Methodological Evaluation And Clinical Application Of Light Chain Assays For Clonal Proliferative Diseases

BackgroundThe analysis of serum free light chains(FLCs)is of clinical relevance for the diagnosis and therapeutic management of clonal proliferative disorders.Quantification of monoclonal immunoglobulins(M proteins)is routinely performed by delineating serum protein electrophoresis(SPE)regions.However,quantification of β-migrating M proteins is hampered by overlapping non-immunoglobulins.Immunosuppression on capillary electrophoresis(ISUB)is currently used to qualitatively subtract(and thus highlight)immunoglobulin isotypes in serum,thereby reducing the obstruction of normal serum proteins.IgG(mainly IgG3)is the most commonly involved isotype in proliferative glomerulonephritis with monoclonal immunoglobulin deposition(PGNMID).ObjectiveIn this study we compare the performance of monoclonal and polyclonal FLC κand λ assays in clinical samples and develop a quantitative immunodepressant(qIS)method that extends the traditional ISUB.We also describe a PGNMID series with only monoclonal immunoglobulin light chain deposition(PGNMID-light chain).Materials and methods(1)A total of 3213 patients hospitalized in our hospital from March to October 2021 were selected as disease group(serum total protein>85 g/L or serum albumin/globulin(A/G)ratio was inverted or serum globulin>40 g/L);In addition,24 patients with multiple myeloma diagnosed in hematology department were selected as positive control group.A total of 200 health examination samples aged 60 and above were randomly collected as normal control group,of which 160 were reference interval establishment cohort and 40 were reference interval validation cohort.Serum FLC was measured and analyzed using Freelite and N Latex on BN ProSpec system(Siemens).FLC results were combined with protein electrophoresis,immunofixation,C-reactive protein,and estimated glomerular filtration rate(eGFR)data to evaluate performance.(2)From 3213 cases of disease group pick the samples of 3 patients with β-region M protein were diluted,and SPE and ISUB were performed on each diluted sample.The differences between ISUB electrophoresis traces and the involved ISUB isotype decompression traces were visualized to distinguish between M proteins and background polyclonal immunoglobulins.The contribution of M protein to the beta region was calculated and applied to the beta region protein concentration to produce quantitative M protein concentrations while minimizing contamination by non-immunoglobulin or polyclonal immunoglobulin.(3)The clinical data of the patients were analyzed,medical records were reviewed,and the diagnosis and treatment of the patients were followed up.From 3213 cases of disease group pick the samples of 3 patients with PCNMID-LC.The clinical and immunopathological characteristics and results of PGNMID-LC were determined by optical microscopy,fluorescence staining,and electron microscopy.Results(1)Poor agreement between methods was observed,including constant and proportional bias and poor correlation.Unlike the FLC κ/λ ratio results from the Freelite assay,the N Latex assay was not affected by the eGFR estimate of renal impairment.With the Freelite assay,98.0%of putative controls without monoclonal haematopoiesis showed κ/λ ratios above the standard diagnostic range or the median of the renal diagnostic range.(2)The measurable range was determined to span the maximum concentration tested(8.3 g/L)to 0.4 g/L with a quality target of 25%error.a Passing-Bablok regression between qIS and the expected M-protein yielded a slope of 1.05(95%CI,0.93-1.10),r=0.99.(3)15 patients presented with nephritis or nephrotic syndrome and their underlying haematological disease was monoclonal genomic disease(n=10)or multiple myeloma(n=5).Monoclonal immunoglobulins were identified by serum and urine immunofixation in 73.3%and 73.0%of patients respectively,serum free light chains were abnormal in 82.0%of patients and bone marrow plasma cell clones were detectable in 86.7%of patients.Renal biopsies showed a pattern of membranous hyperplasia in most patients.By immunofluorescence,the deposits were confined to the glomerulus and consisted of confined light chains and C3,which were granular on electron microscopy and distributed in the subendothelium,submesothelium and subepithelium.Following follow-up,renal response was dependent on haematological response and was seen in five of the nine patients who received plasma cell-directed chemotherapy.Conclusion(1)Unlike the Freelite assay,the κ/λ ratio analysed with the N Latex assay was unaffected by renal failure.Both methods show acceptable performance,but they correlate poorly.(2)qIS is expected to quantify β-migrating M-protein at concentrations an order of magnitude lower than conventional SPE methods,allowing earlier detection of increases or decreases in M-protein.(3)PGNMID-light chain differs from PGNMID-IgG in the higher frequency of detectable pathogenic plasma cell clones.