Taipan snake venom time has high sensitivity for lupus anticoagulants in non-anticoagulated, triple positive antiphospholipid syndrome patients.

INTRODUCTION: Dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) are the mainstay assays in lupus anticoagulant (LA) detection yet they have limitations, particularly in relation to interferences and specificity. The recently validated Taipan snake venom time (TSVT) screening with ecarin time (ET) confirmatory assays overcome many of those limitations due to the innate specificity engendered from direct prothrombin activation, and insensitivity to the effects of vitamin K antagonists (VKA). The present study aimed to further evidence diagnostic utility of TSVT/ET by performing them in samples from 116 nonanticoagulated patients with established triple-positive antiphospholipid syndrome (APS). METHODS: Samples were identified in three expert centres who performed dRVVT, APTT and solid phase antiphospholipid antibody assays with reagents from a variety of manufacturers. All samples additionally received TSVT/ET analysis using standardised reagents. RESULTS: Ninety seven of 116 (83.6%) were dRVVT- and APTT-positive, 85/97 (87.6%) of which were TSVT/ET-positive, 9/116 (7.8%) were dRVVT-positive only, 6 of which were TSVT/ET-positive, and 10/116 (8.6%) were APTT-positive only, 5 of which were TSVT/ET-positive. 96/116 TSVT/ET-positivity returned a high sensitivity for LA of 82.8%. Low coefficients of determination revealed weak relationships between LA potency and anticardiolipin and anti-ß2-glycoprotein I antibody titres for all three LA assays. CONCLUSIONS: TSVT/ET has high sensitivity for the clinically significant LA found in triple positive APS patients. TSVT/ET can establish multiple LA assay positivity in nonanticoagulated patients negative for one of dRVVT or APTT, and is the only assay pairing insensitive to VKAs, the recommended anticoagulation for APS.

Diagnosis and laboratory monitoring of hemophilia A.

Acquired hemophilia A (AHA) is a rare disorder in which autoantibodies against factor VIII (FVIII) lead to a bleeding phenotype that varies from life-threatening to no bleeding at all. Prolonged activated partial thromboplastin times (APTT) in patients with a bleeding phenotype should be investigated to rule out AHA and should never be ignored in a preprocedure patient. Most inhibitors in AHA are heat and time dependent, so mixing studies performed only on an immediate mix are not useful: both lupus anticoagulants and treatment with direct oral anticoagulants can coexist with AHA and confound the diagnosis. Assays for intrinsic coagulation factors and von Willebrand factor should always be performed, regardless of the results of mixing studies. A Bethesda or modified Bethesda assay should be performed to quantify any inhibitor, and if susoctocog alfa (rpFVIII) is available, then an assay for cross-reacting antibodies should also be performed. At diagnosis and until complete remission, if the FVIII in the patient sample is >5 IU/dL, heat inactivation should be performed before the inhibitor assays are performed. While there are no conventional tests available to measure the effects of FVIII bypassing therapies, newer therapies may require monitoring, or their effects may need to be considered when choosing appropriate assays. Measurement of rpFVIII requires a 1-stage clotting assay, and measurement of patient FVIII while on emicizumab requires a chromogenic assay that does not contain human FX. Close communication is required between the treating clinicians and the laboratory to ensure that the correct tests are performed while patients are receiving treatments.

Triple-positive antiphospholipid syndrome does not guarantee positivity in each lupus anticoagulant assay.

BACKGROUND: Triple positivity for all 3 criteria antiphospholipid antibodies confers high risk of symptom development in carriers, and recurrence in antiphospholipid syndrome (APS). Most triple-positivity studies report lupus anticoagulant (LA) testing as positive without distinguishing between positivity with dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) and single-assay positivity or only perform dRVVT. Single LA assay repertoires remain in use in some centers, which risks missing some triple positives. Positivity with both assays may identify higher risk. OBJECTIVES: The aim of this study is to investigate the frequency of single LA assay positivity in triple-positive patients. METHODS: Three hundred forty-two triple-positive profiles from nonanticoagulated patients (237 APS, 45 systemic lupus erythematosus without APS symptoms, and 60 nonclinical criteria) were identified from laboratory databases and assessed for LA positivity by dRVVT and/or APTT. RESULTS: Seventy-three of 237 (30.8%) APS samples were LA-positive with 1 assay, 40/237 (16.9%) by dRVVT only, and 33/237 (13.9%) with APTT only. Nineteen of 45 (42.2%) were LA-positive with 1 assay in the systemic lupus erythematosus cohort; 12/45 (26.7%) with dRVVT only and 7/45 (15.5%) with APTT only. Thirty-three of 60 (55.0%) were LA-positive with 1 assay in the nonclinical criteria cohort; 24/60 (40.0%) with dRVVT only and 9/60 (15.0%) with APTT only. The most common solid-phase assay profile was elevated immunoglobulin G aCL and aß2GPI. CONCLUSION: Up to 55.0% of triple-positive samples were positive in 1 LA assay, representing significant potential for misdiagnosis and inappropriate management via single LA assay repertoires.

Renal transplant and hemostasis: early postoperative changes in recipients and donors.

Background: The benefit of administering pharmacologic thromboprophylaxis following renal transplantation remains uncertain. Objectives: To compare hemostatic parameters before and after renal transplant surgery in both recipients and their donors at predetermined time points. Methods: Blood samples were collected at baseline (T1), immediately after surgery (T2), and at 24 hours after surgery (T3) in both recipients and donors and at 72 (T4) and 120 hours (T5) from recipients only. Assays included in vitro thrombin generation, factor VIII (FVIIIc) activity, von Willebrand factor (VWF) antigen, D-dimer, antithrombin activity, prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin complexes, and plasminogen activator inhibitor-1 (PAI-1) antigen. Results: Fifty-two patients (28 recipients and 24 donors) were enrolled. Both donors and recipients had increased FVIIIc, VWF, F1 + 2, D-dimer, and PAI immediately after surgery but reduced antithrombin. Mixed-model analysis showed that the magnitude of change over time (between T1 and T3) for FVIIIc (mean estimated difference [MED], 72; 95% CI, 41-102; P < .0001), VWF (MED, 89; 95% CI, 35-142; P = .001), F1 + 2 (MED, 283; 95% CI, 144-422; P < .0001), thrombin-antithrombin complexes (MED, 3.5; 95% CI, 1.9-5.1; P < .0001), D-dimer (MED, 2.2; 95% CI, 1.0-3.3; P < .0001), PAI-1 (MED, 9.2; 95% CI, 3.4-14.9; P = .002), and time to peak thrombin generation (MED, 1.5; 95% CI, 0.35-2.7; P = .01) was more significant in recipients than in donors. Conclusion: Persistence of a hypercoagulable state was more prominent in recipients after 24 hours despite recovery in renal function and initiation of thromboprophylaxis.

A new global fibrinolysis capacity assay for the sensitive detection of hyperfibrinolysis and hypofibrinogenemia in trauma patients.

BACKGROUND: Conventional clotting tests are not expeditious enough to allow timely targeted interventions in trauma, and current point-of-care analyzers, such as rotational thromboelastometry (ROTEM), have limited sensitivity for hyperfibrinolysis and hypofibrinogenemia. OBJECTIVES: To evaluate the performance of a recently developed global fibrinolysis capacity (GFC) assay in identifying fibrinolysis and hypofibrinogenemia in trauma patients. METHODS: Exploratory analysis of a prospective cohort of adult trauma patients admitted to a single UK major trauma center and of commercially available healthy donor samples was performed. Lysis time (LT) was measured in plasma according to the GFC manufacturer's protocol, and a novel fibrinogen-related parameter (percentage reduction in GFC optical density from baseline at 1 minute) was derived from the GFC curve. Hyperfibrinolysis was defined as a tissue factor-activated ROTEM maximum lysis of >15% or LT of ≤30 minutes. RESULTS: Compared to healthy donors (n = 19), non-tranexamic acid-treated trauma patients (n = 82) showed shortened LT, indicative of hyperfibrinolysis (29 minutes [16-35] vs 43 minutes [40-47]; p < .001). Of the 63 patients without overt ROTEM-hyperfibrinolysis, 31 (49%) had LT of ≤30 minutes, with 26% (8 of 31) of them requiring major transfusions. LT showed increased accuracy compared to maximum lysis in predicting 28-day mortality (area under the receiver operating characteristic curve, 0.96 [0.92-1.00] vs 0.65 [0.49-0.81]; p = .001). Percentage reduction in GFC optical density from baseline at 1 minute showed comparable specificity (76% vs 79%) to ROTEM clot amplitude at 5 minutes from tissue factor-activated ROTEM with cytochalasin D in detecting hypofibrinogenemia but correctly reclassified >50% of the patients with false negative results, leading to higher sensitivity (90% vs 77%). CONCLUSION: Severe trauma patients are characterized by a hyperfibrinolytic profile upon admission to the emergency department. The GFC assay is more sensitive than ROTEM in capturing hyperfibrinolysis and hypofibrinogenemia but requires further development and automation.

Effect on haemostasis of different replacement fluids during therapeutic plasma exchange-A comparative multicentre observational study.

INTRODUCTION: Therapeutic plasma exchange (TPE) is used for several chronic conditions with little evidence on the efficacy and safety of different choice of replacement fluid. Measurement of haemostasis, particularly in vitro thrombin generation, could play a role in determining the immediate efficacy of different fluid replacement. AIM: To determine the impact of different TPE replacement fluid regimens on haemostatic assays. METHODS: Prospective observational multi-centre cohort study in adult patients 18 years and older evaluating haemostatic changes between four different TPE regimens: (1) 5% human albumin solution (Alb) only, (2) 50:50 mix of 5% Alb + modified gelatin, (3) 70:30 mix of 5% Alb and normal saline (NS), and (4) solvent-detergent, virus-inactivated fresh frozen plasma (FFP) (either alone or combined with other fluids). Twenty-one haemostasis variables were analysed (procoagulant, anticoagulant and fibrinolytic factors) pre and post TPE sessions, including in vitro thrombin generation. Linear mixed modelling and canonical discriminant analyses were used to examine the effect of TPE fluid type on haemostatic variables. RESULTS: A total of 31 patients with up to 5 TPE sessions each (131 sessions in total) were enrolled. Out of 21 markers analysed using linear mixed modelling, the main effects of fluid type were found to be significant for 19 markers (P < 0.05), excluding plasminogen activator inhibitor-1 antigen and thrombin-anti-thrombin. Multivariate Analysis of Variance showed significant differences between the fluid types (Wilks' lambda = 0.07; F63,245.61  = 5.50; P < 0.0001) and this was supported by a canonical discriminant analysis, which identified the 4 most discriminating markers for fluid types as thrombin generation (lag-time, time-to Peak), fibrinogen and Factor V. In our analyses, the effect of FFP on haemostasis was significantly greater compared with other fluid types. Of the non-FFP fluids, 5% Alb + NS had a lower effect on haemostasis compared to other fluid types (Alb and modified gelatin + 5% Alb). CONCLUSION: Thrombin generation and fibrinogen discriminated better the effect of different TPE fluids on haemostasis and should be considered as potential markers to evaluate the immediate haemostatic effect of TPE procedures. The use of NS as a TPE replacement fluid had a distinctive impact on thrombin generation and fibrinogen responses compared to other non-FFP fluids.

Automated measurement of coagulation and fibrinolytic activation markers: Outcomes in coronavirus disease 2019 (COVID-19) patients.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is characterized by marked hypoxaemia and lung oedema, often accompanied by disordered blood coagulation and fibrinolytic systems, endothelial damage and intravascular fibrin deposition. PATIENTS/METHODS: We present a retrospective observational study of 104 patients admitted to hospital with COVID-19. Plasma samples were collected within 72 h of admission. In addition to routine coagulation and haematology testing, soluble thrombomodulin (sTM), thrombin-antithrombin (TAT), tissue plasminogen activator-plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin-α2 antiplasmin complex (PIC) were performed by automated chemiluminescent enzyme immunoassays. RESULTS: Significantly higher levels of D-dimer, TAT, sTM and tPAI-C were observed in non-survivors compared to survivors. To confirm which parameters were independent risk factors for mortality, multiple logistic regression was performed on D-dimer, TAT. sTM, tPAI-C and PIC data. Only increasing sTM was significantly associated with mortality, with an odds ratio of 1.065 for each 1.0 TU/mL increment (95% CI 1.025-1.115). CONCLUSIONS: Of the haemostatic variables measured, sTM, which can be rapidly assayed, is the best independent predictor of mortality in patients hospitalized with COVID-19, and this suggests that endothelial dysfunction plays an important role in disease progression.

International consensus recommendations on the management of people with haemophilia B.

Haemophilia B is a rare X-linked genetic deficiency of coagulation factor IX (FIX) that, if untreated, can cause recurrent and disabling bleeding, potentially leading to severe arthropathy and/or life-threatening haemorrhage. Recent decades have brought significant improvements in haemophilia B management, including the advent of recombinant FIX and extended half-life FIX. This therapeutic landscape continues to evolve with several non-factor replacement therapies and gene therapies under investigation. Given the rarity of haemophilia B, the evidence base and clinical experience on which to establish clinical guidelines are relatively sparse and are further challenged by features that are distinct from haemophilia A, precluding extrapolation of existing haemophilia A guidelines. Due to the paucity of formal haemophilia B-specific clinical guidance, an international Author Group was convened to develop a clinical practice framework. The group comprised 15 haematology specialists from Europe, Australia, Japan, Latin America and North America, covering adult and paediatric haematology, laboratory medicine and biomedical science. A hybrid approach combining a systematic review of haemophilia B literature with discussion of clinical experience utilized a modified Delphi format to develop a comprehensive set of clinical recommendations. This approach resulted in 29 recommendations for the clinical management of haemophilia B across five topics, including product treatment choice, therapeutic agent laboratory monitoring, pharmacokinetics considerations, inhibitor management and preparing for gene therapy. It is anticipated that this clinical practice framework will complement existing guidelines in the management of people with haemophilia B in routine clinical practice and could be adapted and applied across different regions and countries.

Laboratory coagulation tests and recombinant porcine factor VIII: A United Kingdom Haemophilia Centre Doctors' Organisation guideline.

INTRODUCTION: Acquired haemophilia A (AHA) is a rare bleeding disorder caused by development of auto-antibodies to endogenous coagulation factor VIII (FVIII). Recombinant porcine factor VIII (rpFVIII) is currently licensed only for the management of bleeding in patients with AHA. Regular monitoring of rpFVIII is recommended to assess treatment effectiveness. AIM: This guideline from the United Kingdom Haemophilia Centre Doctors' Organisation (UKHCDO) examines the current publications in the area and aims to offer advice for the laboratory monitoring of rpFVIII in patients with AHA. METHODS: A review of the current literature was undertaken followed by critical review by the authors. RESULTS/CONCLUSIONS: A validated one-stage clotting FVIII assay is recommended for the measurement and regular monitoring of rpFVIII. Assessment of cross-reacting rpFVIII inhibitors by one-stage porcine Bethesda assay should be performed as part of the initial diagnosis of AHA or prior to treatment with rpFVIII. Available data show that chromogenic FVIII assays underestimate rpFVIII and this should be considered if measurement of rpFVIII is required in patients receiving Emicizumab.

International multicenter, multiplatform study to validate Taipan snake venom time as a lupus anticoagulant screening test with ecarin time as the confirmatory test: Communication from the ISTH SSC Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies.

BACKGROUND: Lupus anticoagulant (LA) assays are compromised in anticoagulated patients, and existing strategies to overcome the interferences have limitations. The prothrombin-activating Taipan snake venom time (TSVT) screening test and ecarin time (ET) confirmatory test are innately insensitive to vitamin K antagonists (VKA) and direct factor Xa inhibitors (DFXaI). OBJECTIVES: Validate standardized TSVT/ET reagents for LA detection, in a multicenter, multiplatform study. PATIENTS/METHODS: Six centers from four countries analyzed samples with TSVT/ET from 81 nonanticoagulated patients with LA, patients with established antiphospholipid syndrome (APS), and proven persistent LA who were either not anticoagulated (n = 120) or were anticoagulated with VKAs (n = 180) or DFXaIs (n = 71). Additionally, 339 nonanticoagulated LA-negative patients, and 575 anticoagulated non-APS patients (172 VKA, 403 DFXaI) were tested. Anticoagulant spiking experiments were performed and 112 samples containing potential interferences (i.e., direct thrombin inhibitors) were tested. Results were evaluated against locally derived cutoffs. Imprecision was evaluated. RESULTS: Cutoffs were remarkably similar despite use of different analyzers and donor populations. Cutoffs for TSVT ratio, ET ratio, percent correction, and normalized TSVT ratio/ET ratio ranged between 1.08 and 1.10, 1.09 and 1.12, 9.3% and 14.8%, and 1.10 and 1.15, respectively. Coefficients of variation for TSVT and ET ratios were ≤5.0%. TSVT/ET exhibited sensitivity, specificity, and negative and positive predictive values of 78.2%/95.0%/86.3%/91.5%, respectively, with established APS as the LA-positive population, and 86.9%/95.0%/76.8%/97.4%, respectively, with triple-positive APS. Interference was seen with direct thrombin inhibitors, unfractionated heparin, and low molecular weight heparins, but not VKAs or DFXaIs. CONCLUSIONS: TSVT/ET are validated for LA detection in nonanticoagulated patients and those on VKAs or DFXaIs.

Evaluation of laboratory assays for anti-platelet factor 4 antibodies after ChAdOx1 nCOV-19 vaccination.

INTRODUCTION: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following ChAdOx1 nCOV-19 vaccine has been described, associated with unusual site thrombosis, thrombocytopenia, raised D-dimer, and high-titer immunoglobulin-G (IgG) class anti-platelet factor 4 (PF4) antibodies. Enzyme-linked immunosorbent assays (ELISA) have been shown to detect anti-PF4 in patients with VITT, but chemiluminescence assays do not reliably detect them. ELISA assays are not widely available in diagnostic laboratories, and, globally, very few laboratories perform platelet activation assays. METHODS: Assays that are commercially available in the United Kingdom were evaluated for their ability to identify anti-PF4 antibodies in samples from patients with suspected VITT. Four IgG-specific ELISAs, two polyspecific ELISAs, and four rapid assays were performed on samples from 43 patients with suspected VITT from across the United Kingdom. Cases were identified after referral to the UK Expert Haematology Panel multidisciplinary team and categorized into unlikely, possible, or probable VITT. RESULTS AND DISCUSSION: We demonstrated that the HemosIL AcuStar HIT-IgG, HemosIL HIT-Ab, Diamed PaGIA gel, and STic Expert assays have poor sensitivity for VITT in comparison to ELISA. Where these assays are used for heparin-induced thrombocytopenia (HIT) diagnosis, laboratories should ensure that requests for suspected VITT are clearly identified so that an ELISA is performed. No superiority of IgG-ELISAs over polyspecific ELISAs in sensitivity to VITT could be demonstrated. No single ELISA method detected all possible/probable VITT cases; if a single ELISA test is negative, a second ELISA or a platelet activation assay should be considered where there is strong clinical suspicion.

Cross-reacting recombinant porcine FVIII inhibitors in patients with acquired haemophilia A.

INTRODUCTION: Acquired haemophilia A (AHA) is a rare bleeding disorder caused by the development of autoantibodies to endogenous human factor VIII (hFVIII). If treatment of bleeding is required, one option is recombinant porcine FVIII (rpFVIII). Cross-reactivity between factor VIII inhibitors and rpFVIII has previously been described. AIM: The aim of this study was to retrospectively assess the incidence of cross-reacting anti-porcine inhibitors in patients diagnosed with AHA in two UK centres. METHODS: The plasma of fifty-one patients diagnosed with AHA via reduced FVIII:C and positive FVIII inhibitor titre as detected with a Nijmegen-Bethesda assay (NBA) was also tested by a porcine Bethesda assay (PBA). The NBA was modified by replacement of human FVIII with rpFVIII in the PBA, with determination of residual FVIII by one-stage clotting assay. RESULTS: The median FVIII inhibitor titre by NBA was 22.8 BU/mL (range: 0.8-1000 BU/mL). 37% of samples exhibited linear, type 1 kinetics in the NBA. Negative PBA was observed in 26 patients, and 25 were positive (median PBA: 3.5 BU/mL; range: 0.8-120 BU/mL). Type 1 kinetics were observed in 40% of PBA-positive patients. At NBA tires of greater than 100 BU/mL, the positive predictive value for the presence of porcine cross-reactivity was 100%. At NBA below 5 BU/mL, the negative predictive value for the presence of porcine cross-reactivity was 71%. CONCLUSION: Cross-reactivity between FVIII inhibitors and rpFVIII was observed in 49% of patients. The presence of inhibitors to rpFVIII may influence the treatment choice for patients with acquired haemophilia A.