Global coagulation assays detect an early prothrombotic state in children with acute lymphoblastic leukemia.

BACKGROUND: Pediatric patients with acute lymphoblastic leukemia (ALL) are at highest risk of venous thromboembolism (VTE) during the induction phase of treatment (IT). These events are not predictable by conventional coagulation assays. OBJECTIVES: To investigate the utility of global coagulation assays (GCA) for assessing the hemostatic state in children with ALL during IT. METHODS: We included children with ALL (n=15) and healthy controls (n=15). Analyses were performed at different time points during IT of the AIEOP-BFM protocols. In addition to prothrombotic biomarkers, natural anticoagulants proteins and in vivo thrombin generation (TG) markers, ex vivo TG was measured using the gold-standard Calibrated-Automated-Thrombogram method (CAT), the automated-ST Genesia (STG), and Thrombodynamics-analyzer (TD). The latter also provided measurement of fibrin clot formation (FCF). RESULTS: Differently from conventional coagulation assays and in vivo TG markers, ex vivo GCA, detected increasing prothrombotic changes during IT. Particularly, TG measured with TD as expressed by endogenous thrombin potential (ETP) was significantly elevated already at d8-12 (p<0.01) and continued to increase during IT as compared to prior to treatment beginning, indicating a very early shift towards a procoagulant state. A similar pattern was observed for the rate of FCF (V:p<0.01 at d8-12). Remarkably, in patients developing thrombotic complications (n=5), both GCA, STG and TD, showed a significantly higher ETP very early (already at d8-12, p<0.05), well before clinical manifestation. CONCLUSION: GCA capture prothrombotic changes early during IT in ALL pediatric patients. If confirmed, this approach will allow tailoring thromboprophylaxis in children with ALL at highest risk for VTE.

Identification of High Platelet Reactivity Despite ADP P2Y 12 Inhibitor Treatment: Two Populations in the Vasodilator-Stimulated Phosphoprotein Assay and Variable PFA-P2Y Shapes of Curve.

Introduction Response to ADP P2Y 12 receptor inhibition by clopidogrel can be evaluated by various techniques. Here, we compared a functional rapid point-of-care technique (PFA-P2Y) with the degree of biochemical inhibition assessed by the VASP/P2Y 12 assay. Methods Platelet response to clopidogrel was investigated in 173 patients undergoing elective intracerebral stenting (derivation cohort n = 117; validation cohort n = 56). High platelet reactivity (HPR) was defined as PFA-P2Y occlusion time <106 seconds or VASP/P2Y 12 platelet reactivity index (PRI) >50%. Results In the derivation cohort, receiver operator characteristics analysis for the ability of PFA-P2Y to detect biochemical HPR showed high specificity (98.4%) but poor sensitivity (20.0%) and a very low area under the curve (0.59). The VASP/P2Y 12 assay revealed two coexisting platelet populations with different levels of vasodilator-stimulated phosphoprotein (VASP) phosphorylation: a fraction of highly phosphorylated, inhibited platelets and another of poorly phosphorylated, reactive platelets. Analysis of the PFA-P2Y curve shape revealed different types, categorized by time of occlusion (<106 seconds, 106 to 300 seconds, >300 seconds), and pattern (regular, irregular, and atypical). Noteworthy, curves with late occlusion and permeable curves with an irregular or atypical pattern correlated with VASP-PRI >50% and smaller sizes of the inhibited platelet subpopulation. Considering the PFA-P2Y shape of the curve for the detection of HPR improved sensitivity (72.7%) and preserved specificity (91.9%), with a rather high AUC (0.823). The validation cohort confirmed the VASP/P2Y 12 assay data and the usefulness of considering the PFA-P2Y curve shape. Conclusion In patients treated with acetylsalicylic acid and clopidogrel for 7-10 days, the VASP/P2Y 12 assay reveals two coexisting subpopulations of differentially inhibited platelets, whose relative sizes predict global PRI and distinct PFA-P2Y curve patterns, indicating incomplete clopidogrel efficacy. The detailed analysis of both VASP/P2Y 12 and PFA-P2Y is necessary for optimal detection of HPR.

Combinations of rapid immunoassays for a speedy diagnosis of heparin-induced thrombocytopenia.

BACKGROUND: Early recognition and treatment of heparin-induced thrombocytopenia (HIT) are key to prevent severe complications. OBJECTIVE: To assess the diagnostic performance of rapid immunoassays (IA) in detecting anti-PF4/heparin-antibodies. METHODS: Diagnostic performances of lateral-flow IA (LFIA; STic Expert HIT) and latex IA (LIA; HemosIL HIT-Ab) were analyzed in pilot (n = 74) and derivation cohorts (n = 267). Two novel algorithms based on the combination of HIT clinical probability with sequentially performed LIA and chemiluminescent IA (CLIA; HemosIL AcuStar-HIT-IgG) were compared with published rapid diagnostic algorithms: the "Lausanne algorithm" sequentially combining CLIA and particle-gel IA (PaGIA) and the "Hamilton algorithm" based on simultaneously performed LIA and CLIA. RESULTS: LFIA missed 6/30 HIT. The sensitivity and specificity of LIA were 90.9% and 93.5%. The Lausanne algorithm correctly predicted HIT in 19/267 (7.1%), excluded it in 240/267 (89.9%), leaving 8/267 (3%) cases unsolved. The algorithm sequentially combining CLIA and LIA predicted HIT in 19/267 (7.1%) with 1/19 wrong prediction, excluded it in 236/267 (88.4%), leaving 11/267 (4.1%) cases unsolved. The algorithm employing LIA as a first assay predicted HIT in 22/267 (8.2%), excluded it in 235/267 (88%), leaving 9/267 (3.4%) cases unsolved. Finally, the Hamilton algorithm correctly predicted HIT in 10/267 (3.7%), excluded it in 229/267 (85.7%), leaving 28/267 (10.5%) cases unsolved. CONCLUSION: LFIA cannot be used to exclude or predict HIT when using frozen plasma. A Bayesian approach sequentially employing two rapid immunoassays for anti-PF4/heparin antibodies is most effective for the accurate diagnosis of HIT. Based on retrospective data, the combination LIA/CLIA is a candidate for a prospective validation.

Rapid and accurate Bayesian diagnosis of heparin-induced thrombocytopenia.

Prompt diagnostic evaluation of suspected heparin-induced thrombocytopenia (HIT) is critical for guiding initial patient management. We assessed the performance of 3 immunoassays detecting anti-platelet factor 4 (PF4)/heparin antibodies, derived a diagnostic algorithm with a short analytical turnaround time (TAT), and prospectively validated the algorithm. Plasma samples were analyzed by Zymutest-HIA-IgG, HemosIL-AcuStar-HIT-IgG, and ID-H/PF4-PaGIA in retrospective (n = 221) and prospective (n = 305) derivation cohorts. We calculated likelihood ratios of result intervals and cutoff values with 100% negative (NPV) and positive (PPV) predictive values for a positive gold standard functional assay (heparin-induced platelet activation [HIPA]). A diagnostic algorithm was established based on the Bayesian combination of pretest probability and likelihood ratios of first- and second-line immunoassays. Cutoffs with 100% PPV for positive HIPA were >3.0 U/mL (HemosIL-AcuStar-HIT-IgG) and titer ≥16 (ID-H/PF4-PaGIA); cutoffs with 100% NPV were <0.13 U/mL and ≤1, respectively. During the prospective validation of the derived algorithm (n = 687), HemosIL-AcuStar-HIT-IgG was used as unique testing in 566 (82.4%) of 687 cases (analytical TAT, 30 minutes). In 121 (17.6%) of 687 unresolved cases, ID-H/PF4-PaGIA was used as second-line testing (additional TAT, 30 minutes). The algorithm accurately predicted HIT in 51 (7.4%) of 687 patients and excluded it in 604 (87.9%) of 687 patients, leaving only 20 (2.9%) cases unresolved. We also identified 12 (1.7%) of 687 positive predictions not confirmed by HIPA: 10 patients with probable HIT despite negative HIPA and 2 possible false-positive algorithm predictions. The combination of pretest probability with first- and second-line immunoassays for anti-PF4/heparin antibodies is accurate for ruling in or out HIT in ≥95% of cases within 60 minutes. This diagnostic approach improves initial management of patients with suspected HIT.