Quantification of serum free light chain kappa and lambda by the SPAPLUS analyser

ObjectiveClinical assessment of the SPA_PLUS® system for the determination of the serum free light chains kappa (κ FLC) and lambda (λ FLC) compared to the BNII®.Design and methods126 serum specimens from our routine activity were analysed on two different analysers: the BNII® (immunonephelometry, Siemens) and the SPA_PLUS® (turbidimetry, Binding Site). We compared the absolute values of the serum κ FLC and λ FLC, as well as the FLC κ/λ ratio on both analysers. These results were further evaluated together with the clinical history of the patients.ResultsRegression analysis between the BNII® and the SPA_PLUS® for κ FLC and λ FLC did not display any significant differences between both methods in the normal and pathological ranges. Nevertheless, some differences have been observed for some patients in the absolute value of the involved light chain, with potential clinical implications.ConclusionThe results show overall good concordance between both methods. However, it is recommended that the monitoring of patients affected by monoclonal gammapathies by measuring FLC, be performed in the same laboratory and by the same method. Moreover, the FLC results should always be interpreted together with other laboratory tests taking into account the patient's diagnosis.     

kappa/lambda index for confirming urinary free light chain in amyloidosis AL and other plasma cell dyscrasias

Primary systemic amyloidosis (AL), a disease involving the deposition of immunoglobulin light chains in tissue, is caused by a plasma cell dyscrasia. In the case of amyloidosis reported here, no monoclonal component was seen upon routine protein electrophoresis of serum or urine nor was a bone marrow analysis positive for AL. Immunofixation electrophoresis did not show a typical paraprotein band but did show, in the gamma region, two large diffuse bands and a lower concentration of oligoclonal-type bands, all of which stained for free lambda but not for free kappa chain. The ratio of kappa to lambda chains in urine was 0.178, much less than the ratio in serum (1.3). Six other urine samples from a group of patients with documented Bence Jones proteinuria also exhibited kappa/lambda ratios that differed manyfold from the ratios in their corresponding serum samples. On the other hand, the kappa/lambda ratios from seven controls (seven patients with generalized proteinuria unrelated to plasma cell dyscrasia) were similar in serum and urine. This difference between the kappa/lambda ratios from serum and urine can be expressed as a kappa/lambda index. The index was significantly different (P less than 0.01) between the two patient groups compared here, and was useful in confirming the presence of Bence Jones protein in this case with a difficult-to-interpret electrophoretic pattern. Although the kappa/lambda ratio has been widely used for confirmation and identification of monoclonal components in serum, its use in clinical laboratories has not been widely extended to urine. Comparison of serum and urine kappa/lambda ratios as a kappa/lambda index may help reduce the need for more complex immunoelectrophoresis techniques in identifying free light chains in urine.     

Ratio of urinary free immunoglobulin light chain kappa to lambda in the diagnosis of Bence Jones proteinuria.

The aim of this study was to evaluate the diagnostic efficacy of the ratio of urinary free light chain (FLC) kappa to lambda (kappa/lambda ratio) for the detection of Bence Jones protein (BJP). Urine specimens were collected from 243 patients suspected of having BJP. Immunofixation identified 59 BJP-positive specimens among them. The kappa/lambda ratios of all specimens were determined by FLC immunoassays and then the cut-offs for the kappa/lambda ratio were defined as 5.5 for BJP K and 0.1 for BJP lambda by ROC curve analyses. Using the cut-offs, we detected abnormal kappa/lambda ratios in 51 (86%) of the 59 BJP-positives and 11 (6%) of the 184 BJP-negatives identified by the results of immunofixation. High-resolution urinary protein electrophoresis (UPE), a sensitive method for BJP screening, showed almost equal sensitivity to the kappa/lambda ratio, detecting monoclonal band(s) in 52 (88%) of the 59 BJP-positives. However, in UPE analysis these positive specimens should be followed by redundant immunofixation analysis to determine the isotypes. We further evaluated the combination method of FLC assays with UPE that correctly diagnosed 82% of the specimens as positive or negative for BJP, with only two false-negative results. These results suggest that quantitative FLC immunoassays provide an alternative or complementary method for the detection of BJP.     

Chromosomal location of human kappa and lambda immunoglobulin light chain constant region genes

The chromosomal location of human constant region light chain immunoglobulin (Ig) genes has been determined by analyzing a group of human fibroblast/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human kappa and lambda constant region genes. Human chromosomes in each cell line were identified by isoenzyme analysis. The DNA from hybrid cells was digested with restriction endonucleases, size fractionated by gel electrophoresis, transferred to nitrocellulose or DBM paper, and hybridized with (32)P-labeled nucleic acid probes. The C(κ) gene was assigned to human chromosome 2 and the C(λ) genes to chromosome 22, based upon analysis of these hybrid cell lines, and these assignments were confirmed by analysis of subclones. A group of previously unassigned loci can be mapped to chromosome 2 by virtue of their close linkage to C(κ). The λ and κ light chain and heavy chain Ig genes have now been assigned to all three human chromosomes that are involved in translocations with chromosome 8 in human B cell neoplasms. These techniques and probes provide a means to study the detailed arrangement of human Ig genes and their pseudogenes.     

Sensitive competitive-binding ELISAs for quantifying free kappa and lambda light chains in cerebrospinal fluid

Simple, sensitive, and fully standardized solid-phase enzyme-linked competitive binding immunoassays to quantify free kappa and lambda immunoglobulin light chains are described. The assays were developed to measure the concentration of free light chains in cerebrospinal fluid (CSF), in part because elevated levels of free kappa light chains have utility as a diagnostic marker for multiple sclerosis (MS). Polyclonal rabbit antibodies raised against pooled Bence Jones proteins are bound to solid-phase Staphylococcal protein A and used as the primary antisera in this assay. A pool of Bence Jones proteins isolated from the urine of 10 individuals with multiple myeloma are used as a biotin-labeled ligand and to develop a standard curve. The assays as described are sensitive to the low nanogram range and are specific for free kappa or lambda light chains. The assays were found to have acceptable precision, and results correlated highly with concentrations determined using competitive-binding radioimmunoassays previously described.     

Reference values for immunoglobulin kappa and lambda light chains and the kappa/lambda ratio in children's serum.

We analyzed 708 serum samples from healthy children and adolescents by immunonephelometry to obtain reference values for the immunoglobulin kappa (kappa) and lambda (lambda) light chains and for their ratio at a time of life when immunoglobulin synthesis is maturing and continually being stimulated. The lambda chain concentration that is to be maintained throughout the child's life is reached very early, just after 1 year, whereas the concentration of the kappa chains, which increases gradually, reflects the concentration of the immunoglobulins as a whole. These reference values may be useful for studying kappa and lambda chains in illnesses involving the immune system in children.     

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.     

Quantitative real-time PCR method for detection of B-lymphocyte monoclonality by comparison of kappa and lambda immunoglobulin light chain expression

BACKGROUND: An abnormal IgLkappa:IgLlambda ratio has long been used as a clinical criterion for non-Hodgkin B-cell lymphomas. As a first step toward a quantitative real-time PCR-based multimarker diagnostic analysis of lymphomas, we have developed a method for determination of IgLkappa:IgLlambda ratio in clinical samples. METHODS: Light-up probe-based real-time PCR was used to quantify IgLkappa and IgLlambda cDNA from 32 clinical samples. The samples were also investigated by routine immunohistochemical analysis and flow cytometry. RESULTS: Of 32 suspected non-Hodgkin lymphoma samples analyzed, 28 were correctly assigned from real-time PCR measurements assuming invariant PCR efficiencies in the biological samples. Four samples were false negatives. One was a T-cell lymphoma, one was a diffuse large B-cell lymphoma, and one was reanalyzed and found lymphoma-positive by in situ calibration, which takes into account sample-specific PCR inhibition. Twelve of the samples were fine-needle aspirates, and these were all correctly assigned. CONCLUSIONS: This work is a first step toward analyzing clinical samples by quantitative light-up probe-based real-time PCR. Quantitative real-time PCR appears suitable for high-throughput testing of cancers by measuring expression of tumor markers in fine-needle aspirates.     

Guillain-Barré syndrome in kappa light chain myeloma

We have reported a case of Guillain-Barré syndrome developing in a patient with kappa light chain myeloma and acute renal failure. No improvement in acute polyneuritis was observed with serial plasma exchange. At autopsy, peripheral nerves showed no evidence of perivascular lymphocytic infiltration or demyelination, no amyloidosis, and no myelomatous infiltration.     

Monoclonal free light chain detection and quantification: Performances and limits of available laboratory assays

The detection and quantification of immunoglobulin free light chains in serum and urine is recommended for the diagnosis and monitoring of monoclonal gammopathies according to the guidelines of the International Myeloma Working Group (IMWG). Several tests are currently available in the clinical laboratory to detect and quantify free light chains but although quality, efficiency, and effectiveness have been improved, the results are still variable and poorly harmonized and standardized. The present review article wants to analyze these aspects, with a keen eye on techniques, such as mass spectrometry, that could replace in the practical clinical laboratory the current methods including Bence-Jones protein assay and free light chain immunoassays.     

Measurement uncertainty for serum free light chain assays: Estimation and implication on result interpretation

ObjectivesTo estimate the measurement uncertainty (MU) and reference change value (RCV) for serum free light chain assays (sFLC), and to review their implications on result interpretation.Design and methodsData from 6 to 9 months of internal QC and up to 3.5 years of EQA results were collected retrospectively, on the Roche Modular P analyzer and Dade Behring BN II nephelometer from two independent laboratories. MU was estimated from its components related to imprecision and bias determinations, while RCV was calculated from the estimated MU and published values on biological variation.Results and ConclusionThe standard uncertainty related to imprecision for the FLC were similar between two instruments, ranging from ~ 4% to ~ 8%, so were the uncertainty related to bias, ranging between 24% and 44%. The overall MU with 95% coverage for Fκ, Fλ and their ratio (rFLC) were 55%, 87% and 103% (Modular P), and 49%, 76% and 91% (BNII) respectively. The RCVs with biological variation for Fκ, Fλ and rFLC were comparable between two instruments and averaged 106%, 138% and 173% respectively. The relatively method-independent MU reflected the sFLC limitations. For monoclonal gammopathy detection, the universal rFLC reference limits ± MU gave an effective range of 0.02–3.15 (BNII) (as compared to 0.26–1.65), revealing substantial diagnostic grey zones on both ends. When monitoring disease activity, a minimum change (RCV) of ~ 100% in FLC concentrations or ~ 170% in rFLC would be considered significant. Applying MU when interpreting sFLC results puts current diagnostic cut-offs into perspective, and highlights the importance of collating other clinical findings.     

Pediatric and perinatal reference intervals for immunoglobulin light chains kappa and lambda.

In routine analysis for immunoglobulin light chains in pediatric diagnostics, the age-related reference intervals for serum kappa (kappa) and lambda (lambda) light chains were evaluated in 1543 healthy subjects (newborns to age 16 years, including 168 premature infants). Light-chain analysis was performed by rate nephelometry. IgG, IgA, and IgM were measured simultaneously, and heavy- and light-chain differences were calculated for control purposes. Results for IgG, IgA, and IgM generally agreed with reference intervals reported in the literature. kappa showed age-related changes comparable with changes in IgG concentrations, whereas lambda showed moderate fluctuations. The kappa/lambda ratio showed an almost linear increase with age, starting with 0.97 at four months and reaching the highest value of 2.21 at 15 years (mean values). Preterm infants presented with markedly low serum concentrations of IgG and corresponding light chains but with adult-type kappa/lambda ratios because of the maternal-origin IgG.     

血清游离轻链的检测及其在不分泌型多发性骨髓瘤中的临床意义

目的 探讨血清游离轻链(serum free light chains,sFLC)检测在不分泌型多发性骨髓瘤(NSMM)中的临床意义.方法 选择9例NSMM患者,所有患者初诊时行血、尿免疫固定电泳(IFE)检测均为阴性.采用免疫比浊法测定sFLC,以sFLC比值判断单克隆轻链的类型,并与患者同期检测的血清免疫球蛋白水平、总轻链定量以及外周血IgH重链基因重排进行比较,探讨sFLC在NSMM中的临床意义.结果 9例NSMM的免疫球蛋白水平均不同程度降低,血清总轻链比值为1.32～2.20,均在正常参考值范围内;9例患者中6例患者к和(或)λ绝对值明显增高且比值异常.所有患者IgH基因重排阳性.结论 免疫比浊法检测sFLC是一种高度敏感的检测方法,有助于NSMM患者轻链克隆性的判断.