The monoclonal gammopathies constitute a group of

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1 Sequence of Testing for Monoclonal Gammopathies The first test for recognition of monoclonal gammopathies should be serum protein electrophoresis with highresolution agarose gel. Serum protein electrophoresis should be performed whenever multiple myeloma, Waldenström s macroglobulinemia, primary amyloidosis, or a related disorder is suspected. Immunofixation is critical for the differentiation of a monoclonal from a polyclonal increase in immunoglobulins. Quantitation of immunoglobulins should be performed with a rate nephelometer. The viscosity of serum should be measured if the patient has signs or symptoms of hyperviscosity syndrome. A 24-hour urine specimen should be obtained for determination of the total amount of protein excreted each day. Immunofixation of the urine should be performed on every patient who has an M-protein level greater than 1.5 g/dl (15 g/l) in the serum or in whom multiple myeloma, Waldenström s macroglobulinemia, primary amyloidosis, or a related disorder is suspected. (Arch Pathol Lab Med.1999;123: ) The monoclonal gammopathies constitute a group of disorders characterized by the proliferation of a single clone of plasma cells that produce a homogenous monoclonal protein (M-protein). It is extremely important to differentiate between a monoclonal and a polyclonal increase in immunoglobulins because the former is associated with a clonal process that is malignant or potentially malignant, while a polyclonal increase is due to a reactive or inflammatory process. Analysis of serum or urine requires a sensitive, rapid, and dependable screening method that detects the presence of an M-protein, as well as a specific assay to identify the M-protein according to its heavy-chain class and lightchain type. High-resolution agarose gel electrophoresis is a recommended method for the detection of an M-protein. After recognition of a localized band or spike on electrophoresis, immunofixation or immunosubtraction with capillary electrophoresis must be done to confirm the presence of an M-protein. 1 The first test that should be performed is serum protein Accepted for publication August 27, From the Division of Hematology and Internal Medicine, Mayo Medical School, Mayo Clinic and Mayo Foundation, Rochester, Minn. Presented at the College of American Pathologists Conference XXXII, Guidelines for Laboratory Evaluation and Use of Antinuclear Antibodies and Laboratory Diagnosis and Monitoring of Monoclonal Gammopathies, Chicago, Ill, May 29 31, Reprints: Robert A. Kyle, MD, Mayo Clinic, 200 First St SW, Rochester, MN Serum and Urine Assays Robert A. Kyle, MD electrophoresis with high-resolution agarose gel (Figure). This test should be done when multiple myeloma, Waldenström s macroglobulinemia, primary amyloidosis, or a related disorder is suspected. In addition, serum protein electrophoresis is indicated in any patient with unexplained weakness or fatigue, anemia, elevation of erthyrocyte sedimentation rate, unexplained back pain, osteoporosis, osteolytic lesions or fractures, hypercalcemia, Bence Jones proteinuria, renal insufficiency, or recurrent infections. Serum protein electrophoresis should also be performed in adults with any clinical evidence that suggests primary amyloidosis, such as unexplained sensorimotor peripheral neuropathy, carpal tunnel syndrome, refractory congestive heart failure or cardiomyopathy, nephrotic syndrome or renal insufficiency, orthostatic hypotension, or malabsorption. Other features suggestive of primary amyloidosis include unexplained weakness, fatigue, weight loss, light-headedness or syncope, change in voice, paresthesias from carpal tunnel syndrome or sensorimotor peripheral neuropathy, increased bruising, bleeding, or steatorrhea. Even when the serum protein electrophoretic pattern is negative, immunofixation should be done when multiple myeloma, Waldenström s macroglobulinemia, primary amyloidosis, or related disorders are suspected. INTERPRETATION OF SERUM PROTEIN ELECTROPHORESIS An M-protein is usually seen as a discrete band on agarose gel electrophoresis or as a tall, narrow spike or peak in the,, or 2 region of the densitometer tracing. A polyclonal increase in immunoglobulins (having 1 or more heavy-chain classes and both and light-chain types) produces a broad band or a broad-based peak and is limited to the region. Two M-proteins (biclonal gammopathy) occur in 3% to 4% of sera containing an M-protein. 2 Immunoglobulin (Ig)G, IgA, IgM, IgD, and IgE make up the component, but they are also found in the - and regions and may extend to the 2 -globulin area. Hypogammaglobulinemia ( globulin, 0.6 g/dl) is characterized by a decrease in the component and should be documented by quantitation of IgG, IgA, and IgM levels. Hypogammaglobulinemia may be congenital (sex-linked or combined immunodeficiency) 3 or acquired (eg, multiple myeloma, primary amyloidosis, chronic lymphocytic leukemia, lymphoma, nephrotic syndrome, or treatment with corticosteroids). Hypogammaglobulinemia on serum protein electrophoresis occurs in about 10% of patients with multiple myeloma without a serum spike. Most of these patients have a large amount of Bence Jones protein 114 Arch Pathol Lab Med Vol 123, February 1999 Testing for Monoclonal Gammopathies Kyle

2 Protein analysis cascade. * If the M-spike is greater than 1.5 g/dl (15 g/l), a 24-hour urine specimen should be collected for electrophoresis and immunofixation. If the M-spike is greater than 1.5 g/dl (15 g/l), nephelometric measurement of immunoglobulin (Ig)G, IgA, and IgM is indicated. (monoclonal free or light chain) in the urine. Hypogammaglobulinemia on the electrophoretic pattern is seen in approximately 20% of patients with primary amyloidosis. Many with primary amyloidosis have a nephrotic pattern (mainly albumin with nonselective globulin loss) in the urine. A decrease in albumin and an increase in 1 -and 2 - globulins may be present in infections or metastatic malignancy. Reduction of the 1 -globulin component is usually due to a deficiency of 1 -antitrypsin. Two albumin bands (bisalbuminemia) may be found. This familial abnormality produces no symptoms. 4 Other conditions, such as free hemoglobin-haptoglobin complexes from hemolysis may produce a peak suggestive of an M-protein in the 2 -globulin area. Large amounts of transferrin in patients with iron deficiency anemia may produce a prominent band in the region. Fibrinogen (in plasma) is seen as a discrete band between the and regions, and is indistinguishable from an M-protein. In this case, the specimen should be examined for the presence of a clot. If a clot is not seen, the addition of thrombin to the sample will produce a clot, indicating the presence of fibrinogen. If no evidence of fibrinogen is found, immunofixation should be done, because the - band may represent a small M-protein. The nephrotic syndrome produces a decrease in serum albumin and -globulin components and an increase in 2 -globulin and -globulin. This increase may be mistaken for an M-protein. The urinary pattern in nephrotic syndrome consists mainly of albumin and small amounts of globulin. In this situation, an M-protein in the urine does not appear as a tall spike but can be detected with immunofixation if present. The presence of even a small M- protein in the urine of a patient with nephrotic syndrome almost always indicates the presence of primary amyloidosis 5 or light-chain deposition disease. 6 A small M-protein may be present even when the total protein; -, -, and -globulin components; and quantitative immunoglobulins are all within normal limits. Such an M-protein may be concealed in the normal or components. Furthermore, levels of monoclonal light chain (Bence Jones proteinemia), which is often present with Arch Pathol Lab Med Vol 123, February 1999 Testing for Monoclonal Gammopathies Kyle 115

3 concomitant renal failure, are usually too low to be visible as a spike in the agarose gel. In some cases with IgD myeloma, the M-protein is small and easily overlooked. The electrophoretic pattern in gamma, alpha, or mu heavychain diseases is usually nondiagnostic. Chronic liver disease, connective tissue disorders, or chronic infection is characterized by a large, broad-based polyclonal pattern. In chronic hepatitis, the gamma component may reach 6 or 7 g/dl. Some lymphoproliferative disorders, such as angioimmunoblastic lymphadenopathy, may produce a large polyclonal increase in immunoglobulins. In some patients, a polyclonal gammopathy may be present without evidence of an underlying process. IMMUNOFIXATION OF SERUM Immunofixation should be performed when a sharp peak or band is found in the agarose gel or when multiple myeloma, macroglobulinemia, primary amyloidosis, solitary or extramedullary plasmacytoma, or a related disorder is suspected. It should always be done in the presence of a sensorimotor peripheral neuropathy. Immunofixation is critical for the differentiation of a monoclonal from a polyclonal increase in immunoglobulins. Immunofixation may be performed using commercial kits, or laboratory personnel can pour their own plates and cut the appropriate troughs. An M-protein is characterized by the presence of a sharp, well-defined band with a single heavy-chain class and a similar band with either or light-chain antisera. A polyclonal gammopathy is characterized by a broad diffuse band with 1 or more heavy-chain antisera and both and antisera. The presence of an IgM- or IgM- M- protein is consistent with monoclonal gammopathy of undetermined significance of the IgM class, Waldenström s macroglobulinemia, other lymphoproliferative disorders, or primary amyloidosis. A biclonal gammopathy has 2 monoclonal heavy chains with their respective monoclonal light chains. A biclonal gammopathy may also consist of 2 heavy chains of the same class and monoclonal light chains of the same type. In this setting, one must be careful to exclude monomers and aggregates as well as monomers and polymers of an M-protein. If 2 IgA spikes having the same class of light chain are present, the 2 components should be added because they most likely represent monomers and polymers of a monoclonal proliferation of plasma cells. Rarely, 3 M-proteins constituting a triclonal gammopathy may be seen. 7 Immunofixation is useful for detecting a small M-protein in the presence of normal background immunoglobulins or in a polyclonal increase in immunoglobulins. It is needed in patients with multiple myeloma or macroglobulinemia in whom treatment has resulted in disappearance of the band on electrophoresis. It is most helpful for detecting a small M-protein in patients with primary amyloidosis. Immunofixation is particularly useful for the recognition and distinction of biclonal or triclonal gammopathies. If the serum protein electrophoretic pattern is equivocal or if multiple myeloma or a related disorder is suspected, the laboratory should screen the serum with immunofixation using or antisera. If a localized band is found, immunofixation should be performed with IgG, IgA, and IgM antisera. If this testing is negative, immunofixation using IgD and IgE antisera must be done. In all patients with a monoclonal light chain in the serum, the possibility of IgD and IgE monoclonal proteins must be excluded. One can screen with Ouchterlony immunodiffusion for an increased amount of IgD or IgE. Immunofixation with IgD or IgE antisera should be done if either immunoglobulin is elevated to determine if the increase is monoclonal. IMMUNOELECTROPHORESIS Immunoelectrophoresis may also be used for the detection of an M-protein. It is technically easier and less expensive than immunofixation. However, it is not as sensitive as immunofixation. One must use monospecific antisera to each of the heavy and light chains. Most laboratories do not use immunoelectrophoresis. NEPHELOMETRY A nephelometer using monospecific and antiserum may be used to detect the light-chain type of large M- proteins. However, small M-proteins will have a normal - ratio and consequently will not be recognized. Furthermore, this approach will not recognize biclonal gammopathies. Thus, the benefits of this procedure are limited, and it is not recommended. CAPILLARY ELECTROPHORESIS Capillary zone electrophoresis measures protein on-line by absorbance, so protein stains are not necessary and no point of application is seen. The electrophoretograms are similar to those seen with high-resolution agarose gel electrophoresis. Following electrophoresis, immunotyping can be performed by an immunosubtraction procedure in which the serum sample is incubated with Sepharose beads coupled with anti-,,,, and antisera. After incubation with each of the heavy- and light-chain antisera solid phase reagents, the supernatants are reanalyzed to determine which reagents removed the electrophoretic abnormality. Capillary electrophoresis appears to be slightly more sensitive than high-resolution agarose gel electrophoresis. The immunosubtraction procedure is technically less demanding, is automated, and is thus a useful procedure for immunotyping M-proteins. 8,9 QUANTITATION OF IMMUNOGLOBULINS The use of a rate nephelometer is a reliable method for the quantitation of immunoglobulins. The degree of turbidity produced by antigen-antibody interaction is measured by nephelometry in the near-uv regions. Since the method is not affected by molecular size, it will measure 7S IgM, polymers of IgA, or aggregates of IgG accurately. Quantitation may be performed with radial immunodiffusion, but this method may give a spuriously high IgM value because of 7S IgM and a spuriously low IgA level owing to polymers of the IgA monoclonal protein. The standards consist of 19S IgM and 7S IgA. Consequently, radial immunodiffusion is not recommended. The levels of IgM may be 1000 to 2000 mg/dl (10 20 g/l) more than the size of the M-protein in the densitometer tracing. IgG and IgA may also be spuriously increased. 10 EVALUATION AND FOLLOW-UP OF MONOCLONAL GAMMOPATHIES If the patient has no other features of plasma cell dyscrasia and a serum M-spike less than 1.5 g/dl (15 g/l), serum protein electrophoresis should be repeated at an- 116 Arch Pathol Lab Med Vol 123, February 1999 Testing for Monoclonal Gammopathies Kyle

4 nual intervals. A bone marrow examination, skeletal roentgenograms, and a 24-hour urine specimen for immunofixation are not necessary in most instances. If the asymptomatic patient has an M-protein level of 1.5 to 2.5 g/dl (15 25 g/l), additional studies should include nephelometry for quantitation of immunoglobulins and collection of a 24-hour urine specimen for electrophoresis and immunofixation. The serum protein electrophoretic pattern should be repeated in 3 to 6 months, and if stable it should be repeated annually or sooner if any symptoms or complications are present. If the serum M-spike (IgG or IgA) is greater than 2.5 g/dl (25 g/l), there is a greater likelihood of multiple myeloma, so a metastatic bone survey, including single views of the humeri and femurs, should be done. An aspirate and biopsy of the bone marrow should also be seriously considered. Aspirate and biopsy of the bone marrow and a computerized tomographic scan of the abdomen may be helpful in recognition of Waldenström s macroglobulinemia or other lymphoproliferative disorders. Additional studies, such as 2 -microglobulin and C-reactive protein levels, should be performed if clinically indicated. If these studies are satisfactory, serum protein electrophoresis should be repeated in 2 to 3 months; if stable, repeated in 3 to 4 months; and if still stable, it should be repeated annually. A 24-hour urine specimen for electrophoresis and immunofixation should be obtained if the serum M-spike is greater than 1.5 g/dl (15 g/l). A 24-hour urine specimen should also be obtained if the serum M-protein value increases or other evidence of evolving multiple myeloma or macroglobulinemia occurs. These comments serve as a suggested approach, and the physician should alter the studies depending on the total picture. It is of great importance for the physician to be in contact with the laboratory concerning interpretation of the laboratory tests. It is essential that the laboratory know that the physician suspects primary amyloidosis or an extramedullary plasmacytoma or solitary plasmacytoma of bone so that a greater effort can be made for the detection of an M-protein. SERUM VISCOSITY The viscosity of serum should be measured if the patient has signs or symptoms of hyperviscosity syndrome, which include dilation of retinal veins, flame-shaped retinal hemorrhages, blurring or loss of vision, oronasal bleeding, headaches, vertigo, nystagmus, decreased hearing, ataxia, paresthesias, diplopia, congestive heart failure, somnolence, stupor, or coma. 11 Waldenström s macroglobulinemia is the most common cause of hyperviscosity, but it can also occur in patients with large amounts of monoclonal IgA or IgG. The serum viscosity should be determined when the monoclonal IgM protein level is greater than 4 g/dl (40 g/l) or the IgA or IgG protein value is greater than 6 g/dl (60 g/l). An Ostwald-100 viscometer is a satisfactory instrument, but a Wells-Brookfield viscometer (Brookfield Engineering Laboratory, Stoughton, Mass) is preferred because it is more accurate, requires less serum (1 1.5 ml), and can perform at different sheer rates and at variable temperatures. In addition, determinations can be made much more rapidly, especially if the serum viscosity is elevated. The relationship between serum viscosity and symptoms of hyperviscosity is not precise. Symptoms of hyperviscosity rarely occur if the viscosity is less than 4 cp (centipoises). However, some patients have no evidence of hyperviscosity at 10 cp. The clinical features are much more important than the viscosity level. The decision to perform plasmapheresis should be made on the basis of signs and symptoms of hyperviscosity. It is useful to measure the serum viscosity and to perform serum protein electrophoresis before and after plasmapheresis. Patients with hyperviscosity who have been treated with plasmapheresis should be monitored by periodic evaluation of serum protein electrophoresis. If the serum M-protein value increases or if symptoms or signs of hyperviscosity occur, the serum viscosity should be measured. ANALYSIS OF URINE Patients with a serum M-protein should have electrophoresis and immunofixation of an aliquot from a 24-hour urine collection. In addition, immunofixation of the urine should be done initially in all patients with multiple myeloma, Waldenström s macroglobulinemia, primary amyloidosis, heavy-chain diseases, or suspicion of these entities. It is absolutely essential that all patients with a monoclonal light chain in the serum (Bence Jones proteinemia) have electrophoresis of a 24-hour urine specimen, as well as immunofixation. Dipsticks are used in many laboratories to screen for protein. The dipstick is impregnated with a buffered indicator dye that binds to protein in urine and produces a color change proportional to the amount of protein bound to it. However, the dipsticks are often insensitive to Bence Jones protein. 12,13 Screening tests for Bence Jones protein, such as the heat test of Putnam, are unreliable. The heat test may be positive even though the patient s urine reveals no globulin band on electrophoresis and no evidence of an M-protein with immunofixation. Such urine usually produces a broad-based band and normal-appearing and bands or arcs representing an excess of polyclonal light chains. This often occurs in renal insufficiency. 14 In addition, the heat test is often negative when small amounts of monoclonal light chain are detected with immunofixation. Thus, the heat test is not recommended for the detection of monoclonal light chains. Sulfosalicylic acid detects albumin and globulin as well as Bence Jones protein and polypeptides. False-positive reactions may occur from penicillin or its derivatives, tolbutamide or sulfisoxazole metabolites, and certain organic radiographic contrast media. 15 A 24-hour urine specimen must be obtained for determination of the total amount of protein excreted each day. Measurement of the amount of M-protein is best obtained by multiplying the percent of the M-spike by the total protein in the 24-hour specimen. The amount of monoclonal light chain in the urine provides an index of the patient s tumor mass for future monitoring of increasing or decreasing values. The amount of M-protein in the urine is useful in differentiating between monoclonal gammopathy of undetermined significance and multiple myeloma or other malignant plasma cell proliferative disorders. The amount of urine protein can be expressed as mg/dl or mg/l, but it is much more useful to report the M-protein in grams per 24 hours, because the patient may be producing 500 to 4000 ml of urine daily. The 24-hour urine specimen requires no preservative and may be kept at room temperature during collection. An aliquot from the 24-hour urine specimen should be Arch Pathol Lab Med Vol 123, February 1999 Testing for Monoclonal Gammopathies Kyle 117

5 concentrated to approximately 3 g/dl (30 g/l). If less than 100 mg/24 hours is found, the specimen should be concentrated to 100-fold or greater prior to immunofixation. Immunofixation of urine should also be done in all older persons with a nephrotic syndrome of unknown cause, because the presence of a monoclonal light chain is strong evidence of primary amyloidosis or light-chain deposition disease. A urinary M-protein is seen as a dense localized band on agarose gel or as a tall, narrow, homogenous peak on the densitometer tracing. Occasionally, 2 discrete globulin bands are seen. They may represent a monoclonal light chain plus a monoclonal immunoglobulin fragment from the serum M-protein, or they may represent monomers and dimers of the monoclonal light chain. Immunofixation with the appropriate heavy- and light-chain antisera is necessary. A polyclonal increase in light chains is seen as a broad band on the agarose gel or as a broad-based peak in the densitometer tracing and the presence of both and arcs on immunoelectrophoresis or broad bands on immunofixation. IMMUNOFIXATION Immunofixation of the urine should be performed in every patient who has an M-protein level greater than 1.5 g/dl (15 g/l) in the serum or in whom multiple myeloma, Waldenström s macroglobulinemia, primary amyloidosis, or a related disorder is suspected. It should be done even if the routine urinalysis is negative for protein, the 24-hour urine protein value is within normal limits, or if electrophoresis of the concentrated urine specimen shows no globulin spike. The presence of a monoclonal light chain is characterized by a discrete band with either or antisera. Two discrete bands with either or antisera may be found and are due to the presence of monomers and dimers of the monoclonal light chain. A discrete band with heavychain antisera corresponding to the type of heavy chain in the serum coinciding with 1 of the 2 light-chain bands indicates a fragment of the intact immunoglobulin. A monoclonal light chain in the presence of a nephrotic syndrome indicates the presence of primary systemic amyloidosis 5 or light-chain deposition disease 6 in almost all instances. Theoretically, antisera that recognize only free or free should be used rather than antisera that recognize both free light chains and an intact immunoglobulin. However, such antisera are often either nonspecific or insufficiently potent. In addition, a patient may have an immunoglobulin fragment that free or free antisera do not recognize. Consequently, it is advisable to use and antisera that are monospecific and potent for recognizing both free and combined light chains. If electrophoresis of the urine reveals a localized globulin spike and immunofixation does not demonstrate a monoclonal light chain, one must suspect the possibility of gamma heavy-chain disease. Immunofixation should then be performed with antisera to IgG ( heavy chains). Occasionally, regularly spaced, faint but discrete multiple bands are seen in the immunofixation pattern. These restricted bands represent related polyclonal free light chains and are not to be confused with a monoclonal light chain. This has been described as ladder light chain or pseudo-oligoclonal pattern. 16 Free fragments of chains may be seen in the immunofixation pattern. These should not be confused with gamma heavy-chain disease. 17 EXAMINATION OF OTHER BODY FLUIDS The cerebrospinal fluid should be examined by immunofixation if the protein level is elevated. It is particularly important to do so if monoclonal plasma cells or lymphoid cells are present in the cerebrospinal fluid. Since an M- protein crosses the blood-brain barrier, one must perform immunofixation of the serum as well. Rarely, one finds a monoclonal immunoglobulin in the cerebrospinal fluid and none in the serum. In this setting, the possibility of central nervous system lymphoma or, rarely, plasmacytoma of the central nervous system should be considered. Careful cytologic examination of the cerebrospinal fluid is critical in this situation. The presence of an M-protein in the pleural fluid is almost always the result of an M-protein in the serum. It is possible that a plasma cell tumor localized to the pleura could produce an M-protein. Cytologic examination of the pleural fluid is very helpful in this situation. References 1. Kyle RA, Katzmann JA. Immunochemical characterization of immunoglobulins. In: Rose NR, Conway de Macario E, Folds JD, Lane HC, Nakamura RM, eds. Manual of Clinical Laboratory Immunology. 5th ed. Washington, DC: American Society for Microbiology; Kyle RA, Robinson RA, Katzmann JA. The clinical aspects of biclonal gammopathies: review of 57 cases. Am J Med. 1981;71: Rosen FS, Cooper MD, Wedgwood RJ. The primary immunodeficiencies. N Engl J Med. 1995;333: Pola V, Tichy M. Bisalbuminamie: Kritische Übersicht und Bericht über die Beobachtung einer erworbened Form bei einem Myelomkranken. Folia Haematol Int Mag Clin Morphol Blutforsch. 1985;112: Kyle RA, Gertz MA. Primary systemic amyloidosis: clinical and laboratory features in 474 cases. Semin Hematol. 1995;32: Heilman RL, Velosa JA, Holley KE, Offord KP, Kyle RA. Long-term followup and response to chemotherapy in patients with light-chain deposition disease. Am J Kidney Dis. 1992;20: Grosbois B, Jégo P, de Rosa H, et al. Gammapathie triclonale et syndrome immunoprolifératif manlin. Rev Méd Interne. 1997;18: Katzmann JA, Clark R, Wiegert E, et al. Identification of monoclonal proteins in serum: a quantitative comparison of acetate, agarose gel, and capillary electrophoresis. Electrophoresis. 1997;18: Katzmann JA, Clark R, Sanders E, Landers JP, Kyle RA. Prospective study of serum protein capillary zone electrophoresis and immunotyping of monoclonal proteins by immunosubstraction. Am J Clin Pathol. 1998;110: Riches PG, Sheldon J, Smith AM, Hobbs JR. Overestimation of monoclonal immunoglobulin by immunochemical methods. Ann Clin Biochem. 1991;28: Gertz MA, Kyle RA. Hyperviscosity syndrome. J Intensive Care Med. 1995; 10: Hinberg IH, Katz L, Waddell L. Sensitivity of in vitro diagnostic dipstick tests to urinary protein. Clin Biochem. 1978;11: Scarpioni L, Ballocchi S, Bergonzi G, et al. Glomerular and tubular proteinuria in myeloma: relationship with Bence Jones proteinuria. Contrib Nephrol. 1981;26: Perry MC, Kyle RA. The clinical significance of Bence Jones proteinuria. Mayo Clin Proc. 1975;50: Line DE, Adler S, Fraley DS, Burnes FJ. Massive pseudoproteinuria by nafcillin. JAMA. 1976;235: Harrison HH. The ladder light chain or pseudo-oligoclonal pattern in urinary immunofixation electrophoresis (IFE) studies: a distinctive IFE pattern and an explanatory hypothesis relating it to free polyclonal light chains. Clin Chem. 1991;37: Charles EZ, Valdes AJ. Free fragments of gamma chain in the urine: a possible source of confusion with gamma heavy-chain disease. Am J Clin Pathol. 1994;101: Arch Pathol Lab Med Vol 123, February 1999 Testing for Monoclonal Gammopathies Kyle