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.     

Computer-supported interpretation of protein profiles after capillary electrophoresis

BACKGROUND: Electrophoretic patterns of proteins in serum/plasma are useful in the diagnosis and evaluation of many diseases. Capillary zone electrophoresis (CZE) allows rapid and automated protein separation and produces digital absorbance data, appropriate for mathematical analysis. We previously demonstrated success in detection of monoclonal immunoglobulins in such a system. This study tests new algorithms to produce rapid standardized computer-supported interpretation of the entire electropherogram. METHODS: Data from Beckman Paragon CZE 2000 electropherograms were compared with quantitative protein data from >800 routine clinical samples. Algorithms were designed to produce semiquantitative analyses of major proteins and to define different patterns of inflammation based on the electropherogram. RESULTS: The algorithms produced reliable semiquantitative evaluations of prealbumin, albumin, alpha1-antitrypsin, haptoglobin, and transferrin, but were less accurate for alpha1-acid glycoprotein. Some genetic variants of albumin and deficiency variants of alpha1-antitrypsin were easily recognized. Complex clinical traits such as degree and type of inflammation could be evaluated. When used together with previously developed algorithms addressing immunoglobulins, the new algorithms provide relevant clinical interpretation. Selected outputs indicate the need for reflex testing or evaluation by specialists. CONCLUSIONS: Automation of both electrophoresis and interpretation can provide a rapid, inexpensive, standardized analysis that can hopefully improve the diagnostic information and clinical outcome for large groups of patients. It also provides objective criteria for clinical interpretations, to be validated or adjusted in future clinical studies.     

Evaluation of a new capillary zone electrophoresis system for the identification and typing of Bence Jones Protein

OBJECTIVES: Capillary electrophoresis has recently emerged as a new sensitive technique for the separation of urinary proteins. We evaluated a new method for Bence Jones Protein (BJP) detection and characterization on native urine samples by the Paragon CZE 2000 system. To avoid interference in electrophoretic separation, urine samples were preliminarily treated for the selective removal of interfering salt particles. DESIGN AND METHODS: The evaluation was done on a total of 350 fresh 24-h urine samples. The salt particle removal consisted of a manual chromatographic separation, optimized in the course of our evaluation. Capillary zone urinary protein electrophoresis (CZ-UPE) was compared with conventional high-resolution electrophoresis on an agarose gel, while capillary immunosubtraction (CZU-IFE) was compared with agarose gel immunofixation. RESULTS: After finding a consistent protein loss in eluates, the preanalytical treatment was optimized by changing sample dilution and eluate collection. The within- and between-run imprecision values for monoclonal peaks corresponding to BJP ranged from 0.4-12.2% to 3.3-6.3%, respectively. The detection limit for BJP, defined as the lowest measurable monoclonal peak on CZ-UPE, was 0.0012 g/L for kappa BJP and 0.0007 g/L for lambda BJP. CZ-UPE and CZU-IFE sensitivities were significantly lower in urine samples with a total protein level < or = 100 mg/L (67% and 78%, respectively) compared to those with total protein >100 mg/L (92% and 94%, respectively). Comparison between BJP measurements obtained from densitometric scanning with those from absorbance tracing showed a correlation coefficient of 0.994 and a bias of 29.8 mg/L. CONCLUSIONS: Paragon CZE 2000 can be introduced in routine for screening and typing of BJP; in urine samples with a total protein level >100 mg/L, the performance is consistent with results from published validation studies on CZE applied to serum samples.     

ANTIGENIC RELATIONSHIPS OF BENCE JONES PROTEINS,MYELOMA GLOBULINS,AND NORMAL HUMAN γ-GLOBULIN

By means of immunodiffusion and immunoelectrophoresis study has been made of antigenic relationships of Bence Jones proteins, and the three classes of normal and pathological immunoglobulins, 7S γ, β_2A, and β_2M. All thirty-nine Bence Jones proteins studied could be classified into either one of two distinct antigenic types, A or B. Both types are related to the immunoelectrophoretically slow (S) fragment of a papain digest of normal γ-globulin; B is related more closely than A, but neither has antigenic determinants in common with the fast (F) fragment. The 7S γ myeloma globulins were either immunological type I or II. The papain digests of these proteins produced the S and F precipitin lines in immunoelectrophoresis but multiple bands in starch gel electrophoresis, especially in the F region. The S fraction of type I myeloma globulins is antigenically similar to Bence Jones protein of type B, and the S component of type II myeloma globulins has antigenic determinants in common with type A Bence Jones protein. Correspondingly, myeloma patients with type I globulins and proteinuria usually excrete type B Bence Jones proteins, whereas patients with type II excrete type A proteins. The F fragment is the part common to normal 7S γ-globulin and types I and II myeloma globulins but is absent in β_2A and β_2M pathological globulins and in both types of Bence Jones proteins. Papain digests of β_2A myeloma globulins produced a single precipitin line in immunoelectrophoresis. β_2A myeloma globulins appeared to have two antigenic units, one in common with type B Bence Jones protein and normal γ-globulin, and another specific to β_2A. The β_2A myeloma patients excreted type B Bence Jones protein. The papain digest of a macroglobulin produced two precipitin lines, the faster of which had antigenic determinants in common with type B Bence Jones protein, the slower seemed specific for the macroglobulin. Five serum micromolecular globulins proved to be either type A or B Bence Jones proteins. From the above results, an antigenic map was constructed showing which determinants are shared and which are specific for normal 7S γ-globulin, types I and II myeloma globulins, β_2A myeloma globulins, a macroglobulin, and types A and B Bence Jones proteins.     

Analytical performance of serum free light-chain assay during monitoring of patients with monoclonal light-chain diseases

BACKGROUND: Measurement of serum free light chains (FLC) is useful for the diagnosis and monitoring of monoclonal light-chain diseases. It has been suggested that there will be widespread replacement of urine Bence Jones protein measurement by serum FLC assay. We report on our experience with the assay during monitoring of light-chain myeloma (LCMM) and AL amyloidosis (AL). METHODS: Serum FLC immunoassay, serum and/or urine protein electrophoresis and immunofixation were performed on serial samples during monitoring of LCMM. Recovery and immunoreactivity of FLC were tested by sample dilution. Assay precision was determined by repeat assay of samples over several reagent lots. RESULTS: In one of 23 patients with LCMM there was non-reaction of a monoclonal kappa FLC with some reagent lots and the assay did not indicate disease relapse. Samples showed non-linear, non-parallel immunoreactivity on dilution. Several tested monoclonal FLC gave lower values at the assay starting dilution compared with higher sample dilution and non-parallel dose-response curves. The median between-reagent lot variation for FLC measurement was 19-20% CV. CONCLUSIONS: Laboratory staff and clinicians need to be aware of the potential for non-reactivity of individual monoclonal FLC, and the effects of dilution and precision on FLC values and their interpretation.     

Analytical characterization of the urinary proteins from sixty patients with monoclonal gammopathies

The urinary proteins from 60 patients with monoclonal gammopathies were characterized by the combination of conventional cellulose acetate electrophoresis, sodium dodecyl sulphate—polyacrylamide gel electrophoresis, and immunoelectrophoresis. Bence—Jones proteinuria was found in 60% of the patients. Non-specific proteinuria was found in 23 of the 37 patients with Bence—Jones proteinuria, and in 9 patients who did not eliminate monoclonal free light chains in their urines. In most patients this non-specific proteinuria followed a pattern suggestive of predominantly glomerular compromise. κ-chains were eliminated predominantly in the monomeric form, while λ-chains were found to exist mainly as dimers. In two patients the monomer:dimer ratio changed during observation perhaps as a manifestation of “escape” of the neoplastic clone from treatment.     

Monoclonal free light chains in urine and their significance in clinical diagnostics: Are they really tumor markers?

Bence Jones proteins (monoclonal free light chains of immunoglobulins) are the earliest known biological markers of malignant cell dyscrasia; Bence Jones proteinuria is also present in many types of B cell-related neoplasms. Sometimes, it may also occur in Hodgkin's disease. In some cases, benign monoclonal gammapathy was found to be associated nontumorous diseases as well. The type of monoclonal light chain, the degree of polymerization, and the isoelectric point of the molecule may affect the course of the disease. Urine samples from 637 patients with true or suspected lymphoproliferative diseases were investigated over a 2-yr period by different immunochemical methods. Bence Jones proteinuria was identified in 71 cases by isoelectric focusing combined with immunofixation, while the pathological protein was detected only in 63 cases by conventional methods. Bence Jones proteins can be detected by this new method at a level below the sensitivity of conventional procedures. Bence Jones proteins in the urine may signal a malignant tumor or malignant transformation of an earlier disease. The early detection of monoclonal immunoglobulin light chains in the urine may be important in clinical diagnosis, therapy, and follow-up.     

Restricted electrophoretic heterogeneity of immunoglobulin light chains in urine: a cause for confusion with Bence Jones protein

The detection of Bence Jones protein, an important part of the investigation of suspected myeloma, is most commonly done by agarose or cellulose nitrate electrophoresis followed by immunofixation. Bence Jones protein is recognized as single or multiple bands of one type of light chain. Unfortunately, improvements in sensitivity of these techniques (use of high-affinity antisera and higher resolution electrophoresis) frequently allow detection of multiple light chain bands in the urine of patients who do not have a B-cell dyscrasia. The bands are usually kappa, although they may be accompanied by lambda bands. This pattern may lead to the misdiagnosis of Bence Jones protein and oligoclonal light chain production in patients. Here we show that this pattern is produced by polyclonal light chains; it is present in the urine of all patients with a tubular proteinuria of any etiology and may be induced in healthy individuals by blocking their renal tubular protein reabsorption. Polyclonal light chains separate into monomers and dimers on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and into four major bands with many minor bands by isoelectric focusing. This difference in charge and possibly size results in the banding pattern seen on good-quality electrophoresis and immunofixation.     

CLASSIFICATION OF MYELOMA PROTEINS,BENCE JONES PROTEINS,AND MACROGLOBULINS INTO TWO GROUPS ON THE BASIS OF COMMON ANTIGENIC CHARACTERS

Antisera to normal 7S γ-globulin and to Bence Jones proteins permit the grouping of myeloma proteins (gamma and beta 2A types), Bence Jones proteins, and the Waldenström type macroglobulins into two fundamental antigenic groups. The antigenic determinants responsible for this grouping are common to all these proteins which fall in the general category of immunoglobulins. Antisera to Bence Jones proteins were particularly useful for this classification since they failed to react with the proteins of the opposite group. These antisera also permit the grouping of normal 7S γ-globulin into two major types. The Bence Jones proteins from individual patients were found to correspond in antigenic group to that of the serum myeloma protein. Studies with antisera to 7S γ-globulin and to Bence Jones proteins indicated that the Bence Jones proteins were antigenically identical to a portion of the corresponding multiple myeloma protein molecules.     

Trimolecular complexes of lambda light chain dimers in serum of a patient with multiple myeloma

BACKGROUND: Patients with multiple myeloma often have Bence Jones proteins composed of free monoclonal light chains of the kappa or lambda type in their urine. Usually, these light chains exist as monomeric or dimeric forms, but rarely, larger molecules, such as tetramers, have been reported in the serum. METHODS AND RESULTS: We report the presence of trimeric complexes of lambda light chain dimers in a patient who was diagnosed with a free lambda light chain multiple myeloma 2 years earlier and subsequently underwent a stem cell transplant. Recently, the patient presented with a large serum M-spike (23 g/L) by protein electrophoresis. The spike consisted of monoclonal lambda light chains without a heavy chain. The urine contained only 8 mg of lambda light chain in a 24-h specimen. Quantitative analysis of the serum and urinary free light chains (FLCs) indicated the probability of larger aggregates of FLCs. Size-exclusion chromatography, electrophoresis, analytical ultracentrifugation, and mass spectrometric studies of the serum revealed almost exclusively the presence of trimolecular aggregates of lambda light chain dimers without other multimeric species. CONCLUSION: Monoclonal lambda light chains may present as hexameric aggregates that cannot be cleared by renal excretion.     

Simple method for quantification of Bence Jones proteins

BACKGROUND: Quantification of free monoclonal light chains in urine [Bence Jones proteins (BJPs)] is used to diagnose multiple myeloma and to evaluate response to treatment. We have developed and evaluated an optimized approach for quantification of BJPs. METHODS: High-resolution gel electrophoresis of unconcentrated urine and albumin calibrators was carried out on Sebia's Hydrasys instrument with Hydragel HR agarose gels. After staining with acid violet, the gels were scanned densitometrically. The staining intensities of BJP bands relative to the staining intensities of albumin solutions were used to determine the BJP concentrations. Results for patient samples were compared with conventional agarose gel electrophoresis on concentrated samples. RESULTS: The relationships between staining intensity and the protein concentrations of albumin and BJPs were linear up to protein concentrations of approximately 2000 mg/L. The detection limit was approximately 20 mg/L. The interassay imprecision (CV) was approximately 8% (n = 23, duplicate analysis), and the results (y) showed a close positive relationship to the comparison method: slope = 0.82 (confidence interval, 0.75-0.88); y-intercept = 34 (-14 to 81) mg/L; n = 29; r(2) = 0.96. CONCLUSIONS: Agarose gel electrophoresis of unconcentrated urine samples together with a series of albumin calibrators followed by acid violet staining and densitometric scanning is sufficiently reproducible and sensitive to quantify clinically relevant BJPs.     

A STUDY OF IMMUNOGLOBULIN STRUCTURE : II. THE COMPARISON OF BENCE JONES PROTEINS BY PEPTIDE MAPPING

Urinary proteins of patients with myeloma, prepared by precipitation with ammonium sulphate, have been separated by gel filtration on Sephadex G-100 after reduction and aminoethylation. Many specimens separated into a major peak of Bence Jones protein and into minor peaks of albumin, a protein tentatively identified with heavy chain and a smaller molecular weight protein corresponding to the variable portion of the corresponding Bence Jones protein. The Bence Jones protein purified by gel filtration was analyzed by electrophoresis and by peptide mapping after tryptic digestion. The peptide maps of 24 type K and 20 type L Bence Jones proteins were compared. A set of common peptides was identified in the peptide maps of the Bence Jones proteins of the same type; the common peptides of type K proteins were completely different from the common peptides of type L proteins. The patterns of distinctive peptides was compared; no similarities were found between distinctive peptides of type K and of type L proteins. Some similarities were observed in the distinctive peptides of proteins of the same type. The similarities involved in many cases peptides containing cysteine, whereas similarities in other peptides were limited. This observation suggested that the amino acid sequence around the cysteines of the variable NH₂-terminal half of the Bence Jones proteins may show less variability than other sequences. A few proteins of the same type differed in all their distinctive peptides, an indication that multiple amino acid differences exist between individual Bence Jones proteins. The genetic mechanisms responsible for the variability in the amino acid sequence of the NH₂-terminal half of the light chains of immunoglobulins are discussed in view of the results of the comparison by peptide mapping of the Bence Jones proteins.     

Bence Jones proteinuria in multiple sclerosis

We report our findings of Bence Jones proteins (monoclonal free light chains of immunoglobulins) in concentrated urines of patients with multiple sclerosis, by using agarose electrophoresis and immunofixation. The lack of such findings in urines from healthy subjects and patients with other neurological disorders should stimulate further investigation.     

Multiple myeloma with serum IgM kappa and Bence Jones lambda biclonal gammopathy

We report a rare finding: IgM kappa and Bence Jones lambda double gammopathy in serum of a 80-year-old man with untreated symptomatic multiple myeloma. The unusual findings are confined to the laboratory studies demonstrating also a Bence Jones lambda proteinuria, high erythrocyte sedimentation rate (113 mm/h), and anemia. The synthesis of the different light chains seems to occur in separate cellular clones.     

Multiple myeloma in alcoholic liver cirrhosis

A case of multiple myeloma (Bence Jones, lambda) associated with alcoholic liver cirrhosis is reported. A 56-year-old Japanese male died of hepatic failure and hypercalcemia. Autopsy revealed alcoholic liver cirrhosis and plasma cell myeloma. Immunoelectrophoretic analysis of his reserved serum disclosed the presence of M component of lambda Bence Jones protein. IgA and lambda light chain were demonstrated in the cytoplasm of the myeloma cells. Complications such as generalized amyloidosis, metastatic calcification, myeloma kidney and hemorrhagic pancreatitis were noted. The coexistence of multiple myeloma and liver cirrhosis has rarely been reported. On the basis of a review of the reported cases, a possible association between both diseases was discussed.     

IgD-kappa myeloma with unusual manifestations: an exceptional form

The diagnosis of IgD (kappa) multiple myeloma was made in a 59-year-old woman. The disease was characterized by paraproteinemia of IgD (kappa) type, the occurrence of plasma cell leukemia, a polymorphous neoplastic proliferation with lymphoplasmacytoid cells, no Bence Jones proteinuria and a hemorrhagic pleural effusion. Judging from the related literature, it seems likely that the following findings have a great tendency to occur in various combinations: IgD (kappa) paraproteinemia, no evidence of Bence Jones proteinuria, polymorphous features of myeloma cells and extramedullary spread.     

Polyacrylamide-gel disc electrophoresis of unconcentrated urine samples with special reference to bence jones proteins

The polyacrylamide-gel disc electrophoresis has been applied to the fractionation of 27 samples of unconcentrated urine obtained from the patients with monoclonal gammopathy.It was shown that 44% of the whole Bence Jones protein bands examined were detected in the area between 0.70 and 0.90 expressed in terms of relative migration based on transferrin.The method provided a satisfactory separation of native urine proteins with good reproducibility and few technical difficulties. It may become an efficient tool in clinical chemistry.     

Evaluation of the relations between serum proteins electrophoresis and other laboratory tests in monoclonal gammopathies (author's transl)]

We have considered interesting to determine monoclonal gammopathies incidence, in 2191 serum proteins electrophoresis performed in our laboratory from January to December 1974. We have found 15 cases of monoclonal gammopathies, some cases combined with Mieloma (3 cases), some other with other with non specific diseases. We have considered the relations between type of gammopathy and other laboratory tests useful for any other diagnose: they are: immunochemical analysis, E.S.R., red and white count, total proteins, Bence Jones protein.     

Bence Jones Proteins and Light Chains of Immunoglobulins: XV. EFFECT OF CORTICOSTEROIDS ON SYNTHESIS AND EXCRETION OF BENCE JONES PROTEIN

The effect of corticosteroids and cytotoxic chemotherapeutic agents on the excretion of Bence Jones protein was determined for periods of 1 - 62 mo in 29 patients with multiple myeloma and Bence Jones proteinuria. The amount of protein present in 24-h urine specimens collected before treatment and at frequent intervals during monthly treatment cycles was determined. Striking variations occurred in the amount of Bence Jones protein excretion; these changes were especially evident when 75 mg of prednisone were given daily for 7 days as part of a monthly chemotherapeutic regimen. Within the 7-day period seven patients showed essentially no decrease (<25%), whereas 13 and 9 patients had a moderate decrease (25-75%) or a marked decrease (>75%), respectively, in Bence Jones proteinuria as compared to pre-treatment values. The decrease in excretion of Bence Jones protein during this period was attributed mainly to corticosteroid therapy because of the transient nature of the response in most patients and the lack of such response in three patients when the hormone was omitted. Biosynthetic studies were performed to determine in vitro the effect of corticosteroids on Bence Jones protein synthesis. Plasma cells obtained from the bone marrow of 13 patients were incubated in a growth medium containing (14)C-labeled lysine and isoleucine and prednisone in concentrations up to 240 mug/ml, and the amount of Bence Jones protein synthesized was determined immunochemically. No differences in viability were apparent between untreated and prednisone-treated cells. The type of response exhibited by an individual patient in the percent decrease of Bence Jones protein excreted after 7 days of prednisone treatment was comparable to the percent decrease in newly-synthesized Bence Jones protein secreted by tumor cells when cultured in the presence of prednisone at a concentration of 120 mug/ml. The marked differences in the capacity of corticosteroids to affect Bence Jones protein synthesis appear to reflect a biochemical heterogeneity among plasma cell neoplasms.     

Use of immunoglobulin heavy-chain and light-chain measurements in a multicenter trial to investigate monoclonal components: I. Detection

We assessed the combined use of serum protein electrophoresis (SPE) and nephelometric measurement of immunoglobulin heavy- and light-chain components for detecting serum monoclonal immunoglobulins (monoclonal components, MC) in 4788 unselected samples from 4173 patients. MC were detected in 514 samples from 390 patients. In 356 these were detected by SPE; the other 34 had a normal SPE pattern but an abnormal kappa:lambda light-chain ratio (KLR). Only 208 of the 356 (58%) samples with bands by SPE had abnormal KLRs. Samples with MC concentrations greater than 5 g/L had a higher proportion of abnormal KLRs (75%) than those with concentrations less than 5 g/L (42%). The KLR was abnormal in 13% of samples in which no MC were visible by SPE or immunofixation electrophoresis (IFE). Compared with quantitative measurements of immunoglobulin heavy and light chains, high-quality SPE remains the method of choice for the detection of MC. Quantitative methods, however, are able to detect additional MC, especially those containing free light chains, and in the absence of SPE and IFE will detect about 75% of MC present at greater than 5 g/L.