Antiphospholipid antibodies in patients with COVID-19 : a relevant observation?

Background: High incidence of thrombosis in COVID‐19 patients indicates a hypercoagulable state. Hence, exploring the involvement of antiphospholipid antibodies (aPL) in these patients is of interest. Objectives: To illustrate the incidence of criteria (lupus anticoagulant [LAC], anticardiolipin [aCL] immunoglobulin G [IgG]/IgM, antibeta2‐glycoprotein I antibodies [aβ2GPI] IgG/IgM) and noncriteria (anti‐phosphatidyl serine/prothrombin [aPS/PT], aCL, and aβ2GPI IgA) aPL in a consecutive cohort of critically ill SARS‐CoV‐2 patients, their association with thrombosis, antibody profile and titers of aPL. Patients/Methods: Thirty‐one consecutive confirmed COVID‐19 patients admitted to the intensive care unit were included. aPL were measured at one time point, with part of the aPL‐positive patients retested after 1 month. Results: Sixteen patients were single LAC‐positive, two triple‐positive, one double‐positive, one single aCL, and three aCL IgG and LAC positive. Seven of nine thrombotic patients had at least one aPL. Sixteen of 22 patients without thrombosis were aPL positive, amongst them two triple positives. Nine of 10 retested LAC‐positive patients were negative on a second occasion, as well as the double‐positive patient. Seven patients were aPS/PT‐positive associated to LAC. Three patients were aCL and aβ2GPI IgA‐positive. Conclusion: Our observations support the frequent single LAC positivity during (acute phase) observed in COVID‐19 infection; however, not clearly related to thrombotic complications. Triple aPL positivity and high aCL/aβ2GPI titers are rare. Repeat testing suggests aPL to be mostly transient. Further studies and international registration of aPL should improve understanding the role of aPL in thrombotic COVID‐19 patients

Improved standardization of flow cytometry diagnostic screening of primary immunodeficiency by software-based automated gating

Background Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results. Methods FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt (TM) software (Cytognos). For comparison, percentage differences in absolute cell counts/mu L were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples. Results The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/mu L) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I. Conclusion Altogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles

Direct oral anticoagulant removal by a DOAC filter : impact on lupus anticoagulant testing : evaluation on spiked and patient samples

Background: DOAC Filter (DF) is a new device to overcome interference in lupus anticoagulant (LAC) testing by direct oral anticoagulants (DOACs). Objectives: We evaluated DOAC removal from plasma and elimination of DOAC interference in LAC testing by DF, and impact of DF on LAC assays in a representative patient cohort, including a comparison with DOAC-Stop (DS). Methods: Normal pooled plasma (NPP) was spiked with increasing concentrations of apixaban, rivaroxaban, edoxaban, and dabigatran. DOAC and LAC was measured on untreated, DF-treated, and DS-treated spiked samples. Coagulation parameters and thrombin generation were measured on patient samples (n = 20) before and after DF. Patients treated with DOAC, vitamin K antagonist, or heparin and nonanticoagulated patient samples (n = 139) were tested for LAC before and after DF. Results: In spiked NPP, levels were below the lower limit of quantification (LLoQ) after DF/DS treatment for all DOAC concentrations. Following DF, levels were below LLoQ for 53 of 56 DOAC-containing patient samples. Twenty-eight of 33 LAC-positive DOAC-containing samples became negative after filtration, whereas 5 remained LAC-positive (1/5 from a patient with antiphospholipid syndrome [APS)). Four LAC-positive DOAC-containing samples (from patients without APS), became negative after filtration, whereas they remained LAC positive after DS. In the non-DOAC patient groups following DF, LAC changed from positive to negative in 8 (due to a procoagulant effect) and vice versa in 2 cases. Conclusion: DF reduces DOAC interference in LAC testing. As incomplete DOAC removal may occur, DOAC measurements should be performed after filtration. A procoagulant effect after filtration may lead to erroneous LAC results in non-DOAC-containing samples. Therefore, using DF should be restricted to DOAC-containing samples

Improved Standardization of Flow Cytometry Diagnostic Screening of Primary Immunodeficiency by Software-Based Automated Gating

© 2020 Linskens, Diks, Neirinck, Perez-Andres, De Maertelaere, Berkowska, Kerre, Hofmans, Orfao, van Dongen, Haerynck, Philippé and Bonroy.Background: Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results. Methods: FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt™ software (Cytognos). For comparison, percentage differences in absolute cell counts/µL were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples. Results: The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/µL) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I. Conclusion: Altogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles.The coordination and innovation processes of this study were supported by the EuroFlow Consortium. The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA). JN is granted by the Fund for Scientific Research, TBM Funding, Belgium (T000119N). This work was also supported by a grant of the Grand Challenges Program of VIB