Background
Vaccine‐induced immune thrombocytopenia and thrombosis (VITT) following the administration of the AstraZeneca (AZ) ChAdOx1 nCOV‐19 vaccine has recently been reported. The associated ...clinical and laboratory features have included thrombosis at unusual sites, thrombocytopenia, and raised D‐dimers with positivity for IgG anti‐platelet factor 4 (PF4) antibodies.
Objectives
A UK National External Quality Control Assessment Scheme external quality control exercise was carried out by distributing liquid and lyophilized samples from a subject with VITT, a pool of samples from subjects with classical heparin‐induced thrombocytopenia (HIT), and a non‐VITT/non‐HIT case to 85 centers performing HIT testing.
Methods
Participating centers employed their locally validated testing methods for HIT assays.
Results
The lyophilized and liquid samples were found to be commutable for the ELISA assays used in the detection of anti‐PF4 antibodies. The Aeskulisa, Stago, Hyphen, and LIFECODES anti‐PF4 ELISA assays successfully detected the VITT antibody, whereas the Acustar HIT, Werfen LIA, and the Stago STIC assays did not.
Conclusion
It is important that clinical and laboratory teams are aware of the limitations of some anti‐PF4 assays when using them to aid diagnosis of VITT syndrome.
Introduction
Emicizumab (Hemlibra: Roche Switzerland) is a, humanized, bi‐specific monoclonal modified immunoglobulin G4 (IgG4) which binds human FX, FIX and activated FIX (FIXa) to mimic activated ...FVIII activity.
Aim
Evaluate the effects of emicizumab on the APTT, surrogate FVIII activity and FVIII inhibitor results.
Methods
Two samples were provided, one obtained from an emicizumab treated severe haemophilia A patient with FVIII inhibitors and one constructed by in vitro addition of emicizumab using plasma from a severe haemophilia A patient without FVIII inhibitors. An APTT screen, surrogate FVIII and FVIII inhibitor tests were performed on both samples by participating centres.
Results
APTT results were below the lower limit of normal range. Chromogenic FVIII assay results with the Hyphen/Biophen human component‐based assay gave higher than expected coefficient of variation (CV) results, 38%–40%. The modified one‐stage FVIII assay with emicizumab calibrators showed similar results regardless of the APTT reagent. Inhibitor assay median results for sample S18:23 = 1.43 BU (range 0.9‐3.0 BU/ml, CV 38%). S18:24 was classified as below the lower limit of detection.
Conclusion
APTT screens showed a consistent shortening. Unmodified one‐stage Factor VIII assay results were remarkably high. APTT‐based assays are unsuitable for measurement of coagulation factors or inhibitors in patients treated with emicizumab. Bovine origin chromogenic assays are insensitive to emicizumab and should be used to monitor FVIII levels/FVIII inhibitors in emicizumab treated patients. Product‐specific calibrators should be implemented to reduce result variability.
Background:
Patients receiving the direct thrombin inhibitor dabigatran may have selected anticoagulation assays performed as part of routine care. The effect of dabigatran on the international ...normalized ratio (INR), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen requires clarification.
Objective:
To describe the effect of dabigatran on selected assays in North America and the United Kingdom.
Methods:
Pooled normal plasma enriched with dabigatran at concentrations of 25, 50, 75, 100, 125, 150, 200, 300, 400, and 500 ng/mL were sent blinded to 19 centers in the US, the UK, and Canada to assess the effect of dabigatran on routine coagulation screening tests, the INR, aPTT, TT, and fibrinogen.
Results:
Data were returned from 16 centers. For effects on INR, Neoplastine CI Plus and Simplastin HTF were the most sensitive and Thromborel S the least sensitive to increasing dabigatran concentrations. For the aPTT, all reagents demonstrated decreasing sensitivity to increasing dabigatran concentrations. Measured fibrinogen either demonstrated no change or factitious decrease with increasing dabigatran concentrations. Commercial TT methods were very sensitive to low concentrations of dabigatran, with 9 of 10 reporting sites exceeding test limits at dabigatran concentrations of 100 ng/mL.
Conclusions:
The INR, aPTT, and TT rise as dabigatran concentrations increase. Both the INR and aPTT increase in a linear pattern with marginal slopes, creating challenges in using these assays as reliable means for assessing the amount of dabigatran present. The commercial TT assay is very sensitive at low concentrations of dabigatran. Fibrinogen test results may be either unaffected or lower in the presence of dabigatran.
Dabigatran etexilate is a new oral anticoagulant that functions as a direct thrombin inhibitor. An inhibitor of thrombin has the potential to interfere with essentially all clot-based coagulation ...assays and select chromogenic assays, whereas the drug would not be expected to interfere in antigen-based assays. The purpose of this study was to evaluate the effect of dabigatran on various specialized coagulation assays using normal plasma specimens with varying concentrations of dabigatran (the active form of dabigatran etexilate). We have demonstrated that samples containing therapeutic levels of dabigatran may lead to underestimation of intrinsic factor activities with abnormal activated partial thromboplastin time (aPTT) mixing study results and a false-positive factor VIII Bethesda titer; overestimation of protein C and protein S activity and activated protein C resistance ratio when determined using aPTT-based methods; and overestimation of results based on chromogenic anti-IIa assays but no effect on antigen assays and select chromogenic assays.
Prevention of medical errors is a major goal of healthcare, though healthcare workers themselves have not yet fully accepted or implemented reliable models of system error, and neither has the ...public. While there is widespread perception that most medical errors arise from an inappropriate or delayed clinical management, the issue of laboratory errors is receiving a great deal of attention due to their impact on the quality and efficiency of laboratory performances and patient safety. Haemolytic specimens are a frequent occurrence in clinical laboratories, and prevalence can be as high as 3.3% of all of the routine samples, accounting for up to 40%–70% of all unsuitable specimens identified, nearly five times higher than other causes, such as insufficient, incorrect and clotted samples. This article focuses on this challenging issue, providing an overview on prevalence and leading causes of in vivo and in vitro haemolysis, and tentative guidelines on identification and management of haemolytic samples in clinical laboratories. This strategy includes continuous education of healthcare personnel, systematic detection/quantification of haemolysis in any sample, immediate clinicians warning on the probability of in vivo haemolysis, registration of non-conformity, completing of tests unaffected by haemolysis and request of a second specimen for those potentially affected. Clin Chem Lab Med 2008;46:764–72.
Introduction
Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII ...products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half‐life.
Aim
Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one‐stage clotting assays (OSAs) and chromogenic substrate assays (CSAs).
Methods
Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full‐length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N = 35) tested blinded samples using in‐house assays. Data from 51 OSAs (14 activated partial thromboplastin time aPTT reagents) and 42 CSAs (eight kits) were analyzed.
Results
Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under‐ and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two‐ to three‐fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations.
Conclusion
Under‐ or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.
The activity of the factor VIII coagulation protein can be measured by three methods: a one or two-stage clotting assay and a chromogenic assay. The factor VIII activity of most individuals with mild ...hemophilia A is the same regardless of which method is employed. However, approximately 30% of patients show marked discrepancies in factor VIII activity measured with the different methods. The objective of this study was to investigate the incidence of assay discrepancy in our center, assess the impact of alternative reagents on factor VIII activity assays and determine the usefulness of global assays of hemostasis in mild hemophilia A. Factor VIII activity was measured in 84 individuals with mild hemophilia A using different reagents. Assay discrepancy was defined as a two-fold or greater difference between the results of the one-stage and two-stage clotting assays. Rotational thromboelastometry and calibrated automated thrombography were performed. Assay discrepancy was observed in 31% of individuals; 12% with lower activity in the two-stage assay and 19% with lower activity in the one-stage assay. The phenotype could not always be predicted from the individual's genotype. Chromogenic assays were shown to be a suitable alternative to the two-stage clotting assay. Thromboelastometry was found to have poor sensitivity in hemophilia. Calibrated automated thrombography supported the results obtained by the two-stage and chromogenic assays. The current international guidelines do not define the type of assay to be used in the diagnosis of mild hemophilia A and some patients could be misclassified as normal. In our study, 4% of patients would not have been diagnosed on the basis of the one-stage factor VIII assay. Laboratories should use both one stage and chromogenic (or two-stage) assays in the diagnosis of patients with possible hemophilia A.
Introduction
Argatroban is licensed for patients with heparin-induced thrombocytopenia and is conventionally monitored by activated partial thromboplastin time (APTT) ratio. The target range is 1.5 ...to 3.0 times the patients’ baseline APTT and not exceeding 100 s, however this baseline is not always known. APTT is known to plateau at higher levels of argatroban, and is influenced by coagulopathies, lupus anticoagulant and raised FVIII levels. It has been used as a treatment for COVID-19 and Vaccine-induced Immune Thrombocytopenia and Thrombosis (VITT). Some recent publications have favored the use of anti-IIa methods to determine the plasma drug concentration of argatroban.
Methods
Plasma of 60 samples from 3 COVID-19 patients and 54 samples from 5 VITT patients were tested by APTT ratio and anti-IIa method (dilute thrombin time dTT). Actin FS APTT ratios were derived from the baseline APTT of the patient and the mean normal APTT.
Results
Mean APTT ratio derived from baseline was 1.71 (COVID-19), 1.33 (VITT) compared to APTT ratio by mean normal 1.65 (COVID-19), 1.48 (VITT). dTT mean concentration was 0.64 µg/ml (COVID-19) 0.53 µg/ml (VITT) with poor correlations to COVID-19 baseline APTT ratio r2 = 0.1526 p <0.0001, mean normal r2 = 0.2188 p < 0.0001; VITT baseline APTT ratio r2 = 0.04 p < 0.001, VITT mean normal r2 = 0.0064 p < 0.001.
Conclusions
We believe that dTT is a superior method to monitor the concentration of argatroban, we have demonstrated significant differences between APTT ratios and dTT levels, which could have clinical impact. This is especially so in COVID-19 and VITT.
"Over the last decades, major progress has been made in quality assurance of hemostatic laboratory assays. This book will be an indispensable part of every hemostasis laboratory, where, given its ...hands-on nature, it will rarely sit to get dusty on the shelves." -Frits R. Rosendaal, Leiden University Medical Center The hemostasis laboratory has a vital role in the diagnosis and management of patients with familial and acquired hemorrhagic and thrombotic disorders. Its role in the monitoring traditional anticoagulant therapy as well as therapy using new anticoagulants presents new challenges to the laboratory. Quality in Laboratory Hemostasis and Thrombosis not only addresses these important issues, but also covers international guidelines for testing, the development of international standard materials, management of hemostasis testing from the laboratory to the point of care as well as molecular genetic testing. Designed as a guide for all those working in hemostasis laboratories, this book details a quality program that, when put into place, will help to improve standards in testing. All of the authors are internationally recognised for their work in hemostasis and thrombosis. Using their experience, they provide information on standards, equipment and methods that will guide the development of a quality program to support all activities in the hemostasis laboratory.
In a longitudinal retrospective study, we aimed to assess natural protein (NP) tolerance and metabolic control in a cohort of 20 Hereditary Tyrosinaemia type I (HTI) patients. Their median age was 12 ...years (3.2-17.7 years,
= 11 female,
= 8 Caucasian,
= 8 Asian origin,
= 2 Arabic and
= 2 Indian). All were on nitisinone (NTBC) with a median dose of 0.7 g/kg/day (range 0.4-1.5 g/kg/day) and were prescribed a tyrosine (Tyr)/phenylalanine (Phe)-restricted diet supplemented with Tyr/Phe-free L-amino acids. Data were collected on clinical signs at presentation, medical history, annual dietary prescriptions, and blood Phe and Tyr levels from diagnosis until transition to the adult service (aged 16-18 years) or liver transplantation (if it preceded transition). The median age of diagnosis was 2 months (range: 0 to 24 months), with
= 1 diagnosed by newborn screening,
= 3 following phenylketonuria (PKU) screening and
= 7 by sibling screening. Five patients were transplanted (median age 6.3 years), and one died due to liver cancer. The median follow-up was 10 years (3-16 years), and daily prescribed NP intake increased from a median of 5 to 24 g/day. Lifetime median blood Tyr (370 µmol/L, range 280-420 µmol/L) and Phe (50 µmol/L, 45-70 µmol/L) were maintained within the target recommended ranges. This cohort of HTI patients were able to increase the daily NP intake with age while maintaining good metabolic control. Extra NP may improve lifelong adherence to the diet.
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