Understanding and interpreting serum protein electrophoresis

Serum protein electrophoresis is used to identify patients with multiple myeloma and other serum protein disorders. Electrophoresis separates proteins based on their physical properties, and the subsets of these proteins are used in interpreting the results. Plasma protein levels display reasonably predictable changes in response to acute inflammation, malignancy, trauma, necrosis, infarction, burns, and chemical injury. A homogeneous spike-like peak in a focal region of the gamma-globulin zone indicates a monoclonal gammopathy. Monoclonal gammopathies are associated with a clonal process that is malignant or potentially malignant, including multiple myeloma, Waldenstrom's macroglobulinemia, solitary plasmacytoma, smoldering multiple myeloma, monoclonal gammopathy of undetermined significance, plasma cell leukemia, heavy chain disease, and amyloidosis. The quantity of M protein, the results of bone marrow biopsy, and other characteristics can help differentiate multiple myeloma from the other causes of monoclonal gammopathy. In contrast, polyclonal gammopathies may be caused by any reactive or inflammatory process.     

Elimination of the need for urine studies in the screening algorithm for monoclonal gammopathies by using serum immunofixation and free light chain assays

OBJECTIVE: To determine the relative diagnostic contribution of urine assays as part of the screening algorithm for monoclonal gammopathies. PATIENTS AND METHODS: We identified 428 patients with a monoclonal gammopathy and monoclonal urinary protein at initial diagnosis of plasma cell dyscrasia who had also undergone serum immunofixation and serum free light chain quantitation within 30 days of diagnosis. The laboratory results for serum protein electrophoresis, serum immunofixation, serum free light chain, urine protein electrophoresis, and urine immunofixation were reviewed. RESULTS: The patients had diagnoses of multiple myeloma, primary amyloid, monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, solitary plasmacytomas, and other less frequently detected monoclonal gammopathies. All 428 had a monoclonal urine protein, 85.7% had an abnormal serum free light chain kappa/lambda ratio, 80.8% had an abnormal serum protein electrophoresis, and 93.5% had an abnormal serum immunofixation result. All 3 serum assays were normal in only 2 patients, 1 of whom had monoclonal gammopathy of undetermined significance (idiopathic Bence Jones proteinuria) and 1 whose urine sample contained an intact monoclonal immunoglobulin but whose serum and subsequent urine samples showed no evidence of a monoclonal gammopathy. CONCLUSION: Discontinuation of urine studies and reliance on a diagnostic algorithm using only serum studies (protein electrophoresis, immunofixation, and free light chain quantitation) missed 2 (0.5%) of the 428 monoclonal gammopathies with urinary monoclonal proteins, and these 2 cases required no medical intervention.     

Multiple myeloma: recognition and management

Multiple myeloma is the malignant proliferation of plasma cells involving more than 10 percent of the bone marrow. The multiple myeloma cell produces monoclonal immunoglobulins that may be identified on serum or urine protein electrophoresis. Bone pain related to multiple lytic lesions is the most common clinical presentation. However, up to 30 percent of patients are diagnosed incidentally while being evaluated for unrelated problems, and one third of patients are diagnosed after a pathologic fracture, commonly of the axial skeleton. Multiple myeloma must be differentiated from other causes of monoclonal gammopathy, including monoclonal gammopathy of undetermined significance, heavy chain disease, plasmacytoma and Waldenstrom macroglobulinemia. Chemotherapy with melphalan-prednisone is the standard treatment for multiple myeloma. Other treatment modalities include polychemotherapy and bone marrow transplantation. Only 50 to 60 percent of patients respond to therapy. The aggregate median survival for all stages of multiple myeloma is three years.     

Monoclonal gammopathies--from MGUS to myeloma

Monoclonal gammopathies are characterized by the overproduction of a monoclonal immunoglobulin (M-Protein), which may be detected in serum or urine by protein electrophoresis and immunofixation. The presence of an M-Protein results from the proliferation of a single abnormal clone of differentiated B lymphocytes or plasma cells, and is associated with a variety of clinical conditions, ranging from asymptomatic to malignant disease. Recent years have witnessed considerable advances in the treatment of plasma cell myeloma, the most common malignant disorder of the monoclonal gammopathies. As compared with conventional-dose treatments, high-dose chemotherapy with autologous stem-cell transplantation increases response rates and overall survival of patients with myeloma who are younger than 65 years of age. Progress in supportive therapies and the development of promising new drugs such as proteasome inhibitors and thalidomide analogues may provide further benefits for myeloma patients in the future.     

Serum free light chain analysis in the diagnosis and management of multiple myeloma and related conditions

The serum free light chain (FLC) assay is an important tool in the management of patients with monoclonal gammopathies. MEDLINE^®, the Cochrane Central Register of Controlled Trials and the Cochrane Database of Systematic Reviews from January 2000 through July 2013, were used as data sources. The available evidence is rather weak. For screening of multiple myeloma and related conditions, the association of the FLC assay with the traditional serum tests avoids urine study. Screening for immunoglobulin light-chain (AL) amyloidosis or other rare syndromes requires the urine examination. FLC measurement is used in the assessment of the risk of progression of precursor diseases to overt myeloma, and for risk stratification in solitary plasmacytoma, multiple myeloma and AL amyloidosis. In patients with oligosecretory myeloma and AL amyloidosis, the quantification of FLC is essential for monitoring and categorization of response to therapy. Further studies with improved design are warranted to strengthen the available evidence.     

Neuromuscular disorders associated with paraproteinemia

Neuromuscular disorders associated with monoclonal gammopathies are usually uncovered in approximately 10% of patients presenting with peripheral neuropathy complaints. This discovery should prompt further evaluation for underlying plasma cell dyscrasias. The most frequent monoclonal disorders associated with neuropathy are smoldering myeloma, multiple myeloma, Waldenstr?m macroglobulinemia, solitary plasmacytoma, systemic immunoglobulin light chain (AL) amyloidosis, POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes), and cryoglobulinemia. If these are excluded by careful evaluation the patient is classified as having monoclonal gammopathy of undetermined significance. Diagnostic criteria, risk stratification to determine prognosis, and current management for these disorders are reviewed in this article.     

Pathophysiology of the Monoclonal Gammopathies

Two of the lymphoplasmacytic proliferative disorders making up the group of diseases called the monoclonal gammopathies are plasmacytic (or multiple) myeloma and primary (or Waldenstrom's) macroglobulinemia. Myeloma is basically a disease of bone and is marked by plasmacytic proliferation. Characteristic monoclonal changes can be found in almost 100 percent of these cases by combined serum and urine immunoelectrophoresis. In primary macroglobulinemia the predominant cellular expression is lymphocytic.     

Serum free light chains: an alternative to the urine Bence Jones proteins screening test for monoclonal gammopathies

BACKGROUND: Retrospective analyses have established the role of quantitative serum free light chains (FLCs) in the diagnosis of monoclonal light chain disorders. The aims of this study were to assess (a) whether the addition of serum FLCs to serum protein electrophoresis (SPEP) identified additional patients with monoclonal gammopathies; (b) whether serum FLC measurements could replace urinalysis for Bence Jones protein (BJP); and (c) the cost/quality implications of routinely measuring serum FLCs. METHODS: Serum FLCs were added to consecutive requests for SPEP from August to November 2004 and measured by automated immunoassay. RESULTS: Seventy-one of 923 patients had abnormal serum FLC ratios. Seven patients with monoclonal gammopathies and 1 patient with malignant lymphoma (but no monoclonal band) were detected among 43 patients with negative SPEP but positive serum FLC ratios. Thirty-five patients with negative SPEP had false-positive serum FLC ratios. The false-positive rate for a ratio >1.65 was higher than previously described and associated with polyclonal increases in immunoglobulins and renal impairment. Serum FLC ratios were normal in 2 of 13 patients with low-level persistent urine BJP. However, no significant pathology would have been missed by replacing BJP with serum FLCs. Revenue and manpower savings offset 60% of the costs of serum FLCs. CONCLUSIONS: Additional diagnostic information is gained by adding serum FLCs to SPEP as first-line tests for investigating possible B-cell disorders. The quality of the diagnostic service is enhanced by more confident exclusion of light chain disorders and improved interpretive assessment of SPEP and immunofixation electrophoresis.     

Diagnostic performance of quantitative kappa and lambda free light chain assays in clinical practice

BACKGROUND: The quantitative assay for free light chains (FLCs) is a recently introduced commercial test reported to be sensitive and specific for detecting FLC diseases such as primary systemic amyloidosis (AL), light chain deposition disease (LCDD), nonsecretory multiple myeloma (NSMM), and light chain multiple myeloma. We evaluated its diagnostic performance in clinical practice. METHODS: All FLC clinical test results generated in 2003 were abstracted from the Laboratory Information System. Diagnoses were obtained from the Dysproteinemia database and the patient medical history. RESULTS: In 2003, we received samples for FLC assays from 1020 Mayo Clinic patients. The majority of these patients (88%) had bone marrow-derived monoclonal plasma cell disorders (PCDs). The 121 patients who did not have monoclonal gammopathy all had FLC kappa/lambda ratios within the range of values obtained for a reference population in our laboratory. Among the patients with monoclonal gammopathies were patients with multiple myeloma (330), AL (269), monoclonal gammopathy of undetermined significance (114), smoldering multiple myeloma (72), plasmacytoma (22), NSMM (20), macroglobulinemia (9), LCDD (7), and a variety of other PCDs. Among the 110 AL patients who had not been previously treated and who had a FLC assay performed within 120 days of diagnosis, the FLC kappa/lambda ratio was positive in 91% compared with 69% for serum immunofixation electrophoresis (IFE) and 83% for urine IFE. The combination of serum IFE and serum FLC assay detected an abnormal result in 99% (109 of 110) of patients with AL. CONCLUSION: The performance of the FLC assay in this analysis of clinical laboratory data is consistent with results from published retrospective validation studies.     

Classification, staging and prognostic indices for multiple myeloma

The monoclonal gammopathies are a group of heterogeneous disorders associated with monoclonal proliferation of plasma cells. The International Myeloma Working Group proposed the new criteria for diagnosis and classification based on routinely available examinations. According to the criteria, symptomatic myeloma requires evidence of an M -protein in serum and urine, bone marrow plasmacytosis and related end -organ damage. The International staging system (ISS) has been described to estimate prognosis using serum beta-2 microglobulin and serum albumin. The Durie/Salmon plus staging system has also been developed by use of PET/MRI imaging. The use of these staging systems will facilitate available prognostic factors may allow better definition of prognosis.     

Strategy to diagnose monoclonal gammopathies in serum: high-resolution electrophoresis, immunofixation, and kappa/lambda quantification

Identification of monoclonal gammopathies in serum has involved electrophoresis of serum proteins, immunoelectrophoresis (IEP), and quantification of IgG, IgA, and IgM. Recent innovations in technology--including high-resolution electrophoresis (HRE), immunofixation (IFX), and quantification of kappa- and lambda-containing immunoglobulins--allow for more rapid and precise assessment of serum for monoclonal proteins. We present a series of guidelines to determine when high-resolution electrophoresis and quantification of immunoglobulins (including kappa and lambda) are sufficient and when additional IFX is required to characterize the monoclonal gammopathy. Of the samples studied, 88% were correctly diagnosed by HRE with quantification of immunoglobulins and kappa/lambda; only 12% required that IFX be performed. The guidelines allow us to detect monoclonal gammopathies quicker and more efficiently by avoiding redundant IEP or IFX testing. For the vast majority of cases, these guidelines allow for a correct diagnosis within one day. After one year of follow-up since completion of the study, no undetected cases of monoclonal gammopathy have eventuated.     

Multiple myeloma: diagnosis and treatment

Multiple myeloma, the most common bone malignancy, is occurring with increasing frequency in older persons. Typical symptoms are bone pain, malaise, anemia, renal insufficiency, and hypercalcemia. Incidental discovery on comprehensive laboratory panels is common. The disease is diagnosed with serum or urine protein electrophoresis or immunofixation and bone marrow aspirate analysis. Skeletal radiographs are important in staging multiple myeloma and revealing lytic lesions, vertebral compression fractures, and osteoporosis. Magnetic resonance imaging and positron emission tomography or computed tomography are emerging as useful tools in the evaluation of patients with myeloma; magnetic resonance imaging is preferred for evaluating acute spinal compression. Nuclear bone scans and dual energy x-ray absorptiometry have no role in the diagnosis and staging of myeloma. The differential diagnosis of monoclonal gammopathies includes monoclonal gammopathy of uncertain significance, smoldering (asymptomatic) and symptomatic multiple myeloma, amyloidosis, B-cell non-Hodgkin lymphoma, Waldenstr?m macroglobulinemia, and rare plasma cell leukemia and heavy chain diseases. Patients with monoclonal gammopathy of uncertain significance or smoldering multiple myeloma should be followed closely, but not treated. Symptomatic multiple myeloma is treated with chemotherapy followed by autologous stem cell transplantation, if possible. Melphalan, prednisolone, dexamethasone, vincristine, doxorubicin, bortezomib, and thalidomide and its analogue lenalidomide have been used successfully. It is important that family physicians recognize and appropriately treat multiple myeloma complications. Bone pain is treated with opiates, bisphosphonates, radiotherapy, vertebroplasty, or kyphoplasty; nephrotoxic nonsteroidal anti-inflammatory drugs should be avoided. Hypercalcemia is treated with isotonic saline infusions, steroids, furosemide, or bisphosphonates. Because of susceptibility to infections, patients require broad-spectrum antibiotics for febrile illness and immunization against influenza, pneumococcus, and Haemophilus influenzae B. Five-year survival rates approach 33 percent, and the median survival rate is 33 months.     

Identification of malignant plasma cell precursors in the bone marrow of multiple myeloma.

Precursors of plasma cells were studied in the bone marrow of 28 patients with multiple myeloma, plasma cell leukemia, and benign monoclonal gammopathy. Pre-B and B cell populations were analyzed with anti-B monoclonal antibodies corresponding to the clusters standardized at the Leucocyte Typing Workshops in Paris and Boston (CD9, CD10, CD19-22, CD24). In advanced forms of plasma cell malignancies, such as cases of multiple myeloma in stages II and III and of plasma cell leukemia, some cells of lymphoid morphology expressed common acute lymphoblastic leukemia antigen (CALLA, CD10) and HLA-DR, but contained no detectable terminal deoxynucleotidyl transferase enzyme. These CALLA+ cells were absent in benign monoclonal gammopathies. In multiple myeloma, the CALLA+ cells were negative for surface and cytoplasmic immunoglobulins (Ig), and, unlike CALLA+, terminal deoxynucleotidyl transferase (TdT+) pre-B cells in the normal bone marrow also failed to react with antibodies to B cell-associated antigens such as CD9, CD19, CD22, and CD24. The CALLA+, Ig- cells could be regarded as preplasmacytic since, after having been separated and stimulated with the phorbol ester 12-0-tetradecanoyl-phorbol-13 acetate in vitro, they transformed into plasma cells and synthesized the same heavy and light chains as myeloma cells.     

Using quantitative immunoprecipitation mass spectrometry (QIP-MS) to identify low level monoclonal proteins

Mass spectrometry has recently been proposed as a novel sensitive screening tool for monoclonal gammopathies. In a small study we have tested the ability of quantitative immunoprecipitation mass spectrometry (QIP-MS) to identify low level monoclonal immunoglobulins not currently detected by the initial serum protein electrophoresis (SPEP) screen. QIP-MS positively identified the primary monoclonal immunoglobulins in all 11 patient samples alongside additional monoclonal immunoglobulins in a subset of patient samples. We conclude that QIP-MS has clinical utility as a first-line screening tool for monoclonal gammopathy investigation, identifying monoclonality in patients with higher sensitivity and resolution compared to the current standard methods.     

Computer-supported detection of M-components and evaluation of immunoglobulins after capillary electrophoresis

BACKGROUND: Electrophoresis of serum samples allows detection of monoclonal gammopathies indicative of multiple myeloma, Waldenstr?m macroglobulinemia, monoclonal gammopathy of undetermined significance, and amyloidosis. Present methods of high-resolution agarose gel electrophoresis (HRAGE) and immunofixation electrophoresis (IFE) are manual and labor-intensive. Capillary zone electrophoresis (CZE) allows rapid automated protein separation and produces digital absorbance data, appropriate as input for a computerized decision support system. METHODS: Using the Beckman Paragon CZE 2000 instrument, we analyzed 711 routine clinical samples, including 95 monoclonal components (MCs) and 9 cases of Bence Jones myeloma, in both the CZE and HRAGE systems. Mathematical algorithms developed for the detection of monoclonal immunoglobulins (MCs) in the gamma- and ss-regions of the electropherogram were tested on the entire material. Additional algorithms evaluating oligoclonality and polyclonal concentrations of immunoglobulins were also tested. RESULTS: CZE electropherograms corresponded well with HRAGE. Only one IgG MC of 1 g/L, visible on HRAGE, was not visible after CZE. Algorithms detected 94 of 95 MCs (98.9%) and 100% of those visible after CZE. Of 607 samples lacking an MC on HRAGE, only 3 were identified by the algorithms (specificity, 99%). Algorithms evaluating total gammaglobulinemia and oligoclonality also identified several cases of Bence Jones myeloma. CONCLUSIONS: The use of capillary electrophoresis provides a modern, rapid, and cost-effective method of analyzing serum proteins. The additional option of computerized decision support, which provides rapid and standardized interpretations, should increase the clinical availability and usefulness of protein analyses in the future.     

Immunofluorescence labeling indices in myeloma and related monoclonal gammopathies

We measured plasma cell labeling indices (LI) in 52 patients with monoclonal gammopathies. Cytoplasmic reactivity with polyspecific or kappa- and lambda-specific light chain anti-Ig reagents identified monoclonal plasma cells, plasmablasts, and lymphocytoid plasma cells. Among newly diagnosed untreated patients, a high immunofluorescence LI distinguished those with multiple myeloma (MM) from those with stable monoclonal gammopathies (P less than 0.002). Among treated patients with MM, those in the plateau phase of the disease had low LI, whereas patients in the relapse phase or early in treatment had high LI. The immunofluorescence LI correctly classified three more patients with newly diagnosed MM than did the tritiated thymidine LI technique. LI specific for the neoplastic plasma cells resulted in excellent discrimination of patients with active disease. Because results are easily and rapidly obtained, this technique is useful clinically.     

Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine

BACKGROUND: Bence Jones proteins or monoclonal immunoglobulin kappa and lambda free light chains (FLCs) are important markers for identifying and monitoring many patients with B-cell tumors. Automated immunoassays that measure FLCs in urine and serum have considerable clinical potential. METHODS: Sheep antibodies, specific for FLCs, were prepared by immunization with pure kappa and lambda molecules and then adsorbed extensively against whole immunoglobulins. The antibodies were conjugated onto latex particles and used to assay kappa and lambda FLCs on the Beckman IMMAGE protein analyzer. RESULTS: The unconjugated antibodies showed minimal cross-reactivity with intact immunoglobulins or other proteins. With latex-conjugated antibodies, kappa and lambda FLCs could be measured in normal sera and most normal urine samples. Patients with multiple myeloma had increased concentrations of the relevant serum FLC, whereas both FLCs were increased in the sera of patients with systemic lupus erythematosus. CONCLUSIONS: We developed sensitive, automated immunoassays for kappa and lambda FLC measurements in serum and urine that should facilitate the assessment of patients with light chain abnormalities.     

Monoclonal gammopathy in a tertiary referral hospital

ObjectiveMonoclonal gammopathies reflect conditions of plasma B-cell disorders. Our objective was to identify the prevalence and types of these gammopathies in our population.MethodsA 10 year retrospective study was conducted. Serum and/or urine protein electrophoresis were performed on 6624 samples. Positive bands were further tested by immunofixation (IFE).ResultsHomogenous bands were detected in 7% of the patients. IFE method confirmed 6.3% in which 59% were males and 41% were females. The mean age was 64.7 for females and 66.5 for males. The sensitivity and specificity were 91% and 99% respectively. The most common protein was IgG kappa 41%, followed by IgG lambda 19%. Sixty-eight percent of these patients had monoclonal gammopathy of undetermined significance and 14.6% had multiple myeloma.ConclusionThe majority of the studied population had MGUS. This observation is in concord with other western populations. The sensitivity and specificity of protein electrophoresis is diagnostically and reasonably acceptable.     

血清免疫固定电泳在骨髓瘤肾脏病患者诊断中的意义

目的　评价血清免疫球蛋白定量、血清蛋白电泳和血清免疫固定电泳在骨髓瘤肾病诊断和鉴别诊断中的敏感性和准确性。方法　对 4 1份骨髓瘤肾病和 36份其他肾功能损害患者的血清标本 ,采用血清免疫球蛋白定量、血清蛋白电泳和血清免疫固定电泳同时进行检测 ,求其单克隆免疫球蛋白的检出率 ,并将结果用SPSS10 0统计软件进行分析 ,以此来评价方法的敏感性和准确性。结果　免疫球蛋白定量不能检出单克隆成分 ;血清蛋白电泳对IgG和IgM型骨髓瘤检出率达 10 0 % ,其他型有较大缺陷 ,准确性及假阳性率无法判定。除不分泌型骨髓瘤外 ,免疫固定电泳对表现为肾功能损害的各型骨髓瘤检出率达 10 0 % ,准确性 10 0 % ,对照组无一假阳性。对照组与骨髓瘤肾病组比较差异有显著意义 (P <0 0 1)。 3种方法进行两两比较 ,相互间差异均有显著意义 (P <0 0 1)。结论血清免疫固定电泳较血清蛋白电泳和血清免疫球蛋白定量 ,在诊断和鉴别诊断骨髓瘤肾损害中有较高的敏感性和准确性 ,应作为肾脏疾病的常规检查项目在临床使用。     

Serum reference intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains

BACKGROUND: The detection of monoclonal free light chains (FLCs) is an important diagnostic aid for a variety of monoclonal gammopathies and is especially important in light-chain diseases, such as light-chain myeloma, primary systemic amyloidosis, and light-chain-deposition disease. These diseases are more prevalent in the elderly, and assays to detect and quantify abnormal amounts of FLCs require reference intervals that include elderly donors. METHODS: We used an automated immunoassay for FLCs and sera from a population 21-90 years of age. We used the calculated reference and diagnostic intervals to compare FLC results with those obtained by immunofixation (IFE) to detect low concentrations of monoclonal kappa and lambda FLCs in the sera of patients with monoclonal gammopathies. RESULTS: Serum kappa and lambda FLCs increased with population age, with an apparent change for those >80 years. This trend was lost when the FLC concentration was normalized to cystatin C concentration. The ratio of kappa FLC to lambda FLC (FLC K/L) did not exhibit an age-dependent trend. The diagnostic interval for FLC K/L was 0.26-1.65. The 95% reference interval for kappa FLC was 3.3-19.4 mg/L, and that for lambda FLC was 5.7-26.3 mg/L. Detection and quantification of monoclonal FLCs by nephelometry were more sensitive than IFE in serum samples from patients with primary systemic amyloidosis and light-chain-deposition disease. CONCLUSIONS: Reference and diagnostic intervals for serum FLCs have been developed for use with a new, automated immunoassay that makes the detection and quantification of monoclonal FLCs easier and more sensitive than with current methods. The serum FLC assay complements IFE and allows quantification of FLCs in light-chain-disease patients who have no detectable serum or urine M-spike.