Quantification Of Serum Free Light Chains Research Articles

Serum Free Light Chain Quantification Testing: Comparison of Two Methods for Disease Monitoring.

Levels of free immunoglobulin light chains in serum and urine are a sensitive measure of dysregulated immunoglobulin synthesis. The development of an assay for free light chains in serum was a major advance in laboratory testing for monoclonal gammopathies. The original assay by The Binding Site, called Freelite®, has been in common use in laboratory monitoring of monoclonal gammopathies. Two clinical entities, myeloma-defining condition and light chain-predominant multiple myeloma, rely on quantitative measurements of serum free light chains. Using polyclonal antisera specific to free light chains, Diazyme Laboratories developed a latex immunoturbidimetric assay for quantification of human kappa and lambda serum free light chains. We evaluated the Diazyme assay by comparing the results of kappa and lambda free light chain quantification, and kappa/lambda ratio with the results on the same specimens by the Freelite method. We also compared the correlation of the 2 methods to evaluate response to treatment and to changes in clinical status of patients with multiple myeloma. The results of Freelite and Diazyme methods are comparable. There was no statistically significant difference in the performance of the 2 assays for quantification of light chains, kappa/lambda ratio, or correlation of clinical parameters from patients with multiple myeloma at various stages of monitoring the disease in 2 geographically diverse laboratory and clinical environments. The Diazyme method is comparable to Freelite and provides an opportunity to add the test to front-end automation and improvement in efficiency of the assay.

Method comparison for serum free light chain quantification between the Sebia FLC and Freelite assays

Method comparison for serum free light chain quantification between the Sebia FLC and Freelite assays

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.

Long-Term Biological Variation of Serum Protein Electrophoresis M-Spike, Urine M-Spike, and Monoclonal Serum Free Light Chain Quantification: Implications for Monitoring Monoclonal Gammopathies

Long-Term Biological Variation of Serum Protein Electrophoresis M-Spike, Urine M-Spike, and Monoclonal Serum Free Light Chain Quantification: Implications for Monitoring Monoclonal Gammopathies

Long-Term Biological Variation of Serum Protein Electrophoresis M-Spike, Urine M-Spike, and Monoclonal Serum Free Light Chain Quantification: Implications for Monitoring Monoclonal Gammopathies

We analyzed serial data in patients with clinically stable monoclonal gammopathy to determine the total variation of serum M-spikes [measured with serum protein electrophoresis (SPEP)], urine M-spikes [measured with urine protein electrophoresis (UPEP)], and monoclonal serum free light chain (FLC) concentrations measured with immunoassay. Patients to be studied were identified by (a) no treatment during the study interval, (b) no change in diagnosis and <5 g/L change in serum M-spike over the course of observation; (c) performance of all 3 tests (SPEP, UPEP, FLC immunoassay) in at least 3 serial samples that were obtained 9 months to 5 years apart; (d) serum M-spike ≥10 g/L, urine M-spike ≥200 mg/24 h, or clonal FLC ≥100 mg/L. The total CV was calculated for each method. Among the cohort of 158 patients, 90 had measurable serum M-spikes, 25 had urine M-spikes, and 52 had measurable serum FLC abnormalities. The CVs were calculated for serial SPEP M-spikes (8.1%), UPEP M-spikes (35.8%), and serum FLC concentrations (28.4%). Combining these CVs and the interassay analytical CVs, we calculated the biological CV for the serum M-spike (7.8%), urine M-spike (35.5%), and serum FLC concentration (27.8%). The variations in urine M-spike and serum FLC measurements during patient monitoring are similar and are larger than those for serum M-spikes. In addition, in this group of stable patients, a measurable serum FLC concentration was available twice as often as a measurable urine M-spike.

Serum Free Light Chain Ratio (FLCR) at Diagnosis Constitute a Powerful Prognostic Factor of Survival in Multiple Myeloma (MM).

MM is characterized by the presence of a monoclonal immunoglobulin (Ig) in the serum, produced by neoplastic plasma cells infiltrating the bone marrow (BM). Small amounts of free light chains (FLC) are secreted in normal individuals. In case of monoclonality, the FLC ratio increased. The detection and quantification of serum FLCs and of their ratio, was recently introduced as a useful method for the diagnosis and monitoring of light chain MM, amyloidosis, non-secretory MM and MGUS. However, the possible role of FLCR as a prognostic variable has not been investigated yet. We therefore studied the relationship of baseline serum FLCR with disease characteristics and prognosis in MM patients. Eighty-six MM patients (55 kappa-, 31 lambda- ), 16 individuals with MGUS (8 kappa-, 8 lambda-) and 29 healthy individuals (HI) were studied. Serum free light chain levels were measured in frozen sera drawn at diagnosis, before treatment, using a latex-enhanced immunoassay (The Binding Site, Birmingham, UK) on a Behring BN II nephelometric analyzer. Then, the FLCR was calculated, accordingly as kappa/lambda or lambda/kappa, depending on the monoclonal light chain type of the patient. Forty-nine percent of MM patients were males and 65% were ≥ 65 years old. The Ig type was IgG in 61%, IgA in 22%, BJ in 16% and IgD in 1%. Thirty-five percent of patients were in stage I, 31% in stage II and 34% in stage III according to Durie and Salmon while 36% were in stage 1, 22% in stage 2 and 42% in stage 3 of the ISS staging system. Twenty-five percent of patients had Hb < 10g/dL, 13% creatinin ≥2 mg/dl, 10% elevated LDH, 19% serum albumin < 3.5 g/dL, 45% CRP ≥4 mg/L, 41% beta-2-microglobulin ≥5.5 mg/L, 17% presented spontaneous bone fractures and 41% had ≥50% plasma cell BM infiltration. The median serum FLCR at diagnosis was 3.68 in the 55 kappa-MM patients, 38.49 in the 31 lambda-MM patients, 1.24 in kappa-MGUS, 2.14 in lambda-MGUS and 0.43 in HI (κ/λ). Differences between MM and HI and MM and MGUS were significant (p < 0.001). Baseline serum FLCR correlated with creatinin (p=0.03), LDH (p=0.003), the percentage of BM infiltration (p=0.001), the presence of spontaneous fractures (p=0.002) and Durie and Salmon staging (p=0.003). In patients with BJ MM, FLCR was higher then in the ones with other Ig types (p=0.001). Serum FLCR did not correlate with age, gender, hemoglobin, CRP, serum albumin, beta-2-microglobulin, paraprotein level nor with the ISS staging. With a median follow-up of 27.2 months (3.2–89.8), MM patients with serum FLCR < median, had a 3- and 5- year survival of 92±6 and 79±10 respectively, while the corresponding 3- and 5-year survival of patients with FLCR ≥ median were 67±8 and 32±13 (p=0.001). In multivariate analysis, serum FLCR above the median provided prognostic information independent of the value of ISS. In conclusion, baseline serum FLCR appears to be a powerful prognostic factor of survival in MM.

Serum Free Light Chain (SFLC) Elevation Is Associated with High β2-Microglobulin and with a Shorter Time to Treatment in Waldenström Macroglobulinemia (WM).

WM is a rare lymphoproliferative disorder caracterised by bone marrow lymphoplasmacytic infiltration with IgM monoclonal gammopathy. Assessing the tumor burden in WM is challenging and is not directly related to IgM level or bone marrow infiltration. Thus treatment criteria are based on clinical symptoms related to IgM or pancytopenia. SFLC quantification is a useful tool for the diagnosis and follow-up of gammopathies such as amyloidosis and light chain myeloma. So far its quantification has never been studied in WM. We investigated whether SFLC quantification was correlated with monoclonal component value and established prognostic factors, or was predictive of a shorter time to treatment.We analyzed 45 untreated WM patients (IgMκ: 37; IgMλ: 8) at the time of diagnosis (n=23) or during the follow-up (n=22). Patients were assessed for physical examination, body CT scan, serum protein electrophoresis with albumin (g/l) and monoclonal component (g/L) measurements, SFLC level (The Binding Site®, cut-off: SFLCκ 19.4 mg/L and SFLCλ 26.3 mg/L), peripheral blood count and β2-microglobulin (β2-m). Cut-off values of prognostic factors were those defined by the International Prognostic Index for WM (Morel ASH 2006) ie. β2-m > 3mg/L, Hb < 115 g/L, platelets < 100 G/L, albumin < 35 g/L. Treatment was initiated at diagnosis in 7 patients or during the follow-up in 23 patients. Median follow-up for the whole population was 15 months (1 – 74).Median SFLCκ value was 39 mg/L (1.73–399), median SFLCλ was 12.6 mg/L (1.93–19400). 75 % of patients had upper to normal SFLC value (34/45). Table 1 summarizes univariate analysis of (1) the relationship between high SFLC value (SFLC > N) and known biological prognostic factors, and (2) the impact of SFLC > N and other biological parameters on time to treatment. Our results showed that high SFLC correlated with β2-m but not with other biological prognostic factors or with monoclonal IgM value. (2) Patients with SFLC > N had a shorter time to treatment than patients with normal SFLC value. Furthermore, taking SFLC as a continuous variable in patients with SFLC > N, treatment time was strongly correlated to the SFLC quantification (κ: p=0.01; λ: p=0.01). SFLC could thus be used as a new marker of tumor burden in WM and its prognostic value must be tested in larger WM series. The correlation between SFLC variation and response to treatment is still under investigation.Table 1SFLC > N (1)time to treatment (2)SFLC > NNAp=0.03monoclonal IgM valuep=0.08p=0.001β2-m > 3 mg/Lp=0.03p=0.44Hb < 115 g/Lp=0.43p=0.009Platelets < 100 G/Lp=1p=0.36Albumin < 35 g/Lp=0.3p=0.011