Diversity in clinical isolates of Ictalurid herpesvirus 1 (IcHV1) from U.S. farm-raised catfish and virulence assessment in channel and channel × blue catfish hybrids.

Ictalurid herpesvirus 1 (IcHV1) is the most significant viral agent in U.S. catfish aquaculture. Little is known regarding the genetic stability and antigenic variability of IcHV1. Herein, the genetic and antigenic diversity of IcHV1 field isolates was assessed by restriction fragment length polymorphism (RFLP) analysis and serum neutralization assays. RFLP analysis identified two distinct genotypes (IcHV1A and IcHV1B), both discrete from blue catfish alloherpesvirus (BCAHV). Neutralization assays with anti-IcHV1 monoclonal antibody Mab-95 indicate shared antigenic determinants for IcHV1A and IcHV1B that are absent from BCAHV, which Mab-95 did not neutralize. Virulence assessments with representative isolates demonstrate significant differences between isolates within RFLP groups and pooled RFLP group data suggest IcHV1B (pooled survival [mean ± SE]: 58.3% ± 2.6) may be more virulent than IcHV1A (survival: 68.6% ± 2.4). Rechallenges with representative IcHV1A and IcHV1B isolates indicate a cross-protective effect, with fish surviving initial exposure to IcHV1A or IcHV1B showing robust protection when subsequently re-exposed to IcHV1A or IcHV1B. This work demonstrated significant differences in virulence between case isolates, identifying two discrete IcHV1 lineages, distinct from BCAHV, with similar virulence in channel and channel × blue catfish hybrids and a cross-protective effect in catfish surviving exposure to either lineage.

It's all about relationships: Developing nurse-led primary health care in rural communities.

The role of nurses in leading the design and delivery of primary health care services to address health inequities is growing in prominence, specifically in rural Australia. However, limited evidence exists to inform nurse-led primary health care in this context. Based on a focus group with nursing executives and semi-structured interviews with registered nurses we describe nurse experiences of leading the design of a primary health care service in rural Australia and nurse transition to and practice in this service. Nurse experiences were analysed using reflexive thematic analysis. The study reveals the centrality of relational integration in service design and nurse acquisition of relational practice as it relates to nurse to care recipient and nurse to nurse relationships. Tensions between primary health care nurses and their peers, and resultant de-valuing of primary health care practice, are described. The acquisition of nurse professional agency draws attention to investments required to position nurses to lead and sustain care innovations external to hospital settings. The authors propose that relational approaches may provide nurses with the opportunity to reframe their leadership and service contributions towards community literate primary health care provision and provide a pathway to professional emancipation from constrained practice expectations.

Postcesarean Analgesia With Epidural Morphine After Epidural 2-Chloroprocaine: A Randomized Noninferiority Trial.

BACKGROUND: Previous studies have suggested that administration of epidural 3% 2-chloroprocaine (CP) before epidural morphine results in decreased analgesic efficacy of epidural morphine. We sought to determine whether these observations were a result of antagonism or a window period between the conclusion of surgical anesthesia for cesarean delivery and the peak onset time of epidural morphine, and whether a method to preserve the analgesic efficacy of epidural morphine exists. METHODS: Term parturients scheduled for nonemergent, unscheduled cesarean delivery with preexisting labor epidural catheters were recruited for this exploratory, randomized, single-blinded, noninferiority trial. Subjects were randomized to initial dosing to a T4 dermatome surgical anesthetic level with either 3% CP or 2% lidocaine with 1:200,000 epinephrine and sodium bicarbonate (LEB). Subsequent redosing for both groups was performed with LEB at regular intervals. Epidural morphine 3 mg was administered to both groups after delivery. Assessing the difference between the 2 groups in total opioid use for the first 24 hours after epidural morphine administration was the primary objective. The noninferiority margin of 10 oral milligram morphine equivalents was prespecified based on previous noninferiority studies. Secondary end points included time from epidural morphine administration to first rescue opioid request, numerical pain scores, nausea/vomiting, and pruritus. RESULTS: Data were analyzed for 40 parturients, 20 in each group. The median 24-hour opioid consumption for the CP group was 0 (Q1 = 0 and Q3 = 15.6) oral milligram morphine equivalents compared to 15 (6.3-22.5) for the LEB group. The median difference was -7.5, with a 95% confidence interval -15 to 0. Noninferiority was concluded, as the confidence interval was less than the predetermined noninferiority margin of 10 oral milligram morphine equivalents. There was no treatment effect on time to first opioid request and no statistically significant differences in pain scores or nausea, vomiting, or pruritus at all time points (4, 8, 12, and 24 hours after epidural morphine administration). CONCLUSION: While designed as an exploratory study, initial epidural dosing with 3% CP and beginning subsequent redosing with LEB within 30 minutes of the initial CP bolus provided noninferior postcesarean analgesia with epidural morphine compared to initial epidural dosing and redosing with LEB. Previous observations of decreased analgesic efficacy of epidural morphine after epidural CP were likely due to a window period that may be mitigated by redosing with lidocaine; however, larger studies are necessary to confirm these findings.

Strength of cross-sector collaborations in co-designing an extended rural and remote nursing placement innovation: Focusing on student learning in preference to student churning.

AIM: To describe the strength of a cross-sector and multi-university collaboration in co-designing an extended nursing placement innovation in rural and remote Australia. CONTEXT: Registered nurses are Australia's largest health workforce. Short-duration placements can limit nursing student exposure to rural and remote practice, impacting student capacity to tailor and contextualise their practice, navigate complex inequities, establish a sense of belonging and consider rural practice post-registration. Extended nursing placements have been recommended to address these challenges, but there are no guidelines governing their development and limited resources to support implementation. APPROACH: Methods adopted in program development included the following: (1) collaboration establishment; (2) co-defining challenges confronting nurse education in these contexts; (3) co-developing guiding principles; (4) co-designing a new approach to nurse education, the Extended Nursing Placement Program (ENPP); and (5) the co-contribution of stakeholders to program design, implementation and evaluation. Regional stakeholders include a NSW and Victorian Local Health District/Service, three Aboriginal health services and the Royal Flying Doctor Service of Australia. University participants include two metropolitan universities, a University Department of Rural Health and final-year Bachelor of Nursing students. Program implementation in Semester 1 of 2022 with seven final-year nursing students. CONCLUSION: The authors propose that the adoption of collaborative approaches can contribute to re-framing student nurse education and the development of a rural-ready nursing workforce. These approaches can provide regions and universities with the opportunity to avoid student churn whilst promoting the attainment of skills required to work, live and thrive in these locations.

Competition or collaboration in regional Australia? A cross-border and multi-university approach to maximising rural health investments, community health and health workforce outcomes.

AIM: To describe the establishment of a cross-border and multi-university collaboration in rural Australia to mitigate potential competition, maximise Rural Health Multidisciplinary Training (RHMT) Programme investments and regional health workforce outcomes. CONTEXT: Rural Health Multidisciplinary Training programme investments have enabled the establishment of 19 Australian University Departments of Rural Health (UDRH) and 17 Rural Clinical Schools. The importance of these investments is acknowledged. However, in regional settings, due to limited clinical placement and training opportunities, there is potential for heightened competition between universities who are operating within shared geographical footprints. Competition between universities risks focusing RHMT programme activity on individual reporting requirements and activities, in preference to: regional needs; existing community-university relationships; and place-based approaches to health workforce development. PARTICIPANTS: A rural New South Wales and Victorian RHMT-funded departments, collectively known as the Sunraysia Collaboration. APPROACH: Strategic and operational processes, structures and actions underpinning collaboration formation and relationship consolidation will be described. Co-design methodologies employed to collectively define collaboration vision and aims, governance framework and guiding principles, reporting structures and co-contributions to teaching, research and service will be discussed. Collaboration sensitivity to the social, cultural, relationship and economic connectedness within the region and existing health workforce flows will also be explored. CONCLUSION: The Sunraysia collaboration demonstrates one approach towards mitigating potential competition between RHMT Programme funded universities within rural and remote Australia. The collaboration is an exemplar of co-design in action providing an alternative approach to address RHMT Programme parameters and regional needs whilst supporting rural-remote health workforce training and education innovations.

Virulence and immunogenicity of blue catfish alloherpesvirus in channel, blue and blue × channel hybrid catfish.

Blue catfish alloherpesvirus (BCAHV) is a novel virus isolated from the blue catfish (Ictalurus furcatus). To date, the ultrastructure, virulence and immunogenicity of BCAHV have not been reported. Given the importance of blue catfish in producing channel ♀ (I. punctatus) × â™‚ blue (I. furcatus) catfish hybrids and the increasing demand for hybrid catfish in the US catfish industry, the susceptibility of blue, channel and hybrid catfish to BCAHV was assessed. Further, the cross-protective potential of BCAHV against Ictalurid herpesvirus 1 (IcHV1) was investigated in channel and hybrid catfish that survive BCAHV exposure. Neutralization assays revealed BCAHV is refractive (neutralization index [NI] = 0) to anti-IcHV1 monoclonal antibody Mab 95, compared to IcHV1 (NI = 1.8). Exposure of blue catfish fingerling to 1.3 × 105 TCID50 /L BCAHV produced cumulative mortality of 51.67 ± 0.70% and pathologic changes similar to those of channel catfish virus disease. No mortality was observed in channel or hybrid catfish. Twenty-eight days post-challenge, surviving channel and hybrid catfish were exposed to 9.4 × 104 TCID50 /L IcHV1 (LC50 dose), resulting in 100% relative per cent survival compared to naïve cohorts. These data provide baseline information for BCAHV and lay the groundwork for future studies. Data also identify BCAHV as a potential vaccine candidate against IcHV1. Based on host range and immunogenicity evaluations, in addition to genome sequence data from previous studies, BCAHV should be given consideration as a new species of Ictalurivirus.

Real-time resolution of point mutations that cause phenovariance in mice.

With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.

Coaching in self-efficacy improves care responses, health and well-being in dementia carers: a pre/post-test/follow-up study.

BACKGROUND: Maintaining the health and well-being of family carers of people with dementia is vital, given their potential for experiencing burden associated with the role. The study aimed to help dementia carers develop self-efficacy, be less hassled by the caring role and improve their health and well-being with goal-directed behaviour, by participating in an eight module carer coaching program. METHODS: The study used mixed methods in a pre/post-test/follow-up design over 24 months, with assignment of consented dementia carers to either individualised (n = 16) or group coaching (n = 32), or usual carer support services (n = 43), depending on preference. Care-giving self-efficacy and hassles, carer health, well-being and goal-directed behaviours were assessed over time. Analysis of Variance (ANOVA) was used to compare changes over time and the effects of coaching on carer self-efficacy, hassles and health, using the Univariate General Linear Model (GLM). RESULTS: All carers were hassled by many aspects of caring at baseline. Participants receiving coaching reported non-significant improvements in most areas of self-efficacy for caring, hassles associated with caring and self-reported health at post-test and follow-up, than did carers receiving usual carer support. Group coaching had greater success in helping carers to achieve their goals and to seek help from informal and formal support networks and services. CONCLUSION: The study outcomes were generally positive, but need to be interpreted cautiously, given some methodological limitations. It has been shown, however, that health staff can assist dementia carers to develop self-efficacy in better managing their family member's limitations and behaviour, seek help from others and attend to their health. Teaching carers to use goal-directed behaviour may help them achieve these outcomes.

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.

A collaborative primary health care model for children and young people in rural Australia: explorations of cross-sectoral leader action.

BACKGROUND: Cross-sectoral collaborations are considered necessary to address detrimental health, social, educational and economic outcomes that impact marginalised and disadvantaged populations. There is a strong relationship between the health of children and their educational attainment; good health promotes positive learning. This paper reports cross-sectoral executive and senior management level systems changes required to enable the design of a collaborative primary healthcare service model for children and young people in rural Australia. METHODS: A descriptive qualitative design was used. Data were collected from executive and senior managers from three organisations (Education, Health and a University Department of Rural Health [n =6]) through individual semi-structured interviews. Data were analysed using an inductive, thematic approach. The study draws on Lewin's Model of Change. RESULTS: Three overarching themes were generated from the data: an embedded challenge and experimental solutions; building a shared language and understanding; and the role of relationships and trust. Despite the unique geographical and social context of the study area, strategies emerged from the data on how a solution to an embedded challenge, through design of a primary healthcare model, was established and how the strategies described could be transferred and scaled to other rural and remote communities. CONCLUSION: Contextual differences make each rural and remote area unique. In this study, strategies that are described in the managing change literature were evident. The authors conclude that drawing on strong management of change principles could mean that a service model designed for one remote community might be transferrable to other communities.

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.

Direct oral anticoagulants-Remove versus Taipan snake venom time for detection of a lupus anticoagulant in patients taking oral direct factor Xa inhibitors.

BACKGROUND: The optimal method of detecting a lupus anticoagulant (LA) for patients taking direct factor Xa inhibitor (DFXaI) direct oral anticoagulants (DOACs) remains controversial. Methods include charcoal adsorption of the DOACs to allow testing with the activated partial thromboplastin time (APTT) and dilute Russell viper venom time (dRVVT), or use of the DFXaI-insensitive Taipan snake venom time (TSVT) and Ecarin time (ET) assays on neat plasma. OBJECTIVES: The objective was to compare the utility of APTT and dRVVT analysis following DOAC Remove against TSVT/ET on untreated plasma for LA detection in spiked plasmas and routine clinical samples for patients on DFXaIs. PATIENTS/METHODS: Various LA-negative and LA-positive samples were assayed by APTT, dRVVT, and TSVT/ET, and then separately spiked with rivaroxaban, apixaban, and edoxaban calibrators to a concentration of ~190 ng/ml and the assays repeated on spiked plasma before and after DOAC Remove treatment. Testing of 284 consecutive samples from DFXaI-anticoagulated patients by APTT/dRVVT and TSVT/ET before and after DOAC Remove treatment was undertaken. RESULTS: In the spiking model, we found that both TSVT/ET and DOAC Remove strategies generally distinguished LA-negative and LA-positive samples, but some false-positive LA results occurred. In the investigation of 284 consecutive patient samples on DFXaIs, the percentage agreement for LA detection in neat samples tested by TSVT/ET versus APTT and dRVVT after DOAC Remove treatment was 90% (Cohen kappa 0.12). CONCLUSION: Our data highlight uncertainty and disagreement for testing LA in patients on DFXaI. Further studies are required.

Healthy Uses for Garlic.

Garlic originated in West China and has been used for its health qualities since 2600 bc. Garlic was brought to Great Britain in 1548 from the Mediterranean Sea. Early uses of garlic were to treat gastric infections, fevers, and diarrhea. Fresh garlic has the most health benefits through the compound allicin. Health benefits of garlic include the prevention and treatment of cardiovascular disease, antioxidant effects, antimicrobial effects, and reduction of cancer risks.

Investigation of platelet function in patients with chronic kidney disease stages IV-V.

INTRODUCTION: Patients with renal failure are at increased risk of both bleeding and thrombosis. Further descriptions of laboratory investigations in these patients are required. METHODS: Investigation of 24 patients with chronic kidney disease (CKD) stages IV-V with light transmission aggregometry, platelet secretion assays and platelet nucleotide analysis. Patients were in a nonbleeding state and not on antiplatelet medication. Results were compared with our local reference range used within the clinical haematology service. RESULTS: Of the 24 patients, two had decreased responses to arachidonic acid, adenosine diphosphate, collagen, thrombin receptor activator peptide-6 and one had decreased responses to high dose ristocetin, and one had increased response to low dose ristocetin. 11 and 13 out of 24 had abnormal platelet secretion release to collagen and thrombin, respectively. Platelet nucleotide analysis in patients was normal with the exception of a reduction in ADP content in one patient and ATP/ADP ratio in one patient. CONCLUSIONS: In our collection of patients with CKD investigated for platelet function in the nonbleeding state, they generally had normal light transmission aggregometry and nucleotide analysis but around 50% had decreased platelet secretion assays. These results could be important in determining the significance of platelet function tests in patients with bleeding symptoms and renal failure. Further characterization of platelet function tests in future will help characterize haemostasis in renal failure further.

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 COVID-19 coagulopathy; laboratory characterization using thrombin generation and nonconventional haemostasis assays.

INTRODUCTION: Patients with COVID-19 are known to have a coagulopathy with a thrombosis risk. It is unknown whether this is due to a generalized humoral prothrombotic state or endothelial factors such as inflammation and dysfunction. The aim was to further characterize thrombin generation using a novel analyser (ST Genesia, Diagnostica Stago, Asnières, France) and a panel of haematological analytes in patients with COVID-19. METHODS: Platelet poor plasma of 34 patients with noncritical COVID-19 was compared with 75 patients with critical COVID-19 (as defined by WHO criteria) in a retrospective study by calibrated automated thrombography and ELISA. Patients were matched for baseline characteristics of age and gender. RESULTS: Critical patients had significantly increased fibrinogen, CRP, interleukin-6 and D-dimer compared to noncritical patients. Thrombin generation, in critical patients, was right shifted without significant differences in peak, velocity index or endogenous thrombin potential. Tissue plasminogen activator (tPA), tissue factor pathway inhibitor (TFPI) and vascular endothelial growth factor (VEGF) were significantly increased in the critical versus noncritical patients. Critically ill patients were on haemodiafiltration (31%; heparin used in the circuit) or often received escalated prophylactic low-molecular weight heparin. CONCLUSION: These results confirm increased fibrinogen and D-dimer in critical COVID-19-infected patients. Importantly, disease severity did not increase thrombin generation (including thrombin-antithrombin complexes and prothrombin fragment 1 + 2) when comparing both cohorts; counter-intuitively critical patients were hypocoaguable. tPA, TFPI and VEGF were increased in critical patients, which are hypothesized to reflect endothelial dysfunction and/or contribution of heparin (which may cause endothelial TFPI/tPA release).

Investigation of patients with unclassified bleeding disorder and abnormal thrombin generation for physiological coagulation inhibitors reveals multiple abnormalities and a subset of patients with increased tissue factor pathway inhibitor activity.

INTRODUCTION: We have routinely used thrombin generation to investigate patients with unclassified bleeding disorder (UBD). AIMS: To investigate haemostatic abnormalities in patients with UBD that had abnormal thrombin generation on at least one occasion. METHODS: Investigation of 13 known UBD patients with thrombin generation and detailed haemostatic testing was undertaken including TFPI assays but also thrombomodulin and fibrinogen-γ. RESULTS: 12 females and 1 male were included. No patient had a platelet function disorder or coagulation factor deficiency that explained the bleeding phenotype, though 2 patients had factor deficiencies; a factor X of 0.41 IU/mL and a factor XI of 0.51 IU/mL. ThromboGenomics revealed variants for these factors but no other abnormalities. Patients were included who previously had either prolonged lag time or decreased endogenous thrombin potential (ETP) via high dose tissue factor (5 pmol/L) or low dose tissue factor (1.5 pmol/L) with corn trypsin inhibitor (CTI). Tissue factor pathway inhibitor (TFPI) activity was significantly increased (P < .001; increased in 8 patients) compared with controls and abnormalities in soluble thrombomodulin (2 patients), fibrinogen-γ (1 patient) and tPA (4 patients for each) were seen. Total and free TFPI levels were not increased. Mixing studies of patient plasma with 50:50 normal plasma for thrombin generation via low dose tissue factor failed to correct the ETP consistent with ongoing inhibition. Addition of an anti-TFPI antibody partially corrected thrombin generation to normal levels. TFPI sequencing was unremarkable. CONCLUSION: TFPI activity may be increased in a subset of UBD patients. Further research studies are warranted in UBD patients for coagulation inhibitor abnormalities.