Anti-Spike SARS-CoV-2 IgG Assessment with a Commercial Assay during a 4-Month Course after COVID-19 Vaccination

We intended to assess the humoral response induced by the Pfizer/BioNTech Comirnaty COVID-19 vaccine with commercially available immunoassays: anti-spike (S) IgG and IgM, and anti-nucleocapsid (N) IgG antibodies, over a 4-month course. One hundred subjects, including 15 COVID-19 convalescents, comprised the study cohort. The SARS-CoV-2 antibodies concentrations were measured on day 0′ and 10′, 20′, 30′, 60′, 90′, and 120′ after the first dose administration. Over the course of the study, 100% of the participants developed and sustained anti-SARS-CoV-2 S IgG antibodies. The highest concentration, exceeding the quantification range of the test (2080 BAU/mL), was reached by 67% of the subjects on day 30′. The concentration of the antibodies remained stable between days 30′ and 90′ but was followed by a significant decrease between days 90′ and 120′. The stronger and more persistent humoral response was noted for women. The COVID-19 convalescents developed higher antibody levels, particularly 10 days after the first Comirnaty dose. Twenty-three out of the eighty-five naïve vaccinees failed to develop a detectable IgM response. LIAISON® SARS-CoV-2 TrimericS IgG (DiaSorin S.p.A, Saluggia, Italy) may be useful in the assessment of the humoral response to the Comirnaty vaccine. In contrast, Abbott’s anti-S SARS-CoV-2 IgM has a limited utility in this context.

Evaluation of Dried Blood Spot Testing for SARS-CoV-2 Serology Using a Quantitative Commercial Assay

Dried blood spots (DBS) are commonly used for serologic testing for viruses and provide an alternative collection method when phlebotomy and/or conventional laboratory testing are not readily available. DBS collection could be used to facilitate widespread testing for SARS-CoV-2 antibodies to document past infection, vaccination, and potentially immunity. We investigated the characteristics of Roche’s Anti-SARS-CoV-2 (S) assay, a quantitative commercial assay for antibodies against the spike glycoprotein. Antibody levels were reduced relative to plasma following elution from DBS. Quantitative results from DBS samples were highly correlated with values from plasma (r2 = 0.98), allowing for extrapolation using DBS results to accurately estimate plasma antibody levels. High concordance between plasma and fingerpick DBS was observed in PCR-confirmed COVID-19 patients tested 90 days or more after the diagnosis (45/46 matched; 1/46 mismatched plasma vs. DBS). The assessment of antibody responses to SARS-CoV-2 using DBS may be feasible using a quantitative anti-S assay, although false negatives may rarely occur in those with very low antibody levels.

Association of acute graft versus host disease clinical outcomes and the Ann Arbor Biomarker Risk Probability Using the Commercial Assay.

e19023 Background: Acute GVHD (aGVHD) is a major cause of mortality and morbidity following stem cell transplantation (HCT). In effort to prognostic patients with aGVHD, the Mount Sinai Acute GVHD International Consortium (MAGIC) combined two biomarkers, ST2 and REG3α to generate an algorithm known as the Ann Arbor (AA) biomarker risk. This is now available as a commercial test to help predict risk for severe acute GVHD and non-relapse mortality when done at the onset of symptomatic aGVHD. Our institution began utilizing these lab values and here we report our “real world” experience. The primary aim was to correlate AA risk probabilities with recently defined endpoints including day 28 overall response rate (ORR) and 6 month freedom from treatment failure (6mFFTF) and overall survival (OS). Methods: 58 HCT patients who underwent HCT between October 2018 and November 2019 and had the biomarker assay at the onset of aGVHD were identified from the institutional database and clinical information about disease, transplant and outcomes was obtained from the medical records. Clinical staging of aGVHD was documented per standardized published guidelines. Day 28 ORR was defined as complete response by day 28. 6mFFTF was defined as a patient being alive, without relapse of underlying disease or addition of new systemic therapy for aGVHD, and prior to the development of cGVHD. Results: When comparing low (n = 39), intermediate (n = 11), and high (n = 8) AA risk, there was no difference in age ( p =0.254), primary disease ( p =0.10), DRI ( p =0.994), donor source ( p =0.193), conditioning regimen intent ( p =0.537), GVHD prophylaxis ( p =0.351) ATG use ( p =0.06). Low AA risk was associated with lower grades of aGVHD at onset (grade I = 15%, grade II = 80%, grade III = 5%, grade IV = 0%), whereas high AA risk was associated with higher grades of aGVHD at onset (grade I = 0%, grade II = 50%, grade III = 25%, grade IV = 25%) ( p =0.009). For low, intermediate, and high AA risk the day 28 ORR was 69%, 45% and 29% ( p = 0.083); the 6mFFTF was 82%, 82%, and 25% ( p = 0.0001); 6 month OS was 92%, 100%, and 50% ( p = 0.0006) respectively. Conclusions: Our study shows a high correlation between AA risk and outcomes. High risk AA risk was associated with inferior outcomes This study was limited by the retrospective single institutional experience with relatively small numbers. However, our findings using the commercial assay mirror that seen with the MAGIC studies. Our “real world data” can serve as a foundation and continued validation for institutional implementation of AA risk stratification for aGVHD.

A modified clot-based assay to measure negatively charged procoagulant phospholipids

Growing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL). Current commercial PPL-dependent clotting assays use chemically phospholipid depleted plasma to measure PPL activity. The purpose of our study was to modify the PPL assay by substituting the chemically phospholipid depleted plasma with PPL depleted plasma obtained by ultracentrifugation This in order to get readily access to a sensitive and reliable assay to measure PPL activity in human plasma and cell supernatants. The performance of the assay was tested, including the influence of individual coagulation factors and postprandial lipoproteins and compared to a commercial PPL assay (STA-Procoag-PPL). The two PPL assays displayed similar sensitivity to exogenously added standardized phospholipids. The PPL activity measured by the modified assay strongly correlates with the results from the commercial assay. The intraday- and between-days coefficients of variation ranged from 2–4% depending on the PPL activity in the sample. The modified PPL assay was insensitive to postprandial lipoprotein levels in plasma, as well as to tissue factor (TF) positive EVs from stimulated whole blood. Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF+ EVs.

Verification of a rapid von Willebrand factor propeptide assay

Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by a deficiency or defect in von Willebrand factor (VWF). Quantitative defects include, type 1 VWD and type 3 VWD. Type 1 VWD is either due to decreased synthesis and secretion, or increased clearance of VWF. It is essential to diagnose individuals with increased VWF clearance, as treatment of these patients with 1-deamino-8-D-arginine vasopressin is not effective. Currently, there is one commercial assay that measures von Willebrand factor propeptide (VWFpp) levels. This assay is time consuming to perform. With this research we developed and verified a rapid assay to determine VWFpp levels in patient plasma. Methods: The commercial VWF mouse anti-human VWF propeptide matched antibody pair (clones CLB-Pro 35 and CLB-Pro 14.3) was used in enzyme-linked immunosorbent assays of the commercial and the rapid method. While the CLB-Pro commercial assay uses two-hour incubations, our rapid assay uses 30 minute incubations. We compared our assay to the CLB-Pro commercial assay using twenty type 1 VWD patient plasma. Two samples, the World Health Organization (WHO) 6th International Standard (IS) for factor VIII (FVIII)/VWF and a type 1 VWD patient with increased clearance were also tested four times in duplicate for five consecutive days to determine the inter- and intra-assay precision. Results: Our rapid assay showed equal sensitivity to the CLB-Pro commercial assay by detecting 1.5625% VWFpp. The intra- and interassay CVs of our assay were acceptable according to the Food and Drug Administration guideline of 2018. Conclusion: This rapid enzyme-linked immunosorbent assay (ELISA) is as sensitive and precise as the CLB-Pro commercial assay. Therefore, it can be used to rapidly diagnose patients with increased VWF clearance.

An Outbreak of Clostridium (Clostridioides) difficile Infections within an Acute and Long-Term Care Wards Due to Moxifloxacin-Resistant PCR Ribotype 176 Genotyped as PCR Ribotype 027 by a Commercial Assay

We aimed to characterize Clostridioides difficile isolates cultured during a six-month single-center study from stool samples of patients with C. difficile infection (CDI) genotyped by the Xpert®C. difficile/Epi assay by polymerase chain reaction (PCR) ribotyping, toxin genes’ detection and multi-locus variable number tandem repeats analysis (MLVA). The susceptibility to metronidazole, vancomycin and moxifloxacin was determined by agar dilution. In addition, the presence of Thr82Ile in the GyrA and a single nucleotide deletion at position (Δ117) in the tcdC gene were investigated. Between January 1 and June 30, 2016, of 114 CDIs, 75 cases were genotyped as presumptive PCR ribotype (RT) 027 infections using a commercial assay. C. difficile isolates cultured from presumptive RT027 stool samples belonged to RT176. These isolates carried genes for toxin A (tcdA), B (tcdB), binary (cdtA/B) and had Δ117 in the tcdC gene. Using MLVA, the 71/75 isolates clustered into two clonal complexes (CCs). Of these, 39 isolates (54.9%) were from patients hospitalized in acute care and 32 isolates (45.1%) were isolated from patients hospitalized in the long-term care department. All isolates were susceptible to metronidazole and vancomycin, and 105 isolates were resistant to moxifloxacin (92%) carrying Thr83Ile in the GyrA. An outbreak of RT176 CDIs, suspected as RT027, was recognized in a Slovakian hospital. In order to monitor the emergence and spread of RT027-variants, the identification of a presumptive RT027 CDI should be confirmed at a strain level by PCR ribotyping.