Hemostasis Testing in the Emergency Department: A Narrative Review.

Routine laboratory screening is typically performed at initial evaluation of the vast majority of presentations to the emergency department (ED). These laboratory results are crucial to the diagnostic process, as they may influence up to 70% of clinical decisions. However, despite the usefulness of biological assessments, many tests performed are inappropriate or of doubtful clinical relevance. This overutilization rate of laboratory testing in hospitals, which represents a significant medical-economic burden, ranges from 20 to 67%, with coagulation tests at the top of the list. While reviews frequently focus on nonintensive care units, there are few published assessments of emergency-specific interventions or guidelines/guidance to date. The aim of this review is to highlight current recommendations for hemostasis evaluation in the emergency setting with a specific analysis of common situations leading to ED admissions, such as suspected venous thrombosis or severe bleeding. We revisit the evidence related to the assessment of patient's hemostatic capacity based on comprehensive history taking and physical examination as well as best practice recommendations for blood sample collection to ensure the reliability of results. This review also includes an examination of various currently available point of care tests and a comprehensive discussion on indications, limitations, and interpretation of these tests.

Comparison of assays measuring extracellular vesicle-tissue factor in plasma samples: Communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays. MATERIALS AND METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

The use of 1E12, a monoclonal anti-platelet factor 4 antibody, to improve the diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a complication of adenoviral-based vaccine against SARS-CoV-2 due to prothrombotic immunoglobulin (Ig) G antibodies to platelet factor 4 (PF4) and may be difficult to distinguish from heparin-induced thrombocytopenia (HIT) in patients treated with heparin. OBJECTIVES: We assessed the usefulness of competitive anti-PF4 enzyme immunoassays (EIAs) in this context. METHODS: The ability of F(ab')2 fragments of 1E12, 1C12, and 2E1, 3 monoclonal anti-PF4 antibodies, to inhibit the binding of human VITT or HIT antibodies to PF4 was evaluated using EIAs. Alanine-scanning mutagenesis was performed to define the amino acids involved in the interactions between the monoclonal antibodies and PF4. RESULTS: A strong inhibition of VITT IgG binding to PF4 was measured with 1E12 (median inhibition, 93%; n = 8), whereas it had no effect on the binding of HIT antibodies (median, 6%; n = 8). In contrast, 1C12 and 2E1 inhibited VITT (median, 74% and 76%, respectively) and HIT antibodies (median, 68% and 53%, respectively) binding to PF4. When a competitive anti-PF4 EIA was performed with 1E12 for 19 additional VITT samples, it strongly inhibited IgG binding to PF4, except for 1 patient, who had actually developed HIT according to the clinical history. Epitope mapping showed that 1E12 interacts with 5 key amino acids on PF4, of which 4 are also required for the binding of human VITT antibodies, thus explaining the competitive inhibition. CONCLUSION: A simple competitive anti-PF4 EIA with 1E12 could help confirm VITT diagnosis and distinguish it from HIT in patients when both diagnoses are possible.

Interventions to improve appropriateness of laboratory testing in the intensive care unit: a narrative review.

Healthcare expenses are increasing, as is the utilization of laboratory resources. Despite this, between 20% and 40% of requested tests are deemed inappropriate. Improper use of laboratory resources leads to unwanted consequences such as hospital-acquired anemia, infections, increased costs, staff workload and patient stress and discomfort. The most unfavorable consequences result from unnecessary follow-up tests and treatments (overuse) and missed or delayed diagnoses (underuse). In this context, several interventions have been carried out to improve the appropriateness of laboratory testing. To date, there have been few published assessments of interventions specific to the intensive care unit. We reviewed the literature for interventions implemented in the ICU to improve the appropriateness of laboratory testing. We searched literature from 2008 to 2023 in PubMed, Embase, Scopus, and Google Scholar databases between April and June 2023. Five intervention categories were identified: education and guidance (E&G), audit and feedback, gatekeeping, computerized physician order entry (including reshaping of ordering panels), and multifaceted interventions (MFI). We included a sixth category exploring the potential role of artificial intelligence and machine learning (AI/ML)-based assisting tools in such interventions. E&G-based interventions and MFI are the most frequently used approaches. MFI is the most effective type of intervention, and shows the strongest persistence of effect over time. AI/ML-based tools may offer valuable assistance to the improvement of appropriate laboratory testing in the near future. Patient safety outcomes are not impaired by interventions to reduce inappropriate testing. The literature focuses mainly on reducing overuse of laboratory tests, with only one intervention mentioning underuse. We highlight an overall poor quality of methodological design and reporting and argue for standardization of intervention methods. Collaboration between clinicians and laboratory staff is key to improve appropriate laboratory utilization. This article offers practical guidance for optimizing the effectiveness of an intervention protocol designed to limit inappropriate use of laboratory resources.

[Evaluation of blood cell morphology with the RBC Advanced Application: Which cut-offs are most needed for which specific abnormalities?] / Évaluation de la morphologie érythrocytaire avec le RBC Advanced : Quels seuils pour quelles anomalies ?

The detection of erythrocyte morphological abnormalities is a valuable and sometimes overlooked element in the diagnostic management of anemias. The aim of this article is to evaluate the clinical performance of the different detection thresholds tested by our laboratory using the Cellavision RBC Advanced module, after manual reclassification by an experienced operator, and comparing them to the guidelines by the ICSH (International Council for Standardization in Haematology). We arbitrarily set thresholds at 1% for "critical" abnormalities (tear drop cells, target cells, schizocytes and spherocytes) except for sickle cells (threshold set at 0.01%). Our data show excellent sensitivity of 100% for the cut-offs defined by the investigation for tear drop cells and sickle cells, but low specificity for detection of associated clinical pathology compared with ICSH cut-offs, varying from 4% for teardrop cells (detection of myelofibrosis), 26% for target cells (detection of martial deficiency) to 55% for schizyocytes (presence of hemolytic anemia). Our results show a better specificity of the thresholds established by ICSH than our studied thresholds for the detection of the pathologies of concern, suggesting a better clinical relevance.

Multicenter evaluation of light transmission platelet aggregation reagents: communication from the ISTH SSC Subcommittee on Platelet Physiology.

BACKGROUND: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization. OBJECTIVES: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results. METHODS: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied. RESULTS: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine. CONCLUSION: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents.

Minimal requirements for ISO15189 validation and accreditation of three next generation sequencing procedures for SARS-CoV-2 surveillance in clinical setting.

Rapid and recurrent breakthroughs of new SARS-CoV-2 strains (variants) have prompted public health authorities worldwide to set up surveillance networks to monitor the circulation of variants of concern. The use of next-generation sequencing technologies has raised the need for quality control assessment as required in clinical laboratories. The present study is the first to propose a validation guide for SARS-CoV-2 typing using three different NGS methods fulfilling ISO15189 standards. These include the assessment of the risk, specificity, accuracy, reproducibility, and repeatability of the methods. Among the three methods used, two are amplicon-based involving reverse transcription polymerase chain reaction (Artic v3 and Midnight v1) on Oxford Nanopore Technologies while the third one is amplicon-based using reverse complement polymerase chain reaction (Nimagen) on Illumina technology. We found that all methods met the quality requirement (e.g., 100% concordant typing results for accuracy, reproducibility, and repeatability) for SARS-CoV-2 typing in clinical setting. Additionally, the typing results emerging from each of the three sequencing methods were compared using three widely known nomenclatures (WHO, Pangolineage, and Nextclade). They were also compared regarding single nucleotide variations. The outcomes showed that Artic v3 and Nimagen should be privileged for outbreak investigation as they provide higher quality results for samples that do not meet inclusion criteria for analysis in a clinical setting. This study is a first step towards validation of laboratory developed NGS tests in the context of the new European regulation for medical devices and in vitro diagnostics.

Are Viscoelastometric Assays of Old Generation Ready for Disposal? Comment on Volod et al. Viscoelastic Hemostatic Assays: A Primer on Legacy and New Generation Devices. J. Clin. Med. 2022, 11, 860.

With the advent of new viscoelastometric hemostatic assay (VHA) devices, with ready-to-use cartridge reagents allowing for their use by people without special laboratory skills, the appreciation of the actual clinical value of VHAs in settings such as severe trauma, post-partum hemorrhage, cardiac surgery and liver transplantation still needs to be fully validated. While two of the newest versions remain based on a 'cup and pin' system (ROTEM® sigma, ClotPro®), two other new devices (TEG® 6s, Quantra®) rely on very different technologies: clotting blood is no longer in contact with the probe and challenged by oscillation of one of the components but explored with ultrasound exposure. A systematic literature search (including Sonoclot®) retrieved 20 observational studies (19 prospective). Most studies pointed to imperfect agreements, highlighting the non-interchangeability of devices. Only a few studies, often with a limited number of patients enrolled, used a clinical outcome. No study compared VHA results with conventional laboratory assays obtained through a rapid tests panel. Clinical evidence of the utility of the new VHAs largely remains to be proven through randomized clinical trials, with clinically relevant outcomes, and compared to rapid panel hemostasis testing. The availability of new, improved VHA devices provides an impetus and an opportunity to do so.

D-dimer: old dogmas, new (COVID-19) tricks.

D-dimer is a fibrin degradation product encompassing multiple cross-linked D domains and/or E domains present in the original fibrinogen molecule, whose generation is only theoretically possible when hemostasis and fibrinolysis pathways are concomitantly activated. D-dimer measurement has now become a pillar in the diagnosis/exclusion and prognostication of venous thromboembolism (VTE) and disseminated intravascular coagulation (DIC), when incorporated into validated clinical algorithms and especially using age-adjusted diagnostic thresholds. Although emerging evidence is also supporting its use for predicting the duration of anticoagulant therapy in certain categories of patients, the spectrum of clinical applications is constantly expanding beyond traditional thrombotic pathologies to the diagnosis of acute aortic dissection, acute intestinal ischemia and cerebral venous thrombosis among others, embracing also clinical management of coronavirus disease 2019 (COVID-19). Recent findings attest that D-dimer elevations are commonplace in patients with severe acute respiratory syndrome (SARS-CoV-2) infection (especially in those with thrombosis), its value predicts the clinical severity (up to death) of COVID-19 and remains more frequently increased in COVID-19 patients with post-discharge clinical sequelae. Further, D-dimer-based anticoagulant escalation may be associated with a lower risk of death in patients with severe SARS-CoV-2 infection and, finally, D-dimer elevation post-COVID-19 vaccination mirrors an increased risk of developing vaccine-induced thrombocytopenia and thrombosis (VITT).

Assessment of DOAC in GEriatrics (Adage Study): Rivaroxaban/Apixaban Concentrations and Thrombin Generation Profiles in NVAF Very Elderly Patients.

BACKGROUND: Although a growing number of very elderly patients with atrial fibrillation (AF), multiple conditions, and polypharmacy receive direct oral anticoagulants (DOACs), few studies specifically investigated both apixaban/rivaroxaban pharmacokinetics and pharmacodynamics in such patients. AIMS: To investigate: (1) DOAC concentration-time profiles; (2) thrombin generation (TG); and (3) clinical outcomes 6 months after inclusion in very elderly AF in-patients receiving rivaroxaban or apixaban. METHODS: Adage-NCT02464488 was an academic prospective exploratory multicenter study, enrolling AF in-patients aged ≥80 years, receiving DOAC for at least 4 days. Each patient had one to five blood samples at different time points over 20 days. DOAC concentrations were determined using chromogenic assays. TG was investigated using ST-Genesia (STG-ThromboScreen, STG-DrugScreen). RESULTS: We included 215 patients (women 71.1%, mean age: 87 ± 4 years), 104 rivaroxaban and 111 apixaban, and 79.5% receiving reduced-dose regimen. We observed important inter-individual variabilities (coefficient of variation) whatever the regimen, at C max [49-46%] and C min [75-61%] in 15 mg rivaroxaban and 2.5 mg apixaban patients, respectively. The dose regimen was associated with C max and C min plasma concentrations in apixaban (p = 0.0058 and p = 0.0222, respectively), but not in rivaroxaban samples (multivariate analysis). Moreover, substantial variability of thrombin peak height (STG-ThromboScreen) was noticed at a given plasma concentration for both xabans, suggesting an impact of the underlying coagulation status on TG in elderly in-patients. After 6-month follow-up, major bleeding/thromboembolic event/death rates were 6.7%/1.0%/17.3% in rivaroxaban and 5.4%/3.6%/18.9% in apixaban patients, respectively. CONCLUSION: Our study provides original data in very elderly patients receiving DOAC in a real-life setting, showing great inter-individual variability in plasma concentrations and TG parameters. Further research is needed to understand the potential clinical impact of these findings.

Variability among commercial batches of normal pooled plasma in lupus anticoagulant testing.

INTRODUCTION: Lupus anticoagulant (LA) testing requires normal pooled plasma (NPP) in performing mixing studies and can be used for normalized ratios of clotting times (CTs). The aims were to demonstrate whether significant differences in clotting times between two batches of a same commercial NPP (CRYOcheck™) directly affect NPP-based cut-off values. METHODS: Diluted Russell Viper venom time (DRVVT) and activated partial thromboplastin time (aPTT) were used for LA testing. Screening, mixing and confirm tests were performed with Stago® instruments and reagents. Two batches of commercial NPP (A1291 and A1301 from CRYOcheck™; frozen) were compared in the determination of cut-off values. Cut-off values were defined as 99th percentile values of 60 healthy donors and compared with Mann-Whitney U test. RESULTS: Cut-off values obtained with the two NPP batches were significantly different for DRVVT (screen normalized ratio: 1.09 vs. 1.24, screen mix: 41.9 s vs. 38.9 s; index of circulating anticoagulant: 5.0 vs. 8.4; all had p-value <.001). On the contrary, no significant differences were observed for aPTT (screen normalized ratio: 1.32 vs. 1.34; p-value = .4068, screen mix: 37.8 s vs. 38.1 s; p-value = .1153) except for index of circulating anticoagulant: 9.6 versus 10.4 (p-value <.05). CONCLUSION: This study demonstrates that differences between two commercial NPP batches produced by a same manufacturer influenced LA cut-off values used for mixing studies and normalized ratios. Adequate cut-off setting, taking into account NPP CTs, is important to provide accurate conclusion about the presence or absence of a LA and avoid potential clinical impact.

Reassessment of dextran sulfate in anti-Xa assay for unfractionated heparin laboratory monitoring.

Background: Anti-Xa assays are used for unfractionated heparin (UFH) monitoring. Dextran sulfate (DS) is used in some assays to overcome the artifactual preanalytical release of platelet factor 4. However, the practical implications of this test modification have not been studied extensively. Objectives: To investigate the impact of the presence of DS in the anti-Xa assay for UFH laboratory monitoring. Methods: We studied factor Xa inhibition, using an assay without DS (Stago Liquid Anti-Xa), in normal pool plasma spiked with various concentrations of UFH (up to 1 IU/mL) in the presence of increasing concentrations of DS (up to 2560 µg/mL). We also investigated the effect of DS on FXa inhibition measured after the addition of UFH and heparin antagonists (protamine and Polybrene; Sigma Aldrich). Eventually, we compared the anti-Xa levels measured using the assay without DS to those measured with an assay containing DS (BIOPHEN Heparin LRT, Hyphen BioMed). Results: DS per se had a detectable anti-Xa effect. FXa inhibition in UFH-spiked plasma linearly increased with increasing concentrations of added DS, with a plateau at approximately 160 µg/mL DS, at which the apparent anti-Xa level had almost doubled. In the presence of heparin antagonists, the addition of DS increased anti-Xa levels, corresponding to the dissociation of the UFH-antagonists complexes in vitro. With the anti-Xa assay containing DS, UFH inhibition was not detected. Conclusion: In the presence of high concentrations of DS, FXa inhibition was much higher than that predicted from added UFH amounts, presumably related to the greater availability of UFH for interaction with antithrombin. While the relevance of measuring this "masked" heparin has not been demonstrated, the presence of DS renders the result inaccurate in the presence of protamine or Polybrene.