Mass spectrometric-based assessment of the serum kappa to lambda immunoglobulin light chain ratio (κ:λ) in dogs with immunoglobulin secretory diseases.

The ratio of κ light chains to λ light chains (κ:λ) in serum is used as a biomarker of immunoglobulin secreting neoplasia in humans but has not been evaluated in dogs. A mass-spectrometry based method for determining the canine serum κ:λ was developed and used to evaluate samples from control dogs, dogs with an infectious aetiology, dogs with secretory plasma cell tumours (sPCT) and dogs with non-secretory B cell neoplasia. A human-targeted immunoturbidometric κ:λ assay and immunofixation using antisera targeting human κ light chain or λ light chain was also performed on all samples. Using whole serum samples, the MS-based κ:λ method identified 5 sPCT as κ-predominant (mean κ:λ = 3.307) and 5 sPCT as λ-predominant (mean κ:λ = 0.023) and documented differences between these groups and all other groups (p < 0.05 for all). The infectious aetiology group had a lower mean κ:λ ratio (mean κ:λ = 0.069) than control samples (mean κ:λ = 0.103, p = 0.035). Similar results were obtained when samples were enriched for proteins between 10 and 50 kDa using size exclusion chromatography, except for the statistical difference between the control and infectious aetiology group. All λ-predominant cases had only anti-human λ light chain labelling by immunofixation. Three κ-predominant cases had only anti-human κ-light chain labelling and the remaining two cases did not label with either antisera by immunofixation. The immunoturbidometric method had high analytical CV% (λ light chain CV = 13%, κ light chain CV = 50%), was unable to measure light chains in 20.5% of samples and did not distinguish groups. The data suggests that the human-targeted immunoturbidometric method would not be diagnostically useful and that the MS-derived serum κ:λ may be a useful biomarker of canine immunoglobulin secretory neoplasia which may have the ability to distinguish neoplasia from infectious causes of immunoglobulin secretion.

The clinicopathological characteristics and outcomes of IgA nephropathy with predominant lambda or kappa light-chain deposition.

PURPOSE: IgA nephropathy (IgAN) patients with monoclonal light-chain deposition may be at potential risk of hematological progression. However, whether the clinical characteristics of the patients with predominant lambda or kappa light-chain deposition were consistent with monoclonal light-chain deposition is limited to anecdotes. METHODS: We retrospectively studied patients in whom immunofluorescence showed IgA-alone deposits (n = 617) between January 2016 and January 2020. We divided the patients into two groups, the predominant lambda or kappa light-chain deposition group and the control group. Predominant lambda or kappa light-chain deposition was defined as the deposition intensity of kappa or lambda being + - and the other deposition intensity being ≥ 2 + . RESULTS: Nineteen patients had predominant lambda or kappa light-chain deposition. The patients had a median age of 32 years. The median proteinuria was 0.9 g/day. The median eGFR was 79.8 ml/min per 1.73 m2. Two patients had a mildly abnormal FLC ratio, but serum immunofixation electrophoresis showed polyclonal immunoglobulin. Eighteen patients showed lambda light chain-dominated deposition. In electron microscopy, organized structures in dense deposits were not observed in all patients. Nine patients with proteinuria ≥ 1.0 g/day received corticosteroids and immunosuppressants. The median follow-up time was 21 months. The rate of proteinuria remission was 50%. The clinical and pathological characteristics and outcomes were not significantly different between the predominant lambda or kappa light-chain deposition group and the control group. CONCLUSION: The result for IgAN patients with predominant kappa/lambda light-chain deposition seemed to be the same as that of IgAN patients with light-chain codeposition. However, as this was a single-center study with a small size, further multicenter studies and long-term follow-up are needed to confirm our findings.

Pathological review of cardiac amyloidosis using autopsy cases in a single Japanese institution.

AIM: Amyloidosis is a systemic or localized disease of protein deposition characterized by amorphous eosinophilic morphology and positivity of Congo Red staining. The typing of amyloidosis is becoming increasingly important because therapeutic agents for each amyloidosis type have been developed. Herein, the authors review the autopsy cases at an institution to reveal the putative Japanese characteristics of each amyloidosis type and evaluate the clinicopathological significance of each type. MATERIALS AND METHODS: A total of 131 autopsy cases of systemic and localized amyloidosis were retrieved for classification by immunohistochemistry. Immunohistochemistry for transthyretin, amyloid A (AA), immunoglobulin light-chain kappa and lambda, and ß2-microglobulin was performed for all cases. RESULTS: The 131 amyloidosis cases were classified as follows: 71 cases (54.2%) of transthyretin amyloidosis, 32 cases (24.4%) of AA amyloidosis, 8 cases (6.1%) of light-chain amyloidosis, and 5 cases (3.8%) of ß2-microglobulin amyloidosis, along with 15 equivocal cases (11.5%). All cases showed myocardial involvement of amyloidosis. Histopathologically, the transthyretin type was significantly associated with the interstitial and nodular patterns, and with the absence of the perivascular and endocardial patterns. The AA type was significantly associated with the perivascular and endocardial patterns, and with the absence of the nodular pattern. CONCLUSION: The authors revealed the putative characteristics of cardiac amyloidosis in Japan by using autopsy cases. About 90% of amyloidosis cases were successfully classified using only commercially available antibodies.

Clinicopathological significance of light chain deposition in IgA nephropathy.

BACKGROUND: Clinicopathological significance of light chain deposition in IgA nephropathy and the relation of monotypic IgA deposition to bone marrow abnormalities are important issues to be clarified. METHODS: We retrospectively investigated light chain deposition in 526 patients with IgA nephropathy. We divided the patients into 5 groups according to the balance of intensity of both light chain deposition: lambda monotypic, lambda dominant, polytypic, kappa dominant and kappa monotypic. Clinicopathological parameters were compared among the groups. The relation of monotypic IgA deposition to hematological malignancy was also evaluated. RESULTS: The prevalence of monotypic IgA deposition was 6.3%, 33 patients (21 lambda and 12 kappa). Thirty-two (4.0%) and 10 patients (1.9%) were classified into lambda and kappa dominant groups, respectively. Polytypic IgA deposition was observed in 455 patients (85.7%). Age of onset, age at biopsy, urinary protein creatinine ratio, the percentage of global glomerulosclerosis, and the degree of IgA and C3 deposition were different among the groups. However, there was no gradual difference according to the groups. No patient with monotypic IgA deposition showed hematological abnormality at biopsy and during follow-up. CONCLUSIONS: The prevalence of IgA monotypic deposition was extremely low. Clinicopathologically, we could not differentiate patients with monotypic IgA deposition from those with polytypic one and no hematological disorder was documented in patients with monotypic IgA deposition. Whether IgA nephropathy with monotypic IgA deposition and that with polytypic one is the same entity or not, and relation between monotypic IgA deposition and hematological malignancy should be clarified by further investigations.

Kappa and lambda immunohistochemistry and in situ hybridization in the evaluation of atypical cutaneous lymphoid infiltrates.

BACKGROUND: Atypical cutaneous lymphoid infiltrates are challenging lesions in dermatopathology. We present a summary of the literature regarding kappa and lambda immunohistochemistry (IHC) and in situ hybridization (ISH) in the evaluation of atypical cutaneous or mucosal lymphoid infiltrates. METHODS: Relevant articles from 1967 to 2018 in the English language were identified and summarized. In the absence of larger studies, case series of n ≥ 3 were included. RESULTS: Sixty-three articles assessing kappa and lambda IHC and/or ISH were identified. Most focused on marginal zone lymphomas. Other lymphomas included follicle center lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, mantle cell lymphoma, lymphoplasmacytic lymphoma, plasmablastic lymphoma, multiple myeloma, monoclonal gammopathy of undetermined significance, and polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes (POEMS). Non-neoplastic lesions included reactive lymphoid hyperplasia, cutaneous plasmacytosis, connective tissue disease, IgG4-related disease, acrodermatitis chronic atrophicans, Zoon balanitis, dermatitides, and infiltrates around epithelial dysplasias/neoplasias. CONCLUSION: Kappa and lambda IHC and ISH are useful tools in the evaluation of cutaneous B-cell lymphomas and plasma cell neoplasms. The literature supports that the detection of light-chain restriction by IHC and ISH is one of the most useful findings in the differential diagnosis of reactive lymphoid hyperplasia vs B-cell lymphoma with plasmacytic differentiation.

The cut-offs for kappa/lambda ratio in bone marrow immunohistochemistry for the diagnosis of multiple myeloma.

Objective: The previously reported kappa/lambda ratio cut-offs for plasma cell clonality by immunohistochemistry were in different values. This study aimed to identify the cut-offs with the highest accuracy for the diagnosis of multiple myeloma. Methods: Bone marrow specimens consecutively recruited between January 2011 and March 2019. The patients were at least 18 years old with clinical suspicion of multiple myeloma and had bone marrow plasma cell ≥10% by CD138 immunohistochemistry. The index test was immunohistochemical stains for plasma cell kappa/lambda ratio. The reference standard to classify as multiple myeloma required meeting any of the following (1) kappa/lambda ratio ≤1/16 or ≥16, (2) abnormal plasma cell morphology, and (3) monoclonal immunoglobulin. Results: From 147 specimens (70 multiple myelomas and 77 reactive plasmacytosis), our cut-offs kappa/lambda ratio for light chain restriction at ≤1/7 or ≥9 yielded an area under the receiver operating characteristic curve of 1.0000 while ≤1/16 or ≥16 yielded 0.9643 (p-value 0.0212). Conclusion: The cut-offs for kappa/lambda ratio at ≤1/7 or ≥9 for diagnosis of multiple myeloma yielded the highest diagnostic accuracy. The suggested cut-offs could be of potential value in resource-limited laboratories.

Detection of monoclonal free light chains by immunofixation electrophoresis and isoelectric focusing - comparison with the quantitative method of determination.

The study aimed to investigate free light chain (FLC) monoclonality in patients with an abnormal free kappa/lambda ratio (FLC ratio). Seventy serum samples with abnormal FLC ratio were examined using an immunoturbidimetry (Binding Site, SPA) and the two different enzyme-linked immunosorbent assays (1. Sebia diagnostic kit; 2. in house methods), the monoclonal or oligoclonal bands of (FLC) by immunofixation electrophoresis (IE) and isoelectric focusing followed by affinity immunoblotting (IEF/AIB). The reference interval was calculated by non-parametric percentile method. 5.7% of samples examined by IE were suspected of monoclonal character of FLCs, but subsequently monoclonality was refuted by more sensitive IEF/AIB method; 7%, resp. 2.9% of samples showed FLC kappa, resp. FLC lambda oligoclonal character of bands. A statistically significant dependence was found between FLC ratio (Sebia) and FLC ratio (SPA), rs = 0.510, p = .001. Kappa statistic evaluated a fair conformity between the FLC ratio (Sebia) and IEF/AIB (kappa = 0.468) and between FLC ratio (in house) and IEF/AIB (kappa = 0.300). The verified reference interval for FLC ratio (Binding Site) is between 0.35 and 2.18. The IEF/AIB is the most sensitive method to discriminate between monoclonal and oligoclonal bands of FLC. The Binding Site and Sebia diagnostic kits do not give consistent results. The Binding Site diagnostic kit provides more results above reference interval of FLC ratios. For routine decision on monoclonality of the FLC ratio (SPA) it is advisable to use a verified reference interval.

Primary plasma cell leukaemia in a 39-year-old man.

Plasma cell leukaemia (PCL) is an aggressive haematological malignancy which is classified into primary (pPCL) and secondary PCL. A 39-year-old Indian man presented to the Department of Hematology with a 2-week history of fever and lethargy. Clinically, he was pale and febrile. Haemogram revealed bicytopenia with leucocytosis. The peripheral blood film portrayed rouleax formation with 45% of circulating plasma cells. Serum protein electrophoresis and immunofixation revealed IgG lambda paraproteinaemia of 48 g/L. Bone marrow aspirate, flow cytometry and trephine were consistent with IgG lambda pPCL. He was treated with six cycles of bortezomib, thalidomide and dexamethasone combination chemotherapy followed by high-dose melphalan conditioning and autologous stem cell transplant. Currently, he is in complete remission for the past 18 months and is on oral lenalidomide maintenance therapy. Prognosis is often dismal in pPCL with the median overall survival below 1 year if treatment is delayed.

Heavy and light chain (AHL)-type cardiac amyloidosis: first histopathologic-proven case illustrating involvement of the heart.

Cardiac amyloidosis is most commonly comprised of either a monoclonal immunoglobulin or transthyretin; however, in practice, detailing of the former beyond light chain restriction is not typically performed. We present briefly the case of an 80-year-old man with concern for cardiac amyloidosis and a subsequent endomyocardial biopsy revealing significant deposition of amorphous Congo red-positive material. By immunofluorescence microscopy, the amyloidogenic material showed positive expression for IgG heavy chain and kappa light chain, with negative staining for IgM and IgA heavy chains and lambda light chain supporting a diagnosis of heavy and light chain (AHL)-type amyloidosis. Immunofluorescence staining for the IgG heavy chain subclasses supported and further classified the patient's AHL-type cardiac amyloidosis as being IgG4/kappa restricted. The presented case is the first to illustrate AHL-type cardiac amyloidosis via sampling of heart tissue.

Clinicopathological characteristics of light chain proximal tubulopathy in Korean patients and the diagnostic usefulness of immunohistochemical staining for immunoglobulin light chain.

BACKGROUND: Light chain proximal tubulopathy (LCPT) is a rare paraproteinemic renal disease that has been mostly reported in Western patients. LCPT is characterized by the accumulation of immunoglobulin (Ig)-light chain (LC) in the proximal tubule. Immunohistochemical staining for Ig-LC has not been investigated in the context of LCPT. We reported the clinicopathological characteristics and Ig-LC immunoexpression of patients with LCPT for the first time in Korea. METHODS: We reviewed the clinicopathological findings of 5 Korean patients diagnosed with LCPT between 2016 and 2018. In addition, immunohistochemical staining for κ-LC and λ-LC was conducted on paraffin-embedded tissues. RESULTS: The median age was 63 years, and the male-to-female ratio was 3:2. The primary renal manifestations were either azotemia or tubular proteinuria. All patients were diagnosed with multiple myeloma with monoclonal κ-LC (#1-2) or λ-LC (#3-5) in the serum and urine. Kidney biopsies revealed diverse and subtle alterations of the proximal tubule, including crystallization, vacuolization, and/or swelling. Electron microscopy revealed crystals in patients #1-2 and non-crystalline particles within numerous/large/dysmorphic lysosomes in patients #3-5. Ig-LC restriction was demonstrated in the proximal tubule as κ-type in patients #1-2 and as λ-type in patients #3-5 by immunohistochemistry and immunofluorescence. Immunohistochemical staining showed diffuse positivity to κ- and λ-LC, although immunofluorescent staining for κ-LC was focal and weak. LCPT has diverse clinicopathological characteristics and subtle morphological alterations, which necessitate ancillary tests for diagnosis. CONCLUSIONS: We introduced immunohistochemical staining for Ig-LC as a useful tool for the diagnosis of LCPT, especially in the case of κ-type crystals.

Immunoglobulin variable domain high-throughput sequencing reveals specific novel mutational patterns in POEMS syndrome.

Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare multisystem disease resulting from an underlying plasma cell (PC) dyscrasia. The pathophysiology of the disease remains unclear, but the role of the monoclonal immunoglobulin (Ig) light chain (LC) is strongly suspected because of the highly restrictive usage of 2 λ variable (V) domains (IGLV1-40 and IGLV1-44) and the general improvement of clinical manifestations after PC clone-targeted treatment. However, the diagnostic value of Ig LC sequencing, especially in the case of incomplete forms of the disease, remains to be determined. Using a sensitive high-throughput Ig repertoire sequencing on RNA (rapid amplification of cDNA ends-based repertoire sequencing [RACE-RepSeq]), we detected a λ LC monoclonal expansion in the bone marrow (BM) of 83% of patients with POEMS syndrome, including some in whom BM tests routinely performed to diagnose plasma cell dyscrasia failed to detect λ+ monoclonal PCs. Twenty-four (83%) of the 29 LC clonal sequences found were derived from the IGLV1-40 and IGLV1-44 germline genes, as well as 2 from the closely related IGLV1-36 gene, and all were associated with an IGLJ3*02 junction (J) gene, confirming the high restriction of VJ region usage in POEMS syndrome. RACE-RepSeq VJ full-length sequencing additionally revealed original mutational patterns, the strong specificity of which might crucially help establish or eliminate the diagnosis of POEMS syndrome in uncertain cases. Thus, RACE-RepSeq appears as a sensitive, rapid, and specific tool to detect low-abundance PC clones in BM and assign them to POEMS syndrome, with all the consequences for therapeutic options.

Inter-assay variability in automated serum free light chain assays and their use in the clinical laboratory.

Serum κ and λ free light chain levels are markers of plasma cell proliferation, and their measurements have been included in recent guidelines by the International Myeloma Working Group for the management of patients with plasma cellular dyscrasias. Five in vitro diagnostic methods for the immunochemical quantification of serum free light chains (FLC) are available, three based on polyclonal antibodies (Freelite®, The Binding Site; FLC ELISA κ and λ, Sebia; human κ and λ FLC, Diazyme Laboratories) and two on monoclonal antibodies (N Latex FLC, Siemens Healthineers; Seralite®, Sebia). Several studies have shown that these methods cannot be used interchangeably for the follow-up of patients because measured κ and λ FLC concentrations may differ significantly, especially at high levels. Because no international reference material for the measurement of FLC is available, it is not possible to establish which method is the most accurate. For this reason, knowledge about the analytical and diagnostic performances of the assays used is important. The aim of this review is to describe the main analytical features of the κ and λ FLC assays and how they may influence the clinical use of these parameters.

Papel central del laboratorio en el diagnóstico de un caso de infiltración de sistema nervioso central por mieloma múltiple refractario / Central role of the laboratory in the diagnosis of a case of central nervous system infiltration by refractory multiple myeloma

La infiltración leptomeníngea en el mieloma múltiple es una complicación poco frecuente y grave que se presenta generalmente tras recaídas de la enfermedad. Para establecer un correcto diagnóstico es necesario demostrar por citología la presencia de células plasmáticas clonales en el líquido cefalorraquídeo. Desde el laboratorio clínico detectamos esta complicación en una paciente diagnosticada de mieloma múltiple refractario, tras analizar una muestra de líquido cefalorraquídeo. La paciente presentaba diversos síntomas neurológicos como incontinencia fecal y disminución de la movilidad en ambas extremidades inferiores. Inicialmente observamos en el líquido pleocitosis, proteinorraquia y niveles elevados de células de alta fluorescencia, asociadas en ocasiones a células malignas. El proteinograma e inmunofijación del líquido confirmó la presencia del componente monoclonal ya detectado en sangre, y tras procesar la muestra por citometría de flujo pudimos confirmar la infiltración de células plasmáticas malignas en el sistema nervioso central. Nuestro laboratorio desempeñó un papel central y esencial en el diagnóstico de esta infrecuente complicación, mediante el uso combinado del proteinograma, la inmunofijación, la citometría de flujo y el autoanalizador hematológico, incluyendo en este último las células de alta fluorescencia, prometedor biomarcador en el cribado de la presencia de células tumorales en líquidos biológicos

Leptomeningeal involvement in multiple myeloma is a rare and serious complication that usually occurs after relapses of the disease. To establish a correct diagnosis, it is necessary to demonstrate, by cytology, the presence of clonal plasma cells in the cerebrospinal fluid. The clinical laboratory detected this complication in a patient diagnosed with refractory multiple myeloma after analysing a cerebrospinal fluid sample. The patient suffered from several neurological symptoms, such as faecal incontinence and lower limb mobility limitation. Pleocytosis and proteinorachia was initially observed, along with high levels of high-fluorescence cells, which are sometimes associated with malignant cells. The protein electrophoresis and immunofixation of the cerebrospinal fluid confirmed the presence of the monoclonal component, already detected in blood. After processing the sample by flow cytometry it was confirmed that there was infiltration of malignant plasma cells in the central nervous system. This laboratory played a central and essential role in the diagnosis of this uncommon complication, by the combined use of protein electrophoresis, immunofixation, flow cytometry, and the haematology autoanalyser. This latter included the high fluorescence cells as a promising biomarker in the screening for the presence of tumour cells in biological fluids

A study of detection and comparison of immunofluorescence on formalin-fixed paraffin-embedded tissue with fresh frozen renal biopsy specimen.

Direct immunofluorescence on the fresh frozen tissue is established way of demonstrating of immunoglobulins and complement deposition in renal biopsies. IF studies can be done on paraffin-fixed tissue (IF-P) and give comparable results to those obtained on frozen tissue for most pathogenic immunoglobulins and immunoglobulin fragments; although, the detection of C3c may be more problematic. In our study, we used proteinase-K method for antigen retrieval. We aimed to detect immunoglobulins and complements in formalin-fixed paraffin-embedded (FFPE) tissue sections from renal biopsies and have comparison of IF staining intensity on FFPE sections with conventional IF on fresh frozen tissue. Based on our results, we conclude that IF-P can serve as salvage technique and has significant diagnostic utility.

Concentrations of Serum Free Light Chains in Kappa and Lambda Lesions in Light-Chain Myelomas.

BACKGROUND: Concentration of serum free light chains has been promoted as an assay in the evaluation of monoclonal gammopathic manifestations. A high false-positive rate in patients without monoclonal gammopathic manifestations and a high false-negative rate in patients with those manifestations have been reported. The false-negative rate for lambda chain-associated lesions is higher than that for kappa chain-associated lesions. OBJECTIVE: To assess the serum free light chains in light-chain myelomas. METHODS: Concentrations of involved and uninvolved serum free light chains were retrospectively reviewed in patients with light-chain myelomas. RESULTS: The highest recorded levels of involved light chains in kappa-chain myelomas and lambda-chain myelomas were comparable. The levels of uninvolved light chains were higher, although not statistically significantly, in lambda-chain lesions. CONCLUSIONS: The results of serum free light chains in light-chain myelomas support the clinical usefulness of the assay in monitoring patients with light chain myeloma.

[Multifocal chalazodermic amyloidosis: The concept of immunoglobulinemic elastopathy]. / Chalazodermie amyloïde multifocale. Le concept d'élastopathie immunoglobulinémique.

INTRODUCTION: Impairment of dermal elastic tissue occurs in different entities associated with immunoglobulins or immunoglobulin-derived protein-secreting clonal plasma cell proliferations, such as amyloid elastosis, anetodermic nodular amyloidosis or monoclonal gammopathy-associated cutis laxa. We report a case of cutaneous immunoglobulinemic amyloidosis revealed by a unique chalazodermic presentation and we review elastic tissue impairment in patients with monoclonal gammopathies. OBSERVATION: A 67-year-old woman consulted for non-infiltrated anetodermic lesions on the upper left quadrant of her abdomen present for ten years. She also had a chalazodermic plaque with abnormal skin wrinkling and laxity in her right axilla. Biopsies revealed deep dermal and subcutaneous amyloid deposits. Immunohistochemistry with lambda light chain was positive. Orcein staining and electron microscopy showed extensive elastolysis. The patient presented no signs of systemic involvement, but a very small amount of monoclonal IgGλ gammopathy was detected during follow-up. DISCUSSION: This is a unique chalazodermic presentation of immunoglobulinemic amyloidosis that does not fit into a clearly-defined nosological setting. It highlights the complex interactions between immunoglobulin-derived proteins, including light and heavy chains, and elastic tissue components, leading to different types of impairment of the latter. We therefore suggest the unifying concept of immunoglobulinemic elastopathy, underscoring the need to screen for monoclonal gammopathy in patients presenting elastic tissue impairments.