Defects Of Primary Hemostasis Research Articles

Point-of-Care Testing in Patients with Hereditary Disorders of Primary Hemostasis: A Narrative Review.

Inherited disorders of primary hemostasis, such as von Willebrand disease and congenital platelet disorders, can cause extensive, typically mucocutaneous bleeding. Assays to diagnose and monitor these disorders, such as von Willebrand factor activity assays and light transmission aggregometry, are performed in specialized hemostasis laboratories but are commonly not available in local hospitals. Due to the complexity and relative scarcity of these conventional assays, point-of-care tests (POCT) might be an attractive alternative in patients with hereditary bleeding disorders. POCTs, such as thromboelastography, are increasingly used to assess hemostasis in patients with acquired hemostatic defects, aiding clinical decision-making in critical situations, such as during surgery or childbirth. In comparison, the use of these assays in patients with hereditary hemostasis defects remains relatively unexplored. This review aims to give an overview of point-of-care hemostasis tests in patients with hereditary disorders of primary hemostasis. A summary of the literature reporting on the performance of currently available and experimental POCTs in these disorders is given, and the potential utility of the assays in various use scenarios is discussed. Altogether, the studies included in this review reveal that several POCTs are capable of identifying and monitoring severe defects in the primary hemostasis, while a POCT that can reliably detect milder defects of primary hemostasis is currently lacking. A better understanding of the strengths and limitations of POCTs in assessing hereditary defects of primary hemostasis is needed, after which these tests may become available for clinical practice, potentially targeting a large group of patients with milder defects of primary hemostasis.

The effect of recombinant versus plasma-derived von Willebrand factor on prolonged PFA closure times in ECMO patients with acquired von Willebrand syndrome – an observational study

BackgroundExtracorporeal membrane oxygenation (ECMO) is applied in patients with respiratory or cardiopulmonary failure, but bleeding is a frequent complication contributing to the high mortality rates in this patient collective. A major factor predisposing patients to bleeding events is an acquired von Willebrand syndrome (aVWS). So far, specific treatment options for this phenomenon are lacking. In hereditary von Willebrand disease (VWD), treatment with recombinant or plasma-derived von Willebrand factor (rVWF or pVWF) is common practice. Closure time measured by the Platelet Function Analyser-200 (PFA-200) is an established assay to detect defects in primary hemostasis and the method is useful to monitor the effect of hemostatic therapy. The aim of this study was to assess the effect of recombinant (rVWF) vs. plasma-derived von Willebrand factor (pVWF) on closure times measured by PFA in blood obtained from ECMO patients with aVWS.MethodsBlood was sampled from thirteen patients receiving extracorporeal membrane oxygenation and three patients with hereditary VWD. Diagnosis of aVWS was made by conventional coagulation parameters and by multimeric structure analysis. PFA analysis of blood spiked with rVWF or pVWF was performed.ResultsThirteen patients receiving ECMO were recruited. Ten patients survived and three patients suffered major bleeding complications. PFA closure times in ECMO patients with aVWS spiked with rVWF were significantly shorter at all concentrations than with pVWF (e.g., rVWF vs. pVWF: 1 U/ml: 150.4 ± 21.7 s vs. 263.8 ± 11.7 s; 4 U/ml: 97.8 ± 9.8 s vs. 195.8 ± 15.4 s, p<0.001). PFA closure times were also significantly shorter in three patients with hereditary VWD treated with rVWF compared to pVWF (e.g., 1 U/ml rVWF vs. pVWF: 73.7±1.33 s vs. 231.3±43.4 s, p<0.01)ConclusionIn summary, this study shows that rVWF compared to pVWF more effectively reduced PFA closures times in blood samples of ECMO patients with aVWS. Higher doses of VWF are needed to normalize PFA closure time in blood samples of patients with ECMO-induced aVWS compared to hereditary VWD. These data support the use of PFA-200 to monitor hemostatic effects in a future clinical trial involving ECMO patients with aVWS.

Hemostatic profile detailing in apparent VWD cases: A cross sectional study

The von willebrand disease (vWD) accounts to be one of the most common hereditary bleeding ailment that amounts its incidence to almost 1.5% of normal population. It is mostly associated with a defect in primary hemostasis as well as secondary defect in coagulation factor VIII as diagnosis of vwd happened to be challenging with earlier diagnostic criteria’s. Testing Vwd in menorrhagia patients was not at ease. A cross-sectional study was conducted in female patients who have visited obstetrics and gynecology clinic at King Saud University Medical City (KSUMC), Riyadh, Saudi Arabia. The inclusion criteria consist of adult female patients between 16 and 45 years old with menorrhagia. A sample of 45 patients were screened and selected for the above-mentioned study. The SPSS Statistical analysis package was performed to analyze the data’s. The fisher’s exact test was conducted to compare the demographic variables. The independent samples t-test was conducted to compare the means of subjects. The P value of ≤0.05 considered as statistically significant. The cases manifested with a history of bleeding during periods stretching from 7 to 90 days. The vWD was reported in 6.6 % (n = 3) women out of the total 45 patients. The vWF: Ac mean ± SD (51.4 ± 6.3) and vWF: Ag Mean ± SD (93 ± 67) were significantly lesser in vWD patients with that of non-vWD (98.7 ± 22.6) vs (116 ± 42.4) (p = 0.027) (p = 0.032) respectively. WBC, ESR, MCV, MCH, Hemoglobin, PLT count, INR, PT, APTT and FVIII showed no significant difference among the groups (p > 0.05).

A microchip flow-chamber assay screens congenital primary hemostasis disorders.

Von Willebrand disease (VWD) and platelet function disorders (PFDs) are congenital bleeding disorders caused by primary hemostasis defects. Platelet function tests are time-consuming and require considerable amounts of blood sample, and there have been no easy-to-use assays for assessing platelet function quickly and sensitively. We report the usefulness of a microchip flow-chamber system (T-TAS® ) for detecting and/or predicting clinical severity in patients with VWD type 1 and type 2N and platelet storage pool disease. Here, we developed an application of a screening assay for primary hemostasis disorders. Microchips coated with collagen (PL-chip) and collagen/thromboplastin (AR-chip) were utilized to evaluate platelet thrombus formation (PTF) at high shear and fibrin-rich PTF at low shear, respectively, in whole blood samples from 22 patients with VWD (16 type 2A, four type 2B, two type 3) and four patients with PFDs (two BSS, two Glanzmann thrombasthenia). The time-to-increase by 10kPa (T10 ) was calculated from flow pressure curves. Also, whole blood-induced platelet aggregation was assessed using Multiplate® analysis. PL-chip T10 values ≥10min successfully distinguished patients with all types of VWD and PFDs from healthy controls, irrespective of age, bleeding scores, and von Willebrand factor levels. However, AR-chip assay incompletely distinguished between type 2A patients and healthy ones. Multiplate analysis permitted screening of PFDs and type 3 VWD, but values in type 2A partially overlapped with those in controls. PL-chip assay did not reflect the clinical severity in these patients. T-TAS with PL-chip could be a quick screening tool for congenital primary hemostasis disorder, VWD, and PFDs.

Genetic classification and confirmation of inherited platelet disorders: current status in Korea.

Inherited platelet disorders (IPDs), which manifest as primary hemostasis defects, often underlie abnormal bleeding and a family history of thrombocytopenia, bone marrow failure, hematologic malignancies, undefined mucocutaneous bleeding disorder, or congenital bony defects. Wide heterogeneity in IPD types with regard to the presence or absence of thrombocytopenia, platelet dysfunction, bone marrow failure, and dysmegakaryopoiesis is observed in patients. The individual processes involved in platelet production and hemostasis are genetically controlled; to date, mutations of more than 50 genes involved in various platelet biogenesis steps have been implicated in IPDs. Representative IPDs resulting from defects in specific pathways, such as thrombopoietin/MPL signaling; transcriptional regulation; granule formation, trafficking, and secretion; proplatelet formation; cytoskeleton regulation; and transmembrane glycoprotein signaling are reviewed, and the underlying gene mutations are discussed based on the National Center for Biotechnology Information database and Online Mendelian Inheritance in Man accession number. Further, the status and prevalence of genetically confirmed IPDs in Korea are explored based on searches of the PubMed and KoreaMed databases. IPDs are congenital bleeding disorders that can be dangerous due to unexpected bleeding and require genetic counseling for family members and descendants. Therefore, the pediatrician should be suspicious and aware of IPDs and perform the appropriate tests if the patient has unexpected bleeding. However, all IPDs are extremely rare; thus, the domestic incidences of IPDs are unclear and their diagnosis is difficult. Diagnostic confirmation or differential diagnoses of IPDs are challenging, time-consuming, and expensive, and patients are frequently misdiagnosed. Comprehensive molecular characterization and classification of these disorders should enable accurate and precise diagnosis and facilitate improved patient management.

Disease progression and defects in primary hemostasis as major cause of bleeding in multiple myeloma

In patients with multiple myeloma (MM), unexpected bleeding complications remain a major issue. Since routine coagulation parameters are often inconspicuous, diagnosis and treatment of the underlying coagulation disorders are challenging. In our single-center observational study, we analyzed 164 patients with MM for coagulation disorders and bleeding complications. Prolonged closure times (CTs), measured by PFA-100, were the most common, abnormal coagulation test, found in 66% of bleeding patients vs 5% in non-bleeding, followed by qualitative defects of von Willebrand factor (VWF:CB/VWF:Ag ratios), found in 34% vs 1% in the non-bleeding group. Increased serum free light chains (SFLC) and SFLC ratios were significantly associated with prolonged CTs and acquired von Willebrand syndrome (AVWS). Prolonged CTs and AVWS were associated with disease progression, determined by dynamics of SFLC ratios (P<.001), serum creatinine level (P=.013), Beta-2 microglobulin (P=.03), LDH (P=.016), and bone marrow infiltration (P<.001). Of note, response to myeloma therapy was frequently correlated with normalization of coagulation parameters. Bleeding complications in MM are predominantly caused by defects in primary hemostasis and associated with disease progression. In a peri-interventional workup, determination of CTs and VWF:CB/VWF:Ag ratios are of significant importance to assess bleeding risk.

Animal Models Demonstrate a Critical Role of Factor VIII in Par4- and Platelet-Mediated Pathology

Recent studies suggest that Tissue factor (TF)-initiated pathway can directly and rapidly yield FVIIIa-FIXa intrinsic tenase complex prior to coagulation amplification by thrombin (PMID 28729433). FVIII may also mediate TF/FVIIa-dependent FXa generation in the initiation phase of coagulation (PMID 30756344). These new insights prompted us to evaluate whether FVIII plays an important role in generating the minimum level of thrombin required for platelet activation in vivo. We used two distinct murine models of platelet- and Par4-mediated pathology to address this question. We have previously shown that mice homozygous for a hypomorphic thrombomodulin allele (ThbdPro/Pro) are aborted in pregnancies of FV Leiden (FVQ/Q) females before day 12.5 of gestation (Table 1, row 1; PMID 23325830). Par4-activation on maternal platelets plays a key role in fetal death in this model; its absence allows embryonic development and restores Mendelian frequency of live ThbdPro/Pro embryos found at term (Table 1, row 2; PMID 17438064). We examined the effect of reduced TF or genetic deletion of FVIII on embryonic survival of ThbdPro/Pro embryos. We demonstrate that inhibition of TF or the absence of FVIII, each significantly protect the fetus from maternal platelet-mediated death (Table 1, rows 3 &amp; 4). Treatment of the mother with anti-TF antibody 1H1 resulted in 11 ThbdPro/Pro embryos (30 analyzed, 50% or 15 expected; 37% observed, 95% CI 20 to 56%). In the absence of FVIII, 10 ThbdPro/Pro embryos were observed (30 analyzed, 50% or 15 expected; 37% observed, 95% CI 20 to 56%). The outcome of TF inhibition was comparable to the absence of FVIII (P=0.79 Χ2 test of independence). In a second model, Endothelial Protein C Receptor null mice (ProcR-/-) die in midgestation (Table 2, row 1), but reducing TF expression restores Mendelian frequency of ProcR-/- pups (PMID 15956290). We find that ProcR-/- embryos survive past midgestation if the mother is injected with platelet depleting antibodies. Genetic absence of Par4 allows ProcR-/- mice to survive embryonic development (Table 2, row 2; 25% or 18 ProcR-/- neonates expected, 13 observed, 95% CI 10.3 to 29.7%). We investigated the role of FVIII in the midgestational demise of ProcR-/- mice. We demonstrate that genetic absence of FVIII also protects ProcR-/- mice from intrauterine demise (Table 2, row 3; 25% or 12 out of 48 ProcR-/- expected, 12 observed, 95% CI 13.6 to 29.6%). Similar to TF reduction, the absence of FVIII restores Mendelian frequency of ProcR-/- embryos at term. Thus, the outcome from the genetic absence of FVIII was comparable to the absence of Par4 (P=0.4 Χ2 test of independence). In a third model, we have established that epiblast restricted deletion results in normal Mendelian frequency of thrombomodulin-deficient embryos (Meox2Cretg Thbdδ/δ) at term, but about 1/3rd survive the trauma of birth. Most surviving mice later succumb to severe tail and limb thrombosis and several exhibit circling behavior indicative of stroke. Genetic absence of Par4 does not improve perinatal survival, but protects thrombomodulin-deficient mice from stroke and thrombotic disease, significantly improving adult survival. We tested whether genetic absence of FVIII affords protection similar to the absence of Par4. We report that similar to Par4-/- Meox2Cretg Thbdδ/δ mice, FVIII-/- Meox2Cretg Thbdδ/δ mice do not exhibit thrombosis or stroke (current age range 2 to 8 months). Thus, the absence of FVIII is protective in Par4-mediated thrombosis and stroke observed in thrombomodulin-deficient mice. These studies establish an essential role of FVIII in Par4-mediated pathologies. They strongly support the notion that FVIII activation is required for sufficient accumulation of thrombin for platelet activation. Notably, these are models of physiological thrombosis or platelet-mediated fetal death, without the use of artificial injury or insult. All three models that we describe lack a fully functional system for generating activated protein C (aPC). Our results suggest that inactivation of FVIII by aPC may be necessary to tightly regulate thrombin-mediated platelet activation. Reduced platelet activation observed in hemophilia A mice, albeit in the context of prevention of thrombosis, also lends support to the notion that defects in primary hemostasis due to reduced platelet activity could be an important component of bleeding diathesis in hemophilia A patients. Disclosures No relevant conflicts of interest to declare.

An update on quality control for the PFA-100/PFA-200

Testing of platelet function comprises a crucial element of hemostasis assessment, particularly for investigations into bleeding and/or bruising. The Platelet Function Analyzer (PFA)-100 is the most utilized primary hemostasis-screening test system available, as recently remodeled/upgraded to the PFA-200. Internal quality control (IQC) and external quality assessment (EQA) (including proficiency testing) represent critical elements of ensuring test practice quality. Although true for all tests, IQC and EQA are logistically challenging for platelet function testing, inclusive of the PFA-100/200. We accordingly update our experience with novel yet feasible approaches to both IQC and EQA of PFA-100/200. Over the past 10 years, a total of 43 challenges have been tested, with most challenges designed to mimic moderate or severe primary hemostasis defects. The current report is restricted to the last four years and has also differentially assessed PFA-100 vs. PFA-200 EQA results to identify potential variance. Numerical results for closure times (CTs) and participant-supplied interpretive comments were analyzed. Reported CTs for each challenge were within limits of expectation, and good reproducibility was evidenced by repeated challenges. Coefficients of variation (CVs) for challenges, generally ranging from 15% to 25%, were similar or better than those obtained using native whole blood and consistent with past reports. Participant interpretations were generally consistent with test data and expectations. There was no evident difference in PFA-100 vs. PFA-200 EQA test results. The EQA material has also been successfully evaluated from the perspective of potential IQC. To conclude, IQC and EQA processes for the PFA-100/200 have been established that are highly reproducible, supporting the concept of EQA/IQC for platelet function testing, and also facilitating monitoring and improvement in its performance. In terms of EQA, PFA-100 and PFA-200 instruments appear to behave similarly.

Usefulness of multiple electrode aggregometry as a screening tool for bleeding disorders in a pediatric hospital

Platelet function testing is a cornerstone in the diagnostic investigation of patients with a bleeding history. Multiple electrode aggregometry (MEA) has been shown to detect von Willebrand disease (VWD), platelet function disorders, and drug-induced bleeding disorders. However, there are few studies supporting its successful use in children. We have implemented and used MEA over 3 years in our hemostasis laboratory in order to study its usefulness to supplement and expedite diagnosis. This is a retrospective, single-center, cohort study of 109 hospitalized children who underwent a laboratory investigation of hemostasis and either had a reported bleeding history or an abnormal bleeding episode. Plasmatic coagulation testing, blood counts, plasmatic von Willebrand testing, platelet function analyzer (PFA-100), and impedance aggregometry (MEA) were performed in all children. Light transmission aggregometry testing was performed as needed. In 41 cases (37.6%), a working diagnosis was made; a primary hemostatic disorder was detected in 35 children (VWD (n = 16), platelet disorder (n = 15), and valproic acid therapy-induced bleeding disorder (n = 3), acetylsalicylic acid-related bleeding (n = 1). In patients diagnosed with VWD, MEA ristocetin-induced platelet aggregation test (RISTO) high test revealed abnormally low aggregation in six patients (43.8%); whereas in patients diagnosed with a platelet function disorder, abnormally low values were found by MEA in only three children (20%). Three of the four children with laboratory evidence of drug-induced platelet dysfunction had abnormalities on MEA. There were no cases in which an abnormal MEA result was used to make a previously undetermined diagnosis. Retrospectively, MEA has demonstrated limited additional diagnostic value beyond standard laboratory testing for detecting defects of primary hemostasis in children.

NADPH Oxidase-2 and Atherothrombosis: Insight From Chronic Granulomatous Disease.

The phagocytic cell enzyme NADPH oxidase-2 (Nox2) is critical for killing micro-organisms via production of reactive oxygen species and thus is a key element of the innate immune system. Nox2 is also detectable in endothelial cells and platelets where it has vasoconstrictive and aggregating properties, respectively. Patients with X-linked chronic granulomatous disease with hereditary Nox2 deficiency not only have impaired bacterial killing but, in association with loss of Nox2 function, also have enhanced carotid artery dilation, impaired platelet-related thrombosis, and reduced carotid atherosclerotic burden. Experimental studies corroborated these reports in chronic granulomatous disease by demonstrating (1) Nox2 is upregulated in atherosclerotic plaque, and this upregulation significantly correlates with oxidative stress and (2) pharmacological inhibition of Nox2 is associated with a delayed atherosclerotic progression in animal models. Furthermore, the role of Nox2 in platelet-associated thrombosis was substantiated by experiments showing impaired platelet activation in animals treated with a Nox2 inhibitor or impaired platelet aggregation along with reduced platelet-related thrombosis in the mouse knockout model of Nox2. Interestingly, in chronic granulomatous disease patients and in the mouse knockout model of Nox2, no defects of primary hemostasis were detected. This review analyses experimental and clinical data suggesting Nox2 is a potential target for counteracting the atherothrombotic process.

Clinical audit on the management of acquired platelet disorders in Assiut University Children Hospital

IntroductionPlatelet disorders lead to defects in primary hemostasis and produce signs and symptoms different from coagulation factor deficiencies (disorders of secondary hemostasis). Primary hemostatic disorders are characterized by prolonged bleeding time and the characteristic physical examination in the form of mucocutaneous bleeding. Initial laboratory evaluations for patients with acquired platelet disorders include complete blood count and peripheral blood smear. Treatment of patients with suspected acquired platelet disorder is generally specific to the underlying disorder.Patients and methodsData of children older than 1 month of age admitted with bleeding tendency caused by acquired platelet disorders during 1 year were collected and analyzed, and clinical management was compared with the standard management guidelines according to the American Society of Hematology 2011.ResultsIn all patients, complete blood count was performed, whereas peripheral blood smear was performed only for 46 (60.5%) cases. Bone marrow examination was performed in 30 (39.5%) cases and it was indicated in 23 (77%) of these cases, whereas in seven (23%) cases it was done for purpose of research with guardian consent. Medical treatment (in the form of intravenous immunoglobulin and/or corticosteroids) was provided to seven (9%) cases in whom treatment was not indicated. Intravenous immunoglobulin was administered to 22 (29%) cases, and there was a delay in its administration to 10 (45%) of these cases because of unavailability. Corticosteroids were administered to 26 (34%) cases; eight (31%) of these cases received it for a longer time than indicated. Anti-D immunoglobulin was not administered to any patient.

Defective Platelet Activation and Bleeding Complications upon Cholestasis in Mice

Background/Aims: Platelets are essential mediators of hemostasis to avoid excessive blood loss. Cirrhosis and chronic liver diseases are characterized by alterations in hemostasis. Alterations in the secondary hemostasis have been well studied, while defects in primary hemostasis, especially the consequences of cholestatic liver disease on platelet function are not well defined. Methods: After bile duct ligation (BDL) platelet activation and thrombus formation were analyzed in mice. Results: BDL in mice had a moderate effect on platelet counts; however, intrinsic platelet activation was strongly reduced upon activation of the collagen receptor GPVI at early time points. 7 days after bile duct ligation, platelets displayed an almost complete loss of activation with reduced agonist-triggered release of alpha and dense granules and expression of integrin α_IIbβ₃ on the platelet surface. This activation defects resulted in strongly reduced thrombus formation under flow, reduced platelet adhesion to fibrinogen and bleeding complications in BDL mice as measured by tail bleeding experiments. Mechanistically, elevated nitric oxide and prostacyclin levels induced phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), an established inhibitor of platelet activation. Furthermore increased tissue plasminogen activator in plasma of BDL mice led to enhanced plasmin levels that might be responsible for reduced glycoprotein expression of BDL platelets. Besides, high amounts of bile acids contribute to defective signal transduction as shown in platelets from mice fed with a cholic acid diet. Conclusions: Cholestatic liver disease induces multiple platelet activation defects and impairs thrombus formation responsible for bleeding complications at least in mice.

All too common: bleeding and genetic variation

Inherited bleeding disorders are both common and challenging to diagnose, particularly when the disorder is mild (reviewed in references [1, 2]). Most mild bleeding disorder patients are not routinely bleeding in their daily lives, and it can be difficult to differentiate bleeding events common in healthy populations from events which rise to the level of pathology. Fundamental to the diagnosis of bleeding disorders is a detailed assessment of the history of the patient and the family. This article is protected by copyright. All rights reserved.

Prevalence of acquired von Willebrand syndrome during essential thrombocythemia: a retrospective analysis of 170 consecutive patients.

The identification of patients with essential thrombocythemia (ET) who are at increased risk of acquired von Willebrand syndrome (AVWS) would likely facilitate individualization of treatment and improve its outcomes. The aim of the study was to determine the prevalence of AVWS in patients with ET and to verify whether individuals with and without this bleeding disorder differ in terms of their baseline clinical parameters. The study included 170 consecutive patients with ET. AVWS was diagnosed on the basis of reduced levels of von Willebrand factor and abnormal results of other routine tests. Patients with and without concomitant AVWS were compared in terms of their demographic characteristics, past and current medical histories, and laboratory parameters. Concomitant AVWS was found in 34 patients (20%). Individuals with AVWS were diagnosed with ET at a significantly younger age than those without the syndrome. In addition, these patients significantly less often were in remission at the time of testing, had significantly higher erythrocyte and platelet counts, and showed abnormalities of the coagulation profile corresponding to defects of primary hemostasis as well as abnormal values of most parameters used i n the routine diagnosis of AVWS. Even every fifth patient with ET may develop AVWS. Young age at diagnosis of ET and the lack of response to its previous treatment are potential risk factors for AVWS that should be considered during the management of the primary condition. All patients with ET and signs of a bleeding disorder, irrespective of the platelet count, should be tested for the presence of AVWS.

Coagulopathy in liver disease: a balancing act.

Liver disease results in complex alterations of all 3 phases of hemostasis. It is now recognized that hemostasis is rebalanced in chronic liver disease. The fall in clotting factor levels is accompanied by a parallel fall in anticoagulant proteins. High von Willebrand factor levels counteract defects in primary hemostasis. Conventional coagulation tests do not fully reflect the derangement in hemostasis and do not accurately predict the risk of bleeding. Global coagulation assays (thrombin generation, thromboelastography) reflect the interaction between procoagulant factors, anticoagulant factors, platelets, and the fibrinolytic system and show promise for assessing bleeding risk and guiding therapy. These assays are not yet commercially approved or validated. Prevention of bleeding should not be aimed at correcting conventional coagulation tests. Thrombopoietin receptor agonists were shown to increase the platelet count in cirrhotic patients undergoing invasive procedures but may increase the risk of thrombosis. Rebalanced hemostasis in liver disease is precarious and may be tipped toward hemorrhage or thrombosis depending on coexisting circumstantial risk factors. Bacterial infection may impair hemostasis in cirrhosis by triggering the release of endogenous heparinoids. There are no evidence-based guidelines for hemostatic therapy of acute hemorrhage in liver disease. There is currently inadequate evidence to support the use of recombinant FVIIa, prothrombin complex concentrates, or tranexamic acid in acute variceal or other hemorrhage.

Defects in Primary Hemostasis and Acquired Von Willebrand Disease As Cause of Bleeding in Patients with Multiple Myeloma

Introduction. Paraproteinemia has been associated with both thromboembolic and bleeding complications. Previous retrospective analyses and case reports report on a direct correlation of serum immunoglobulin levels (especially IgM and IgA) with critical bleeding events. Underlying mechanisms include interference with platelet function, immune thrombocytopenia, clotting factor deficiencies, and acquired von Willebrand disease (AVWD).Methods. Laboratory data, including qualitative and quantitative analyses of von Willebrand Factor (vWF), collagen binding assay (CBA) and PFA-100 closure time from 132 patients with diagnosis of multiple myeloma between 2003 and 2015 were retrieved, and cases were retrospectively evaluated for bleeding complications. Coagulation abnormalities were correlated with clinical history and disease parameters.Results. 70 out of 132 patients (53%) displayed bleeding complications including multiple hematomas (n=41), epistaxis (n=10), or perioperative bleeding (n=9). Bleeding vs. non-bleeding patients showed no differences regarding age, gender, previous chemotherapy, or immunoglobulin isotypes. Special coagulation lab results of patients with bleeding events are shown in table 1.Table 1Pathological coag labs in 70 patients with bleedingNormalAbnormalSensitivity(95% CI)Specificity(95% CI)PPV*(95% CI)NPV*(95% CI)PLT (<50x103/µl)7000100.47 (0.38-0.56)PT, aPTT6820.06 (0.02-0.1)0.95 (0.85-0.99)0.57 (0.2-0.88)0.47 (0.38-0.56)vWF:Ag or RCoF6460.09 (0.04-0.18)0.95 (0.86-0.99)0.67 (0.3-0.9)0.48 (0.39-0.9)PFA-10027430.61 (0.49-0.73)0.97 (0.88-0.99)0.96 (0.84-99)0.69 (0.58-0.78)CBA/vWF45250.35 (0.25-0.48)0.98 (0.9-0.99)0.96 (0.78-1)0.58 (0.48-0.67)PFA-100 or CBA/vWF52180.74 (0.62-0.84)0.95 (0.86-0.99)0.94 (0.84-1)0.76 (0.65-0.85)*PPV = positive predictive value, NPV = negative predictive valueSerum free light-chain, but not complete Ig paraprotein levels were significantly associated with bleeding events (p<0.001), prolonged PFA-100 closure time (p<0.001), and pathological CBA/vWF-ratio (p<0.05).Conclusions. Bleeding events were most commonly due to defects of primary hemostasis. Global coagulation tests as well as vWF:Ag and RCo were insufficient in predicting bleeding. When the results of both PFA-100 and CBA/vWF-ratio were considered, 74% of the bleeding patients could be detected. We suggest that both PFA-100 and CBA/vWF-ratio should be determined in patients with multiple myeloma and signs of bleeding in order to rule out AVWD with sufficient sensitivity. Serum free light-chain levels were correlated with hemorrhage and detectable AVWD. Therefore, free Ig light-chains may play a role in the pathophysiology of bleeding. DisclosuresWeisel:Janssen Pharmaceuticals: Consultancy, Honoraria, Other: Travel Support, Research Funding; Noxxon: Consultancy; Onyx: Consultancy, Honoraria; Novartis: Other: Travel Support; Celgene: Consultancy, Honoraria, Other: Travel Support, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel Support; BMS: Consultancy, Honoraria, Other: Travel Support.

Factors predisposing to acquired von Willebrand syndrome during the course of polycythemia vera – retrospective analysis of 142 consecutive cases

IntroductionThe aim of this study was to verify if PV patients with and without avWS differ in terms of their baseline clinical parameters. Material and MethodsThe study included 142 consecutive patients with PV. avWS was diagnosed on the basis of abnormally low levels of von Willebrand factor and other routine tests. Patients with and without concomitant avWS were compared in terms of their demographic characteristics, present and past medical histories and laboratory parameters. ResultsConcomitant avWS was found in 17 PV patients (12.0%). Individuals with avWS have been diagnosed with PV at significantly younger age than those without, and significantly less often were in remission at the time of testing for bleeding disorders. Most of them (58.8%) presented with typical signs of bleeding disorder. Moreover, they showed significantly higher erythrocyte, leukocyte and platelet counts, abnormalities of coagulation profile corresponding to defects of primary hemostasis and abnormal values of all parameters used in the routine diagnosis of avWS. ConclusionsEven every tenth patient with PV may develop avWS. Young age at diagnosis of PV and poor response to previous treatment of this condition are potential risk factors of avWS that should be considered during history taking. Sings of bleeding disorder observed in a person with PV necessitate evaluation for avWS.

Laboratory testing for platelet function disorders.

Platelet function testing is both complex and labor intensive. A stepwise approach to the evaluation of patients with suspected platelet disorders will optimize the use of laboratory resources, beginning with an appropriate clinical evaluation to determine whether the bleeding is consistent with a defect of primary hemostasis. Bleeding assessment tools, evaluation of platelet counts, and review of peripheral blood cell morphology can aid the initial assessment. For patients requiring further laboratory testing, platelet aggregometry, secretion assays, and von Willebrand factor assays are the most useful next steps and will direct further specialized testing including flow cytometry, electron microscopy, and molecular diagnostics. Guidelines and recommendations for standardizing platelet function testing, with a particular focus on light transmission aggregometry, are available and can provide a template for clinical laboratories in establishing procedures that will optimize diagnosis and assure quality results. This review outlines an approach to platelet function testing and reviews testing methods available to clinical laboratories.

External Quality Assessment/Proficiency Testing and Internal Quality Control for the PFA-100 and PFA-200: An Update

Platelet function testing is an essential component of comprehensive hemostasis evaluation within the framework of bleeding and/or bruising investigations, and it may also be performed to evaluate antiplatelet medication effects. Globally, the platelet function analyzer (PFA)-100 (Siemens Healthcare, Marburg, Germany) is the most used primary hemostasis-screening instrument and has also been recently remodeled/upgraded to the PFA-200. The PFA-100 is sensitive to a wide range of associated disorders, including platelet function defects and von Willebrand disease (VWD), as well as to various antiplatelet medications. The PFA-100 is also useful in therapy monitoring, especially in VWD. External quality assessment (EQA) (or proficiency testing) and internal quality control (IQC) are critical to ensuring quality of test practice, inclusive of all hemostasis tests. However, both EQA and IQC for platelet function testing, including the PFA-100, is logistically challenging, given theoretical requirements for production, storage, and shipment of large volumes of "stabilized" normal and pathological blood/platelets covering both normal function plus a wide variety of potential defects. We accordingly describe the development and testing of novel feasible approaches to both EQA and IQC of PFA-100/PFA-200 instruments, whereby a range of formulated "platelet function antagonist" materials are utilized. For EQA purposes, these are distributed to participants, and citrated normal whole blood collected on site is then added locally, thereby creating test material that can be locally evaluated. Several exercises have been conducted by the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) over the past 6 years. A total of 26 challenges, with most designed to mimic moderate to severe primary hemostasis defects, have been tested in 26 to 50 laboratories depending on the year of dispatch. Numerical results for PFA-100/PFA-200 closure times (CTs) and interpretive comments supplied by participants are analyzed by the RCPAQAP. During this period, reported CTs for each challenge were within limits of expectation and good reproducibility was evidenced by repeated challenges. Coefficients of variation (CVs) generated for challenges using the two major PFA-100/PFA-200 cartridge types (collagen/adenosine diphosphate and collagen/epinephrine) are always similar to those obtained using native whole blood, and in general range from 15 to 25%. Interpretations are also in general consistent with expectations and test data provided by laboratories. The EQA created material has also been assessed within the context of possible IQC material. In conclusion, EQA and IQC processes for the PFA-100/PFA-200 have been developed that include highly reproducible test challenge processes, not only supporting the concept that EQA/IQC is possible for platelet function testing but also providing a valuable mechanism for monitoring and improving laboratory performance in this area.

Concurrent Rectus Sheath Hematoma and Iliopsoas Hematoma in a Cirrhotic Patient

Spontaneous muscle hematomas are rare in patients with cirrhosis and are associated with a high mortality rate. Hemostatic imbalances associated with cirrhosis are very complex and include primary and secondary hemostasis defects. Here we report a case of bilateral rectus sheath hematoma (RSH) with unilateral iliopsoas hematoma in a cirrhotic patient and review the literatures reported cases.