ISTH bleeding assessment tool and platelet function analyzer in children with mild inherited platelet function disorders.

OBJECTIVES: To evaluate the diagnostic performance of platelet function analyzer (PFA) and The International Society on Thrombosis and Hemostasis bleeding-assessment-tool (ISTH-BAT) in detecting mild inherited platelet function disorders (IPFDs) in children with suspected bleeding disorders. METHODS: Prospective single-center diagnostic study including consecutive patients <18 years with suspected bleeding disorder and performing a standardized workup for platelet function defects including ISTH-BAT, PFA, platelet aggregation testing, blood smear-based immunofluorescence, and next-generation sequencing-based genetic screening for IPFDs. RESULTS: We studied 97 patients, of which 34 von Willebrand disease (VWD, 22 type-1, 11 type-2), 29 IPFDs (including delta-/alpha-storage pool disease, Glanzmann thrombasthenia, Hermansky-Pudlak syndrome) and 34 with no diagnosis. In a model combining PFA-adenosine diphosphate (ADP), PFA-epinephrine (EPI), and ISTH-BAT overall performance to diagnose IPFDs was low with area under the curves of 0.56 (95% CI 0.44, 0.69) compared with 0.84 (95% CI 0.76, 0.92) for VWD. Correlation of PFA-EPI/-ADP and ISTH-BAT was low with 0.25/0.39 Spearman's correlation coefficients. PFA were significantly prolonged in patients with VWD and Glanzmann thrombasthenia. ISTH-BAT-scores were only positive in severe bleeding disorders, but not in children with mild IPFDs or VWD. CONCLUSION: Neither ISTH-BAT nor PFA or the combination of both help diagnosing mild IPFDs in children. PFA is suited to exclude severe IPFDs or VWD and is in this regard superior to ISTH-BAT in children.

Assessment and Monitoring of Coagulation in Patients with COVID-19: A Review of Current Literature.

Coagulation abnormalities are common in patients with COVID-19 and associated with high morbidity and mortality. It became a daily challenge to navigate through these abnormal laboratory findings and deliver the best possible treatment to the patients. The unique character of COVID-19-induced coagulopathy necessitates not only a dynamic follow-up of the patients in terms of hemostatic findings but also the introduction of new diagnostic methods to determine the overall function of the coagulation system in real time. After the recognition of the high risk of thromboembolism in COVID-19, several professional societies published their recommendations regarding anticoagulation in patients with COVID-19. This review summarizes common hemostatic findings in COVID-19 patients and presents the societal recommendations regarding the use of coagulation laboratory findings in clinical decision-making. Although several studies have investigated coagulation parameters in patients with COVID-19, the methodological shortcomings of published studies as well as the differences in employed anticoagulation regimens that have changed over time, depending on national and international guidelines, limit the applicability of these findings in other clinical settings. Accordingly, evidence-based recommendations for diagnostics during acute COVID-19 infection are still lacking. Future studies should verify the role of coagulation parameters as well as viscoelastic methods in the management of patients with COVID-19.

Standardization of Light Transmission Aggregometry for Diagnosis of Platelet Disorders: An Inter-Laboratory External Quality Assessment.

Several in vitro platelet function tests are available for the diagnosis of inherited platelet function disorders. Currently, the light transmission aggregometry (LTA) is recommended as one of the first-step tests. LTA is available in most specialized hemostasis laboratories. Although the LTA is accepted as a 'gold standard' assay for the evaluation of platelet function, its standardization in the clinical practice is still challenging. The GTH-based THROMKID-Plus Study Group has performed an inter-laboratory trial in Germany and Austria. Five different agonists were selected according to the Scientific and Standardization Committee/International Society on Thrombosis and Haemostasis recommendations and shipped in 3 different sets (one should represent a healthy control and two should simulate platelet function disorders) to 15 specialized laboratories in Germany and Austria. Agonists were analyzed by APACT or PAP4/8 aggregometer using platelet-rich plasma from healthy donors. In addition, laboratory-internal platelet agonists were tested in platelet-rich plasma from a healthy donor. All laboratories (9 used APACT, 6 used PAP4/PAP8) showed very consistent data regarding the maximum percentage of aggregation induced by the tested agonists and identified the differential diagnosis of the simulated platelet function disorders with one exception, which was due to technical problems. In contrast, there was a high variability of the laboratory-internal inductors regarding reagent type, concentrations and pathological cut-off values. Our study showed that the shipment of agonists is suitable for an inter-laboratory survey of LTA. However, there is still a remarkable need for standardization of agonist reagents and their concentration as well as for definition of reference ranges.

An international external quality assessment for laboratory diagnosis of heparin-induced thrombocytopenia.

Essentials A pilot study for External Quality Assessment for testing of HIT is described. The qualitative accordance for the PF4/heparin IgG test was 97.6%. The qualitative accordance for functional HIT tests was considerably lower. External Quality Assessment for functional HIT tests is required. SUMMARY: Objective Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening complication of heparin exposure. Diagnosis is most reliable using a combination of an enzyme immunoassay (EIA) that detects antibodies against platelet factor 4 (PF4)/heparin complexes ("antigen" assay) and a "functional" assay that detects platelet-activating properties of the pathogenic HIT antibodies. No External Quality Assessment (EQA) is available for a combination of the tests. Here we report on the results of the first international EQA. Methods The pilot EQA was organized by the Department of Transfusion Medicine, Universitätsmedizin Greifswald, Germany. Six serum samples of patients, which were referred to Greifswald for HIT diagnosis, and one negative control sample were distributed to seven participants in Germany, Canada, and Singapore. Participants were asked to report the optical density (OD) values of their local EIA test for IgG-specific antibodies against the PF4/heparin complexes and the results for a functional assay (HIPA or SRA). Consensus was defined as a minimum 70% agreement, i.e., agreement among at least five of the seven participating laboratories. Results and conclusion Six out of seven participants reported results for EIA, with a high quantitative accordance (97.6%). For the functional assay, consensus was reached for all samples except the negative control, for which some participants reported nonspecific reactivity. All HIT-negative samples were correctly diagnosed by all participants; for HIT-positive samples, consensus of 70% was reached. Although the limited availability of sample material is an obstacle to overcome, an EQA combining both EIA and functional testing is feasible.

Assessment of human platelet survival in the NOD/SCID mouse model: technical considerations.

BACKGROUND: The NOD/SCID mouse model is a unique and sophisticated method to study the survival of human platelets (PLTs) in vivo. Meanwhile, several research groups adopted this model to analyze a wide range of PLT antibodies. Differences exist between the research groups regarding the method of PLT injection, the amount and route of antibody injection, and the preparation of blood samples collected from the animal, making it difficult to compare results between studies. STUDY DESIGN AND METHODS: We compared the survival of human PLTs infused into NOD/SCID mice via the tail vein or the retro-orbital plexus. The percentage of circulating human PLTs in the mouse circulation was determined by flow cytometry. Murine blood samples were prepared using two different methods: 1) direct fixation of whole blood samples and 2) isolation of PLTs by density gradient centrifugation. RESULTS: Recovery of human PLTs after tail vein injection was comparable to retro-orbital injection (13% vs. 11% of all circulating PLTs, p = 0.401). However, the survival rate of tail vein-infused PLTs was higher than that of retro-orbitally injected PLTs (median PLT survival after 5 hr 84% vs. 56%, p = 0.025). Moreover, we observed that determination of circulating human PLTs in directly fixed murine whole blood samples shows better reproducibility compared to the density gradient centrifugation method. CONCLUSIONS: Tail vein injection of human PLTs into the NOD/SCID mice is superior to retro-orbital injection in terms of human PLT survival. Direct fixation of whole blood samples allows better reproducibility of results compared to the density gradient centrifugation method.

Evaluation of a new nanoparticle-based lateral-flow immunoassay for the exclusion of heparin-induced thrombocytopenia (HIT).

Heparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies (abs) that recognise platelet factor 4-heparin (PF4/hep) complexes. Several laboratory tests are available for the confirmation and/or refutation of HIT. A reliable and rapid single-sample test is still pending. It was the objective of this study to evaluate a new lateral-flow immunoassay based on nanoparticle technology. A cohort of 452 surgical and medical patients suspected of having HIT was evaluated. All samples were tested in two IgG-specific ELISAs, in a particle gel immunoassay (PaGIA) and in a newly developed lateral-flow immunoassay (LFI-HIT) as well as in a functional test (HIPA). Clinical pre-test probability was determined using 4T's score. Platelet-activating antibodies were present in 34/452 patients, all of whom had intermediate to high clinical probability. PF4/hep abs were detected in 79, 87, 86, and 63 sera using the four different immunoassays. The negative predictive values (NPV) were 100% for both ELISA tests and LFI-HIT but only 99.2% for PaGIA. There were less false positives (n=29) in the LFI-HIT compared to any other test. Additionally, significantly less time was required to perform LFI-HIT than to perform the other immunoassays. In conclusion, a newly developed lateral-flow assay, LFI-HIT, was capable of identifying all HIT patients in a cohort in a short period of time. Beside an NPV of 100%, the rate of false-positive signals is significantly lower with LFI-HIT than with other immunoassay(s). These performance characteristics suggest a high potency in reducing the risk and costs in patients suspected of having HIT.

Performance characteristics of two commercially available IgG-specific immunoassays in the assessment of heparin-induced thrombocytopenia (HIT).

BACKGROUND: The in vitro demonstration of antibodies against platelet factor-4/heparin (PF4/hep) complexes is an important contribution to the diagnosis of heparin-induced thrombocytopenia (HIT). The use of PF4/hep IgG-specific immunoassays enhances the specificity of HIT-investigations without any impairment of the sensitivity. Several IgG-specific immunoassays with different origin and structure of the target antigen-complex are commercially available. METHODS: Using a retrospective cohort consisting of 459 patients suspected to have HIT, we compared the performance characteristics of two commercially available IgG-specific immunoassays, GTI- (Genetic Testing Institute) and HIA-IgG-ELISA (Hyphen Biomed Research). RESULTS: PF4/hep antibodies were detected in 85 and 81 sera using GTI- and HIA-IgG-ELISA, respectively. OD values and clinical likelihood of patients who tested positive in one assay only were significantly lower than in those who tested positive in both immunoassays. Both IgG-specific assays showed high negative predictive values (100%) and similar but unsatisfactory positive predictive values, determined by a minimum clinical score of 5 and a positive HIPA result (41% and 43%, respectively). The implementation of a confirmatory step using excessive heparin increased the PPV of both assays, but results in a reduction of NPV in HIA-IgG-ELISA. CONCLUSIONS: The detection of IgG antibodies alone improves the clinical usefulness of immunoassays. However, functional assays remain indispensable to avoid the overdiagnosis of HIT caused by the detection of IgG non-platelet activating antibodies. The OD value in IgG immunoassays appears to correlate with the clinical relevance of the antibodies and might be used as a predictive parameter in the assessment of HIT.