Normal Reference Values Evaluation of Rapid Chemiluminescent Adamts 13 Activity Assay

Introduction: Acquired thrombotic thrombocytopenic purpura (TTP) is a severe, rare, thrombotic microangiopathy (TMA). A diagnosis of acquired TTP is confirmed by a severe deficiency (< 10%) of ADAMTS13 activity. Recently, maybe because of Sars-COV2 Pandemic, in our laboratory we had the impression that normal reference ranges of our ADAMT13 activity assay HemosiL Acustar ADAMTS13 rapid immunoassay, could be larger than manufacturer's. The objective of this study was to evaluate whether manufacturer's are suitable normal reference values for this assay in our laboratory Methods: To evaluate manufacturer's reference limits (60-130.6%) in our laboratory we decided to assay with HemosIL Acustar ADAMTS13 activity test 30 plasma samples from normal subjects. Result(s): 3 out of 20 normal subjects tested showed ADAMTS13 activity outside manufacturer's limits (1 below 60.6% and 2 above 130.6%), therefore even if calculated in a small number of subjects (30 individuals) we decide to try to calculate in house ADAMTS13 reference values. They ranged from 47.5 (10th perc.), 72.5 (25th perc.) and 41.6 (mean- 2sd) to 150.0 (99th perce.), 119,6 (75th perc.) and 152.7 (mean+2sd). To investigate the analytical performance of manufacturer's and calculated cut-offs limits, we re-evaluate one year of ADAMTS13 activity results (95 subjects). ADAMTS13 activity was severely reduced (< 0.7%) in 10 acute TTPs;20 patients with Hemolytic Uremic Syndrome showed ADAMTS13 activity above manufacturer's and in all calculated cut-offs;21/45 patients with TTPs in remission phase showed ADAMTS activity below all in house calculated cut off and 24/45 showed ADAMTS activity below manufacturer's;12/20 other patients showed ADAMTS activity below all in house calculated and manufacturer's cut offs, all of them were Sars COV2 positive subjects Conclusion(s): In conclusion HemosiL Acustar ADAMTS13 activity calculated cut-offs in our laboratory were larger than manufacturer's reference range. By the analysis of one year ADAMTS13 activity dosages both manufacturer's and calculated cut-offs showed similar performances but our calculated reference ranges even if obtained by the analysis of a small number of normal subjects were found to be more similar to literature ELISA (40-130%) and FRET (45-147%) ADAMTS13 activity normal values.

Anti-Platelet4 Antibodes Testing in Pandemic: Which Test to Use

Introduction: Heparin-induced thrombocytopenia (HIT) is a severe adverse reaction to heparin caused by heparin-dependent, platelet-activating anti-plateletfactor 4 (PF4)/heparin antibodies. Vaccine-induced thrombocytopenia and thrombosis (VITT) following Astrazeneca vaccine has been described, associated with IgG anti-PF4 antibodies. Only ELISA immunoassays have been shown to detect anti-PF4 antibodies in these patients. Diagnosis of both HIT and VITT requires confirmation of heparin-dependent, platelets activating antibodies to avoid overdiagnosis and overtreatment Anti-PF4 laboratory assay requests during pandemic are in most of the cases of COVID positive vaccinated subjects, vaccinated healed from COVID19 subjects and COVID negative vaccinated subjects. Aim of our study was to investigate which laboratory test to use in patients with suspected HIT in this particular historical period Methods: Thirty patients with suspected HIT/ VITT were tested with three anti-PF4 immunoassays (HIT Ab Latex Immunoassay-Werfen, Acustar HITIgG CliA-Werfen, HPIA ELISA-Stago): 8 Astrazenca suspected VITT;1 Moderna and 1 Pfizer suspected VITT, 20 suspected HIT (9/20 COVID positive and 11/20 COVID negative patients). In order to confirm immunoassay positivity Platelet Aggregation Test (PAT) functional test was performed in all patients found to be positive for at least one assay. Result(s): 3/8 suspected Astrazenca VITT tested positive only by ELISA assay 2/3 tested positive by PAT (confirmed VITT). Both patients with suspected Pfizer and Moderna VITT tested negative for all immunoassays. 4/9 COVID patients with suspected HIT tested positive only by ELISA assay, only 1 of them tested positive by PAT. 4/11 COVID negative suspected HIT tested positive by all immunoassays performed and 1/11 tested positive only by ELISA and CliA immunoassays, among these 5 subjects only 2 were confirmed HIT by PAT functional assay Conclusion(s): With the only exception of suspected Astrazeneca VITT no superiority of IgG-ELISA over CliA or Latex immunoassay in sensitivity to HIT was observed. No single immunoassay method detected all probable HIT cases;if a single test is negative, a second immunoassay or a platelet activation assay should be considered where there is strong clinical suspicion Interestingly by using Bayesan diagnosis of HIT and CliA conservative negative cut-off < 0.13 U/ml suggested in the literature (Marchetti et al. 2020) all our CliA negative (results < manufacturer's cut-off of 1.0 U/mL) and ELISA positive suspected HIT/VITT were correctly classified as positive by CliA assay.

Mononucleosis: Peculiarity of Blood Count Scattergram Performed on Abbott Alinity Hq

Introduction: Mononucleosis is an infectious disease caused by Epstein-Barr virus (EBV, human herpes virus type 4, HHV-4) and is characterized by asthenia, fever pharyngitis, and lymphadenopathy. In our laboratory diagnosis is made by rapid test and Epstein-Barr virus antibody assay. The presence of Epstein-Barr virus (VCA) specific IgM antibodies indicates primary infection. A marked lymphocytosis with inversion of the formula can be seen on the blood count. The smear shows numerous activated lymphocytic elements By examining the complete scattergram of patients with confirmed primary infection we noticed a peculiar arch arrangement of the lymphocytes in the FL1 x ALL specific leukocyte scatters. Method(s): In this study Vircell's Virapid mono M&G is used, an immunotest for the qualitative determination of 4 serological markers of EBV: two IgM, VCA and heterophile, and two IgG, VCA and EBNA. The presence of anti-VCA IgM antibodies and the absence of anti-EBNA antibodies are indicative of primary infection. CBC was performed on Abbott Alinity hq, which uses a combination of photometry optical counting and fluorescence analysis in order to enumerate cells and cellular constituens. The instrument utilizes eight light scatter detectors which include ALL (axial light loss), IAS (intermediate angles of light scatter), PSS (polarized side scatter), DSS (depolarized side scatter) and FL1 ( fluorescent channel). Result(s): The sixteen cases examined, all of which resulted positive for primary infection on the rapid test, showed a peculiar FL1 x ALL scattergram (see Fig.1). In the lymphocytes' scattergram cloud, we observed an archshaped trend going upwards and rightwards, thus highlighting cells with greater fluorescence and size Often these lymphocytes are identified as monocytes In cases of lymphocytosis from other causes (CLL lymphomas) we can see how the lymphocytes' scattergram cloud is totally different. In such a case the cloud seems like a short bar due to lymphocytes with increased fluorescence signal though with small size (see Fig.2). Conclusion(s): In the 16 cases of primary EBV infection examined, the blood count shows a peculiar FL1 x ALL scattergram, which compared with the scattergram of other causes of lymphocytosis highlights a substantial difference that could support the laboratory technician in the diagnostic differentiation of a lymphocytosis: lymphocytic response to viral infection (EBV, SARS-Cov19, ecc) or monoclonal lymphocyte proliferation.

Thrombin generation test in COVID-19 related coagulopathy

Introduction: critically ill COVID-19 patients are known to have a coagulopathy characterized by increased levels of D-dimer (DD) associated to a thrombotic risk and a significant increase in mortality. However, it is not known whether the associated COVID-19 coagulopathy is due to a prothrombotic state or is caused by endothelial dysfunction and inflammation. Aim of our study, was to better characterize the hypercoagulability state of COVID-19 patients using Thrombin Generation analyser (ST Genesia, Diagnostica Stago, Asnieres, France). Method(s): a total of 46 non-critically ill hospitalized COVID-19 patients were compared to 19 critically ill COVID-19 patients utilizing calibrated automated thrombography and other biochemical, hematological and coagulation parameters. Result(s): critically ill patients had a significant increase in C reactive protein (CRP), interleukin-6 (IL-6), prothrombin time (PT), DD and a significant decrease in lymphocytes count. No significant differences in Thrombin Generation Test (TGT) parameters were observed between the two groups of patients with the only exception of the "Lag Time" parameter. Discussion(s): the obtained results confirmed increased levels of DD and PT in critically ill COVID-19 patients. Of note, disease severity did not cause an increase in Thrombin Generation when compared to non-critically COVID-19 patients. The significantly prolonged Lag Time in critically ill COVID-19 patients without decreased endogenous thrombin potential suggests an hypocoagulability state in these patients. The relevance of this finding is uncertain and may appear counterintuitive since these patients are expected to have a hypercoagulability status, and requires further research. Copyright © 2022 Biomedia. All rights reserved.

Anti-platelet factor 4 pathologic antibodies after chadox1 ncov-19 vaccination mauriziano hospital diagnosis and findings

Introduction.Vaccine induced immune thrombocytopenia and thrombosis (VITT) following ChAdOx1 nCOV-19 vaccine has been described, associated with unusual site thrombosis, thrombocytopenia, raised D-dimer and high titre immunoglobulin-G (IgG) class anti-Platelet Factor 4 (PF4) antibodies. Laboratory management of suspected cases begins with a sensitive anti PF4 antibodies binding assay (PF4-ELISA). If the PF4 binding assay is negative, this patient does not have Heparin induced Thrombocytopenia (HIT) or VITT. If the PF4 binding assay is positive, the positivity should be confirmed with one or multiple HIT functional assays as available, such as the serotonin release assay (SRA), heparin-induced platelet activation assay, platelet aggregation (PAT) test, flow cytometry test.Methods.We summarized clinical and laboratory findings of 7 patients in Piedmont who developed thrombosis and thrombocytopenia following AZD1222 vaccination. Plasma from all patients was used to test for anti PF4 antibodies by 2 different ELISA assays (Immucor and Stago) and by 2 different HIT functional assays, PAT and flow cytometry (HIT alert test) both performed in the presence of heparin, PF4 or both.Results.The 7 patients [6 males and 1 female, median age: 38 (range:31-76)] presented with thrombosis 2 to 17 days post vaccination: 5 males had deep vein thrombosis not in unusual sites, 1 male had stroke and the female had cerebral venous thrombosis (CVT). None had received heparin prior symptoms onset. Only 2 out of 7 patients tested positive for anti PF4 ELISA antibodies with both assays: the men with stroke showed low positivity (OD = 0,56 and 0,41) and the female with CVT strong positivity (OD = 3,2 and 3,87). Only the female patient with CVT tested positive with both HIT functional assays, PAT and HIT alert cytometry test in the presence of PF4 independently of heparin. Both assays were inhibited by high concentrations of heparin.Conclusions. In our limited experience VITT demonstrated to be an extremely rare event in the context of AZD1222 COVID-19 vaccination even in the subset of patients with thrombosis and thrombocytopenia.

SARS-2-Cov 2 induced coagulopathy: An antiphospholipid cohort study

Introduction Various coagulation alterations have been reported in COVID-19 patients, especially in those with the most severe forms: A) elevated d-dimer (x6 time normal value) correlates with poor prognosis;b) low-molecular weight heparins have been reported as an effective drug to support intensive care;c) thrombosis of the pulmonary vessels has often been found at the autoptic investigation of COVID-19 patients. It is well-known that bacterial and viral infections can induce antiphospholipid antibodies (aPL), which is often not associated to thrombotic events. A recent study reported the presence of aPL mainly of IgA isotype (not part of laboratory work-out for APS), in COVID-19 positive patients, they did not specify the aPL titres and LAC testing was not available. Most importantly from a clinical point of view, all the reported patients have suffered in the past for vascular manifestations. In order to better understand the SARS-2-Cov 2 coagulopathy, we performed an observational study aiming to investigate the status of haemostasis in these patients, performing a panel of coagulation markers. Patients and methods 101 consecutive PCR-confirmed COVID-19 infected patients admitted at the AO Ordine Mauriziano Hospital, Torino, Italy, were included in this study. Patients were tested for aPL profile, D-Dimer, von Willebrand factor, and IL-6 levels. Patients defined as critically ill were those hospitalized in an intensive care unit at the time of the blood withdrawal. Results Our data showed positivity for any aPL in about half of the patients (48.5%). In detail, we found that a positivity for Lupus Anticoagulant (LAC) can be detected in up to 1 out of 3 symptomatic COVID-19 patients when tested according to the ISTH. However, the so called triple aPL positivity, the profile most strongly associated with a thrombotic event in patients with APS (the concomitant presence of LAC, anticardiolipin IgG and anti-beta2GPI IgG antibodies) has been observed in only one patient (1%). When including any triple positivity (for LAC plus anticardiolipin and anti-beta2-GPI either IgG/IgM) we were able to identify 3 patients (3%). More importantly, most of aPL positivity was detected at the low-medium titre;aPL positivity are known to be detectable during infections, to include viral diseases such as HIV and hepatitis C. The presence of aPL in these contexts is often transient and almost always non-specific (nonthrombosis-related). Discussion In conclusion, although isolated, LAC positivity is present in about a third of patients. Their clinical role in the pathogenesis of a coagulopathy and pulmonary thrombosis during COVID-19 infection still need to be demonstrated.