Keren, David F.

Abstract Introduction Multiple myeloma (MM) is characterized by the abnormal proliferation of plasma cells, resulting in the overproduction of distinctive monoclonal proteins (M-protein). Suspected MM necessitates screening for M-protein through a combination of serum protein electrophoresis, serum immunofixation (SIFE), and serum free light chain (SFLC) determination. An M-protein appears as a relatively restricted band on agarose gel, where migration in ɑ-2 is rare. Methods A 55-year-old man with pulmonary tuberculosis presented with severe lower back pain. On examination, he appeared chronically ill, with conjunctival pallor. X-rays revealed vertebral compression fractures. The full blood count confirmed anemia; however, serum calcium and creatinine levels did not meet myeloma-defining event criteria. Results The serum protein electrophoresis revealed hypogammaglobulinemia, with the SIFE demonstrating unusual unrestricted κ staining in the ɑ-2 region. A markedly elevated κ SFLC and κ:λ ratio were found. Bone marrow examination demonstrated approximately 90% plasmacytosis. Urine immunofixation revealed a small, restricted κ band disproportionate to the κ SFLC. Notably, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified only polyclonal κ SFLC. Discussion Given the absence of a discernible M-protein on SIFE, a small κ restriction on urine immunofixation, and a polyclonal increase in κ SFLCs, the patient’s condition is being managed as an oligosecretory MM.

Abstract Background THES is a rare autosomal recessive syndromic enteropathy caused by pathogenic variants in two genes, the Tetratricopeptide Repeat Domain 37 (TTC37) and Ski2 Like RNA Helicase (SKIV2L), that encode components of the human SKI complex involved in RNA degradation. This disease is characterized by several clinical features with variable penetrance including intractable congenital diarrhea, wooly hair abnormalities, intrauterine growth restriction, facial dysmorphism, immune dysfunction, liver abnormalities, and skin hyperpigmentation. Hypogammaglobulinemia with low IgG and normal to low IgM levels have been described in these cases which may require intravenous immunoglobulin (IVIG) replacement therapy. Some cases have a reduced population of switched memory B cells, T cells with reduced IFN-γ secretion, and hyporesponsive NK cells. Our patient is a 4-year-old female reported to have a homozygous TTC37 mutation with clinical manifestations of TPN dependence, liver fibrosis with portal hypertension, splenomegaly, developmental delay, thrombocytopenia and anemia requiring transfusion, and a history of hypogammaglobinemia requiring IVIG replacement. Patient would go on to develop elevated immunoglobin levels leading to additional testing described below. Methods Serum total protein analysis performed using the using Siemens Atellica CH Total Protein II test (Weichselbaum method using biuret reagent). Serum immunoglobin (IgA, IgG and IgG), IgG subclass, and kappa/lambda free light chain level analysis performed by turbidimetric photometry assay using a Binding Site Optilite analyzer. Serum protein electrophoresis and immunotyping performed using CAPILLARYS 2 capillary zone electrophoresis and immunosubtraction with antisera. Serum protein immunofixation performed using Sebia Hydrasys 2 with Hydragel 9 IF Maxi kit. Confirmation and final determination of serum immunoproteins performed at our reference laboratory using MASS-FIX (MALDI-TOF MS) matrix-assisted laser desorption/ionization-time of flight mass spectrometry after patient serum applied to 5 separate immunoaffinity resins specific to immunoglobulin G, A, M, K, and L for purification. Results Initial testing showed a broadly migrating beta-gamma region IgG kappa monotypic restriction measuring 1.7 g/dL, with predominantly kappa subtraction and trivial lambda subtraction by immunosubtraction; serum free kappa/lambda light chain ratio was increased at 22.94. Serum immunoglobins and free light chains measured (mg/dL): IgA 738 (15 - 160), IgM 48 (50 - 240), IgG 2,805 (405 - 1,160), free kappa 20.42 (0.33 - 1.94), and free lambda 0.89 (0.57 - 2.63). IgG subclasses measured (mg/dL): IgG1 533 (320 - 950), IgG2 174 (50 - 340), IgG3 16.9 (10.0 - 120.0), IgG4 1,221.1 (2.0 - 110.0). Additional serum protein values included (g/dL): total protein 8.10 (6.00 - 8.30), albumin 2.97 (3.43 - 4.84), alpha-1 0.32 (0.21 - 0.44), alpha-2 0.52 (0.54 - 0.97), beta globulin 3.42 (0.65 - 1.03), and gamma globulin 0.87 (0.70 - 2.30). The polyclonal nature of the IgG4 spike was later demonstrated by the MASS-FIX mass spectrograph. Conclusions MASS-FIX is a useful addition to the toolset used in the diagnosis of possible monoclonal gammopathies and protein disorders, especially where broad monotypic restrictions are observed or in patients with abnormal presentations for monoclonal gammopathies. Using current standard techniques, the above case could have been incorrectly classified as a monoclonal process resulting in unnecessary cost and incorrect treatment.

Abstract This guideline provides the recommendations of an expert panel for the clinical and laboratory evaluation of patients suspected of having a clinical condition that produces a monoclonal protein in serum or urine. The recommendations describe the clinical conditions in which a monoclonal protein should be sought, the optimal sequence of testing to diagnose and monitor these patients, and the most effective laboratory procedures.

Abstract Background Electrophoretic methods to detect, characterize and quantify M-proteins play an important role in the management of patients with monoclonal gammopathies (MGs). Significant uncertainty in the quantification and limit of detection (LOD) is documented when M-proteins are <10 g/L. Using spiked sera, we aimed to assess the variability in intact M-protein quantification and LOD across 16 laboratories. Methods Sera with normal, hypo- or hyper-gammaglobulinemia were spiked with daratumumab or elotuzumab, with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Laboratories blindly analyzed samples according to their serum protein electrophoresis (SPEP)/isotyping standard operating procedures. LOD and intra-laboratory percent coefficient of variation (%CV) were calculated and further specified with regard to the method (gel/capillary electrophoresis [CZE]), gating strategy (perpendicular drop [PD]/tangent skimming [TS]), isotyping (immunofixation/immunosubtraction [ISUB]) and manufacturer (Helena/Sebia). Results All M-proteins ≥1 g/L were detected by SPEP. With isotyping the LOD was moderately more sensitive than with SPEP. The intensity of polyclonal background had the biggest negative impact on LOD. Independent of the method used, the intra-laboratory imprecision of M-protein quantification was small (mean CV = 5.0%). Low M-protein concentration and high polyclonal background had the strongest negative impact on intra-laboratory precision. All laboratories were able to follow trend of M-protein concentrations down to 1 g/L. Conclusions In this study, we describe a large variation in the reported LOD for both SPEP and isotyping; overall LOD is most affected by the polyclonal immunoglobulin background. Satisfactory intra-laboratory precision was demonstrated. This indicates that the quantification of small M-proteins to monitor patients over time is appropriate, when subsequent testing is performed within the same laboratory.

Abstract Background Serum protein electrophoresis (SPEP) is used to quantify the serum monoclonal component or M-protein, for diagnosis and monitoring of monoclonal gammopathies. Significant imprecision and inaccuracy pose challenges in reporting small M-proteins. Using therapeutic monoclonal antibody-spiked sera and a pooled beta-migrating M-protein, we aimed to assess SPEP limitations and variability across 16 laboratories in three continents. Methods Sera with normal, hypo- or hypergammaglobulinemia were spiked with daratumumab, Dara (cathodal migrating), or elotuzumab, Elo (central-gamma migrating), with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Provided with total protein (reverse biuret, Siemens), laboratories blindly analyzed samples according to their SPEP and immunofixation (IFE) or immunosubtraction (ISUB) standard operating procedures. Sixteen laboratories reported the perpendicular drop (PD) method of gating the M-protein, while 10 used tangent skimming (TS). A mean percent recovery range of 80%–120% was set as acceptable. The inter-laboratory %CV was calculated. Results Gamma globulin background, migration pattern and concentration all affect the precision and accuracy of quantifying M-proteins by SPEP. As the background increases, imprecision increases and accuracy decreases leading to overestimation of M-protein quantitation especially evident in hypergamma samples, and more prominent with PD. Cathodal migrating M-proteins were associated with less imprecision and higher accuracy compared to central-gamma migrating M-proteins, which is attributed to the increased gamma background contribution in M-proteins migrating in the middle of the gamma fraction. There is greater imprecision and loss of accuracy at lower M-protein concentrations. Conclusions This study suggests that quantifying exceedingly low concentrations of M-proteins, although possible, may not yield adequate accuracy and precision between laboratories.

Abstract Appropriate utilization of clinical laboratory services is important for patient care and requires institutional stewardship. Clinical laboratory stewardship programs are dedicated to improving the ordering, retrieval, and interpretation of appropriate laboratory tests. In addition, these programs focus on developing, maintaining, and improving systems to provide proper financial coverage for medically necessary testing. Overall, clinical laboratory stewardship programs help clinicians improve the quality of patient care while reducing costs to patients, hospitals, and health systems. This document, which was created by a new multiinstitutional committee interested in promoting and formalizing laboratory stewardship, summarizes core elements of successful hospital-based clinical laboratory stewardship programs. The core elements will also be helpful for independent commercial clinical laboratories.

Context.— Serum tests used for the screening and diagnosis of monoclonal gammopathies include serum protein electrophoresis (SPE; agarose gel or capillary zone), immunofixation (IFE) and immunosubtraction capillary electrophoresis, serum free light chains, quantitative immunoglobulins, and heavy/light–chain combinations. Urine protein electrophoresis and urine IFE may also be used to identify Bence-Jones proteinuria. Objective.— To assess current laboratory practice for monoclonal gammopathy testing. Design.— In April 2016, a voluntary questionnaire was distributed to 923 laboratories participating in a protein electrophoresis proficiency testing survey. Results.— Seven hundred seventy-four laboratories from 38 countries and regions completed the questionnaire (83.9% response rate; 774 of 923). The majority of participants (68.6%; 520 of 758) used agarose gel electrophoresis as their SPE method, whereas 31.4% (238 of 758) used capillary zone electrophoresis. The most common test approaches used in screening were SPE with reflex to IFE/immunosubtraction capillary electrophoresis (39.3%; 299 of 760); SPE only (19.1%; 145 of 760); SPE and IFE or immunosubtraction capillary electrophoresis (13.9%; 106 of 760); and SPE with IFE, serum free light chain, and quantitative immunoglobulins (11.8%; 90 of 760). Only 39.8% (305 of 767) of laboratories offered panel testing for ordering convenience. Although SPE was used by most laboratories in diagnosing new cases of myeloma, when laboratories reported the primary test used to follow patients with monoclonal gammopathy, only 55.7% (403 of 724) chose SPE, with the next most common selections being IFE (18.9%; 137 of 724), serum free light chain (11.7%; 85 of 724), and immunosubtraction capillary electrophoresis (2.1%; 15 of 724). Conclusions.— Ordering and testing practices for the screening and diagnosis of monoclonal gammopathy vary widely across laboratories. Improving utilization management and report content, as well as recognition and development of laboratory-directed testing guidelines, may serve to enhance the clinical value of testing.

Summary Automated high performance liquid chromatography and Capillary electrophoresis are used to quantitate the proportion of Hemoglobin A 2 (HbA 2 ) in blood samples order to enable screening and diagnosis of carriers of β‐thalassemia. Since there is only a very small difference in HbA 2 levels between people who are carriers and people who are not carriers such analyses need to be both precise and accurate. This paper examines the different parameters of such equipment and discusses how they should be assessed.