Applied capillary electrophoresis system affects screening for monoclonal gammopathy in serum: verification study of two eight-capillary systems.

Capillary electrophoresis is a method with a long history of developments which enables monitoring of several pathological processes and has an irreplaceable role in screening for presence of M-protein. The aim of this study was to assess analytical performance of Sebia and Helena systems, as well as their screening efficiency for M-protein by identifying characteristic electrophoretic pattern abnormalities. The controls were analyzed in triplicate over a five-day period. Comparability testing was performed on 46 (Capillarys3Octa) and 49 (V8Nexus) serum samples with routinely used Capillarys2. Electropherograms (EPGs) were reviewed by two specialists independently to select samples for further processing by immunofixation. All precision test results met the eligibility criteria by Ricos et al. The correlation coefficients higher than 0.8 indicated excellent comparability although the results were slightly more comparable among the same manufacturer systems. There were no variations in observed abnormalities in EPGs when Capillarys systems were compared, but a disparity was detected in 11/49 EPGs on comparing Capillarys2 and V8Nexus. The cause of the detected difference could be in a different graphical presentation of the findings and in a lesser resolution of the applied buffer. The impression is that the V8Nexus system combined with the utilized buffer provides greater resolution in the alpha-1 and alpha-2 globulin fractions, but that it declines in the gamma globulin fraction. The precision and estimated accuracy criteria were met by both systems. Comparison results implied that capillary systems are not equally effective in M-protein screening, highlighting the necessity to include system screening efficiency in analytical evaluation.

Unusual total anti-SARS-CoV-2 antibody kinetics observed during longitudinal monitoring after BNT162b2 vaccination.

The present study aimed to clarify unusual total antibody kinetics in three female individuals observed during longitudinal monitoring of antibody response to BNT162b2 COVID-19 vaccine in 54 healthy volunteers. Total and IgG antibodies against the SARS-CoV-2 spike glycoprotein were measured using Roche and Abbott quantitative assays, respectively, a day before and 8, 71, 135 and 217 days after the second dose. Samples showing unusual kinetics were additionally tested with Beckman Coulter and Euroimmun IgG assays, as well as IgA assay. Antibody levels peaked 8 days after the second dose (total:2769 U/mL; IgG:20022 AU/mL) and declined to 611 U/mL (total) and 783 AU/mL (IgG), after 217 days. A delayed increase of total but not IgG antibodies evidenced in three females, was in two cases coupled with an increase in IgA antibodies. This study identified a previously unknown contribution of anti-SARS-CoV-2 IgA antibodies to a delayed total antibody increase in a subgroup of vaccinated individuals. It also emphasizes that different commercially available serological assays do not provide uniform information about the post-vaccination immune status and that thorough understanding the assays' features is crucial for the proper interpretation of antibody response monitoring.

Analytical validation of 39 clinical chemistry tests and 17 immunoassays on the Alinity analytical system.

The aim of this study was to perform the analytical validation of Alinity c and i analyzers (Abbott Laboratories, Chicago, IL, USA) for 39 clinical chemistry tests and 17 immunoassays. Precision was evaluated at least at two concentration levels for 5 days in quintuplicate, following CLSI EP15-A3. Method comparison included parallel analysis of leftover routine samples on Alinity analyzers and the previously used Cobas c501 and e601 (Roche Diagnostics, Mannheim, Germany). Linearity was tested by preparing sequential sample dilutions with high analyte concentration, following the CLSI EP6 document. For clinical chemistry tests, within-run coefficients of variation (CV) were up to 6.0% (beta-2-microglobulin), while between-run CVs up to 5.4% (immunoglobulin M). Among immunoassays, the highest within-run CV was obtained for vitamin B12 (6.9%), while between-run for CA 19-9 (4.3%). Complete agreement with Roche analyzers was observed for 16 (41%) clinical chemistry assays and 6 (35%) immunoassays. Half of all evaluated assays did not meet the desirable biological variation criteria for bias, being especially exceeded for alpha1-antitrypsin, apolipoprotein A1, ceruloplasmin, complement C3 and C4, hemoglobin A1c, lipoprotein (a) and myoglobin, as well as some tumor markers (CA 125, CEA, fPSA, AFP, and ferritin), hormones (cortisol, DHEA-S, insulin) and vitamins (25-OHD). Linearity in the tested ranges was confirmed. Overall, this study revealed that precision criteria derived from manufacturer's claims were not satisfied for all assays while comparison study for some assays yielded differences that imply the need for additional assay evaluation prior to introduction into routine practice.

The reliability of SARS-CoV-2 IgG antibody testing - a pilot study in asymptomatic health care workers in a Croatian university hospital.

AIM: To evaluate three fully automated serological assays in terms of reactivity to SARS-CoV-2 immunoglobulin G (IgG) and perform SARS-CoV-2 IgG antibody testing among asymptomatic health care workers (HCW) at the University Hospital Center Zagreb. METHODS: Three IgG serological assays (Abbott SARS-CoV-2 IgG, Elecsys Anti-SARS-CoV-2, and MAGLUMI 2019-nCoV IgG) were initially evaluated by analyzing 42 samples from confirmed COVID-19-recovered patients and 48 negative individuals. A total of 1678 HCW (~30% of all hospital employees) were screened for SARS-CoV-2 IgG with the Abbott assay, run on Abbott Architect i2000SR. The samples exceeding the predefined cut-off (1.4 S/C) were reanalyzed with the Elecsys, MAGLUMI, and VIDAS SARS-COV-2 IgG assays. RESULTS: Initially, the MAGLUMI 2019-nCoV IgG produced 26.2% false negatives and the Elecsys Anti-SARS-CoV-2 produced one false positive. Among 1678 HCW, the Abbott assay showed only 10 (0.6%) positive results, with mostly mildly elevated signals. Nine of these samples were non-reactive when they were retested with the Elecsys, MAGLUMI, and VIDAS assays. As for the one remaining sample, it was positive when tested with the Elecsys assay, while the other two assays yielded negative results. CONCLUSIONS: SARS-CoV-2 IgG seroprevalence among asymptomatic HCW in our hospital setting was low, with different assays indicating a different number of positive samples. One of the assays yielded a large false negative rate. These findings can be attributed to differences in assay formulation but also to heterogeneity and diverse reactivity of antibodies against SARS-CoV-2 antigens.

Immunoglobulin heavy/light chain analysis enhances the detection of residual disease and monitoring of multiple myeloma patients.

AIM: To evaluate the clinical utility of incorporating a novel heavy/light chain immunoassay (HLC) into the existing methods for the assessment of multiple myeloma (MM) patients. METHODS: Convenience sera samples from 90 previously treated IgG and IgA MM patients in different disease stages were analyzed. The study was conducted in Clinical Hospital Center Zagreb between 2011 and 2013. The collected sera were analyzed by standard laboratory techniques (serum protein electrophoresis, quantification of total immunoglobulins, serum immunofixation, serum free light chain [FLC] assay) and HLC assay. RESULTS: HLC ratios outside the normal range were found in 58 of 90 patients, including 28 out of 61 patients with total immunoglobulin measurements within the normal range and 5 out of 23 patients in complete response. Both elevated HLC isotype level and abnormal HLC ratio correlated with the parameters of tumor burden, including percentage of plasma cells in the bone marrow (P<0.001 and P=0.002, respectively) and an abnormal serum FLC ratio (for both P<0.001). In addition, abnormal HLC isotype level correlated with serum beta-2-microglobulin level (P=0.038). In terms of prognosis, abnormal HLC isotype level and abnormal HLC ratio were significantly associated with shorter overall survival (P<0.001 and P=0.002, respectively). Interestingly, suppression of the uninvolved (polyclonal) isotype pair, but not other non-myeloma immunoglobulin isotypes, was also associated with a shorter overall survival (P=0.021). In a multivariate analysis, an abnormal HLC ratio and ß2-microglobulin level >3.5mg/L were independent risk factors for survival. CONCLUSION: The new HLC assay has greater sensitivity in detecting monoclonal protein, correlates with tumor burden markers, and affects patients' outcome.

Antibody response and self-reported adverse reactions following vaccination with Comirnaty: a pilot study from a Croatian university hospital.

This study aimed to determine antibody responses against SARS-CoV-2 spike (S) after both BioNTech-Pfizer Comirnaty vaccine doses and study the correlation with self-perceived adverse reactions. Antibodies determination with Elecsys anti-SARS-CoV-2 S assay was performed a day prior to or just before administration of the second dose and 8-13 days after the second dose. Participants selected from a predefined list of the experienced local (injection site reactions) and/or systemic (fatigue, headache, myalgia, arthralgia, chills and fever) post-vaccination adverse reactions. An average 100-fold increase in antibody titre in naive vaccinees was observed between the two time points (median 67 U/mL vs 2841 U/mL, p<0.001). Participants aged below 50 had higher antibody titres (median 99 U/mL vs 26 U/mL, p=0.003 after the first dose; median 3617 U/mL vs 2556 U/mL, p=0.026 after the second dose). All reported adverse reactions were mild-to-moderate, with more participants declaring systemic reactions after the second dose (p=0.001), without a clear correlation with antibody titre.

A novel approach for more precise quantification of M-protein using variables derived from immunosubtraction electropherogram and associated biochemistry analytes.

Introduction: Due to limitations in currently used methodologies, the widely acknowledged approach for quantifying M-protein (MP) is not available. If employed as a source of quantitative data, the immunosubtraction electropherogram (IS-EPG), a qualitative analysis of MP, has the potential to overcome known analytical issues. The aim of this study is to explore measured and derived variables obtained from immunosubtraction electropherogram as a tool for quantifying MP and to compare the derived results to currently available methods. Materials and methods: Measurands were amplitudes of MP and albumin fractions. Assessed derived variables included also immunoglobulin (Ig) G, IgA, IgM and total protein data. Capillary electrophoresis was used for determination of MP (in % of total protein concentration, or concentration of MP in g/L) by perpendicular drop and tangent skimming method. Results: Passing-Bablok analysis showed the most comparable results in D1Ig and D1nIg variables, and the largest discrepancies in AD1nIg and AD2nIg variables. The background presence had greater impact on D1nIg comparison results than did on D1Ig results. The contribution of albumin fraction data did not improve the comparability of the results. The coefficients of variation of derived variables were lower (maximum 3.1%) than those obtained by densitometric measurements, regardless of MP concentration, polyclonal background, or migration pattern (2.3-37.7%). Conclusion: The amplitude of MP spike in IS-EPG is an valuable measurand to compute derived variables for quantifying MP. The most comparable results were achieved with the D1Ig variable. Patients with monoclonal gammopathy can benefit from increased precision employing an objective and background independent measurand, especially during longitudinal follow-up.

Humoral immune response to COVID-19 vaccines in people with secondary progressive multiple sclerosis treated with siponimod.

OBJECTIVE: The aim of this study is to determine a development of humoral response after COVID-19 vaccination in persons with secondary progressive multiple sclerosis (pwSPMS) on siponimod, compared to healthy controls (HC). METHODS: In 13 pwSPMS taking siponimod and 11 HC, testing for SARS-CoV2 antibodies was performed after vaccination against COVID-19. RESULTS: pwSPMS taking siponimod had a significantly lower titer of SARS-CoV2 antibodies compared to healthy controls (19.4 (0-250) vs. 250 (250), p>0.001). Two (15.4%) pwSPMS on siponimod had unmeasurable titers of SARS-CoV2-2 antibodies, while all HC had positive titers. CONCLUSION: Although the results of this study are limited by a small sample size, results have consistently shown low titers of SARS-CoV-2 IgG after COVID-19 vaccinations in pwSPMS on siponimod.

Non-Myelofibrosis Chronic Myeloproliferative Neoplasm Patients Show Better Seroconversion Rates after SARS-CoV-2 Vaccination Compared to Other Hematologic Diseases: A Multicentric Prospective Study of KroHem.

Disease- and treatment-mediated immunodeficiency might render SARS-CoV-2 vaccines less effective in patients with hematologic diseases. We performed a prospective non-interventional study to evaluate humoral response after one and two doses of mRNA-1273, BNT162b2, or ChAdOx1 nCoV-19 vaccine in 118 patients with different malignant or non-malignant hematologic diseases from three Croatian treatment centers. An electrochemiluminescent assay was used to measure total anti-SARS-CoV-2 S-RBD antibody titers. After one vaccine dose, 20/66 (33%) achieved seropositivity with a median antibody titer of 6.1 U/mL. The response rate (58/90, 64.4%) and median antibody titer (>250 U/mL) were higher after two doses. Seropositivity varied with diagnosis (overall p < 0.001), with the lowest rates in lymphoma (34.6%) and chronic lymphocytic leukemia (52.5%). The overall response rate in chronic myeloproliferative neoplasms (CMPN) was 81.3% but reached 100% in chronic myeloid leukemia and other non-myelofibrosis CMPN. At univariable analysis, age > 67 years, non-Hodgkin's lymphoma, active treatment, and anti-CD20 monoclonal antibody therapy increased the likelihood of no vaccine response, while hematopoietic stem cell recipients were more likely to respond. Age and anti-CD20 monoclonal antibody therapy remained associated with no response in a multivariable model. Patients with the hematologic disease have attenuated responses to SARS-CoV-2 vaccines, and significant variations in different disease subgroups warrant an individualized approach.

Humoral immune response in convalescent COVID-19 people with multiple sclerosis treated with high-efficacy disease-modifying therapies: A multicenter, case-control study.

AIM: To determine the influence of high-efficacy disease modifying therapy (DMT) on the development of IgG SARS-CoV-2 antibody response in COVID-19 convalescent people with multiple sclerosis (pwMS). METHODS: Seventy-four pwMS taking high-efficacy DMTs (specifically natalizumab, fingolimod, alemtuzumab, ocrelizumab, cladribine and ublituximab) and diagnosed with COVID-19 and 44 healthy persons (HC) were enrolled. SARS-CoV2 antibodies were tested with Elecsys® Anti-SARSCoV-2 S assay. RESULTS: pwMS taking high-efficacy DMTs had a significantly higher chance of having negative titer of SARS-CoV2 antibodies compared to healthy controls (33 negative pwMS [44.6%] compared to one negative HC [2.3%], p < 0.001). pwMS taking B-cell depleting therapy (ocrelizumab and ublituximab) had a significantly higher chance of having negative titer of SARS-CoV2 antibodies compared to pwMS on all other DMTs (29 negative pwMS on B-cell therapy [64.4%] compared to four negative pwMS on all other DMTs [13.8%], p < 0.001). Out of other DMTs, two (33.3%) pwMS taking fingolimod and two (16.7%) pwMS taking cladribine failed to develop IgG SARS-COV-2 antibodies. B-cell depleting therapy independently predicted negative titer of IgG SARS-CoV-2 antibody (Exp[B] =0.014, 95%CI 0.002-0.110, p < 0.001). CONCLUSIONS: A significant proportion of convalescent COVID-19 pwMS on high-efficacy DMTs will not develop IgG SARS-CoV-2 antibodies. B-cell depleting therapies independently predict negative and low titer of IgG SARS-CoV-2 antibody.

Verification study of free light chains assays on reagent-optimized analysers.

INTRODUCTION: Our aim was to compare analytical specifications of two assays (monoclonal vs. polyclonal) for free light chains (FLCs) quantification optimized for two different analytical platforms, nephelometer ProSpec (Siemens, Erlangen, Germany) and turbidimetric analyser Optilite (The Binding Site, Birmingham, UK). MATERIALS AND METHODS: The evaluation included verification of the precision, repeatability and reproducibility, estimation of accuracy and method comparison study with 37 serum samples of haematological patients. Kappa and lambda FLC were measured in each sample by both methods and kappa/lambda ratio was calculated. RESULTS: Results show satisfactory precision of both methods with coefficients of variation for ProSpec of CVwr = 2.20% and CVbr = 3.44%, and for Optilite CVwr = 2.82% and CVbr = 4.15%. Estimated bias for FLC lambda was higher on the ProSpec analyser, but bias for FLC kappa was higher on the Optilite analyser. Correlation coefficients were 0.98; P < 0.001 for FLC kappa and 0.97; P < 0.001 for FLC lambda. Considering normal/pathological FLC ratio moderate agreement within assays was detected (κ = 0.621). When the results were categorized according to criteria for progressive disease, 4/37 (0.10) cases were differently classified. Lambda FLC values by Optilite in three samples with monoclonal FLC lambda were more than twelve times higher than by ProSpec. A 25% difference in FLC ratio was detected in 16/37 (0.43) and 50% difference in 13/37 (0.35) patients. CONCLUSIONS: All manufacturers' precision claims could not be achieved in the verification study. The comparison of results to biological variations data showed that coefficients of variations are acceptable for both assays. The assays should not be used interchangeably in haematological patients.

Unusual pattern in haemoglobin electrophoresis in Croatian population: a case report.

Haemoglobinopathies are hereditary disorders of globin chain synthesis and are the most common inherited diseases worldwide. Haemoglobin E is a structural haemoglobin variant characteristic for South East Asian population. We present a rare and unusual finding of haemoglobin E detected in University Hospital Centre Zagreb by capillary zone electrophoresis. Detection of haemoglobin structural variant helped to avoid misdiagnosis of sideropenic anemia and thus potentially harmful therapeutic intervention. In today's European multiethnic population haemoglobinopathies are a public health issue and Croatian laboratory professionals should be aware of a possibility of finding an unusual haemoglobin pattern.