large kappa Bence-Jones protein peak and a small intact IgG-kappa paraprotein peak. 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.
178
Multiple Spikes in Urine - Myeloma % 21.9 5.2 6.5 8.2 58.1 = 1.0 g mg/dl 3 1 1 1 8 14 URINE
This 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 IgGlambda 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 macroglobulinemia.
179
Biclonal Gammopathy
% 39.6 5.0 14.6 26.1 14.7
g/dl 2.2 0.3 0.8 1.4 0.8 5.5
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 IgGkappa 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.
180
181
Triclonal Gammopathy - Myeloma % 18.0 2.5 5.4 71.1 3.0 g/dl 1.6 0.2 0.5 6.3 0.3 8.8 PRE-CHEMOTHERAPY
% 53.2 3.0 8.7 21.7 13.5
g/dl 3.4 0.2 0.6 1.4 0.9 6.3
POST-CHEMOTHERAPY
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 182
in the gamma. The beta spike was typed as IgG-lambda. The smaller spikes are more readily visible 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.
% 26.8 8.7 10.2 52.0 2.3 = 1.16 g
mg/dl 8 3 3 16 1 30
URINE
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 postchemotherapy pattern shown was obtained after a year of outpatient follow-up. 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.
183
Hepatitis in Myeloma
% 38.0 4.0 6.1 28.3 23.7
g/dl 2.7 0.3 0.4 2.0 1.7 7.1
Patients with multiple myeloma are highly susceptible to viral hepatitis of all types. Despite the partial immune suppression, myeloma patients 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 the beta and gamma regions caused by production of IgA and some polyclonal rise in IgG. Although susceptibility 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.
184
Gamma Heavy Chain Disease
% 63.8 2.9 9.6 12.5 11.2
g/dl 4.0 0.2 0.6 0.8 0.7
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 185
Serum Immunofixation
Urine Electrophoresis 186
Urine Immunofixation
Purified Gamma Chain
187
OTHER M-COMPONENT PATTERNS Biclonal M-components IgG and IgA
Bence-Jones Protein and Kappa Ladder Urine
188
Bone Marrow Transplant with Oligoclonal Gammopathy
189
Monoclonal Cold Agglutinin Disease
Incomplete IgA M-component
Biclonal M-components with Deposition Disease 190
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
IgG-Lambda and Free Lambda M-components
IgM Anti-IgA M-component
195
Kappa in Alpha-2 Region Urine
Papular Mucinosis IgM M-component 196
197
Sebia Electrophoresis
Immunofixation Urine in Track 14
198
REFERENCES
U U
1. Alexanian R., Barlogie B., and Dixon D.: Prognosis of asymptomatic multiple myeloma. Arch. Intern. Med. 1988; 148:19631965. 2. Bakerman S.: ABC's of Interpretive Laboratory Data. 2nd edition, Interpretive Laboratory Data Inc., Greenville NC, 1984.
U U
3.
Bakerman S.: Clinical Chemistry Notes. Greenville NC, 1986.

U U
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.: Introduction to High Resolution Protein Electrophoresis and Associated Techniques. Helena Laboratories, Austin TX, 1986.
U U
7. Fauchier P., and Catalan F.: Interpretive Guide to Clinical Electrophoresis. Important Normal and Abnormal Patterns. Helena Laboratories, Beaumont TX, 1988.
U U
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.: High-Resolution Electrophoresis and Immunofixation. Butterworths, Boston, 1987.
U U
12. Killingsworth L.M.: High Resolution Protein Electrophoresis: A Clinical Overview with Case Studies. Helena Laboratories, Austin Tx, 1985.
U U
199
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.: Monoclonal Gammapathies: Clinical Significance and Basic Mechanisms. Eurage, Rijswijk, 1985.
U U
15. Radl J., and Van Camp B., eds.: Monoclonal Gammapathies II: Clinical Significance and Basic Mechanisms. Eurage, Rijswijk, 1989.
U U
16. Raine C.S., ed.: Advances in Neuroimmunology. Annals N.Y. Acad. Sci., 1988; 540.
U U
17. Ritzmann S.E., ed.: Protein Abnormalities Volume 1 Physiology of Immunoglobulins: Diagnostic and Clinical Aspects. Alan R. Liss, Inc., New York, 1982.
U U
18. Ritzmann S.E., ed.: Protein Abnormalities Volume 2 Pathology of Immunoglobulins: Diagnostic and Clinical Aspects. Alan R. Liss, Inc., New York, 1982.
U U
19. Ritzmann S.E., and Killingsworth L.M., eds.: Protein Abnormalities Volume 3 Proteins in Body Fluids, Amino Acids and Tumor Markers: Diagnostic and Clinical Aspects. Alan R. Liss, Inc., New York, 1983.
U U
200
Additional Cases
Alpha-1 Anti-Trypsin PiMS Heterozygote
HTU UTH HTU
Alpha-1 Anti-Trypsin Deficiency PiZZ with Cirrhosis

UTH
AL-Amyloidosis Serum
HTU UTH HTU
AL-Amyloidosis Urine
UTH HTU
AL-Amyloidosis IgG Serum

UTH
HTU
AL-Amyloidosis IgG Urine

UTH HTU
Biclonal M-components IgG and IgA

UTH HTU
Bisalbuminemia
UTH HTU
Bisalbuminemia enlarged
UTH HTU
Bence-Jones Protein and Kappa Ladder - Urine

UTH HTU
Bone Marrow Transplant with Oligoclonal Gammopathy

UTH HTU
Monoclonal Cold agglutinin Disease

UTH HTU
Common Variable Hypogammaglobulinemia

UTH HTU
Cryofibrinogenemia
UTH HTU
CSF Cervical Myelitis

UTH HTU
CSF Oligoclonal Bands Immunofixation

UTH
CSF Oligoclonal Bands Scan

HTU UTH
CSF CNS Myeloma

HTU UTH
CSF CNS Sarcoid

HTU UTH HTU
CSF CNS Systemic Lupus Erythematosus

UTH
CSF CNS Toxoplasmosis Encephalitis

HTU UTH
201
CSF Transverse Myelitis

HTU UTH
Diabetic Nephrosis
HTU UTH HTU
Incomplete IgA M-component

UTH HTU
Biclonal M-components with Deposition Disease

UTH
Gamma Heavy Chain Disease Serum Electrophoresis

HTU UTH HTU
Serum Scan
UTH HTU
Serum Immunofixation
UTH HTU
Urine Electrophoresis
UTH HTU
Urine Immunofixation
UTH
HTU
Purified Gamma Chains

UTH
Haptoglobin-Hemoglobin Complex
HTU UTH HTU
Human Immunodeficiency Virus Gammopathy

UTH
IgA M-component Broad Spike

HTU UTH
IgA M-component small serum

HTU UTH HTU
IgA M-component CSf from previous

UTH HTU
IgA M-component very small

UTH HTU
IgA M-component in Alpha-2 Serum

UTH HTU
IgA M-component in Alpha-2 Urine

UTH HTU
IgA-Lambda M-component with Hemolysis

UT H
IgG-Lambda Myeloma
HTU UT H HTU
IgG-lambda and Free Lambda M-components

UTH
202
IgM Anti-IgA M-component

HTU UTH HTU
Immunodeficiency Case 1
UTH
Immunodeficiency Case 2
HTU UTH HTU
Immunodeficiency with Hyper IgM

UTH
Immunodeficiency Secondary
HTU UTH HTU
Iron Deficiency with Low Transferrin

UTH
Kappa in Alpha-2 region Urine

HTU UTH HTU
Kappa Chains in Serum

UTH HTU
Myeloma with Nephrosis Serum

UTH
HTU
Myeloma with Nephrosis Urine

UTH HTU
Myeloma Untreated Natural Course

UTH
HTU
Nephrosis
UTH HTU
Oligoclonal Banding Carcinoma

UTH HTU
Oligoclonal Banding Serum

UTH HTU
Oligoclonal Banding Urine

UTH HTU
Papular Mucinosis IgM M-component

UTH
HTU
Pleural Effusion
UTH
HTU
Pleural Fluid Transudate

UTH
HTU
Polyclonal Increase in Gamma

UTH
HTU
Secondary Malignancy
UTH HTU
Sickle Cell Crisis

UTH HTU
Serum Immunoelectrophoresis M-components

UTH
203
Sprue with Malnutrition

HTU UTH
Staphylococcal Sepsis
HTU UTH HTU
Synovial Fluid Exudate

UTH
Synovial Fluid Gout

HTU UTH HTU
Type 2 Mixed Cryoglobulinemia

UTH HTU
Urine Immunoelectrophoresis M-components

UTH
Urine Beta Spike

HTU UTH HTU
Urine with Hemoglobin

UTH HTU
Urine Inflammatory Proteinuria

UTH
Urine Sepsis
HTU UTH HTU
Urine Spike and Ladder

UTH HTU
Urine Tubular and Inflammatory Proteinuria

UTH HTU
UTH
204

SPEP UPEP Interp

Transféré par

Informations du document

Description originale:

Copyright

Formats disponibles

Partager ce document

Partager ou intégrer le document

Options de partage

Avez-vous trouvé ce document utile ?

Ce contenu est-il inapproprié ?

Droits d'auteur :

Formats disponibles

SPEP UPEP Interp

Transféré par

Droits d'auteur :

Formats disponibles

AN ATLAS OF PROTEIN ELECTROPHORESIS

Serum, Urine, Cerebrospinal Fluid Donald R. Hoffman, Ph.D.

Copyright 2002, 2006, Donald R. Hoffman

High Resolution Serum Electrophoresis

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 46.1 6.0 10.1 13.2 24.6

g/dl 3.00 0.39 0.66 0.86 1.60 6.50

Normal Serum Pattern

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 62.8 3.6 9.4 14.7 9.5

g/dl 4.46 0.26 0.67 1.04 0.67 7.1

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 63.9 3.5 10.4 12.8 9.4

g/dl 3.96 0.22 0.64 0.79 0.58 6.2

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 60.8 2.3 9.3 16.6 11.0

g/dl 4.6 0.2 0.7 1.2 0.8 7.5

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 34.2 6.9 9.0 15.4 34.5

g/dl 2.26 0.46 0.59 1.02 2.28 6.6

Polyclonal Elevation of Gamma

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 49.6 3.1 8.7 13.1 25.4

g/dl 3.87 0.24 0.68 1.02 1.98 7.8

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 23.0 4.5 10.4 11.6 50.5

g/dl 1.68 0.33 0.76 0.85 3.69 7.3

Infection with Immune Complexes

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 26.4 7.0 16.7 9.1 40.7

g/dl 1.95 0.52 1.24 0.67 3.01 7.4

Infection with Oligoclonal Response

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 34.5 6.7 13.1 15.5 30.2

g/dl 2.38 0.46 0.90 1.07 2.08 6.9

IgG = 2450 mg/dl IgA = 453 mg/dl IgM = 363 mg/dl

Monoclonal Gammopathy of Undetermined Significance

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 60.9 7.3 11.1 10.1 10.6

g/dl 3.41 0.41 0.62 0.57 0.59 5.6

IgG = 571 mg/dl IgA = 110 mg/dl IgM = 216 mg/dl

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 72.6 7.2 3.5 3.5 13.2

mg/dl 122 12 6 6 22 168

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 61.2 4.1 19.0 10.2 3.6

g/dl 3.68 0.23 1.14 0.61 0.20 5.9

IgG = 255mg/dl IgA = 10mg/dl IgM = 22mg/dl

% 66.0 6.0 18.0 7.9 2.0

g/dl 3.96 0.36 1.08 0.47 0.12 6.0

mg/dl mg/dl mg/dl

% 52.4 6.3 14.3 12.7 14.3

g/dl 3.3 0.4 0.9 0.8 0.9 6.3

mg/dl mg/dl mg/dl

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 62.2 0 12.3 9.0 16.4

g/dl 4.7 0 0.9 0.7 1.2 7.6

Alpha-1-Antitrypsin Deficiency with Cirrhosis

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein

% 31.1 0.6 6.6 11.4 50.2

g/dl 1.97 0.04 0.42 0.72 3.16 6.3

Albumin Alpha-1 Alpha-2 Beta Gamma Total Protein