Smock, Kristi J.

Kanack and colleagues analyze anti-platelet factor 4 antibodies from 5 patients with vaccine-induced thrombotic thrombocytopenia (VITT) secondary to COVID-19 adenoviral vaccination and antibodies from patients with spontaneous heparin-induced thrombocytopenia (HIT) and classical HIT. VITT antibodies are monoclonal or oligoclonal, similar to spontaneous HIT, whereas classical HIT antibodies are polyclonal. Heparin inhibits antibody-induced platelet activation in VITT, suggesting that heparin should be considered for the treatment of VITT.

AbstractRare cases of COVID‐19 vaccinated individuals develop anti‐platelet factor 4 (PF4) antibodies that cause thrombocytopenia and thrombotic complications, a syndrome referred to as vaccine‐induced immune thrombotic thrombocytopenia (VITT). Currently, information on the characteristics and persistence of anti‐PF4 antibodies that cause VITT after Ad26.COV2.S vaccination is limited, and available diagnostic assays fail to differentiate Ad26.COV2.S and ChAdOx1 nCoV‐19‐associated VITT from similar clinical disorders, namely heparin‐induced thrombocytopenia (HIT) and spontaneous HIT. Here we demonstrate that while Ad26.COV2.S‐associated VITT patients are uniformly strongly positive in PF4‐polyanion enzyme‐linked immunosorbent assays (ELISAs); they are frequently negative in the serotonin release assay (SRA). The PF4‐dependent p‐selectin expression assay (PEA) that uses platelets treated with PF4 rather than heparin consistently diagnosed Ad26.COV2.S‐associated VITT. Most Ad26.COV2.S‐associated VITT antibodies persisted for >5 months in PF4‐polyanion ELISAs, while the PEA became negative earlier. Two patients had otherwise unexplained mild persistent thrombocytopenia (140‐150 x 103/µL) 6 months after acute presentation. From an epidemiological perspective, differentiating VITT from spontaneous HIT, another entity that develops in the absence of proximate heparin exposure, and HIT is important, but currently available PF4‐polyanion ELISAs and functional assay are non‐specific and detect all three conditions. Here, we report that a novel un‐complexed PF4 ELISA specifically differentiates VITT, secondary to both Ad26.COV2.S and ChAdOx1 nCoV‐19, from both spontaneous HIT, HIT and commonly‐encountered HIT‐suspected patients who are PF4/polyanion ELISA‐positive but negative in functional assays. In summary, Ad26.COV2.S‐associated VITT antibodies are persistent, and the un‐complexed PF4 ELISA appears to be both sensitive and specific for VITT diagnosis.

Abstract Background: ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen Johnson & Johnson) vaccines against COVID-19 have been associated with thrombotic thrombocytopenic reactions referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT) characterized by the presence of platelet-activating, anti-PF4 antibodies. While VITT shares key clinical features with a similar but separate entity, Heparin-induced thrombocytopenia (HIT), there appear to be important differences: 1) VITT patients have extremely high thrombosis rates and are very strongly positive in PF4-polyanion ELISAs, and 2) Many patients with VITT frequently present with refractoriness to therapy or have disease recurrence that suggests distinct antibody characteristics due to a strong autoimmune anti-PF4 response. Aims: The goal of this study was to characterize anti-PF4 antibodies in VITT. Methods: Five VITT patients were studied, one after ChAdOx1 nCoV-19 vaccination and four after Ad26.COV2. Reactivity of VITT anti-PF4 antibodies to uncomplexed PF4, PF4-Polyvinyl sulfonate (PVS), and PF4-heparin targets was evaluated, and the platelet-activating ability of these antibodies was examined in the PF4-dependent P-selectin Expression assay (PEA). Anti-PF4 antibodies were isolated from patient blood samples using PF4-treated heparin sepharose beads, and isolated antibodies were subject to mass spectrometric evaluation (Liquid Chromatography Electrospray Ionization Quadrupole time-of-flight mass spectrometry [LC-ESI-QTOF MS]). Results: Antibodies from all VITT patients recognized both uncomplexed and complexed PF4 (Fig. 1A). Interestingly, recognition of PF4 by VITT antibodies was lower if PF4 targets were complexed with polyanions, PVS, or heparin (Fig. 1A). These results contrasted with those obtained in a "classical" HIT patient which showed reactivity to PF4/polyanion complexes, but not to uncomplexed PF4 (Fig 1A). All samples activated platelets in the PEA (data not shown). Mass spectrometric evaluation of anti-PF4 antibodies isolated from VITT patients demonstrated monoclonal anti-PF4 antibodies in three patients, and bi- and tri-clonal antibodies in one patient each (a representative monoclonal antibody anti-PF4 antibody is shown in Fig 1B). Consistent with current dogma, polyclonal anti-PF4/polyanion antibodies were seen in "classical" HIT (Fig 1C). Evaluation of anti-PF4 antibodies in spontaneous HIT, a type of autoimmune HIT seen in pro-inflammatory milieus such as orthopedic surgery and infectious prodromes also demonstrated monoclonal anti-PF4 antibodies (Fig 1D). Eluates from control heparin-sepharose beads did not reveal any immunoglobulins (data not shown). Conclusion: Although development of platelet-activating anti-PF4 antibodies and the thrombotic thrombocytopenia syndrome seen after ChAdOx1 nCoV-19 and Ad26.COV2.S vaccination resembles HIT, these findings demonstrate that clonally restricted anti-PF4 antibodies mediate VITT while polyclonal anti-PF4 antibodies mediate HIT. In addition, we noted clonally-restricted anti-PF4 antibodies in another condition that does not require proximate heparin exposure, spontaneous ("autoimmune") HIT. In VITT, the strong immune response after vaccine administration may result in the activation of a single or few pre-existing anti-PF4 reactive clones, and development of clonally restricted anti-PF4 antibodies with a similar pathophysiology to Spontaneous HIT. It is also likely that high levels of monoclonal/oligoclonal anti-PF4 antibodies cause the severe thrombotic phenotypes seen in VITT and Spontaneous HIT. The high mortality rate and reports of disease refractoriness to therapy in VITT may warrant consideration of additional therapeutic modalities like rituximab and therapeutic plasma exchange in select cases. Figure Legends: (A): VITT (Patient 1-ChAdOx1 nCoV-19; Patients 2-5, Ad26.COV2.S) patient samples were tested in ELISA against uncomplexed PF4 (white), and PF4 in complex with polyvinyl sulfonate (light grey), or unfractionated heparin (dark gray). (B-D) Mass spectrometric evaluation of anti-PF4 antibodies isolated from VITT (B), HIT (C) and spontaneous HIT patient sera (D). "Relative Intensity" refers to abundance of the Ig light chain relative to the polyclonal background. Numbers above Ig light chain peaks depict mass/charge ratios. NC- Normal control. Figure 1 Figure 1. Disclosures Murray: Mayo Clinic: Other: Has received patents for the Mass-Fix technology which has been licensed to the Binding Site with potential royalties.. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Context.— Assessing direct oral anticoagulant (DOAC) drug levels by reliable laboratory assays is necessary in a number of clinical scenarios. Objective.— To evaluate the performance of DOAC-specific assays for various concentrations of dabigatran and rivaroxaban, assess the interlaboratory variability in measurement of these DOACs, and investigate the responsiveness of the routine clotting assays to various concentrations of these oral anticoagulants. Design.— College of American Pathologists proficiency testing survey data from 2013 to 2016 were summarized and analyzed. Results.— For dabigatran, the interlaboratory coefficient of variation (CV) of ecarin chromogenic assay was broad (ranging from 7.5% to 29.1%, 6.3% to 15.5%, and 6.8% to 9.0% for 100-ng/mL, 200-ng/mL, and 400-ng/mL targeted drug concentrations, respectively). The CV for diluted thrombin time for dabigatran was better overall (ranging from 11.6% to 17.2%, 9.3% to 12.3, and 7.1% to 11.2% for 100 ng/mL, 200 ng/mL, and 400 ng/mL, respectively). The rivaroxaban-calibrated anti-Xa assay CVs also showed variability (ranging from 11.5% to 22.2%, 7.2% to 10.9%, and 6.4% to 8.1% for 50-ng/mL, 200-ng/mL, and 400-ng/mL targeted drug concentrations, respectively). The prothrombin time (PT) and activated partial thromboplastin time (aPTT) showed variable dose- and reagent-dependent responsiveness to DOACs: PT was more responsive to rivaroxaban and aPTT to dabigatran. The undiluted thrombin time showed maximum prolongation across all 3 dabigatran concentrations, making it too sensitive for drug-level monitoring, but supporting its use as a qualitative screening assay. Conclusions.— DOAC-specific assays performed reasonably well. While PT and aPTT cannot be used safely to determine DOAC degree of anticoagulation, a normal thrombin time excludes the presence of dabigatran.

Context.— As pharmacogenetic testing is incorporated into routine care, it is essential for laboratories to provide accurate and consistent results. Certified laboratories must successfully complete proficiency testing. Objectives.— To understand and examine trends in participation and performance of laboratories participating in the College of American Pathologists pharmacogenetic proficiency testing surveys. Design.— Results from College of American Pathologists pharmacogenetic proficiency testing challenges from 2012 through 2017 were reviewed for concordance with expected genotype and phenotype for each sample (intended responses). Results.— Laboratories correctly reported results for 96.7% to 100% of samples with no variants. Excluding CYP2D6, laboratories correctly detected and reported variant alleles for each gene (93.7%–99.2% correct). CYP2D6 showed lower concordance, with 83.1% of laboratories reporting the intended genotype across all samples; however, in many cases, the laboratories that did not report a variant allele did not test for that allele. Among laboratories reporting the intended genotype, most successfully reported the intended phenotype (85.9%–99.0%). Conclusions.— Although laboratories are generally performing well, there is room for additional improvement, particularly for challenging genes, such as CYP2D6. Efforts in the field of pharmacogenomics to recommend alleles that should be included in clinical tests, identify reference materials, and standardize translation from genotype to phenotype may address some of the remaining variability in results.

Context.— The College of American Pathologists (CAP) developed proficiency testing for platelet function assays by using blood collected by the participant added to challenge tubes containing either saline (normal) or tirofiban (abnormal). Objective.— To analyze platelet function proficiency testing for Platelet Function Analyzer PFA-100, platelet aggregation, PlateletWorks, and PlateletMapping. Design.— Proficiency testing data from 2012–2016 were analyzed. Results.— For PFA-100, a total of 1200 laboratories participated; the coefficient variation (CV) of cartridge closure times was 22% (saline); 44,952 of 45,616 survey responses (99%) provided an interpretation, and 42,934 of 44,952 (96%) were correct. For optical platelet aggregation, 190 laboratories participated; the CV was 17% (saline), 7444 of 7813 survey responses (95%) provided an interpretation, and 7015 of 7444 (94%) were correct. For PlateletWorks, 60 laboratories participated; the CV was 3% to 11% (saline); 2412 of 2454 survey responses (98%) provided an interpretation, and 1207 of 1276 (95%) were correct for adenosine diphosphate (ADP) and 936 of 1136 (82%) for collagen. For PlateletMapping, 200 laboratories participated. For ADP, 1128 of 2697 survey responses (42%) provided an interpretation, but only 927 of 1128 (82%) were correct. For arachidonic acid, 1139 of 2604 survey responses (44%) provided an interpretation and 964 of 1139 (85%) were correct. Conclusions.— CAP is the first to provide proficiency testing for platelet aggregation, PlateletWorks, and PlateletMapping. Platelet aggregation, PFA-100, and PlateletWorks using ADP as an agonist performed well with more than 90% of laboratories providing an interpretation and a similar number providing correct results. PlateletWorks using collagen and PlateletMapping showed worse interpretive accuracy than the other methods.

Rivaroxaban and dabigatran are among the newest anticoagulants, and measuring their concentration in patients is a new challenge for clinical laboratories. We analyzed data from the ECAT proficiency program to determine how well the assays are performing in clinical laboratories internationally. Most laboratories received a passing grade (Z score <3) for the results of their dabigatran and rivaroxaban testing. Failing Z scores were not associated with any particular method. With dabigatran, some homemade calibrators gave higher results than the commercially available calibrators. There were no significant differences among the instruments or the 5 reagents in use, but results showed inter‐laboratory variability that could have clinical significance. The 3 reagents with the lowest number of users had poor inter‐laboratory precision. Ten different anti‐Xa reagents were in use for rivaroxaban testing. One reagent gave lower results than other reagents at 100 ng/mL but not at 300 ng/mL. There were no significant differences among the different rivaroxaban calibrators or instruments. In conclusion, inter‐laboratory precision could be improved for both dabigatran and rivaroxaban assays. Homemade dabigatran calibrators differed from commercially available calibrators, and there was a statistically significant difference between some of the rivaroxaban reagents. About 10% of results received failing Z scores or passed but fell in a range that require the laboratory to investigate for bias or other inaccuracy in their method. Am. J. Hematol. 91:E464–E467, 2016. © 2016 Wiley Periodicals, Inc.

SummaryIntroductionThe performance of factor XI activity (FXI) by laboratories in the North American Specialized Coagulation Laboratory Association proficiency testing program was analyzed.MethodsOver 10 years (2003–2013), 80 samples were distributed; 33–55 laboratories participated per exercise providing 3833 total responses. Analysis was performed on numeric results and qualitative classification of results.ResultsThe sample FXI levels ranged from 3.8 to 154.0 IU/dL. The overall interlaboratory average coefficient of variation (CV%) was 17.5%; the CV was higher for a sample with low (3.8 IU/dL) FXI. Results were correctly classified as abnormal (100%) for a sample with 3.8 IU/dL FXI and normal/borderline normal (97.7%) for 45 samples with 80 to < 140 IU/dL FXI. The classification was heterogeneous for samples with FXI of 50 to < 80 IU/dL. Six specimens were repeat‐tested from 2007 to 2013. The mean FXI was not significantly different in laboratories using the same method on both exercises, suggesting good intralaboratory precision over time. Univariate analysis of data from 2011 to 2012 did not find a consistent significant difference among the activators, analyzers, calibrators, and FXI‐deficient plasmas.ConclusionLaboratories generally performed well in assessment of FXI based on interlaboratory precision when FXI >30 IU/dL and on classification of samples with very low or normal FXI.