Oral Anticoagulant Use in Patients With Atrial Fibrillation and Chronic Kidney Disease: A Review of the Evidence With Recommendations for Australian Clinical Practice.

Chronic kidney disease is common in patients with atrial fibrillation (AF) and is associated with heightened risks of stroke/systemic embolisation and bleeding. In this review we outline the evidence for AF stroke prevention in kidney disease, identify current knowledge gaps, and give recommendations for anticoagulation at various stages of chronic kidney disease. Overall, anticoagulation is underused. Warfarin use becomes increasingly difficult with advancing kidney disease, with difficulty maintaining international normalised ratio (INR) in therapeutic range, increased risk of intracranial and fatal bleeding compared to non-vitamin K oral anticoagulants (NOACs), and high rates of discontinuation. Similarly, the direct thrombin inhibitor dabigatran is not recommended as it is predominantly renally excreted with consequent increased plasma levels and bleeding risk with advanced kidney disease. The Factor Xa inhibitors apixaban and rivaroxaban have less renal excretion (25-35%), modest increases in plasma levels with advancing kidney disease, and are the preferred first line choice for anticoagulation in moderate kidney disease based on strong evidence from randomised clinical trials (RCTs). In severe kidney disease there is a paucity of RCT data, but extrapolation of the pharmacokinetic and RCT data for moderate kidney disease, and observational studies, support the considered use of dose-adjusted Factor Xa inhibitors unless the bleeding risk is prohibitive. In Australia, apixaban is approved for creatinine clearance down to 25 mL/min, and rivaroxaban down to 15 mL/min. For end-stage kidney disease warfarin is the only agent approved, but we recommend against anticoagulation (except in selected cases) due to high bleeding risk, multiple co-morbidities, and questionable benefit.

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.

Comparison of flow cytometry with other modalities in the diagnosis of myelodysplastic syndrome.

INTRODUCTION: The myelodysplastic syndromes (MDSs) are heterogeneous myeloid malignancies, conventionally diagnosed by cytomorphology and cytogenetics, with an emerging role for flow cytometry. This study compared the performance of a 4-parameter flow cytometry scoring system, the Ogata Score, with other modalities in the diagnosis of MDS. METHODS: Bone marrow aspirate and trephine biopsies from 238 patients performed to assess for possible MDS were analysed, and the flow cytometry score was retrospectively applied. The sensitivity and specificity of the flow cytometry score, the aspirate microscopy, the trephine microscopy with immunohistochemistry, and cytogenetic and molecular results were determined relative to the final diagnosis. RESULTS: The medical records of the 238 patients were reviewed to determine the final clinical diagnosis made at the time of the bone marrow examination. This final diagnosis of MDS, possible MDS or not MDS, was based on clinical features and laboratory tests, including all parameters of the bone marrow investigation, except for the flow cytometry score, which was only determined for this study. The flow cytometry score was 67.4% sensitive and 93.8% specific. Aspirate microscopy had higher sensitivity (83.7%) and similar specificity (92.0%), whereas trephine microscopy had similar sensitivity (66.3%) and specificity (89.4%) to flow cytometry. Although the flow cytometry score had a lower sensitivity than aspirate microscopy, in 18 patients (7.6% of the total) the flow cytometry score was positive for MDS, whereas aspirate microscopy was negative or inconclusive. CONCLUSION: The flow cytometry score and trephine microscopy exhibited reasonable sensitivity and high specificity, and complement aspirate microscopy in the assessment of MDS.

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.

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.

Polychromatic flow cytometry is more sensitive than microscopy in detecting small monoclonal plasma cell populations.

BACKGROUND: There is an emerging role for flow cytometry (FC) in the assessment of small populations of plasma cells (PC). However, FC's utility has been questioned due to consistent underestimation of the percentage of PC compared to microscopy. METHODS: A retrospective study was performed on bone marrow samples analysed by 8-colour FC. Plasma cell populations were classified as polyclonal or monoclonal based on FC analysis. FC findings were compared with microscopy of aspirates, histology and immunohistochemistry of trephine biopsies, and immunofixation (IFX) of serum and/or urine. RESULTS: FC underestimated PC compared to aspirate and trephine microscopy. The 10% diagnostic cutoff for MM on aspirate microscopy corresponded to a 3.5% cutoff on FC. Abnormal plasma cell morphology by aspirate microscopy and clonality by FC correlated in 229 of 294 cases (78%). However, in 50 cases, FC demonstrated a monoclonal population but microscopy reported no abnormality. In 15 cases, abnormalities were reported by microscopy but not by FC. Clonality assessment by trephine microscopy and FC agreed in 251/280 cases (90%), but all 29 discordant cases were monoclonal by FC and not monoclonal by microscopy. These cases had fewer PC and proportionally more polyclonal PC, and when IFX detected a paraprotein, it had the same light chain as in the PC determined by FC. CONCLUSIONS: FC was more sensitive in detecting monoclonal populations that were small or accompanied by polyclonal PC. This study supports the inclusion of FC in the evaluation of PC, especially in the assessment of small populations. © 2016 International Clinical Cytometry Society.

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.

Flow cytometry demonstrates differences in platelet reactivity and microparticle formation in subjects with thrombocytopenia or thrombocytosis due to primary haematological disorders.

BACKGROUND: Traditional methods for the assessment of platelet function require a minimum number of platelets. As flow cytometry is independent of platelet number, we measured platelet activation and microparticle formation in thrombocytopenia and thrombocytosis. MATERIALS AND METHODS: Blood was obtained from normal subjects or subjects with immune thrombocytopenic purpura (ITP), myelodysplasia (MDS) or essential thrombocythaemia (ET). Platelet activation and microparticle formation were assessed in resting and agonist stimulated samples. RESULTS: Platelet activation was significantly decreased in MDS in agonist-stimulated platelets when compared to normals and ITP, however increased microparticle-to-platelet ratios were found. Absolute platelet-derived microparticle counts were significantly higher in ET when compared to normals, but there was no significant difference in microparticle-to-platelet ratios between ET and normals. CONCLUSIONS: Decreased platelet activation was demonstrated in MDS when compared to normal subjects and ITP. Platelet-derived microparticle counts are increased in ET, reflecting increased platelet counts rather than an increase in platelet reactivity. Flow cytometric analysis of platelets may aid the diagnosis and management of these conditions.

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.

Detection of the procoagulant activity of microparticle-associated phosphatidylserine using XACT.

One of the mechanisms by which platelet-derived microparticles elicit procoagulant activity is by an increased exposure of phosphatidylserine on their surface. We have previously demonstrated the utility of an activated factor X-based assay for the detection of procoagulant phospholipid activity [Xa clotting time (XACT)]. The objective of this study was to further characterize the specificity of the XACT to detect microparticle-associated procoagulant phospholipid activity. XACT testing for procoagulant phospholipid was measured using an ST4 machine and microparticle counting was performed using flow cytometry for Annexin V binding. Plasma microparticle counts were significantly correlated to XACT times (P = 0.0001). The XACT assay was insensitive to tissue factor, whereas the addition of microparticles to a whole blood sample shortened XACT times. Procoagulant phospholipid activity could be detected in both citrate and EDTA anticoagulated samples; however, XACT times and microparticle counts were more stable in EDTA anticoagulated samples over a 60 min period. The procoagulant phospholipid activity of microparticles generated by collagen stimulation was significantly impaired in EDTA anticoagulated samples when compared with citrate. Microparticles were capable of higher degrees of thrombin generation than equivalent concentrations of phosphatidylserine (as assessed by XACT times), suggesting that other factors bound to the microparticle surface enhance the procoagulant response. In conclusion, the XACT assay is a specific method for the detection of procoagulant phospholipid activity arising from phosphatidylserine on the microparticle surface; however, other factors presumably bound to the surface of the microparticle may also contribute to enhanced thrombin generation detectable by prothrombinase assays.

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.