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Background: Free light chain (FLC) quantification is the most analytically sensitive blood-based method commercially available to diagnose and monitor patients with plasma cell disorders (PCDs). However, instead of directly detecting monoclonal FLCs (mFLCs), FLC assays indirectly assess clonality based on quantifying κ and λ FLCs and determination of the к/λ FLC ratio. Often an abnormal FLC ratio is the only indication of a PCD, and confirmation by a direct method increases diagnostic confidence. The aim of this study was to develop an analytically sensitive method for direct detection of mFLCs., Methods: Patient sera (n = 167) previously assessed by nephelometric FLC quantification and immunofixation electrophoresis (IFE) were affinity enriched for IgG, IgA, and total and free κ and λ light chains and subjected to MALDI-TOF MS. Relative analytical sensitivity of these methods was determined using serially diluted sera containing mFLCs., Results: In sera with abnormal FLC ratios (n = 127), 43% of monoclonal proteins were confirmed by IFE, 57% by MALDI-TOF MS without FLC enrichment, and 87% with FLC enrichment MALDI-TOF MS. In sera with normal FLC ratios (n = 40), the FLC MALDI-TOF MS method identified 1 patient with an mFLC. Serial dilution and analysis of mFLC containing sera by IFE, nephelometry, and FLC MALDI-TOF MS demonstrated that FLC MALDI-TOF MS analysis had the highest analytical sensitivity., Conclusions: FLC immunoenrichment coupled to MALDI-TOF MS enables direct detection of mFLCs and significantly increases the confirmation of abnormal serum FLC ratios over IFE and MALDI-TOF MS without FLC enrichment, thereby providing added confidence for diagnosing FLC PCDs., (© 2019 American Association for Clinical Chemistry.)

Background: 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., Methods: 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., Results: 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%)., Conclusions: 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.

Background: Currently, monoclonal immunoglobulins are identified and quantified from bands on electrophoretic gels. As an alternative, clonality might be determined by measuring the separate light chain types of each Ig class to allow numerical assessment of Ig'kappa/Ig'lambda ratios, analogous to free light chain kappa/lambda ratios., Methods: Using immunization, tolerization, and adsorption procedures, we prepared sheep antibodies against each of the 6 separate molecules, IgGkappa, IgGlambda, IgAkappa, IgAlambda, IgMkappa, and IgMlambda. Antibody targets comprised the junctional epitopes between the heavy chain and light chain domains. After purification, we assessed the antisera on a Siemens Dade-Behring BN II nephelometer for analytical quality and clinical utility., Results: High-avidity, specific antibodies allowed the production of automated nephelometric immunoassays for each Ig light chain type. Laboratory comparison with serum protein electrophoresis, using dilution experiments, showed lower analytical sensitivity for monoclonal IgG detection but similar or greater sensitivity for IgA and IgM, particularly when the monoclonal bands overlaid transferrin. Results obtained from typing of monoclonal proteins into IgG, A, or M types were comparable with results obtained by immunofixation-electrophoresis methods. Initial clinical studies, in multiple myeloma patients, indicated that Ig'kappa/Ig'lambda ratios were sometimes more sensitive than immunofixation electrophoresis, provided numerical results, and correlated with changes in disease., Conclusions: Immunoassays for intact Ig kappa/lambda pairs are possible and should assist in the management of patients with monoclonal gammopathies.

Background: Although a homogeneous assay for serum LDL-cholesterol (LDL-C) has become a routine clinical procedure, problems remain in assay performance characteristics., Methods: We examined the performance of a recently developed automated homogeneous assay (New-Daiichi assay) for serum LDL-C and compared the results with those obtained by the current homogeneous method (Denka-Seiken assay) or by ultracentrifugation as a control., Results: The New-Daiichi assay showed satisfactory basic performance characteristics such as reproducibility, linearity, and stability. There was no interference in the assay by various substances examined. The LDL-C values obtained with this method correlated well with those obtained by ultracentrifugation. In samples from patients with obstructive jaundice, both methods detected cholesterol from abnormal lipoproteins (such as lipoprotein-X and -Y), but the New-Daiichi assay was less reactive and more specific for LDL-C., Conclusion: The new method has improved performance for the accurate measurement of LDL-C in clinical practice.

Background: Myeloma-induced bone loss is related to an uncoupling of bone formation and bone resorption. The aim of the present study was to assess the potential clinical value of biochemical markers of bone formation in the work up of patients with plasma cell dyscrasias., Methods: Serum total alkaline phosphatase, bone-specific alkaline phosphatase (BAP), and osteocalcin (OC) were measured in 43 patients with newly diagnosed multiple myeloma (MM), in 40 patients with monoclonal gammopathy of undetermined significance (MGUS), in 40 patients with untreated benign vertebral osteoporosis (OPO), and in 48 healthy adults., Results: In MM and MGUS patients, serum BAP, but not serum OC, was lower than in healthy controls (P<0.05). Serum OC was higher in patients with OPO than in healthy controls (P<0.05). The strongest associations between markers were found in OPO patients and in healthy adults. MM patients with early-stage disease or without detectable osteolysis had decreased serum BAP values (P<0.05). Serum OC was higher in MM patients with stage III disease (P<0.05) than in healthy controls. MM patients with OPO-like bone involvement had lower BAP values than sex- and age-matched MGUS patients with OPO-like bone involvement and patients with benign OPO (P<0.05)., Conclusions: In patients with plasma cell dyscrasias, serum BAP, rather than serum OC, appears to reflect a suppressed bone formation rate and may be helpful in the differentiation between benign and myeloma-induced OPO. However, the overall clinical use of biochemical markers of bone formation in patients with plasma cell dyscrasia appears limited.

Background: The measurement of immunoglobulin E (IgE) in serum is widely used in the diagnosis of allergic reactions and parasitic infections. We describe here a fully automated assay for human IgE suitable for routine application in a general chemistry analyzer., Methods: We used an ILab 900 analyzer. This instrument automates a particle-enhanced immunoturbidimetric assay with an analysis time of 9 min., Results: The assay was linear in the range 4-1000 kIU/L (r = 0.9998). The intra- and interassay CVs at 57, 235, and 434 kIU/L were <3.5% and <7.4%, respectively. The detection limit was 4 kIU/L. Hemoglobin (

We evaluated the performance of a homogeneous assay for the automated measurement of high-density lipoprotein cholesterol (HDL-C) and compared it with a conventional precipitation technique in the following groups of people: control subjects (group A), clinically-healthy elderly (group B), myocardial infarction patients (group C), nephrotic syndrome patients (group D), and liver cirrhosis patients (group E). The performance of the technique was acceptable with respect to precision, accuracy, linearity, and detection limit. Triglycerides up to 40 mmol/L and bilirubin up to 150 micromol/L did not cause interferences. Hemoglobin decreased HDL-C measurements. Samples were stable at -20 degrees C for up to four months. Bland-Altman plots showed a good agreement between both techniques in the control group but with a progressive divergence in the patient groups B to E. Results indicate limitations of the technique in certain clinical conditions and, coincidentally, the need for reliable calibration materials.

Capillary electrophoresis (CE) and immunosubtraction capillary electrophoresis (IS-CE) were compared with the conventional methods agarose gel electrophoresis (AGE) and immunofixation electrophoresis (IFE) for detection and identification of paraproteins. In total, 74 paraproteins out of 468 serum samples were detected by both methods. Seventy-three monoclonal bands with concentrations ranging from 0.6 to 50.9 g/L were detected by the routine method. With CE, 70 paraproteins were detected and quantified on the electropherogram. Four paraproteins were not detected by CE; three of these were IgG (0.6, 1.1, and 2.2 g/L, respectively), and one was a IgM paraprotein (20.3 g/L) that could be visualized by minor changes in the running conditions. In comparison with IFE, 69 paraproteins were typed identically using IS-CE; only one paraprotein (IgMkappa, 14.9 g/L) could not be identified. On the other hand, a monoclonal IgA band that had not been detected by AGE was identified by CE and IS-CE. We conclude that, in general, CE could be a useful method for detection of paraproteins and that IS-CE is a good alternative to IFE. Additional studies are required to investigate the ionic strength and pH of the running buffer, because these prove to be the most crucial factors for routine CE separation of paraproteins.

Semiautomated agarose electrophoresis and immunofixation performed with Hydrasys-Hyrys (Sebia) were compared with conventional, manually performed methods, including cellulose acetate electrophoresis, immunoelectrophoresis, and immunofixation. Reference intervals for agarose electrophoresis with Hydrasys-Hyrys were determined. Within-run imprecision (CV) for fraction quantitation with the semiautomated system was between 1% (albumin) and 4.5% (beta-globulin). Total imprecision (CV) was between 2.7% (albumin) and 7.3% (beta-globulin). Semiautomated agarose electrophoresis showed linear correlation with cellulose acetate electrophoresis. Thirty-four specimens with monoclonal components were analyzed by manual immunoelectrophoresis and immunofixation and by Hydrasys. In one case, a light-chain disease was missed with Hydrasys when the sample was diluted 1:3 (the routine dilution) but not when the sample was assayed undiluted. In another case, the Hydrasys system revealed a small IgGA monoclonal component in addition to the IgA monoclonal component detected by the manual methods. In the other cases, no differences between the manual methods and the semiautomated method were seen with respect to paraprotein identification.

We compared the automated Paragon 2000 clinical capillary zone electrophoresis (CZE) system with two manual methods, agarose electrophoresis (AGE) and cellulose acetate electrophoresis (CAE). Reference intervals in healthy adults were determined for each method. When compared with AGE and CAE, CZE gave substantially higher reference values for the alpha1-globulin fraction. With CZE, within-run precision for fraction quantitation was between 0.5% (albumin) and 4.1% (alpha1-globulin). Total precision was between 0.8% (albumin) and 5.3% (beta-globulin). Data obtained from CZE showed poor linear correlation with results obtained by AGE but good linear correlation with data from CAE. Analysis of serum from patients with inter alia inflammation, nephrotic syndrome, or polyclonal gammopathy showed that clinical information obtained by CZE is comparable with information obtained by AGE and CAE. We conclude that CZE offers a clinically reliable alternative to AGE and CAE and has the advantages of automation, higher precision, and faster turnaround time.

A selection of 58 specimens with a monoclonal component identified by immunoelectrophoresis and/or immunofixation was analyzed with the immunosubtraction procedure on the Paragon 2000 capillary electrophoresis system. The capillary system detected 93% of the paraproteins and, using immunosubtraction, correctly identified 91% of the paraproteins. Paraproteins that were detected by immunofixation and/or immunoelectrophoresis but not by capillary electrophoresis were also missed by agarose electrophoresis and cellulose acetate electrophoresis. Cellulose acetate electrophoresis was the least sensitive method for detection of paraproteins. Only 74% of the monoclonal components were detected by this technique, whereas 86% were revealed by agarose electrophoresis. In addition to monoclonal paraproteins, we also studied biclonal paraproteins and oligoclonal banding. Capillary electrophoresis and immunosubtraction correctly detected and identified three specimens containing biclonal paraproteins. In one specimen, capillary zone electrophoresis detected only one band, whereas agarose gel electrophoresis detected two bands. The sensitivity for detection and identification of oligoclonal banding by capillary electrophoresis was inferior to immunofixation.

Phosphate concentrations were determined in 52 cases of paraproteinemia. The unmodified acidic ammonium molybdate method produced 19% spuriously high results. The false increase of phosphate concentration was attributable to formation of precipitate in the reaction mixture. The precipitate was formed by interaction between immunoglobulins and the unmodified acidic ammonium molybdate reagent. The magnitude of interference bore no relation to the type, concentrations, or isoelectric point of the paraproteins or to the presence or absence of free light chains. Diluting the sample to approximately 40 g/L total protein reduced but did not always eliminate the interference. In some cases paraprotein concentration as low as 8 g/L falsely increased plasma phosphate results. Apparently, only IgG and IgM but not IgA paraproteins produced the interference. Deproteination by ultrafiltration or by treatment with trichloroacetic acid removed the interference. The Kodak slide method and the new modified Boehringer Mannheim phosphate test were found to be interference-free. However, in some cases the latter new formulation is sensitive to substantial changes in ionic concentration of the reaction mixture.

Each monoclonal protein (M-protein, or myeloma protein) consists of two heavy polypeptide chains of the same class and subclass and two light polypeptide chains of the same type. Electrophoresis on cellulose acetate membranes is satisfactory for screening, although agarose electrophoresis is more sensitive for detecting small M-proteins. Immunoelectrophoresis should be performed when myeloma, macroglobulinemia, amyloidosis, or a related disorder is suspected. Immunofixation is useful when results of immunoelectrophoresis are equivocal. The recognition of a Bence Jones protein depends on the demonstration of a monoclonal light chain by immunoelectrophoresis or immunofixation of an adequately concentrated urine specimen. The differential diagnosis of an M-protein includes monoclonal gammopathy of undetermined significance (benign monoclonal gammopathy), multiple myeloma, solitary plasmacytoma of bone or extramedullary plasmacytoma, macroglobulinemia, lymphoma, chronic lymphocytic leukemia, and primary systemic amyloidosis.

alpha 1-Microglobulin (alpha 1m), a glycoprotein (Mr = 30,000) found in serum and urine, is also present in serum conjugated to monomeric IgA (alpha 1-m-IgA). We have developed a simultaneous enzyme-linked immunoenzyme/immunoradiometric assay that involves three different monoclonal antibodies. Assay of serial dilutions of serum and urine demonstrated parallelism. Normal mean concentrations in serum (n = 75) were: total alpha 1m, 2.33 mumol/L; alpha 1m-IgA, 1.24 mumol/L; unconjugated (free) alpha 1m, 1.09 mumol/L; molar ratio (alpha 1m-IgA/total alpha 1m), 0.53. The mean concentration of alpha 1m in eight urine specimens from normal individuals was 0.19 mumol/L, with no detectable alpha 1m-IgA. A low urinary pH does not significantly affect assay results, unlike assays of beta 2-microglobulin. In patients with myeloma-related renal disease, total and free alpha 1m values for serum correlated well with values for creatinine and beta 2-microglobulin in serum.

Human sera were subjected to analytical ultracentrifugation and "high-pressure" gel-permeation chromatography on a system of combined TSK Gel G5000 PW and G3000 SW columns. The chromatographic method produced remarkably superior resolution of the proteins, especially those exceeding 100 000 Da. We calculated the molecular masses of eluted fractions on the basis of their detection by low-angle laser light scattering and their differential refractive index. We discuss the results in relation to the clinical data.

Pregnancy-specific beta 1-glycoprotein (SP1) was assayed by particle-counting immunoassay in serum from 86 healthy blood donors and 236 patients with various types of gammopathy. A concentration of 1 microgram/L was taken as the upper normal limit. Abnormally high values were found in one of 10 patients with monoclonal gammopathy of undetermined significance, in 65% of 152 patients with multiple myeloma, in 84% of 64 patients with Waldenström's macroglobulinemia, and in seven of 10 patients with monoclonal gammopathies associated with other myeloproliferative disorders. In a study of 90 myeloma patients, the SP1 value correlated (p less than 0.001) with the concentration of beta 2-microglobulin in serum, a value which had been corrected for possible renal dysfunction, but not with the concentration of the monoclonal component. SP1 was detected by direct immunofluorescence in myeloma cells of bone-marrow smears from six of 10 patients with myelomatosis. These six patients had serum SP1 values greater than 1 microgram/L, whereas the four patients with fluorescence-negative myeloma cells had SP1 values less than 1 microgram/L.

Our study of 95 serum samples from 37 patients with monoclonal gammopathy revealed distorted irregular monoclonal (M) protein bands after serum protein electrophoresis (SPE) on cellulose acetate membrane. In 71 (75%) of the 95 sera, the M-protein was underestimated and the albumin concentration overestimated. Dilution of the serum sample before SPE eliminated the abnormality of the M-protein bands. By SPE, the mean albumin concentration in these 71 undiluted sera was 45.8 (SD 7.4) g/L vs 37.9 (SD 5.8) g/L for the diluted sera; moreover, this was true of individual samples: measured albumin concentration in each diluted serum sample was always less than in the undiluted serum. As measured by the bromcresol green dye-binding method, the albumin concentration was 32.8 (SD 5.9) g/L. Similarly, the M-protein concentration in SPE was 49.5 (SD 12.3) g/L for the undiluted sera vs 61.8 (SD 15.1) g/L for the diluted sera, and the M-protein concentration in each diluted serum sample always exceeded that in the undiluted serum. Underestimation of M-protein limits the usefulness of M-protein measurement in evaluating the patient's response to therapy and for early detection of disease progression. SPE strips should be carefully inspected visually, and sera with M-protein band abnormalities should be diluted and re-assayed if SPE is to quantify concentrations of M-protein and albumin accurately.

We have modified the periodic acid-Schiff staining technique for glycoproteins for use with thin agarose-film electrophoresis membranes. With this procedure, carbohydrate-rich proteins can be detected with negligible background staining and insignificant staining of nonglycoproteins such as albumin and nonglycosylated Bence Jones proteins (free light chains). On the other hand, carbohydrate-rich M components of immunoglobulins M and A in serum and in cerebrospinal fluid from patients with plasma cell dyscrasia are readily detected. The technique is two- to threefold more sensitive than Ponceau S. Glycoproteins in serum and body fluids can be determined as a routine analytical procedure.

A direct mehtod [Clin. Chim. Acta 46, 113 (1973)] for determination of inorganic phosphate in serum was adapted for use with a centrifugal analyzer. Contamination is minimized and analysis rate maximized by doing the reaction in the reagent wells of the transfer disc and by utilizing the high-speed spectrophotometric and data-reduction capabilities of the centrifugal analyzer. Hemolysis, icterus, and moderate lipemia cause no interference. Grossly lipemic sera and sera from patients with plasma cell dyscrasias can be analyzed by incorporating appropriate blanking and dilution techniques. The method exhibits excellent sensitivity and precision and results correlate well with those from a continuous-flow procedure.