Heparin-induced Platelet Aggregation Research Articles

Proteomic profiling for biomarker discovery in heparin-induced thrombocytopenia

AbstractNew analytical techniques can assess hundreds of proteins simultaneously with high sensitivity, facilitating the observation of their complex interplay and role in disease mechanisms. We hypothesized that proteomic profiling targeting proteins involved in thrombus formation, inflammation, and the immune response would identify potentially new biomarkers for heparin-induced thrombocytopenia (HIT). Four existing panels of the Olink proximity extension assay covering 356 proteins involved in thrombus formation, inflammation, and immune response were applied to randomly selected patients with suspected HIT (confirmed HIT, n = 32; HIT ruled out, n = 38; and positive heparin/platelet factor 4 [H/PF4] antibodies, n = 28). The relative difference in protein concentration was analyzed using a linear regression model adjusted for sex and age. To confirm the test results, soluble P-selectin was determined using enzyme-linked immunosorbent assay (ELISA) in above mentioned patients and an additional second data set (n = 49). HIT was defined as a positive heparin-induced platelet activation assay (washed platelet assay). Among 98 patients of the primary data set, the median 4Ts score was 5 in patients with HIT, 4 in patients with positive H/PF4 antibodies, and 3 in patients without HIT. The median optical density of a polyspecific H/PF4 ELISA were 3.0, 0.9, and 0.3. Soluble P-selectin remained statistically significant after multiple test adjustments. The area under the receiver operating characteristic curve was 0.81 for Olink and 0.8 for ELISA. Future studies shall assess the diagnostic and prognostic value of soluble P-selectin in the management of HIT.

Performance evaluation of heparin-induced platelet aggregation vs serotonin release assay.

Heparin-induced thrombocytopenia (HIT) is a rare but life-threatening condition that requires rapid diagnosis for proper management. Laboratory testing should only be performed on patients with intermediate- or high-risk pretest probability. The platelet factor 4 (PF4) enzyme-linked immunosorbent assay (ELISA) is the screening test that should be confirmed by higher specificity testing such as the heparin-induced platelet aggregation (HIPA) or the serotonin release assay (SRA). This study aims to evaluate the performance of the HIPA in comparison to the SRA, establish cutoffs of the PF4 ELISA to predict positivity for HIPA/SRA, and study the mortality rate between patients with suspected HIT confirmed as HIT positive vs negative. All positive PF4 ELISA cases with confirmatory HIPA and SRA testing were included. As the SRA was considered the gold standard, the HIPA performance was evaluated in comparison to SRA before and after the implementation of a new standardized interpretation guide in 2022. The mortality of these cases was also documented by chart reviews. In total, 232 cases with positive or indeterminate anti-PF4 IgG ELISA had confirmatory testing with HIPA and SRA. The sensitivity of the HIPA improved from 55.4% in 2018 to 2021 to 73.8% in 2022. The specificity remained similarly high in 2018 to 2021 vs 2022 (94.9% vs 87.5%). The negative predictive value (NPV) improved in 2022. The PF4 optical density cutoff to predict the positivity of SRA was 0.85 vs 1.47 to predict the positivity of HIPA but decreased to 0.83 when combining HIPA and/or SRA. There was no significant difference in mortality between patients with suspected HIT confirmed positive vs negative. Although the HIPA has a lower sensitivity than the SRA, the new standardized interpretation guide improved its sensitivity and NPV in 2022. Future improvements are needed to use the HIPA as a stand-alone confirmatory test with the goal to shorten hospital length of stay and expedite proper anticoagulation management.

Diagnostic Performance of a Particle Gel Immunoassay in Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious complication of adenoviral vector-based COVID-19 vaccines. Similar to heparin-induced thrombocytopenia (HIT), antibodies reacting to platelet factor 4 (PF4) are responsible for platelet activation in VITT. The diagnosis of VITT includes the detection of anti-PF4 antibodies. Particle gel immunoassay (PaGIA) is one of the rapid immunoassays that is commonly used in the diagnosis of HIT to detect anti-PF4 antibodies. The aim of this study was to investigate the diagnostic performance of PaGIA in patients suspected of VITT. In this retrospective, single-center study, the correlation between PaGIA, enzyme immunoassay (EIA), and modified heparin-induced platelet aggregation assay (HIPA) in patients with findings suggestive of VITT was investigated. A commercially available PF4 rapid immunoassay (ID PaGIA H/PF4, Bio-Rad-DiaMed GmbH, Switzerland) and an anti-PF4/heparin EIA (ZYMUTEST HIA IgG, Hyphen Biomed) were used according to manufacturer's instructions. Modified HIPA was accepted as the gold standard test. Between March 8 and November 19, 2021, a total of 34 samples from clinically well-characterized patients (14 males, 20 females, mean age: 48.2 ± 18.2 years) were analyzed with PaGIA, EIA, and modified HIPA. VITT was diagnosed in 15 patients. Sensitivity and specificity of PaGIA were 54 and 67%, respectively. Anti-PF4/heparin optical density values were not significantly different between PaGIA positive and negative samples (p = 0.586). The sensitivity and specificity of EIA, on the other hand, were 87 and 100%, respectively. In conclusion, PaGIA is not reliable in the diagnosis of VITT because of its low sensitivity and specificity.

Heparin-induced thrombocytopenia following Shiga-toxin-associated hemolytic uremic syndrome: a case report

BackgroundUp to 50% of cases of Shiga-toxin-producing Escherichia coli hemolytic uremic syndrome occur in adults, and the clinical presentation is variable. Microbiological analyses must be performed in all patients with thrombotic microangiopathy to identify Shiga-toxin-producing Escherichia coli, even in the absence of diarrhea.Case presentationA 79-year-old Caucasian woman was admitted to hospital because of severe proctitis. In the following days, the patient’s level of consciousness declined, and she developed acute kidney injury, thrombocytopenia, and hemolytic anemia. Shiga-toxin-producing Escherichia coli was found in fecal cultures, suggesting the diagnosis of hemolytic uremic syndrome. In the following days, her clinical conditions improved, but thrombocytopenia worsened, and the patient developed posterior tibial vein thrombosis. The discordant evolution of thrombocytopenia compared with other clinical and laboratory parameters prompted a new evaluation of its causes. Diagnosis of heparin-induced thrombocytopenia was confirmed by heparin-induced platelet aggregation assay and positive antibodies to platelet factor 4.ConclusionsA discordant evolution of platelet count in patients with thrombotic microangiopathy requires a systematic reevaluation of the thrombocytopenia.

Similarities of antiphospholipid antibodies in HIT and APS patients with heparin-platelet factor 4 antibodies

Heparin-induced thrombocytopenia (HIT) is a prothrombotic autoimmune disorder confirmed by the existence of Heparin-Platelet Factor 4 (HPF4) antibodies. The aim of this work is to study the possible relationship between anti-HPF4 and antiphospholipid antibodies (aPLs) that may explain the discrepancies observed in patients with a suspected HIT (HIT group) with positive immunoassay (HPF4-Elisa) and negative functional assay (heparin-induced platelet aggregation test). So, we performed H-PF4 antibodies research in 31 APS confirmed patients (APL group). All tests performed have been compared to normal controls (n = 34). We found anti-H-PF4 in 7/31 patients of APL group. In parallel, we search for aPLs in 9/34 patients tested positive for anti-HPF4 in HIT group, all of them were positive. All specificities were observed in the two anti-HPF4 positive groups (aβ2GP1 IgM/IgG/IgA, aCL (IgM/IgG/IgA, aPS-PT IgM/IgG). The most associated antibodies with anti-HPF4 are the anti ß2Glycoprotein1 (Odds ratio = 50.1). We suggest that the presence of aPLs in HIT group with anti-HPF4 could be the cause of the discrepancies. In addition, we performed the Heparin Neutralization Assay (HNA) which is specific for anti-HPF4, neutralization was obtained for patients exposed to heparin. Furthermore, we suggest that we should performed a larger cohort to confirm the causal relationship of aPLs and also to expand the tests allowing the differentiation between these autoantibodies.

Managing argatroban in heparin-induced thrombocytopenia: Aretrospective analysis of 729 treatment days in 32 patients with confirmed heparin-induced thrombocytopenia.

Managing argatroban in heparin-induced thrombocytopenia: Aretrospective analysis of 729 treatment days in 32 patients with confirmed heparin-induced thrombocytopenia.

Platelet ACKR3/CXCR7 favors antiplatelet lipids over an atherothrombotic lipidome and regulates thromboinflammation

AbstractPlatelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation. Pharmacological CXCR7 agonist (VUF11207) significantly reduced prothrombotic platelet response in blood from acute coronary syndrome patients ex vivo. CXCR7 agonist administration reduced thrombotic functions and thromboinflammatory plateletleukocyte interactions post–myocardial infarction and arterial injury in vivo. ACKR3/CXCR7 ligation did not affect surface availability of surface receptors, coagulation profile, bleeding time, plasma-dependent thrombin generation (thrombinoscopy), or clot formation (thromboelastography) but counteracted activation-induced phosphatidylserine exposure and procoagulant platelet-assisted thrombin generation. Targeted (micro-UHPLC-ESI-QTrap-MS/MS) and untargeted (UHPLCESI-QTOF-MS/MS) lipidomics analysis revealed that ACKR3/CXCR7 ligation favored generation of antithrombotic lipids (dihomo-γ-linolenic acid [DGLA], 12-hydroxyeicosatrienoic acid [12-HETrE]) over cyclooxygenase-1 (COX-1) or 12-lipoxygenase (12-LOX) metabolized prothrombotic and phospholipase-derived atherogenic lipids in healthy subjects and CAD patients, contrary to antiplatelet therapy. Through 12-HETrE, ACKR3/CXCR7 ligation coordinated with Gαs-coupled prostacyclin receptor to trigger cyclic adenosine monophosphate/protein kinase A–mediated platelet inhibition. ACKR3/CXCR7 ligation reduced generation of lipid agonists and lipid signaling intermediates, which affected calcium mobilization, intracellular signaling, and consequently platelet interaction with physiological matrices and thromboinflammatory secretome. This emphasized its functional dichotomy from prothrombotic CXCR4. Moreover, CXCR7 agonist regulated heparin-induced thrombocytopenia–sera/immunoglobulin G–triggered platelet and neutrophil activation, heparin-induced platelet aggregation, generation of thromboinflammatory lipids, platelet-neutrophil aggregate formation, and thromboinflammatory secretion ex vivo. Therefore, ACKR3/CXCR7 may offer a novel therapeutic strategy in acute/chronic thromboinflammation exaggerated cardiovascular pathologies and CAD.

High Incidence of True Heparin-Induced Thrombocytopenia and Heparin-Induced Thrombocytopenia Antibody Development in Critically Ill Medical Patients

Background: Thrombocytopenia is very prevalent in critically ill medical patients. Heparin-induced thrombocytopenia (HIT), which is a serious immunologic complication of heparin administration, is one of the major concerns. Diagnosis of HIT in critically ill patients is challenging due to concomitant illnesses. We previously reported the HIT cohort with the high proportion of critically ill medical patients (61.7%) in Thailand (Uaprasert N, etal. Blood Coagul Fibrinolysis 2013;24:261-8). However, data of the true incidence and risk factors for HIT in this particular population in Thailand are still lacking. In addition, the confirmatory assay is not readily available and unlikely to be useful for timely clinical management. Therefore, clinical predictors and screening assays are important for immediate clinical judgment. This study aimed to determine the incidence and define risk factors for HIT and HIT antibody (HIT-Ab) development as well as an appropriate clinical predicting model for HIT in critically ill medical patients.Methods: Consecutive critically ill medical patients who received heparins and were admitted in the intensive care unit (ICU), coronary care unit (CCU) and intensive coronary care unit (ICCU) at King Chulalongkorn Memorial Hospital between October 2013 and May 2017 were included in this study. Patients admitted in general wards who met one of the following criteria: 1) on mechanical ventilator 2) receiving inotropic agents 3) recent undergoing renal replacement therapy due to acute kidney injury and/or 4) APACHE II score > 25, were also included for analysis. Blood samples were collected between 6 and 8 days after heparin exposure. Patients died or discharged before day 6th after heparin exposure were excluded from the study. The rapid particle gel immunoassay (PaGIA) was used as the screening test for detecting antibodies against the heparin/platelet factor 4 complex. Subjects yielding positive PaGIA were sent for the confirmatory test using the in-house platelet aggregometry measuring heparin-induced platelet aggregation (HPA). Details of the medical history and hospital course of individual patient were reviewed to obtain risk factors for HIT as well as to compute two well-defined clinical probability scores (4Ts and HIT expert probability, HEP).Results: There were 217 patients included for analysis. The average age was 64.9 ± 18.7 (mean ± standard deviation) years. The baseline platelet counts were 217 ± 116 x 109/L. A platelet fall ≥ 50% from baseline was found in 135 (62.2%) patients. The most common type of heparin administered was unfractionated heparin (165; 76%). HIT-Ab using PaGIA was detected in 52 (24%) patients, while true HIT was confirmed using HPA in 3 patients (1.38%). Most common underlying illnesses found in patients developing HIT-Ab were hypertension, coronary arterial disease and active infection. Acute stroke, a platelet fall ≥ 50% from baseline, 4Ts score of > 3 and HEP score of ≥ 2 were significantly associated with development of HIT-Ab in univariate analysis, while heparin types and hemodialysis were not statistically associated with HIT-Ab development. Notably, only acute stroke was significantly associated with HIT-Ab development in multivariate analysis (Odds ratio 6.56; 95% confidence interval 1.42-30.31, p=0.016).Conclusion: The incidence of HIT-Ab detected in critically ill medical patients in our cohort was comparable to most studies, while the incidence of true HIT in this cohort was apparently higher than previous reports, which their incidences were ≤ 0.5%. The reasons underlying the high incidence of true HIT in critically ill medical patients in Thailand remains elusive. Acute stroke is a significant risk for HIT-Ab development in critically ill medical patients receiving heparin. DisclosuresNo relevant conflicts of interest to declare.

Antibody-mediated procoagulant platelets in SARS-CoV-2-vaccination associated immune thrombotic thrombocytopenia.

The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. In order to prevent severe infection, mass COVID-19 vaccination campaigns with several vaccine types are currently underway. We report pathological and immunological findings in eight patients who developed vaccine-induced immune thrombotic thrombocytopenia (VITT) after administration of SARS-CoV-2 vaccine ChAdOx1 nCoV-19. We analyzed patient material using enzyme immune assays, flow cytometry and heparin-induced platelet aggregation assay and performed autopsies on two fatal cases. Eight patients (five females, three males) with a median age of 41.5 years (range, 24-53) were referred to us with suspected thrombotic complications 6 to 20 days after ChAdOx1 nCoV-19 vaccination. All patients had thrombocytopenia at admission. Patients had a median platelet count of 46.5x109/L (range, 8-92). Three had a fatal outcome and five were successfully treated. Autopsies showed arterial and venous thromboses in various organs and the occlusion of glomerular capillaries by hyaline thrombi. Sera from VITT patients contain high-titer antibodies against platelet factor 4 (PF4) (optical density [OD] 2.59±0.64). PF4 antibodies in VITT patients induced significant increase in procoagulant markers (Pselectin and phosphatidylserine externalization) compared to healthy volunteers and healthy vaccinated volunteers. The generation of procoagulant platelets was PF4 and heparin dependent. We demonstrate the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.

The complicated relationships of heparin‐induced thrombocytopenia and platelet factor 4 antibodies with COVID‐19

COVID‐19 (coronavirus disease 2019) represents a prothrombotic disorder, and there have been several reports of platelet factor 4/heparin antibodies being present in COVID‐19‐infected patients. This has thus been identified in some publications as representing a high incidence of heparin‐induced thrombocytopenia (HIT), whereas in others, findings have been tempered by general lack of functional reactivity using confirmation assays of serotonin release assay (SRA) or heparin‐induced platelet aggregation (HIPA). Moreover, in at least two publications, data are provided suggesting that antibodies can arise in heparin naïve patients or that platelet activation may not be heparin‐dependent. From this literature, we would conclude that platelet factor 4/heparin antibodies can be observed in COVID‐19‐infected patients, and they may occur at higher incidence than in historical non‐COVID‐19‐infected cohorts. However, the situation is complex, since not all platelet factor 4/heparin antibodies may lead to platelet activation, and not all identified antibodies are heparin‐dependent, such that they do not necessarily reflect “true” HIT. Most recently, a “HIT‐like” syndrome has reported in patients who have been vaccinated against COVID‐19. Accordingly, much more is yet to be learnt about the insidious disease that COVID‐19 represents, including autoimmune outcomes in affected patients.

Heparin-Induced Thrombocytopenia in COVID-19: A Systematic Review.

Background:It has been more than a year since the whole world is struggling with COVID-19 pandemic and may experience resurgences in the near future. Along with severe pneumonia, this disease is notorious for extensive thromboembolic manifestations. That is why experts advocated aggressive anticoagulation as a part of the therapy since the beginning. However, from May 2020 onwards, cases of heparin-induced thrombocytopenia (HIT) are being reported. HIT in itself is an autoimmune entity leading to life-threatening thrombosis in the setting of thrombocytopenia. Continuation of heparin can have disastrous consequences in case of unrecognized HIT. Hence, timely recognition of HIT is of utmost value to modify the anticoagulation strategy and salvaging lives. We performed a systemic review trying to find all reported cases of HIT in COVID-19.Methods:It involved extensive search of the databases including PubMed, Google Scholar, Scopus, and Embase in an attempt to find all reported literature in the last 1 year (November 1, 2019–December 25, 2020) using keywords in various combinations. Literature search resulted in a total of 27 articles and 12 articles were finally selected based on the study design and their relevance pertaining to the intervention done and the outcome of interest. Results:A total of 35 patients were included (mean age 56.7 ± 12.8 years, male-to-female ratio = 2:1). The most frequent comorbidity was hypertension. Fifty-seven percent of cases were with low-molecular weight heparin and the rest with unfractionated heparin. Confirmatory functional assay was done in 85.7% of cases (67% by serotonin-release assay [SRA] and 33% by heparin-induced platelet aggregation [HIPA]). All cases tested with HIPA were positive, while with SRA, only 30% were positive. The most common alternate anticoagulation used was argatroban infusion. The new arterial thrombotic event was seen in only 5.7% of cases as repeat myocardial infarction, stroke, and splenic infarction, while clinically significant bleeding was seen in 17.1% of cases. Fifty percent of bleeding episodes were seen where conventional doses of argatroban were used, while no mortality was seen with low-dose argatroban infusion. However, only 45.7% of patients were discharged, 31.4% of patients died, while the outcome was pending for 23% of patients.Conclusion:Severe endotheliitis and immune dysregulation giving rise to HIT antibodies and antiphospholipid antibodies have been demonstrated in COVID-19 and modifying our therapy becomes indispensable when it is pathogenic with potentially fatal consequences. In the light of interim results of REMAP-CAP study in severe COVID-19 cases where heparin does not improve the outcome, the present anticoagulation strategy needs re-evaluation. Unrecognized HIT can be catastrophic and close clinical monitoring is required for patients on heparin therapy.

Heparin Induced Thrombocytopenia in Patients with COVID-19

BackgroundThe Coronavirus disease-2019 (COVID-19) is a global pandemic caused by novel coronavirus SARS-CoV-2. Acute respiratory and renal failure as well as systemic coagulopathy are critical aspects of the morbidity and mortality in patients with COVID-19. Heparin Induced Thrombocytopenia (HIT) occurs when IgG antibodies form against platelet factor 4-Heparin complex, resulting in platelet activation and removal, leading to a prothrombotic state. HIT is suspected when there is a platelet count decrease of more than 50% after exposure to heparin, along with hypercoagulability. Clinical Scoring systems like 4T Score (Thrombocytopenia, Timing, Thrombosis, no other cause of Thrombocytopenia) have been developed to assess the pretest probability of HIT. The use of unfractionated heparin, post-orthopedic and post-cardiac surgery state, female gender, and old age are recognized as risk factors for HIT. There is a nine-fold increased risk of developing HIT in patients requiring hemodialysis. ICU patients and patients with VTE without thrombocytopenia are considered to have low pre-test probability for HIT. Studies have shown that only 6% who are investigated serologically for HIT actually have the diagnosis. We conducted this study to assess the incidence and risk factors for HIT in COVID-19 positive patients and its impact on mortality.MethodsA retrospective analysis was performed on all patients who were COVID-19 positive and hospitalized between March 1, 2020 and June 26, 2020 at our institution. Patients with intermediate or high suspicion for HIT, based on 4T score of 4 or higher, underwent IgG-specific platelet factor 4(PF4)-dependent enzyme immune assay (EIA). Washed platelet assays such as serotonin release assay (SRA) and heparin-induced platelet aggregation (HIPA) were used as confirmatory tests in cases with intermediate or low optical density (OD) with EIA. The incidence of HIT, its impact on mortality, and positivity of IgG-specific PF4-EIA in COVID-19 patients were studied, and statistical analysis was done with X2 testing. Subgroup analysis was performed based on demographic factors and risk factors for HIT, including exposure to heparin or low molecular weight heparin (LMWH), history of or cancer, recent orthopedic or cardiac surgery, exposure to renal replacement therapy (RRT), and severity of disease (D-dimer >6 ULN, Acute Kidney Injury, ICU admission, and mechanical ventilation requirement). These factors were analyzed by Fisher’s exact test to determine their impact on mortality. The hospital course for HIT antibody-positive patients was further analyzed to study the impact of COVID-19 related therapy, such as Remdesivir, Tocilizumab, Hydroxychloroquine, Steroids, and anticoagulation after diagnosis of HIT.ResultsWEight out of 1265 hospitalized COVID-19 positive patients tested positive for IgG-specific platelet factor 4(PF4)-dependent enzyme immune assay (EIA+). Incidence of EIA+ in COVID-19 patients was 0.6%, which is significantly higher than in the general population 0.2% (p<.0001, 95% CI 0.25-1.20%). The incidence of thromboembolic events in EIA+ patients was 87.5%, significantly higher than the rate of 10.90% in all COVID-19 patients (p<.0001, CI 41.96- 86.98%). The mortality rate in EIA+ patients was 50%, significantly greater than the mortality rate of 12% in all hospitalized COVID-19 patients (p=.0011, CI 9.46-66.53). Serological confirmation of HIT diagnosis was 37.5% (2 had OD>1, 1 was SRA positive) which is significantly higher than confirmation of HIT in nonCOVID-19 patients 6% (p<.0001, 95% CI 29.57-85.32%). All 3 confirmed HIT patients (100%) had severe disease (3/3 required ICU admission, 2/3 required mechanical ventilation, the one not requiring mechanical ventilation required RRT).ConclusionOur study indicates incidence of HIT is higher in the COVID-19 population. The incidence of positive EIA for patients with intermediate to high 4T scores is also higher in COVID-19 positive patients. This can be attributed to the cytokine storm and severe sepsis seen in critically ill COVID-19 patients. Our study also suggests that development of HIT can contribute to increased risk for thromboembolic events as well as mortality of COVID-19 patients, however, our study is limited due to small sample size. Therefore, prospective studies are needed to analyze the impact of HIT on morbidity, mortality and long-term outcomes in COVID-19 patients.TableDisclosuresNo relevant conflicts of interest to declare.

Genome-Wide Association Study Identifies Variation in ABO As Risk Factor for Platelet Reactivity in Heparin-Induced Thrombocytopenia

Background: Heparin-induced thrombocytopenia (HIT) is an unpredictable, potentially catastrophic adverse effect of heparin treatment resulting from an immune response to platelet factor 4 (PF4)/heparin complexes. Genome-wide association studies (GWAS) have the potential to identify genetic risk factors and generate insights into biological mechanisms of adverse drug reactions. However, published HIT GWAS are underpowered and do not include confirmation of HIT using functional assays. We aimed to identify genetic associations with HIT using a GWAS approach in two European ancestry populations tested for functional assay and PF4/heparin antibody levels. Methods: We performed a GWAS with positive functional assay as the outcome in a large discovery cohort of patients, including heparin-induced platelet aggregation (HIPA) positive cases (n=1243), antibody positive but functional assay negative controls (n=1091), and antibody negative controls (n=1718). Logistic regressions were adjusted for age, gender, and first two principal components. Significant associations from the discovery cohort (alpha=5.00x10-8) were then investigated in a replication cohort of serotonin-release assay (SRA)-confirmed HIT cases (n=173), antibody positive controls (n=125), and antibody negative controls (n=488). All patients were genotyped with the HumanOmniExpressExome BeadChip, including 9,065,510 variants after imputation and quality control. Genes near significantly associated loci were resequenced. Results: In the discovery cohort, a genome-wide significant association was observed between common variation in the ABO gene and HIPA positive status (rs505922 odds ratio 0.738 [0.668-0.816], p=2.813x10-9). This variant was also significantly associated with SRA-confirmed HIT in the replication cohort (odds ratio 0.392 [0.172-0.891], p=0.025). Meta-analysis indicated strong association of ABO variation with positive functional assay (Figure 1). Sequencing and fine-mapping of the ABO locus indicated this effect was driven by ABO blood groups, with the O blood group being a risk factor for HIT (odds ratio 1.42 [1.25-1.61], p=6.11x10-8). Conclusions: We identify common variation in ABO as a risk factor for platelet reactivity and HIT. Strong association of ABO variation may have important implications for prediction and understanding of HIT pathogenesis. Figure 1 Disclosures No relevant conflicts of interest to declare.

Potential and Limitations of the New P2Y12 Inhibitor, Cangrelor, in Preventing Heparin-Induced Platelet Aggregation During Cardiac Surgery: An In Vitro Study.

Heparin-induced thrombocytopenia (HIT) can put cardiac surgery patients at a high risk of lethal complications. If anti-PF4/heparin antibodies (anti-PF4/Hep Abs) are present, 2 strategies exist to prevent intraoperative aggregation during bypass surgery: first, using an alternative anticoagulant, and second, using heparin combined with an antiaggregant. The new P2Y12 inhibitor, cangrelor, could be an attractive candidate for the latter strategy; several authors have reported its successful use. The present in vitro study evaluated cangrelor's ability to inhibit heparin-induced platelet aggregation in the presence of anti-PF4/Hep Abs. Platelet-poor plasma (PPP) from 30 patients with functional anti-PF4/Hep Abs was mixed with platelet-rich plasma (PRP) from 5 healthy donors.Light transmission aggregometry was used to measure platelet aggregation after adding 0.5 IU·mL of heparin (HIT) to the plasma, and this was compared with samples spiked with normal saline (control) and samples spiked with cangrelor 500 ng·mL and heparin 0.5 IU·mL (treatment). Friedman test with post hoc Dunn-Bonferroni test was used for between-group comparisons. Heparin 0.5 IU·mL triggered aggregation in 22 of 44 PPP-PRP mixtures, with a median aggregation of 86% (interquartile range [IQR], 69-91). The median aggregation of these 22 positive samples' respective control tests was 22% (IQR, 16-30) (P < .001). Median aggregation in the cangrelor-treated samples was 29% (IQR, 19-54) and significantly lower than the HIT samples (P < .001). Cangrelor inhibited heparin-induced aggregation by a median of 91% (IQR, 52-100). Cangrelor only reduced heparin-induced aggregation by >95% in 10 of the 22 positive samples (45%). Cangrelor inhibited heparin-induced aggregation by <50% in 5 of the 22 positive samples (22%) and by <10% in 3 samples (14%). This in vitro study found that cangrelor was an unreliable inhibitor of heparin-induced aggregation in the presence of anti-PF4/Hep Abs. We conclude that cangrelor should not be used as a standard antiaggregant for cardiac patients affected by HIT during surgery. Unless cangrelor's efficacy in a particular patient has been confirmed in a presurgery aggregation test, other strategies should be chosen.

Accuracy of heparin-induced platelet aggregation test for the diagnosis of heparin-induced thrombocytopenia

IntroductionWhereas the utility of washed platelet assays such as the heparin-induced platelet activation test (HIPA) for the diagnosis of heparin-induced thrombocytopenia (HIT) is regarded as high, the performance of simpler assays such as the heparin-induced platelet aggregation test (PAT) is still elusive. Using well-characterized samples of a large cohort study, we aimed to assess the diagnostic accuracy of PAT for the diagnosis of HIT. Material and methodsOne-hundred twenty-two immunoassay-positive serum samples of a previous, prospective single-center cohort study including consecutive patients with suspected HIT (n = 1291) were used. HIPA was determined as reference gold standard; samples were previously analyzed using PAT as well as polyspecific platelet factor 4/heparin enzyme-linked immunosorbent assay (ELISA). 4Ts score was calculated using the patient documentation. Diagnosis of HIT was defined as a positive HIPA, which is a positive reaction in 2 out of 4 donor platelets within 30 min. ResultsHIT was diagnosed in 39 out of 122 patients corresponding to a prevalence of 32%. Median optical density (ELISA) was 2.8 (inter-quartile range 2.3, 3.0) in patients with HIT and 0.7 (0.5, 1.3) in patients without HIT. PAT was positive in 27 out of 39 HIT patients and it was negative in 83 out of 83 HIT-negative patients. Thus, the sensitivity of PAT for the diagnosis of HIT was 69% (95% confidence interval 52%, 83%) and the specificity 100% (96%, 100%). ConclusionsOur results demonstrate that PAT is a valuable test to confirm HIT but cannot be applied to rule-out HIT in clinical practice.

The new P2Y12 inhibitor cangrelor unreliably inhibits heparin-induced platelet aggregation in the presence of hit antibodies, an in vitro study

Introduction Cardiac surgery in patients with HIT puts the patient at high risk of lethal thrombotic complications if heparin is used during surgery. Two strategies exist to prevent intraoperative platelet aggregation during cardio-pulmonary bypass if anti-PF4/heparin antibodies (HIT-Abs) are present. The first is to use an alternative anticoagulant, the second is to use heparin combined with an antiaggregant agent, iloprost or tirofiban. The new P2Y12 inhibitor cangrelor could be an attractive candidate in this setting and several authors report its successful use. In this in vitro study we evaluated the capacity of cangrelor to inhibit platelet aggregation induced by heparin in the presence of HIT-Abs. Methods Platelet poor plasma (PPP) from 30 patients with functional HIT-Abs was mixed with platelet rich plasma (PRP) from healthy donors. Heparin-induced platelet aggregation (HIPA) was measured by light transmission aggregometry (LTA) after adding heparin to achieve a final concentration of 0.5 IU ml-1 and compared to samples with normal saline only (negative control) or cangrelor (final concentration 500 ng ml-1) added prior to heparin (treatment). Results Heparin 0.5 IU ml-1 triggered platelet aggregation in 22 out of 44 PPP-PRP mixtures, with a median aggregation of 85.9 % (IQR 69.2 to 90.9). For these 22 HIPA positive samples, the median aggregation in the corresponding negative control was 22.1% (IQR 15.9 - 29.7) (p Discussion In this in vitro study we found that cangrelor unreliably inhibits heparin-induced platelet aggregation in the presence of HIT-Abs. We conclude that cangrelor cannot be used as a standard antiaggregant agent in combination with heparin for cardiac surgery in HIT patients, unless its efficacy has been confirmed in a functional test prior to surgery.

Evaluation of a flow cytometer-based functional assay using platelet-rich plasma in the diagnosis of heparin-induced thrombocytopenia

BackgroundHeparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4/heparin (PF4/hep)-complexes. The in vitro demonstration of PF4/hep antibodies using functional assays is essential for an optimal management of patients suspected to have HIT. However, conventional functional assays are technically challenging and limited to specialized laboratories. In contrast, flow cytometers are commonly used in routine laboratories. The aim of this study is to investigate the performance characteristics of a commercially available, flow cytometer based assay in the diagnosis of HIT. Study designSera of consecutive patients with suspected HIT were investigated using the Emo-test HIT Confirm® assay and compared to the standard method consisting of an IgG-specific enzyme immunoassay (EIA) for anti-PF4/hep antibodies and the heparin induced platelet aggregation (HIPA) test. Results390 sera were included in the study, 164 sera tested IgG EIA-positive, of which 33 also tested HIPA-positive. No HIPA-positive samples were EIA-negative. In the Emo-test HIT Confirm® assay, 112 sera revealed positive results (%Hepla > 13); however, 51 (45.5%) were EIA-negative. Of the 33 HIPA-positive/EIA-positive HIT sera, 23 tested positive in the Emo-test HIT Confirm® assay, 2 gave ambiguous results, and 8 sera yielded false-negative results. Accordingly, the HIT Confirm® assay showed a sensitivity of 69.7% with a slightly better specificity of 75.4% compared to the EIA (sensitivity 100%, specificity 63.3%). An increase in diagnostic specificity for HIT to 85% was found when positive results were obtained in both the Emo-test HIT Confirm® assay and EIA. ConclusionThe Emo-Test HIT Confirm® assay may improve the specificity of laboratory investigations of HIT. However, the assay can only be recommended in combination with an immunoassay due to the high rate of false negativity. Our observation indicates a need to establish external quality assessment for functional assays to avoid such clinically relevant pitfalls.

Heparin-induced thrombocytopaenia diagnostic testing after adult cardiac surgery: a single-centre experience and development.

To assess the incidence and outcome of heparin-induced thrombocytopaenia (HIT) in patients after cardiac surgery on cardiopulmonary bypass (CPB) and to review the time course of platelet counts and the use of different immunological diagnostic tests. All patients ≥18 years of age who underwent CPB from 2006 to 2015 and who were postoperatively admitted to our cardiac intensive care unit (ICU) were included in this retrospective study. Screening for heparin/platelet factor-4 antibodies was performed using an antibody test, which was later replaced with a screening test specific for IgG antibodies without IgA/IgM cross-reactivity. The enzyme immunoassay (EIA) for the detection of antibodies of all immunoglobulin classes against heparin/PF4 complexes was replaced with an IgG-specific EIA. HIT was confirmed by a heparin-induced platelet aggregation test until 2014. Among 4978 patients admitted between 2006 and 2015, 539 (11%) patients were evaluated for HIT. Patients were excluded because of age <18 years (n = 9), non-cardiac surgery without CPB (n = 10) or incomplete data (n = 3). Of the remaining 517 patients, 43 (8.3%) patients were HIT-positive. HIT incidence was 0.86%. The proportion of HIT-positive patients was similar in men and women (8.4% and 8.2%, respectively). Men and women with suspected HIT also had similar in-hospital mortality (odds ratio ≈ 1; P = 0.926). The incidence of HIT was lower in our study than previously reported. Novel immunological tests have improved to specifically detect IgG antibodies. Furthermore, they are able to detect anti-protamine antibodies, which may be present in patients with high clinical probability of testing negative for HIT. Incidence and clinical relevance of heparin/protamine antibodies will be subjects of future investigation.

Evaluation of STic Expert® HIT Kit and Its Comparison with ID-PaGIA™ Test in Suspected Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction caused by heparin. HIT occurs due to IgG antibodies directed against heparin-bound platelet factor 4 (PF4). The aim of this study was to evaluate the STic® Expert HIT for detection of anti-PF4/heparin antibodies in comparison with ID-PaGIA™ kit. The results were further confirmed by Heparin-induced platelet aggregation test (HIPA). A total of 17 patients with a suspected diagnosis of HIT were enrolled. The 4T scoring of each case was performed. Testing for HIT was carried out by ID-PaGIA™, STic® Expert HIT, and HIPA. Testing by STic® Expert HIT test demonstrated positivity in three cases while testing by ID-PaGIA™ test kit revealed four positive cases. Two of these cases were confirmed as HIT by HIPA. The study suggests that STic® Expert HIT an equally effective test, in combination with the 4T scoring system for detecting/excluding the diagnosis of HIT. A large number of cases are needed for validation.

Idiopathic catastrophic thrombosis with happy ending

A 59-year-old male patient suffered three life-threatening instent thromboses after an initial resuscitation due to an ST-segment elevation myocardial infarction of the anterior cardiac wall. With a high-risk profile for...