Estimation of serum M-protein concentration from polyclonal immunoglobulins: an alternative to serum protein electrophoresis and standard immunochemical procedures

BACKGROUND: Serum monoclonal proteins (M-proteins) are usually quantified by electrophoresis or immunochemical measurement. A third alternative involves immunochemical measurement of the monoclonal isotype by subtraction of the polyclonal part of the isotype calculated from the other polyclonal isotypes and light chain. We experimentally calculated heavy chain/light chain equivalence factors for three immunoglobulin (Ig) isotypes and compared serum M-protein concentrations obtained using the three approaches. METHODS: Equivalence factors were calculated for 1427 samples from 322 patients with a clinical diagnosis of monoclonal gammopathy of undetermined significance and validated in serum samples with and without a monoclonal component. Serum light chain recovery by nephelometry was compared to estimates using factors for serum immunoglobulin concentrations. Some 3735 samples were measured by electrophoresis and nephelometry for comparison of M-protein concentrations. RESULTS: The experimentally calculated factors were 3.3059 (IgGkappa), 3.5204 (IgGlambda), 3.4567 (IgAkappa), 3.5308 (IgAlambda), 4.9104 (IgMkappa) and 4.8020 (IgMlambda). Light chains quantified by nephelometry in 984 serum samples without M-protein gave recovery of 100.97%+/-6.89% compared to immunoglobulin concentration estimates. Light chain recovery of <80% was observed more often for IgMkappa (61.0%) and IgMlambda (42.5%). Method differences for M-protein were >25% for 37.4%-52.3% of the serum samples. Electrophoresis results were higher than nephelometry results for 5% of serum samples and 23.7% of the estimates, especially for M-protein <20 g/L. Method differences for concentrations >40 g/L were <25% for most IgG and IgA isotypes, but not IgM. CONCLUSIONS: No method can accurately measure serum M-proteins over the entire concentration range. Estimation using polyclonal immunoglobulins and light chains represents a simple alternative to electrophoresis that is applicable to any serum M-protein concentration, regardless of electrophoretic migration and particularly for M-protein serum <20 g/L.     

Retrospective study of monoclonal gammopathies detected in the clinical laboratory of a Spanish healthcare district: 14-year series.

BACKGROUND: We studied the incidence, classification and isotype distribution of monoclonal gammopathies and M-protein detected between 1992 and 2005 inclusive in the clinical laboratory of a healthcare district in Madrid (Spain) with an average population of 280,574 inhabitants. METHODS: Serum electrophoresis was carried out on a cellulose acetate support up until 1997, and then using capillary zone electrophoresis systems, with M-protein identification carried out by agarose gel immunofixation. The age-adjusted incidences were standardized with respect to the WHO World Standard Population Distribution, based on the world average population between 2000 and 2025. The clinical diagnosis was recorded from the patient case history. RESULTS: M-protein was detected in a total of 537 patients; of these, 42 had been diagnosed before 1992, representing a 0.19% prevalence in our population. The mean age-adjusted incidence of monoclonal gammopathy was 10.72 per 100,000 inhabitants/year (SE 1.31), ranging from 4.85/100,000 in 1992 to 14.28/100,000 in 2003 and 2004. The median patient age at diagnosis was 73 years (range 25-96 years), with males accounting for 46.8% of all cases of monoclonal gammopathy, and 57.8% of all malignant monoclonal gammopathies. A total of 54.1% of the patients were clinically defined as presenting monoclonal gammopathy of undetermined significance, 31.3% presented multiple myeloma, and the remaining 14.6% presented malignant gammopathies. The most frequent M-protein isotype was IgG (55.8%), followed by IgA (20.8%) and IgM (13.6%). A total of 88% of the light chain M-proteins, 54% of isotype IgM, 51% of isotype IgA and 36% of isotype IgG were associated with B lymphoproliferative diseases. CONCLUSIONS: We conclude that the clinical laboratory should play an important role in the study of monoclonal gammopathies, since it is the only location where all M-protein patients are observed. On the other hand, studies of this type should be carried out over long-term periods, owing to the variations we have noted in the detection of M-proteins.     

Detection of Plasma Cell Disorders by Mass Spectrometry: A Comprehensive Review of 19,523 Cases

ObjectivesTo verify the analytical performance of a new mass spectrometry–based method, termed MASS-FIX, when screening for plasma cell disorders in a routine clinical laboratory.Patients and MethodsResults from 19,523 unique patients tested for an M-protein between July 24, 2018, and March 6, 2020, by a combination serum protein electrophoresis (SPEP) and MASS-FIX were examined for consistency with pretest implementation performance. MASS-FIX’s ability to verify abnormal results from SPEP and free light chain measurements was then compared with that of immunofixation electrophoresis (IFE) using a separate cohort of 52,586 patients tested by SPEP/IFE during the same period.ResultsOverall, 62.4% of our cohort was negative for an M-protein. Importantly, 7.3% of all specimens had an M spike on SPEP (0.1 to 8.5 g/dL) and MASS-FIX detected an M-protein in all these samples. Of all samples, 30.3% had M-proteins that were detected by MASS-FIX but the SPEP finding was too small for quantification. Of the positive samples, 5.7% contained a therapeutic monoclonal antibody. Of the positive samples, 4.1% had an N-glycosylated light chain (biomarker of high-risk plasma cell disorders). MASS-FIX confirmed a higher percentage of SPEP abnormalities than IFE. MASS-FIX was slightly more sensitive than IFE when confirming an M-protein in samples with an abnormal free light chain ratio. MASS-FIX had a very low sample repeat rate (1.5%). MASS-FIX was highly automatable resulting in a higher number of samples/technologist/day than IFE (～30% more).ConclusionOverall, MASS-FIX was successful in maintaining validation characteristics. MASS-FIX was more sensitive in confirming SPEP abnormalities when compared with IFE. Ability to detect therapeutic monoclonal antibodies and glycosylated light chains was distinctly advantageous.     

Measurement of serum monoclonal components: comparison between densitometry and capillary zone electrophoresis.

Quantitative measurement of serum monoclonal protein (M-protein) is one of the most important tools for monitoring disease activity in monoclonal gammopathies. The aims of this study were to evaluate serum M-protein quantification by capillary zone electrophoresis (CZE) and to compare results with those obtained by densitometric scanning of high-resolution agarose gel electrophoresis (HRE-AGE). The evaluation was carried out on 82 samples from patients with various monoclonal gammopathies. All the suspected M-proteins were confirmed and characterised by immunofixation on agarose gel (IFE). CZE was performed on a Paragon CZE 2000 system (Beckman Coulter). Passing-Bablok regression was: y (CZE)=1.27 x(HRE-AGE)-5.21 g/L. The correlation coefficient was 0.92. Bland-Altman analysis demonstrated a mean difference of -1.83 g/L (95% CI -0.76 to -2.90) with clear evidence of a concentration-related bias. Densitometry gave higher values at low M-spikes (<20 g/L), whereas CZE gave higher values at large M-spikes (>20 g/L). The concentration-related bias was found to be independent of the immunoglobulin isotype. In conclusion, to compare previous results obtained by M-protein densitometric scanning with those obtained by direct measurement of CZE peaks, the calculation of a univocal transforming factor appears to be unreliable.     

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.     

Detection of monoclonal proteins by capillary electrophoresis using a zwitterion in the running buffer

Some cases have been reported in which a small monoclonal protein (M-protein) cannot be detected by conventional cellulose acetate membrane electrophoresis (CAE) or capillary zone electrophoresis (CZE) using a short fused-silica capillary. This is probably because these methods do not have the necessary sensitivity or resolution. To overcome this problem, we improved the CZE system by using a longer capillary and adding a zwitterion to the running buffer (pH 10.0). A comparison of CZE and CAE demonstrated that with the exception of alpha(1)- and alpha(2)-globulin, the correlation was satisfactory in serum samples from 34 patients with M-proteins which had been detected by immunoelectrophoresis. In addition, a comparison of CZE electropherograms with those from CAE showed that small M-proteins that went undetected by CAE could be detected by CZE in four patients whose diseases included epipharyngeal carcinoma, solitary plasmacytoma, Crow-Fukase syndrome and macroglobulinemia. The improved resolution produced by a longer capillary may be effective for the detection of small M-proteins.     

Determination of the length of sedimentation reaction in blood using the TEST 1 system: comparison with the Sedimatic 100 method, turbidimetric fibrinogen levels, and the influence of M-proteins.

BACKGROUND: Determination of the length of sedimentation reaction in blood (LSRB) is frequently used in daily practice to assess disease intensity. Recently, a micro-sedimentation method was introduced (TEST 1) that uses EDTA anti-coagulated blood samples. The aim of this study was to characterize this method by comparing it to a conventional Westergren method (Sedimatic 100). Furthermore, correlation between fibrinogen and the LSRB and the influence of M-proteins on the LSRB was investigated. METHODS: Unselected paired samples were used for comparison between the TEST 1 and Sedimatic 100 methods (n=733); fibrinogen was measured in EDTA samples (n=765) using a turbidimetric method. Furthermore, LSRB was measured in 29 EDTA samples in paired serum tubes from patients in whom an M-protein was detected. RESULTS: TEST 1 showed excellent correlation with the Sedimatic 100 method (y=1.00x; n=733; r=0.92, 95% CI 0.90-0.93; p<0.0001), and had no significant bias (0.15 mm/h, 95% CI -0.48 to 0.75 mm/h). Furthermore, TEST 1 LSRB showed satisfactory correlation with the fibrinogen content (y=3.13+0.06x; n=765; r=0.78, 95% CI 0.75-0.80; p<0.0001). In samples containing M-proteins, satisfactory correlation between the M-protein content and TEST 1 LSRB was found (y=0.69+0.22x; n=29; r=0.71, 95% CI 0.45-0.85; p<0.0001), while excellent correlation was found when only M-proteins of the IgM type were taken into account (y=-0.95+0.23x; n=9; r=0.93, 95% CI 0.71-0.99; p<0.0002). CONCLUSIONS: The results confirm previous reports that TEST 1 is a reliable method to measure the LSRB, and shows for the first time the quantitative relationship between TEST 1 LSRB and M-proteins, particularly those of the IgM type.     

Treatment of multiple myeloma with monoclonal antibodies and the dilemma of false positive M-spikes in peripheral blood

ObjectivesTo characterize the effect of three humanized IgG κ monoclonal antibodies (daratumumab, isatuximab, and elotuzumab) on the interpretation of results generated by protein electrophoresis, immunofixation, free light chain, and heavy/light chain assays performed on human serum.MethodsHealthy volunteer serum and serum from multiple myeloma patients were supplemented with clinically relevant concentrations of each of the three monoclonal antibodies. These specimens then underwent analysis via serum protein electrophoresis, immunofixation, serum free light chain quantification, heavy/light chain quantification, total IgG, and total protein. In addition, serum specimens from patients who had undergone treatment with elotuzumab for multiple myeloma underwent similar analysis.ResultsAddition of the study drugs to serum from both the healthy donor as well as multiple myeloma patients resulted in a visible and quantifiable M-protein on SPEP and a visible IgGκ band by IFE. Increases were also noted in total IgG, IgGκ, and IgGκ/IgGλ-ratios. Analysis of serum from multiple myeloma patients receiving study drug showed similar findings with an additional IgGκ band and quantifiable M-protein with similar migration patterns in specimens drawn after administration.ConclusionThe treatment of multiple myeloma patients with monoclonal antibodies results in a visible and quantifiable M-protein that has the potential to falsely indicate poor response to therapy.     

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.     

The predictive power of serum kappa/lambda ratios for discrimination between monoclonal gammopathy of undetermined significance and multiple myeloma.

The predictive power of serum kappa/lambda ratios on initial presentation of immunoglobulin G (IgG) or IgA monoclonal component was studied to differentiate between monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients. The retrospective study involved 145 patients clinically diagnosed with monoclonal gammopathy of undetermined significance or multiple myeloma, who had serum M-protein IgG <35 g/L or IgA <20 g/L at M-protein detection. Serum light chains kappa and lambda were measured by fixed-time nephelometry. Test performance indices, predictive values and likelihood ratios were calculated according to the Weissler recommendation. MM patients were considered as diseased and MGUS patients as non-diseased in order to estimate the performance characteristics of serum kappa/lambda ratios. There was a statistically significant difference in kappa/lambda ratios distribution between both groups of patients, in both M-protein kappa-type (Mann-Whitney U=168, p<0.001) and in M-protein lambda-type (Mann-Whitney U=143, p<0.001). Negative likelihood ratios at threshold levels of 0.6 and 4.2 were 2.17- and 3.32-fold greater, respectively, than positive likelihood ratios, so that the predictive power of a serum kappa/lambda ratio within these limits is better in ruling out (negative predictive power) than ruling in disease (positive predictive power). The post-test characteristics of a serum kappa/lambda ratio interval between 0.6 and 4.2 in discriminating MGUS from MM in our geographic population were: sensitivity 0.96 (0.93-0.99 95% CI); specificity 0.70 (0.63-0.77); positive predictive value 0.68 (0.64-0.73); negative predictive value 0.96 (0.94-0.99); likelihood ratios (+)LR 3.23 (2.68-4.04); and (-)LR 17.16 (11.00-63.00). Thus, serum M-protein with a kappa/lambda ratio between 0.6 and 4.2 increases the posterior probability of MGUS from 0.60 to 0.96 in asymptomatic patients, for whom only monitoring may be suggested when the serum kappa/lambda ratio is within these limits.     

Underestimation of monoclonal proteins by serum protein electrophoresis on cellulose acetate

Our study of 95 serum samples from 37 patients with monoclonal gammopathy revealed distorted irregular monoclonal (M) protein bands after serum protein electrophoresis (SPE) on cellulose acetate membrane. In 71 (75%) of the 95 sera, the M-protein was underestimated and the albumin concentration overestimated. Dilution of the serum sample before SPE eliminated the abnormality of the M-protein bands. By SPE, the mean albumin concentration in these 71 undiluted sera was 45.8 (SD 7.4) g/L vs 37.9 (SD 5.8) g/L for the diluted sera; moreover, this was true of individual samples: measured albumin concentration in each diluted serum sample was always less than in the undiluted serum. As measured by the bromcresol green dye-binding method, the albumin concentration was 32.8 (SD 5.9) g/L. Similarly, the M-protein concentration in SPE was 49.5 (SD 12.3) g/L for the undiluted sera vs 61.8 (SD 15.1) g/L for the diluted sera, and the M-protein concentration in each diluted serum sample always exceeded that in the undiluted serum. Underestimation of M-protein limits the usefulness of M-protein measurement in evaluating the patient's response to therapy and for early detection of disease progression. SPE strips should be carefully inspected visually, and sera with M-protein band abnormalities should be diluted and re-assayed if SPE is to quantify concentrations of M-protein and albumin accurately.     

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.     

Bortezomib-dexamethasone combination in a patient with refractory multiple myeloma and impaired renal function

BACKGROUND: Multiple myeloma (MM) is a hematologic neoplasia characterized by the monoclonal proliferation of bone marrow plasma cells. Renal failure occurs in 20% to 30% of MM patients at diagnosis and in >50% with an advanced form of the disease. For those with advanced MM, often a high-risk group of patients with poor prognosis, salvage treatment for renal failure needs to avoid nephrotoxic drugs. CASE SUMMARY: We report a case of a 78-year-old white male (weight, 90 kg) presented to the Department of Medical Oncology and Hematology, Istituto Clinico Humanitas, Rozzano, Milan, Italy, with refractory MM immunoglobulin G kappa (IgGkappa), Durie-Salmon Stage IIA, with progressive renal failure after an oral melphalan, prednisone, and thalidomide regimen (4 mg/m2.d, 40 mg/m2.d for 7 days every 6 weeks, and 100 mg/d, respectively). The patient had documented increments of serum monoclonal component (M-protein), anemia, and renal failure with Bence Jones proteinuria (serum creatinine, 2.9 mg/dL; creatinine clearance, 30 mL/min; hemoglobin, 10.9 g/dL; serum IgGkappa M-protein, 3.9 g/dL; proteinuria 3.5 g/d;light-chain level in urine, 1.2 g/L). After 2 cycles with bortezomib at a reduced dose (1.0 mg/m2 twice weekly for 2 weeks followed by a 10-day rest period) to evaluate tolerability and renal toxicity, the patient completed another 3 cycles at the standard dose (1.3 mg/m2) in combination with dexamethasone (20 mg on the day of bortezomib administration and the day after). This led to an improvement of the renal function with a reduction of serum and urinary M-protein (serum creatinine 1.4 mg/dL; serum IgGkappa M-protein, 2.9 g/dL; proteinuria, 2 g/d; kappa light-chain level in urine, 0.7 g/L). The patient developed thrombocytopenia but did not suffer from some of the more severe adverse events associated with bortezomib, such as infection or peripheral neuropathy, even at full dose. CONCLUSION: We report an elderly patient with refractory MM and progression with renal failure who responded to bortezomib treatment. Bortezomib was well tolerated in this patient.     

Prevalence of Monoclonal Gammopathy in Wild-Type Transthyretin Amyloidosis

Objective

To evaluate the prevalence of monoclonal gammopathy (MG) in patients with wild-type transthyretin amyloidosis (ATTRwt) (formerly known as senile amyloidosis).

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.     

Gammopathy interference in clinical chemistry assays: mechanisms, detection and prevention.

Monoclonal gammopathy is characterized by the presence of an M-protein in serum or urine that has homogeneous structural and functional properties. It can occur in very high concentrations and may cause significant interference in clinical chemistry assays. Examples of gammopathy interference for the analytes glucose, bilirubin, gamma-glutamyltransferase, urea and ferritin are presented. Various mechanisms of interference are described, such as the production of turbidity by the M-protein and the binding of the M-protein to a component of the test system or analyte. In immunoglobulin tests, the M-protein is the analyte itself and may not be completely bound by the test antibody owing to its structural properties. Modern analyzers can detect unusual changes in absorption during the course of a reaction, and thus the formation of turbidity due to M-proteins. This interference may be prevented by optimizing the buffering conditions of the reagents to avoid the formation of turbidity or by removal of the M-protein prior to analysis of the sample. Owing to the unique properties of each M-protein, it is impossible to protect common clinical chemistry test systems completely from gammopathy interference. Therefore, efficient ways for the detection of such interference are needed.     

Beta 2-microglobulin determined by radioimmunoassay with monoclonal antibody

In this sensitive radioimmunoassay for beta 2-microglobulin (beta 2-M) involving a commercially available monoclonal antibody, we used a second monoclonal antibody, produced in our laboratory, for affinity-chromatographic purification of beta 2-M. Both monoclonal antibodies bound to all of the charge isomers of beta 2-M identified by two-dimensional gel electrophoresis. Polyethylene glycol was used to separate the phases after incubation for 3 h at room temperature. Sensitivity was 0.25 ng of beta 2-M. Within-assay CVs were less than 5.4%, between-assay CVs less than 7.3%. Analytical recovery was 95-102%. The reference interval was 1.2-2.2 mg/L for 49 healthy subjects. The correlation coefficient for comparison with a polyclonal antibody assay was 0.997 for 44 patients. In a study correlating serum beta 2-M and creatinine concentrations with glomerular filtration rate for 50 patients, correlation coefficients for log-log transformed data were -0.94 and- 0.95, respectively. Concentrations of beta 2-M were increased in serum of patients with imparied renal function and also in patients with normal renal function who had disorders involving the immunologic response. We found no clear-cut advantage of measuring serum beta 2-M over serum creatinine in the estimation of glomerular filtration rate.     

Serum free light chain reference values: A critical approach

ObjectivesThe clinical usefulness of serum free light chain (FLC) measurement in the management of patients with plasma cell proliferative disorders has been reported in several papers, and most clinical studies use the reference ranges declared by the manufacturer. The aim of the present study was to evaluate the reproducibility of FLCs immunoassay and to validate the reference range, before introducing it in routine setting.Design and methodsInternal quality control materials and a pool of fresh serum samples were used to evaluate imprecision; 162 fresh sera from healthy blood donors were analyzed to evaluate the reference range for FLCs. In order to verify the κ/λ FLC ratio, 43 sera from patients with polyclonal hypergammaglobulinemia were tested. The FLC immunoassay was performed using a nephelometer with the Freelite reagents.ResultsThe imprecision studies performed using a serum pool tested with two different lots of reagents showed a mean CV of 16.09% for κFLC and of 16.72% for λFLC. Lower CV%s and different mean values were found by calculating the results from each specific lot separately, while different results were obtained using the control materials provided by the manufacturer. In reference subjects, the 2.5–97.5th percentiles were found to be 4.52–22.33 and 4.84–21.88 mg/L for κFLC and λFLC, respectively. The range for κ/λ ratio (0.65–2.36) was validated with the values obtained from subjects with polyclonal hypergammaglobulinemia. In retesting 15 samples from blood donor subjects with a different lot of reagents, mean bias percentages of 17.60 for κFLC and 15.26 for λFLC were obtained.ConclusionsThese findings confirm the lot-to-lot variability of the FLC assays also in the measurement of polyclonal light chains, as well as the need to carefully validate the reference values.     

Clearing drug interferences in myeloma treatment using mass spectrometry

ObjectiveTo explore the possibility of using a combination of a rapid MALDI-TOF-MS method (Mass-Fix) in conjunction with higher resolution LC-ESI-QTOF-MS (miRAMM) measurements to discriminate the IgG kappa M-protein from daratumumab, elotuzumab and isatuximab in myeloma patients.Design & Methods86 patients with an IgG kappa M-protein were spiked with therapeutic levels of the drugs and examined by Mass-Fix and miRAMM to establish the percent of cases that could be resolved by each method. The method was then applied to 21 samples from patients receiving one of the drugs.ResultsMass-Fix was capable of resolving the t-mAb from M-protein for 87 percent of the spiked samples. For the cases unresolved by Mass-Fix, miRAMM was capable of resolving the remaining drug interferences. The 21 IgG kappa myeloma patients that were receiving the drugs were all resolved by Mass-Fix.ConclusionThis proposed algorithm allows use of a clinical available assay (Mass-Fix) while maximizing the number of cases that can accurately resolve the t-mAb from the M-protein.     

Antigen-specific free immunoglobulin light-chain antibodies: could it be a new diagnostic marker for patients with allergy?

OBJECTIVES: Human free immunoglobulin light chains (FLCs) have long been considered as nonmeaningful spillover remnants from the process of immunoglobulin production; however, recent findings suggest that the antibody activity of FLCs may be involved in the pathology of allergic responses. We therefore assessed the antigen-binding ability of FLCs to evaluate their usefulness as diagnostic markers for patients with allergy. DESIGN AND METHODS: FLCs were separated from the serum samples of patients seropositive against cedar pollen and mice immunized with bovine serum albumin and 1-fluoro-2,4-dinitrobenzene by ultrafiltration and protein G absorption. A sensitive immunoassay confirmed the absence of any IgG in the separated FLC fractions from the human serum samples. RESULTS: Solid-phase immunoassay for cedar pollen showed that none of the human serum samples possessed any antibody activity against the antigen after the removal of whole immunoglobulins. Furthermore, while the immunized mice also showed high antibody titers against the antigens, but the serum specimens showed no residual antibody activity against the antigens after the FLCs were separated from the whole immunoglobulins. CONCLUSIONS: The results of the present study suggested that the FLC fractions may possess little or no antigen-binding activity, and that therefore, they may not serve as useful diagnostic markers in patients with allergy.