Biosensing based on surface-enhanced Raman spectroscopy as an emerging/next-generation point-of-care approach for acute myocardial infarction diagnosis.

Cardiovascular disease is a major global health issue. In particular, acute myocardial infarction (AMI) requires urgent attention and early diagnosis. The use of point-of-care diagnostics has resulted in the improved management of cardiovascular disease, but a major drawback is that the performance of POC devices does not rival that of central laboratory tests. Recently, many studies and advances have been made in the field of surface-enhanced Raman scattering (SERS), including the development of POC biosensors that utilize this detection method. Here, we present a review of the strengths and limitations of these emerging SERS-based biosensors for AMI diagnosis. The ability of SERS to multiplex sensing against existing POC detection methods are compared and discussed. Furthermore, SERS calibration-free methods that have recently been explored to minimize the inconvenience and eliminate the limitations caused by the limited linear range and interassay differences found in the calibration curves are outlined. In addition, the incorporation of artificial intelligence (AI) in SERS techniques to promote multivariate analysis and enhance diagnostic accuracy are discussed. The future prospects for SERS-based POC devices that include wearable POC SERS devices toward predictive, personalized medicine following the Fourth Industrial Revolution are proposed.

Whole exome sequencing identifies a novel SCN1A mutation in genetic (idiopathic) generalized epilepsy and juvenile myoclonic epilepsy subtypes.

INTRODUCTION: Genetic (idiopathic) generalized epilepsy (GGE) is a common form of epilepsy characterized by unknown aetiology and a presence of genetic component in its predisposition. METHODS: To understand the genetic factor in a family with GGE, we performed whole exome sequencing (WES) on a trio of a juvenile myoclonic epilepsy/febrile seizure (JME/FS) proband with JME/FS mother and healthy father. Sanger sequencing was carried out for validation of WES results and variant detection in other family members. RESULTS: Predictably damaging variant found in affected proband and mother but absent in healthy father in SCN1A gene was found to be associated with generalized epilepsy and febrile seizure. The novel non-synonymous substitution (c.5753C>T, p.S1918F) in SCN1A was found in all family members with GGE, of which 4/8 were JME subtypes, and/or febrile seizure, while 3 healthy family member controls did not have the mutation. This mutation was also absent in 41 GGE patients and 414 healthy Malaysian Chinese controls. CONCLUSION: The mutation is likely to affect interaction between the sodium channel and calmodulin and subsequently interrupt calmodulin-dependent modulation of the channel.

Heat shock response and metabolic stress in the tropical estuarine copepod Pseudodiaptomus annandalei converge at its upper thermal optimum.

Heat shock response (HSR), in terms of transcription regulation of two heat shock proteins genes hsp70 and hsp90), was analysed in a widespread tropical copepod Pseudodiaptomus annandalei. The mRNA transcripts of both genes were quantified after copepods at a salinity of 20 underwent an acclimation process involving an initial acclimation temperature of 29 °C, followed by gradual thermal ramping to the target exposure temperature range of 24-36 °C. The respective cellular HSR and organismal metabolism, measured by respiratory activity at exposure temperatures, were compared. The fold change in mRNA expression for both hsp70 and hsp90 (8-9 fold) peaks at 32 °C, which is very close to 32.4 °C, the upper thermal optimum for respiration in the species. Unexpectedly, the modelled HSR curves peak at only 3 °C (hsp90) and 3.5 °C (hsp70) above the mean water temperature (29.32 °C) of the copepod in the field. We propose that copepods in tropical waters adopt a preparative HSR strategy, early at the upper limit of its thermal optimum, due to the narrow thermal range of its habitat thus precluding substantial energy demand at higher temperatures. However, the model suggests that the species could survive to at least 36 °C with short acclimation time. Nevertheless, the significant overlap between its thermal range of hsp synthesis and the narrow temperature range of its habitat also suggests that any unprecedented rise in sea temperature would have a detrimental effect on the species.

Ethnic variation of genetic (idiopathic) generalized epilepsy in Malaysia.

PURPOSE: Ethnic variation in epilepsy classification was reported in the Epilepsy Phenome/Genome Project. This study aimed to determine the ethnic variation in the prevalence of genetic (idiopathic) generalized epilepsy (GGE) and GGE with family history in a multi-ethnic Asian population in Malaysia. METHOD: In this cross-sectional study, 392 patients with a clinical diagnosis of GGE were recruited in the neurology outpatient clinic, University of Malaya Medical Centre (UMMC), from January 2011 till April 2016. RESULTS: In our epilepsy cohort (n=2100), 18.7% were diagnosed to have GGE. Of those, 28.6% >(N=112) had family history of epilepsy with a mean age of seizure onset of 16.5 years old, and 42.0% had myoclonic seizures (N=47). The lifetime prevalence of epilepsy among first-degree relative of those with GGE and positive family history was 15.0%. Analysis according to ethnicity showed that Malaysian Chinese had the lowest percentage of GGE among those with epilepsy (12.3%), as compared with Indian and Malay (25.3% and 21.3%, p<0.001). In addition, 32.1% of these Indian patients with GGE had positive family history, which is more than the Malay (26.4%) and Chinese (27.5%) ethnic groups. Consanguineous marriage was noted in 5 Indian families with positive family history (9.6%). CONCLUSION: There was ethnic variation in the prevalence of GGE, whereby the Malaysian Chinese had the lowest percentage of GGE as compared with Indian and Malay. A substantial proportion of GGE had positive family history among the three ethnics groups.

Effects of antiplatelet therapy on platelet extracellular vesicle release and procoagulant activity in health and in cardiovascular disease.

Dual antiplatelet therapy with aspirin and clopidogrel is commonly used to prevent recurrent ischemic events in patients with cardiovascular disease. Whilst their effects on platelet reactivity are well documented, it is unclear, however, whether antiplatelet therapy inhibits platelet extracellular vesicle (EV) release. The aim of this study was to investigate the effects of antiplatelet therapy on platelet EV formation and procoagulant activity. Blood samples from 10 healthy controls not receiving antiplatelet therapy were incubated in vitro with aspirin or a P2Y12 inhibitor (MeSAMP). Blood samples from 50 patients receiving long-term dual antiplatelet therapy and undergoing coronary angiography were also studied. Platelet reactivity was assessed by Multiplate™ impedance aggregometry. Platelet EV formation and procoagulant activity of pretreated and untreated blood samples in response to arachidonic acid (AA), adenosine diphosphate (ADP), ADP+PGE1, and thrombin receptor-activating peptide (TRAP) stimulation were assessed by flow cytometry and Procoag-PL assays, respectively. Incubation of normal platelets with aspirin significantly inhibited AA-induced platelet reactivity, EV formation, and procoagulant activity, whilst MeSAMP significantly inhibited platelet reactivity and EV formation in response to AA, ADP, and TRAP, but had minimal effect on procoagulant activity. Most patients receiving dual antiplatelet therapy showed an appropriate reduction in platelet reactivity in response to their treatment; however there was not complete inhibition of increased platelet and EV procoagulant activity in response to ADP, AA, or TRAP. In addition, we could not find any correlation between platelet reactivity and procoagulant activity in patients receiving dual antiplatelet therapy.

Longitudinal changes in hemostatic parameters and reduced pulsatility contribute to non-surgical bleeding in patients with centrifugal continuous-flow left ventricular assist devices.

BACKGROUND: Bleeding and thromboembolic events are identified complications in patients supported with newer centrifugal continuous-flow left ventricular assist devices (cfLVADs). Bleeding events have been associated with acquired von Willebrand syndrome (vWS) in these patients, though longitudinal changes and the effect of pulsatility remain unquantified. We evaluated longitudinal effects of third-generation cfLVADs on hemostatic biomarkers, non-surgical bleeding, and thromboembolic events. We investigated the association between pulsatility (as defined by aortic valve opening) on von Willebrand Factor (VWF) profile and bleeding. METHODS: We prospectively studied 28 patients implanted with the HeartWare (HeartWare International, Framingham, MA) cfLVAD for up to 360 days. We performed bleeding and coagulation assays 8 times from pre-implant to Day 360 (D360) post-implant, including platelet aggregometry, VWF collagen binding activity-to-antigen (CBA/Ag) ratio, thromboelastography, soluble P-selectin, platelet-specific marker soluble glycoprotein VI (sGPVI), and platelet microparticles. Aortic valve opening was assessed by echocardiography at each assessment. Bleeding and thromboembolic events were documented. RESULTS: Bleeding events occurred in 14 patients (50%). Maximal platelet inhibition occurred by D30. VWF profile impairment (VWF CBA/Ag < 0.8) was demonstrated in 89% of patients at D30, with subsequent recovery but further deterioration after D180. Bleeding was associated with elevated pre-implant sGPVI (p = 0.008). Pulsatility was associated with higher VWF CBA/Ag (p = 0.02) and a trend to less bleeding. CONCLUSIONS: Third-generation cfLVADs were associated with longitudinal changes in hemostatic markers, and bleeding was associated with elevated pre-implant plasma sGPVI. Further, pulsatility may contribute to recovery of the VWF profile and potentially lower bleeding risk.

A Genome Wide Study of Copy Number Variation Associated with Nasopharyngeal Carcinoma in Malaysian Chinese Identifies CNVs at 11q14.3 and 6p21.3 as Candidate Loci.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a neoplasm of the epithelial lining of the nasopharynx. Despite various reports linking genomic variants to NPC predisposition, very few reports were done on copy number variations (CNV). CNV is an inherent structural variation that has been found to be involved in cancer predisposition. METHODS: A discovery cohort of Malaysian Chinese descent (NPC patients, n = 140; Healthy controls, n = 256) were genotyped using Illumina® HumanOmniExpress BeadChip. PennCNV and cnvPartition calling algorithms were applied for CNV calling. Taqman CNV assays and digital PCR were used to validate CNV calls and replicate candidate copy number variant region (CNVR) associations in a follow-up Malaysian Chinese (NPC cases, n = 465; and Healthy controls, n = 677) and Malay cohort (NPC cases, n = 114; Healthy controls, n = 124). RESULTS: Six putative CNVRs overlapping GRM5, MICA/HCP5/HCG26, LILRB3/LILRA6, DPY19L2, RNase3/RNase2 and GOLPH3 genes were jointly identified by PennCNV and cnvPartition. CNVs overlapping GRM5 and MICA/HCP5/HCG26 were subjected to further validation by Taqman CNV assays and digital PCR. Combined analysis in Malaysian Chinese cohort revealed a strong association at CNVR on chromosome 11q14.3 (Pcombined = 1.54x10-5; odds ratio (OR) = 7.27; 95% CI = 2.96-17.88) overlapping GRM5 and a suggestive association at CNVR on chromosome 6p21.3 (Pcombined = 1.29x10-3; OR = 4.21; 95% CI = 1.75-10.11) overlapping MICA/HCP5/HCG26 genes. CONCLUSION: Our results demonstrated the association of CNVs towards NPC susceptibility, implicating a possible role of CNVs in NPC development.

Low concentration detergent sclerosants induce platelet activation but inhibit aggregation due to suppression of GPIIb/IIIa activation in vitro.

INTRODUCTION: Sclerotherapy is associated with thromboembolic and ischemic neurological adverse events but the effects of sclerosants on platelet function are unknown. The aim of this study was to investigate the in vitro effects of detergent sclerosants Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on platelet activation and aggregation. MATERIALS AND METHODS: Whole blood and platelet rich plasma samples were incubated with sclerosants. Platelet and platelet microparticle (PMP) counts were measured by flow cytometry. Platelet activation was examined by ELISA for soluble factors (sP-selectin, von Willebrand factor, sCD40L and serotonin) and by flow cytometry for membrane-bound markers (CD62p, CD63) and cytoplasmic calcium. Platelet aggregation was assessed by PFA-100®, light transmission and impedance (Multiplate®) aggregometry, and by flow cytometry for glycoprotein (GP) Ib and GPIIb/IIIa subunits, heterodimer expression and activation (PAC-1 binding). RESULTS: Both agents lysed platelets at high concentrations (≥ 0.1%) but induced platelet activation at lower concentrations as evident by a rise in membrane-bound and soluble markers, cytoplasmic calcium and release of phosphatidylserine+PMP. Agonist-stimulated platelet aggregation was inhibited by both sclerosants. Membrane expression of GPIb and GPIIb/IIIa individual subunits or heterodimer was not affected by sclerosants but the activation of GPIIb/IIIa was suppressed. CONCLUSION: Low concentration sclerosants activated platelets and released microparticles but inhibited platelet aggregation due to suppression of GPIIb/IIIa activation.

Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene.

CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock-out mice. This represents creation of the first gene knock-out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock-in (Clic1(FN)) allele, followed by Clic1 knock-out (Clic1(-/-)) mice by crossing Clic1(FN) allele with TNAP-cre mice, resulting in germline gene deletion through Cre-mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1(-) (/-) mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y(12) receptor signaling.

The effects of Asian population substructure on Y STR forensic analyses.

A total of 3046 males of Chinese, Malay, Thai, Japanese, and Indian population affinity were previously typed for the Y STR loci DYS19, DYS385 (counted as two loci), DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS456, DYS458, DYS635, DYS448, and Y GATA H4 using the AmpFlSTR Yfiler kit. These samples were assessed for population genetic parameters that impact forensic statistical calculations. All population samples were highly polymorphic for the 16 Y STR markers with the marker DYS385 being the most polymorphic, because it is comprised of two loci. Most (2677 out of a total of 2806 distinct haplotypes) of the 16 marker haplotypes observed in the sample populations were represented only once in the data set. Haplotype diversities were greater than 99.57% for the Chinese, Malay, Thai, Japanese, and Indian sample populations. For the Y STR markers, population substructure correction was considered when calculating the rarity of a Y STR profile. An F(ST) value, rather than a R(ST) value, is more appropriate under a forensic model. Because the F(ST) values are very small within the Asian populations, the estimate of the rarity of a haplotype comprised of 10-16 markers does not need substructure correction. However haplotypes with fewer markers may require F(ST) corrections when calculating the rarity of the profile.

Timely diagnosis and management of heparin-induced thrombocytopenia in a frequent request, low incidence single centre using clinical 4T's score and particle gel immunoassay.

Clinicians must promptly decide which patients suspected of having heparin-induced thrombocytopenia (HIT) warrant a change in anticoagulation. This single-centre series of 246 HIT testing referrals assessed the combination of clinical score (thrombocytopenia, timing, thrombosis, other causes of thrombocytopenia not evident; 4T's), Diamed ID-Heparin-PF4 immunoassay (PaGIA) and 14C Serotonin Release Assay (SRA) to develop a practical and safe diagnostic strategy for HIT. A total of 142/256 (58%) referrals were in patients with a low 4T's score, with 12/246 (5%) in the high scoring group. PaGIA was positive in 24/246 (9.7%) patients, whilst SRA was positive in 9/246 (3.6%). The overall positive predictive value of a positive PaGIA test alone was 37.5%, however this reached 80% for the high scoring group. Both negative PaGIA and low clinical score independently had negative predictive values of 100%. We subsequently developed an algorithm that, when applied to this cohort, would have resulted in 18/246 patients (7%) definitely requiring alternative anticoagulation, whilst a further 7/246 (2.8%) patients would have been considered on an individual basis. Ultimately (based on SRA) this would have resulted in 16/246 (6.5%) patients unnecessarily having a change in their anticoagulation, with 9/246 (3.6%) patients being 'correctly treated'. The combination of 4T's scoring and PaGIA permitted a practical and safe approach to rapid HIT diagnosis and management.

Mis-identification of factor inhibitors by diagnostic haemostasis laboratories: recognition of pitfalls and elucidation of strategies. A follow up to a large multicentre evaluation.

AIMS: We previously reported the ability of diagnostic haemostasis facilities to identify coagulation factor abnormalities and inhibitors, through a large multi-centre study conducted on behalf of the Royal College of Pathologists of Australasia (RCPA) Quality Assurance Program (QAP). In the current report, additional data evaluation aims to (1) help identify the reasons behind the failures in inhibitor identification, (2) highlight the pitfalls in inhibitor testing, and (3) help elucidate some strategies for overcoming these problems and to assist in better identification and characterisation of inhibitors. METHODS: Forty-two laboratories blind tested a set of eight samples for the presence or absence of inhibitors. These included true factor inhibitors (FVIII and FV), and other samples that reflected potential pre-analytical variables (e.g., heparin contamination, serum, EDTA plasma, aged plasma) that might arise and complicate inhibitor detection or lead to false inhibitor identification. RESULTS: There was a wide scatter of inhibitor results, with false positive and false negative inhibitor identification, and mis-identification of inhibitors (e.g., FVIII inhibitor identified where FV inhibitor present). Further analysis of the pattern of reported laboratory results, including routine coagulation testing and factor profiles, allowed some additional interpretative power to provide strategies that will assist laboratories to improve the accuracy of inhibitor identification in the future. CONCLUSIONS: There are currently occasional lapses in factor inhibitor identification, which this report will hopefully help address.

Identification of factor inhibitors by diagnostic haemostasis laboratories: a large multi-centre evaluation.

We have assessed the proficiency of diagnostic haemostasis facilities to correctly identify coagulation factor abnormalities and inhibitors. Forty-two laboratories participating in the external Quality Assurance Program (QAP) conducted by the RCPA agreed to participate and were each sent a set of eight samples (each 3 x 1 ml) for evaluation. They were asked to blind test these samples for the presence or absence of inhibitors, and where identified, to perform further analysis (including specific inhibitor analysis). In order to make the exercise more challenging, in addition to true factor inhibitors, samples were provided that reflected potential pre-analytical variables that might arise and complicate inhibitor detection or lead to false inhibitor identification. In brief, the sample set comprised a true high level factor (F) V inhibitor, a true moderate level FVIII inhibitor (but sample was defibrinogenated), a true lupus anticoagulant (LA), a normal (but slightly aged) plasma sample, a normal serum sample, a normal EDTA sample, an oral anticoagulant/vitamin K deficiency sample, and a gross heparin ( approximately 10 U/ml) contaminated sample. Sixty-three percent of participants correctly identified the true FV inhibitor as such, although the reported range varied greatly [10 to >250 Bethesda units (BU/ml)] and 46% correctly identified the true FVIII inhibitor, despite the complication of the sample presentation, although the reported range also varied (7 to 64 BU/ml). Some laboratories either failed to identify the inhibitor present, or misidentified the inhibitor type. The LA, the oral anticoagulant/vitamin K deficiency, the normal serum sample, and the normal (aged) sample were also correctly identified by most laboratories, as was the absence of specific factor inhibitors in these samples. However, a small subset of laboratories incorrectly identified the presence of specific factor inhibitors in some of these samples. The heparin sample was also correctly identified by most (68%) laboratories. In contrast, the normal EDTA sample was misidentified as a FV and/or FVIII inhibitor by most (68%) laboratories, and only one laboratory correctly identified this as an EDTA sample. Thus, we conclude that although laboratories are able, in most cases, to identify the presence of true factor inhibitors, there is a large variation in identified inhibitor levels and there are also some significant errors in identification (i.e. false negatives and misidentifications). In addition, there is a significant false positive error rate where some laboratories will identify the presence of specific factor inhibitors where no such inhibitor exists (i.e. false positives).

An international survey of current practice in the laboratory assessment of anticoagulant therapy with heparin.

AIMS: We conducted a survey of laboratory practice for assessment of heparin anticoagulant therapy by participants of the Royal College of Pathologists of Australasia Quality Assurance Program (RCPA QAP). METHODS: A questionnaire was sent to 646 laboratories enrolled in the Haematology component of the QAP, requesting details of tests used for monitoring heparin therapy. RESULTS: Seventy laboratories (10.8%) returned results that indicated that they performed laboratory monitoring of heparin therapy. Most laboratories (69/70 = 98.6%) use the activated partial thromboplastin time (APTT) to monitor unfractionated heparin, with eight (11.4%) also using the APTT for monitoring low molecular weight (LMW) heparin. Five (7.1%) laboratories use the thrombin time (TT) test to help monitor heparin therapy and 37 (52.9%) laboratories use an anti-Xa assay to monitor heparin (either LMW or unfractionated). Normal reference ranges (NRR) for APTT differed considerably between laboratories, even those using the same reagent. Therapeutic ranges (TR) also differed considerably between laboratories, for both APTT and the anti-Xa assay. Laboratory differences in NRR and TR using the same reagents could only be partly explained by the use of different instrumentation. CONCLUSIONS: There is a large variation in current laboratory practice relating to monitoring of heparin anticoagulant therapy. This finding is similar to that of a similar survey conducted by the RCPA QAP almost a decade ago. This study suggests that better standardisation is still required for laboratory monitoring of heparin therapy.

How useful is the monitoring of (low molecular weight) heparin therapy by anti-Xa assay? A laboratory perspective.

We have conducted a series of laboratory-based surveys to assess variability in assay results utilized to monitor heparin anticoagulant therapy. These surveys involved laboratories participating in the Haematology component of the Royal College of Pathologists of Australasia Quality Assurance Program (RCPA QAP). Thirty five of 646 laboratories that were sent a preliminary questionnaire indicated that they performed anti-Xa assays and these laboratories were sent a panel of four plasma samples. These plasma samples contained respectively: (i) no added heparin, (ii) low molecular weight heparin (LMWH), enoxaparin, added to a level of approximately .5 U/mL, (iii) unfractionated heparin added to a level of approximately .5 U/mL, and (iv) LMWH added to a level of approximately 1.0 U/mL. Tests to be performed were the activated partial thromboplastin time (APTT), the thrombin time (TT), fibrinogen, and anti-Xa. As expected, returned results for APTT and TT showed some elevation in heparinized samples while fibrinogen assays were not affected. Anti-Xa assays yielded the following results (median [range]): (i) .01 [0-.11], (ii) .43 [.33-.80], (iii) .23 [.10-.49], and (iv) .90 [.60-1.30]. Thus, although median values were close to those anticipated, there was a wide variation in returned results. In a repeat exercise a few months later laboratories were also asked about their therapeutic ranges (TRs) and provided with an additional vial of LMWH-spiked (1.0 U/mL) plasma labeled as 'heparin-standard' to be used as an assay calibrant. TRs varied substantially between laboratories, from low ranges of .2-.4 to high ranges of .8-1.2. Anti-Xa assay results were similar to those of the first survey: (median [range]): (a) repeat testing: (i) .02 [0-.28], (ii) .47 [.34-.80], (iii) .25 [.14-.58], (iv) .95 [.65-1.31]; (b) repeat testing using survey provided 'heparin-standard': (i) .02 [0-.24], (ii) .55 [.4-.83], (iii) .28 [.10-.63], (iv) 1.00 [.9-1.16]. Thus using the provided 'heparin-standard' yielded lower variability in results for LMWH. In conclusion, the high variability of anti-Xa assay results coupled with the widely variable TRs suggests that therapeutic heparin monitoring is poorly standardized, and this raises some concerns over the clinical value of such monitoring.

Increased procoagulant phospholipid activity in blood from patients with suspected acute coronary syndromes: a pilot study.

Increased platelet activation is well documented in patients with acute coronary syndromes and can be detected by various methods, including flow cytometry and enzyme-linked immunosorbent assay. However, such techniques require several steps and cannot provide quick results. Platelet activation ultimately results in procoagulant phospholipid exposure and we have previously described a simple activated factor X-activated clotting time (XACT) test that is insensitive to resting platelets but that is significantly shortened by activated platelets, microparticles and procoagulant phospholipids. Our aim was to determine whether the XACT test could be used to distinguish patients with chest pain due to cardiac ischaemia from those having chest pain due to non-cardiac causes. We thus carried out XACT tests on ethylenediamine tetraacetic acid whole blood and plasma samples obtained from 46 patients presenting to the emergency department with chest pain and from 30 controls. Sixteen cases (30%) were subsequently diagnosed as acute coronary syndromes. Blood samples from these patients displayed overall significantly shortened XACT results relative to both healthy controls (P<0.001) and chest pain not due to cardiac ischaemia (P<0.004). This discrimination was much better with whole blood samples than when platelet-poor plasmas were tested (P=0.153), suggesting that free microparticles were not the only factors responsible. Thus, the detection of increased procoagulant phospholipid activity in whole blood by shortened XACT results may be a simple and rapid diagnostic marker of some cardiac ischaemic events.

A single-centre prospective study of clinical and haemostatic risk factors for venous thromboembolism following lower limb arthroplasty.

Previous studies report conflicting results concerning the potential significance of thrombophilic genotypes in postarthroplasty venous thromboembolism (VTE). This study assessed thrombophilic genotypes, haemostatic and clinical variables as independent risk factors for VTE postarthroplasty. A total number of 569 patients undergoing elective lower limb arthroplasty at a single centre were prospectively studied. All patients were interviewed and had blood samples collected preoperatively. Bilateral lower limb ultrasonography was performed at day 7 +/- 2 postoperatively in all patients (ventilation/perfusion lung scanning in symptomatic patients only). The incidence of inhospital postoperative VTE was 26%. In univariate analysis - increased age, knee arthroplasty, recent surgery, general anaesthesia, shorter operation time, non-receipt of blood transfusion and differences in surgical practice (including use of pneumatic calf compression, surgical drains and postoperative bandaging techniques) were significantly associated with VTE. Factor V Leiden, prothrombin G20210A and MTHFR C677T mutations were not significant risk factors for VTE, and of all haemostatic variables tested, only median activated partial thromboplastin time showed significant difference between VTE and non-VTE patients (34 s vs. 33 s). Multiple logistic regression analysis demonstrated that increased age, knee arthroplasty and individual surgeon's routine practices were the only significant independent risks for VTE; hence routine preoperative blood screening for a potential hypercoaguable state is not indicated in this surgical setting.

Multilaboratory testing of thrombophilia: current and past practice in Australasia as assessed through the Royal College of Pathologists of Australasia Quality Assurance Program for Hematology.

We have conducted a series of multilaboratory surveys during the last 6 years to evaluate testing proficiency in the detection of congenital and acquired thrombophilia. For lupus anticoagulant (LA) testing, participant laboratories used a panel of tests, including activated partial thromboplastin time (aPTT; 100% of laboratories), kaolin clotting time (26 to 70%), and Russell's viper venom time (RVVT; 75 to 100%). Coefficients of variation (CVs) for assays ranged from 5 to 40%. RVVT assays appeared to be most sensitive and specific for detection of LA (fewer false-negatives or -positives), although laboratories performed best when they used a panel of tests. For congenital thrombophilia, tests evaluated comprised protein C (PC), protein S (PS), antithrombin (AT), and activated protein C resistance (APCR). Most participant laboratories performed PC using chromogenic (approximately 75%), or clot based (approximately 15%) assays, with few (< 10%) performing antigenic assessments. PS was most often assessed (approximately 60%) by immunological or antigenic assays, usually of free PS, or by functional or clot-based assays (approximately 40%). AT is usually assessed by functional chromogenic assays (approximately 95%). APCR was assessed using aPTT (approximately 50%) or RVVT (approximately 50%) clot-based assays, with the aPTT APCR typically performed using factor V-deficient plasma predilution, but the RVVT APCR typically performed without. Laboratories using the RVVT APCR generally performed better in detection of factor V Leiden-associated APCR, with the aPTT method group yielding higher false-negative and/or false-positive findings (approximately 5% of occasions). Some clot-based PC and PS assays appeared to be influenced by APCR status, and yielded lower apparent PC and PS levels with positive APC resistance. The overall error rate for PC, PS, and AT was approximately 2 to 8% (i.e., false-normal interpretations for deficient plasma or false-abnormal interpretations for normal plasma). The CVs for these assays ranged from 5 to 40%, with highest CVs typically obtained with PS assays.

Factor V inhibitors: rare or not so uncommon? A multi-laboratory investigation.

Acquired deficiencies of, or inhibitors to, factor V are considered rare events. We report a series of 14 acquired factor V deficiencies, 10 of which were confirmed to have inhibitors to factor V, as identified within Australia in the past 5 years following a multi-laboratory investigation. The initial index case seen by one laboratory was followed within 4 months by a separate similar case. This prompted local contact with colleagues (n = 20) working in other haemostasis referral laboratories to identify the current case series. In total, nearly one-half of all haemostasis referral laboratories contacted had seen a case within the past 5 years. Clinical features and the apparent associated risk of bleeding complications generally varied, as did laboratory findings and the likely causal event. There were three females and 11 males. Age ranged from 44 to 95 years (median, 81 years). The level of inhibitor ranged from undetectable to over 250 Bethesda units. The probable cause leading to development of the inhibitors ranged from exposure to bovine thrombin, exposure to antibiotics, surgery and malignancy. Of additional interest was the apparent association of anti-phospholipid antibodies in many of the cases. For example, in the two similar index cases, with factor V inhibitor titres > 200 Bethesda units, high levels of anti-cardiolipin antibodies (> 70 GPL units) were also detected. Although less clear because of inhibitor interference, many of the cases also showed evident co-associated lupus anticoagulant activity. In conclusion, we report a series of factor V inhibitors recently identified within our geographic region that would represent an annual incidence of around 0.29 cases per million Australians. Although considered a rare finding, there is a high likelihood that most haemostasis referral laboratories will see a case every five or so years.