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.     

Medical and technical usefulness of measurement of kappa and lambda immunoglobulin light chains in serum with an M-component

Kappa and lambda-immunoglobulin light chains were measured to evaluate their usefulness for identifying monoclonal components in serum. Reference concentrations of the Ig light chains were evaluated by the analysis of the serum of 50 blood donors. Two hundred and fifty patients were selected for the presence of an M-component in their serum, which was detected by serum electrophoresis on agarose. The heavy and light chains of the M-component were identified by immunofixation. The concentrations of the three major immunoglobulin classes and of the two light chain types were measured by immunonephelometry. On the basis of the Ig kappa/lambda ratio alone, the Ig light chain type was correctly identified in 76% of the M-components, and there were no misidentifications. By comparing the calculated concentrations of M-component and residual polyclonal Ig with the measured concentrations of the 3 major Ig classes, it was possible to identify the heavy chain type of 58% of the M-components. These observations suggest that M-components can often be identified by the measurement of Ig light chains, a procedure involving less work and time than immunofixation. Moreover, the follow up of gammapathies could benefit from quantitative parameters evaluated from the Ig light chain concentrations: the concentration of the M-component, the ratio of M-component vs total Ig of the same class, and the ratio of M-component vs the sum of the 3 major Ig classes.     

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.     

Chromosomal location of structural genes encoding murine immunoglobulin lambda light chains. Genetics of murine lambda light chains

To determine the chromosomal localization of murine lambda light (L) chain structural genes, DNA from a panel of 11 mouse x hamster somatic cell hybrids was scored for the presence of sequences homologous to cloned lambda DNA probe molecules. Six of the hybrids had detectable lambda I and lambda II gene sequences. In all six, the full complement of murine sequences was present, and in its germline configuration. The remaining hybrids lacked any detectable murine lambda L chain gene sequences. The only mouse chromosome present in all of the positive hybrids and absent from the negative ones was number 16, allowing the assignment of lambda L chain structural genes to this chromosome. Together with the previous assignments of the kappa L chain genes to chromosome 6 and heavy chain genes to chromosome 12, this finding completes the mapping of Ig structural genes in the mouse at the chromosomal level.     

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.     

IgG-cryoglobulin consisting of both kappa and lambda light chains and beta 1C/1A.

A 72-year-old woman was diagnosed as multiple myeloma. Her plasma contained IgG-cryoglobulin consisting of both kappa and lambda light chains and beta 1C/1A. Such unique cryoglobulinemia has been reported in 2 cases in the literature, but never in Japan.     

A new variable region in mouse immunoglobulin lambda light chains

A series of lambda+ murine hybridomas were derived from a BALB/c mouse after a single injection of anti-lambda 2 antibodies coupled to LPS. Nine lambda B cell clones (five lambda 2 and four lambda 3) were expected and seven reacted with antibodies specific for the C lambda 2 constant region but showed a particular isoelectric spectrum. Their RNA products did not hybridize with the V lambda probe. The partial DNA sequence of gene segments coding the unexpected light chain of one hybridoma shows that the V gene segment has only 55% homology with the V lambda 2 gene segment sequence and that J lambda 2 and probably C lambda 2 gene segments are used. Taken together, these results demonstrate the existence of a new lambda light chain.     

Nephelometry of the kappa/lambda light-chain ratio in serum of normal and diseased children

We determined, immunonephelometrically, the ratio between the two types of light chains of immunoglobulins, kappa and lambda, in serum of 94 children, ages 0.4 to 14 years, with no manifest immunological disorders. Children with an abnormal protein pattern by immunoelectrophoresis show other values for this ratio than do children in this reference group. We also determined the ratios for children with IgA deficiency (I), juvenile rheumatic arthritis (II), and acute lymphoblastic leukemia (III). Children with I show the same kappa/lambda ratios as for the children in the reference group. Children with II also show the same mean kappa/lambda ratios, but a significantly wider range of ratios. In children with III, the ratio during chemotherapy is slightly depressed, significantly lower than after cessation of therapy. All groups--healthy children and patients--show an increase in kappa-chain-bearing immunoglobulins with age, but the concentration of lambda-chain-bearing immunoglobulins remains relatively constant.     

Normal values for free light chains in serum different age groups.

The concentration of free light chains from the immunoglobulins was measured in twelve paired sera from mothers and newborns and from 149 sera from normal individuals in various age groups. Variations in concentration during life are correlated to the variations in the concentration of 'regular' immunoglobulins. A concentration of light chains in cord blood of 35% of mean normal adult level (MNA) together with a rapid passage of light chains across the placenta is interpreted as indicating catabolization of maternal light chains in the fetus. This is further supported by the finding of a lower concentration of light chains in maternal serum than in normal adult serum. The investigation shows that the concentration of light chains falls rapidly from 35 to 24% of MNA during the first few days of life. From the first week of life the concentration of light chains increases and low normal adult values are attained by one year of age. Except for difference in concentration, the elution pattern for light chains from Sephadex G-100 columns was similar for normal, adult and cord blood. The relationship between kappa and lambda chains--the K/L ratio--is 1.2 for normal and maternal serum and 1.0 for cord serum.     

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.     

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.     

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.     

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.     

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.     

Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains

Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow cytometry and were clonal by Southern blotting. Surprisingly, two of the mu/lambda-expressing cell lines contained both kappa alleles in germline configuration, and synthesis/expression of conventional lambda L chains was directly proven by immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in one of them. Thus, human fetal bone marrow B lineage cells harbor the capacity to make functional lambda L chain gene rearrangements without rearranging or deleting either kappa allele. A third unusual cell line, designated 30.30, was observed to coexpress cell surface kappa and lambda L chains associated with mu H chains. The 30.30 cell line had a diploid karyotype, a single H chain rearrangement, both kappa alleles rearranged, and a single lambda rearrangement. Immunoprecipitation/SDS-PAGE confirmed that 30.30 cells synthesized and expressed kappa and lambda L chains. Multiparameter flow cytometry was used to demonstrate the existence of kappa+/lambda+ cells in fetal bone marrow and fetal spleen at frequencies of 2-3% of the total surface Ig+ B cell population. The flow cytometry data was confirmed by two-color immunofluorescence microscopy. The existence of normal human B cells expressing cell surface kappa and lambda refutes the widely accepted concept that expression of a single L chain isotype is immutable. The kappa+/lambda+ cells may represent transients undergoing L chain isotype switching.