Platelet Function Analyzer Research Articles

Point of Care Management of Perioperative Coagulopathy Reduces Blood Loss and Consumption of Blood Transfusion Products (Preliminary Results)

Purpose Lung transplantation is very often accompanied with significant bleeding and coagulopathy. Standard perioperative management consists of administering fresh frozen plasma (FFP) and red blood cells. These transfusion products are generally administered according to anesthesiologist's decision based on his personal experience. However, point of care (POC) monitoring of coagulopathy is available nowadays such as thrombolestometry (ROTEM), platelet function analyzer (PFA 200) and agregometry (Multiplate) to conduct administering of blood products or coagulation factors. The purpose of this study is to find out if POC management of bleeding and coagulopathy can reduce perioperative blood loss and consumption of blood products compared to standard approach. Methods Eighteen patients undergoing bilateral lung transplantation were randomized into 2 groups (from January 2018). In the first group (9 patients), management of bleeding and coagulopathy was done by anesthesiologist based on his clinical experience - standard approach, in the second group (9 patients), management of bleeding and coagulopathy was based on results of POC methods such as ROTEM, PFA 200, Multiplate. Results In POC group we found significantly reduced consumption of FFP and red blood cells during surgery and 24 hours after surgery. Also, perioperative blood loss was decreased in POC group comparing to standard approach group. Results are summarized in Table. Conclusion Management of bleeding and coagulopathy based on results of POC methods such as ROTEM, PFA 200 and Multiplate significantly reduces consumption of blood products and decreases perioperative blood loss, despite a small number of patients. Lung transplantation is very often accompanied with significant bleeding and coagulopathy. Standard perioperative management consists of administering fresh frozen plasma (FFP) and red blood cells. These transfusion products are generally administered according to anesthesiologist's decision based on his personal experience. However, point of care (POC) monitoring of coagulopathy is available nowadays such as thrombolestometry (ROTEM), platelet function analyzer (PFA 200) and agregometry (Multiplate) to conduct administering of blood products or coagulation factors. The purpose of this study is to find out if POC management of bleeding and coagulopathy can reduce perioperative blood loss and consumption of blood products compared to standard approach. Eighteen patients undergoing bilateral lung transplantation were randomized into 2 groups (from January 2018). In the first group (9 patients), management of bleeding and coagulopathy was done by anesthesiologist based on his clinical experience - standard approach, in the second group (9 patients), management of bleeding and coagulopathy was based on results of POC methods such as ROTEM, PFA 200, Multiplate. In POC group we found significantly reduced consumption of FFP and red blood cells during surgery and 24 hours after surgery. Also, perioperative blood loss was decreased in POC group comparing to standard approach group. Results are summarized in Table. Management of bleeding and coagulopathy based on results of POC methods such as ROTEM, PFA 200 and Multiplate significantly reduces consumption of blood products and decreases perioperative blood loss, despite a small number of patients.

Point of Care Management of Perioperative Bleeding or Coagulopathy and Volume Replacement Strategy with Albumin 5% Seems to Reduce Primary Graft Disfunction after Lung Transplantation (Preliminary Results)

Purpose Primary Graft Dysfunction (PGD) is the most common complication during the first 72 hours after lung transplant. Amount of perioperative blood loss and blood product administration may be some of the causes. Intravascular volume during surgery and in ICU is usually maintained by the administration of crystalline and colloids solutions and by administration of fresh frozen plasma (FFP) due to coagulopathy. In the field of the allogenic ischemic organ FFP essentially becomes another allogenic material and can cause undesired immunomodulation and contribute to the development of PGD. Nowadays point of care (POC) management of coagulopathy and bleeding is available using methods such as thromboelastometry (ROTEM), platelet function analyzer (PFA 200) and agregometer (Multiplate) which contribute to the optimalization of blood products and coagulation factors administration. The purpose of this study is to find out if POC management of perioperative bleeding and coagulopathy can reduce postoperative PGD development and length of artificial ventilation. Methods 18 patients undergoing bilateral lung transplantation were randomized into 2 groups. In the first group (9 subj) the management of perioperative bleeding or coagulopathy and volume replacement strategy were based on clinical experience of the anesthesiologist - standard approach. In the second group (9 subj) the management of perioperative bleeding and coagulopathy was based on the results of POC methods. Albumin 5% solution was preferably used for volume replacement therapy. Results In the POC management group PGD development, length of artificial ventilation and langht of stay was significantly reduced. Conclusion POC management of perioperative bleeding or coagulopathy and volume replacement strategy with Albumin 5% significantly reduce PGD development and decrease length of stay and artificial ventilation.

Hemostatic properties of cold-stored whole blood leukoreduced using a platelet-sparing versus a non-platelet-sparing filter.

Whole blood (WB) is an appealing alternative to component-based transfusion in patients with significant bleeding. Historically, WB was transfused less than 48 hours after collection and was not leukoreduced (LR). However, LR components are now standard in many hospitals and LR WB is desirable. We investigated the effect of the type of LR filter used, as well as storage duration, on coagulation laboratory testing of WB. Ten units of LR WB-5 units manufactured with a Food and Drug Administration (FDA)-approved platelet (PLT)-sparing filter (WB-PS) and 5 units manufactured with an FDA-approved non-PLT-sparing filter (WB-NPS)-underwent complete blood count, PLT function analyzer (PFA [PFA-100]), thromboelastography (TEG), prothrombin time (PT), partial thromboplastin time (PTT), Factor (F)V activity, chromogenic FVIII, thrombin generation, and microparticle quantification on Storage Days 3, 5, 7, 10, and 14. WB-PS contains more PLTs than WB-NPS (mean, 71 × 109 /L vs. 1 × 109 /L, p < 0.001). WB-PS yielded essentially normal TEG tracings, while TEG tracings of WB-NPS were grossly abnormal (mean reaction time, 7.0 min for WB-PS vs. 9.7 min for WB-NPS, p < 0.001; mean alpha-angle 54.9° vs. 38.1°, p < 0.001; mean maximum amplitude, 54.9 mm vs. 13.9 mm, p < 0.001). PFA-100 closure was more common among units of WB-PS compared to units of WB-NPS (72% vs. 4%, p < 0.001). PT, PTT, and factor activities were not dramatically affected by the LR filter. The choice LR filter has a major impact on the hemostatic properties of WB. Although storage of WB is associated with a rapid decline in PLT count, hemostasis as assessed by TEG and PFA-100 is not diminished over a 2-week storage period.

Platelet dysfunction after Out of Hospital Cardiac Arrest. Results from POHCAR: A prospective observational, cohort study

AimCoagulation and platelet function following out of hospital cardiac arrest (OHCA) at admission to a UK cardiology centre were investigated prospectively in this observational feasibility study, and compared to that of patients receiving percutaneous coronary intervention (PCI) for ST segment elevation myocardial infarction (STEMI). MethodBlood samples taken immediately at emergency department admission from patients after OHCA of probable cardiac origin were analysed using near-patient thromboelastometry and a platelet function analyser. Physiological parameters, demographic information, bleeding rates and 30-day survival were recorded, and compared to that of patients undergoing PCI for STEMI. ResultsThirty patients were enrolled into each group. Platelet activation with thrombin receptor stimulation was reduced in OHCA patients compared to STEMI patients; mean TRAP AUC OHCA 79.3 (95% CI 63.7–94.9) vs STEMI 101.6 (95% CI 87.4–115.8), p = 0.03. The maximum clot firmness time was prolonged in the OHCA group compared to the STEMI group; 1718s (1545s–1906s) vs 1544s (1387s–1709s), p = 0.01. Other measures of clot formation and strength were comparable between groups. Hyperfibrinolysis (maximum lysis > = 15%) was common in both groups (57% in STEMI; 50% in OHCA) but did not increase 30-day bleeding risk. ConclusionOHCA patients demonstrated reduced thrombin receptor function at hospital admission but overall clot formation dynamics comparable to STEMI patients, indicating no gross coagulopathy post OHCA in our cohort. Hyperfibrinolysis was common both post OHCA and after STEMI. The results of this small feasibility study cannot draw clinical conclusions but will inform power calculations for future studies.

Association Between Prolonged Closure Time on the Platelet Function Analyzer-200 and Risk of Perioperative Blood Transfusion.

Association Between Prolonged Closure Time on the Platelet Function Analyzer-200 and Risk of Perioperative Blood Transfusion.

Novel approaches to quality control and external quality assessment for platelet function testing with a focus on the platelet function analyser (PFA-100 and PFA-200)

Platelet function testing is a key diagnostic activity within hematology laboratories associated with advanced hemostasis diagnostics. Platelets represent a key component of primary hemostasis, and deficiency and/or defects, either congenital or acquired, leads to bleeding diathesis in affected individuals. There are various levels of platelet function tests, from simple screens, to complex functional assays, as well as molecular analysis. Platelet function testing has evolved to now incorporate a variety of processes, such as whole blood aggregometry, light transmission aggregometry (LTA), testing by platelet function analyser (PFA) -100 (or -200), flow cytometry, and many other methodologies. Despite some of these tests being available now for decades, and/or continuously evolving, internal quality control (IQC) and external quality assessment (EQA) is limited and made difficult by the nature of the tests and test material, the latter typically representing functional cellular material (i.e., platelets). The current review looks at platelet function testing from the perspective of diagnostic screening, and highlights current limitations, as well as potential solutions that will enable more effective and accurate testing in the future. The primary focus of the review, however, is on IQC and EQA for the platelet function analyser (PFA-100 and PFA-200).

Individualized Antithrombotic Therapy in Heartware HVAD Recipients.

There are no evidence-based guidelines for antithrombotic therapy in left ventricular assist device patients. We evaluated the efficacy of a multi-agent, test-guided protocol, which remained consistent throughout our use of the Heartware HVAD. Thrombelastography and platelet function analyzer (PFA)-100 guide antiplatelet titration. Goals are normal kaolin thrombelastography maximum amplitude and prolonged PFA-100. We analyzed incidence and rates of nonprocedural bleeding, stroke, and pump thrombosis for all 81 primary Heartware left ventricular assist devices implanted since 2011. For each event, we examined whether each test was within goal range. We also compared medication burden and dosage between patients with and without events. Pump thrombosis and hemorrhagic stroke each occurred in two patients (2.5%; 0.03 events/patient-year [EPPY]), and ischemic stroke in six (7.4%; 0.10 EPPY). Nonprocedural bleeding occurred 75 times in 34 patients (42%; 1.24 EPPY), primarily gastrointestinal (28%; 0.66 EPPY). Subtherapeutic international normalized ratio (INR) was the most common at thrombotic events; supratherapeutic partial thromboplastin time, international normalized ratio (INR), and PFA-adenosine diphosphate (ADP) were common at bleeding events. Medication burden and dosage were lower in patients with events than in stable patients. Protocol-guided antithrombotic therapy resulted in low rates of pump thrombosis and hemorrhagic stroke. Ischemic stroke and gastrointestinal bleeding rates were higher than in a comparable HVAD population. Optimization of our protocol to decrease its complexity and to reduce bleeding is underway.

High Residual Platelet Reactivity during Aspirin Therapy in Patients with Non-St Segment Elevation Acute Coronary Syndrome: Comparison Between Initial and Late Phases.

BackgroundHigh platelet reactivity (HPR) during therapy with acetylsalicylic acid (ASA) is a poor prognostic factor in acute coronary syndromes (ACS). The prevalence of HPR during ACS is greater than that reported in stable diseases. However, it is unclear whether this prevalence of HPR is a transient phenomenon or a characteristic of this high-risk population.ObjectiveThe main objective is to compare the effects of ASA on platelet function in the initial and late phases of ACS in a single population. Secondary objectives are: correlation between the tests between themselves and the relationship between the tests and the variation of the inflammatory markers (C-reactive protein and interleukin-6).MethodsSeventy patients with non-ST segment elevation (NSTE) ACS in use of 100-200 mg of ASA per day for at least 7 days were prospectively studied. Platelet function was assessed in the first 48 hours and subsequently after 3 months using four methods: VerifyNow™ (VFN), whole blood platelet aggregation (WBPA) with arachidonic acid (AA) and collagen as agonists, and platelet function analyzer (PFA). The level of statistical significance considered was < 0.05.ResultsAccording to the more specific methods (WBPA with AA and VFN), the incidence of HPR was significantly higher in the early phase than in the late phase: WBPA with AA: 31% versus 13%, p = 0.015; VFN: 32% versus 16%, p = 0.049. The other methods tested, which were less specific for ASA, did not show significant differences between phases. The correlation between the methods was weak or moderate (r ranging from 0.3 to 0.5, p < 0.05), and there were no significant associations between HPR and inflammatory markers.ConclusionThe prevalence of HPR during AAS therapy, assessed by specific methods for cyclooxygenase 1 (COX-1), is higher during the acute phase than in the late phase of NSTE ACS.

Superiority of the Rapid Von Willebrand Factor (VWF) VWF:GPIbR and VWF:GPIbM Assays in Type 2A, 2B and 2M Von Willebrand Disease

A complete set of rapid activity and classical von Willebrand factor (VWF) assays for Willebrand disease (VWD) diagnosis was used in the present study to characterize VWD type 1, 2A, 2B and 2M patients due to mutations in the A1, A2 and A3 domains. The VWF:RCo/VWF:Ag, VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF:Ag ratios at cuttt off value of 0.7 separated VWD type 1 and LowVWF from VWD type 2. The results from the Brno cohort of VWD 2A patients with the G1579R mutation in the A2 domain in sixteen affected member from five families and in one case with the G1609R in the A2 domain revealed that the VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF: Ag ratios are marked decreased (range 0.03-0.27) to a similar degree as compared to VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios (range 0.03-0.27) due to the proteolytic loss of large and intermediate VWF multimers. The results in VWD 2B patients due to gain of ristocetin induced platelet agglutination (RIPA) function mutations R1306W, R1308C and R1341 in the A1 domain demonstrated that the ratios for VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF:Ag as compared to VWF:RCo/VWF:Ag ratio were markedly decreased in VWD 2B, whereas the VWF:GPIbM/VWF:Ag ratio was somewhat higher (range 0.32 to 0.36) in VWD 2M. VWD 2M patients due to loss of RIPA function mutation R1359K in the A1 domain are featured by decreased VWF ratios for WVF:RCo/Ag and VWF:GPIbR/Ag, but less decreased for the VWF:GPIbM/Ag ratio and normal VWF ratio for VWF:CB/Ag ratio the need to retain the VWF:CB assay to make a correct diagnosis of VWD 2M for its differentiation from VWD type 1. The G1415D mutation in the A1 domain is featured by decreased RIPA and decreased VWF:RCo/VWF:Ag ratio (VWD 2M) but normal values for VWF:CB/VWF:Ag, VWF:GPIbM/VWF:Ag and VWFGPIbR/VWF:Ag ratios consistent with VWD 2M. Double heterozygous P1266L/V1278I mutation in two patients and heterozygous E1292D/WT mutation in three patients in the A1 domain were diagnosed as VWD 2M or 1M associated with a secretion defect (SD). The Platelet Function Analyzer Closure Times (PFA-CT) are strongly prolonged in VWD 2A, 2B and 2M. and moderately prolonged between the upper limit of normal to 300 seconds in heterozygous mutated VWD type 1 patients.

Comparison of farmacodynamic properties of three different aspirin formualtions in patients with stable coronary disease

Background/Aim. The platelet aggregation, as a laboratory test for assessment of platelet function, is very efficient for optimal antiplatelet treatment and also to identify individuals who have suboptimal response to antiplatelet drugs, such as aspirin and clopidogrel. The aim of this study was to determine the level of inhibition of platelet aggregation using impedance aggregometry in the patients receiving different preparations of acetylsalicylic acid (ASA) in a dose of 100 mg per day. Methods. The examination included 215 patients (110 men and 105 women), treated with one of three different ASA preparations after acute myocardial infarction, as a single therapy or with clopidogrel. Among them, 89 patients were on Aspirin protect? (Bayer, Germany) ? Group 1 and 66 patients were on Cardiopirin? (GL Pharma GMBH, Austria) ? Group 2, while 60 patients were taking Andol? (Pliva, Croatia) ? Group 3. The groups were equal in the presence of factors that can influence platelet aggregation (age, gender, smoking, diabetes, taking other drugs). The platelet function was measured using the impedance aggregometer Multiplate (Multiplate Platelet Function Analyzer, Roche) in the blood samples with heparin for the platelet aggregation activated by the arachidonic acid (ASPI) and by thrombin (TRAP) tests [the area under the aggregation curve (AUC) was used to express the aggregation response over the measured time (AU*min)]. Results. Efficacy of ASA preparations showed statistically significant differences among the three investigated groups (?KW2 = 46.279; p &lt; 0.001), and it was also observed separately in the patients undergoing single therapy (?KW2 = 26.344; p &lt; 0.001) and dual therapy (?KW2 = 23.498; p &lt; 0.001). It was found that the patients who were taking Aspirin protect? obtained significantly better antiplatelet efficiency compared to the patients receiving Cardiopirin? (Z = 5.472; p &lt; 0.001) and Andol? (Z = 5.387; p = 0.022). There is reduced efficiency of all ASA preparations in smokers, while patients receiving Aspirin protect? were 10.5 times more likely to be responders. Conclusion. Different ASA preparations observed in this study showed different efficiency on the platelet function as measured by the method of impedance aggregometry.

Platelet aggregometry assay for evaluating the effects of platelet agonists and antiplatelet compounds on platelet function in vitro

Platelet aggregometry assays are generally used for the analysis of platelet function but can also be adapted for further research and therapy focused applications. This method describes the procedures for the preparation of human platelet-rich plasma (PRP) and platelet-poor plasma (PPP) for the assessment of human platelet aggregation induced by agonists such as platelet-activating factor (PAF), thrombin, collagen, adenosine diphosphate (ADP), arachidonic acid, etc.•This method can be applied in vitro to evaluate the aggregatory effects of these agonists and to assess the antiaggregatory effects of several bioactive antiplatelet agents (compounds of natural or pharmacological origin) in human PRP.•This versatile method can be used in both basic and clinical research for the assessment of platelet aggregation (a major cardiovascular risk factor), platelet agonists, and inhibitors, in physiological or pathological conditions.•This method can be adapted to assess platelet activity in postprandial and intervention studies ex vivo.

External Quality Assurance of Platelet Function Assays: Results of the College of American Pathologists Proficiency Testing Program.

The College of American Pathologists (CAP) developed proficiency testing for platelet function assays by using blood collected by the participant added to challenge tubes containing either saline (normal) or tirofiban (abnormal). To analyze platelet function proficiency testing for Platelet Function Analyzer PFA-100, platelet aggregation, PlateletWorks, and PlateletMapping. Proficiency testing data from 2012-2016 were analyzed. For PFA-100, a total of 1200 laboratories participated; the coefficient variation (CV) of cartridge closure times was 22% (saline); 44,952 of 45,616 survey responses (99%) provided an interpretation, and 42,934 of 44,952 (96%) were correct. For optical platelet aggregation, 190 laboratories participated; the CV was 17% (saline), 7444 of 7813 survey responses (95%) provided an interpretation, and 7015 of 7444 (94%) were correct. For PlateletWorks, 60 laboratories participated; the CV was 3% to 11% (saline); 2412 of 2454 survey responses (98%) provided an interpretation, and 1207 of 1276 (95%) were correct for adenosine diphosphate (ADP) and 936 of 1136 (82%) for collagen. For PlateletMapping, 200 laboratories participated. For ADP, 1128 of 2697 survey responses (42%) provided an interpretation, but only 927 of 1128 (82%) were correct. For arachidonic acid, 1139 of 2604 survey responses (44%) provided an interpretation and 964 of 1139 (85%) were correct. CAP is the first to provide proficiency testing for platelet aggregation, PlateletWorks, and PlateletMapping. Platelet aggregation, PFA-100, and PlateletWorks using ADP as an agonist performed well with more than 90% of laboratories providing an interpretation and a similar number providing correct results. PlateletWorks using collagen and PlateletMapping showed worse interpretive accuracy than the other methods.

Dark chocolate modulates platelet function with a mechanism mediated by flavan-3-ol metabolites.

Cocoa is a rich source bioactive compounds, i.e., flavan-3-ols, and its consumption has been associated with several beneficial effects, such as the positive modulation of the hemostasis targeted by the platelet function. However, these phenolic compounds have a very low bioavailability and extensively undergo phase I and II metabolism, with the appearing into the bloodstream of (epi)catechin conjugates and phenyl-γ-valerolactones and their conjugates, at different times.The aims of this study were to explore the effect of dark chocolate on platelet function and to investigate the relationship between this interplay and flavan-3-ol derived metabolites.Eighteen healthy male volunteers ingested 50 g of 90% cocoa chocolate within 5 minutes. Blood samples were collected immediately before chocolate ingestion (T0) and 4 hours afterwards (T1). Platelet function analyzer (PFA)-100 closure time was assessed using collagen/adenosine-5′-diphosphate (COL/ADP) and collagen/epinephrine (COL/EPI) cartridges. Plasma flavan-3-ol metabolites were identified and quantified by means of liquid chromatography coupled to a triple quadrupole mass spectrometer (UHPLC-ESI-MS/MS).Results evidenced a significant increase of COL/ADP-induced PFA-100 closure time, but not COL/EPI, 4 hours after ingestion of dark chocolate. Total plasma structurally-related (epi)catechin metabolite (SREM) concentration significantly increased at T1, together with 4 out of the 6 detected metabolites. Total phenyl-γ-valerolactone concentrations remained unchanged. Spearman correlations evidenced a strong correlation between COL/ADP closure time and SREMs, mainly led by (epi)catechin-sulfate isomers.These data confirm that the potential beneficial effect of dark chocolate on primary hemostasis may be mediated by flavan-3-ol circulating metabolites.

Poor Platelet Function on Sonoclot Signature Is Associated with High Incidence of Bleeding in Severe Immune Thrombocytopenia

Introduction: Patients of immune thrombocytopenia (ITP) are at an high risk of bleeding, and the risk of bleeding is maximum when platelet counts fall below 30,000/μL (severe ITP). Though it is not known not all patients have a similar risk of bleeding at a given platelet count. In 59th ASH annual meeting, 2017 (poster 2320) Mishra K et al showed that the platelet function measured by ‘Sonoclot coagulation and platelet function analyzer’ is significantly different in bleeders as compared to non-bleeders severe ITP patients. We aimed to investigate and follow up the non-bleeder severe ITP patients, whose platelet count remains <30,000 with or without treatment, and find out the incidence of bleed.Method: The study was conducted at the department of internal medicine, postgraduate institute of medical education and research, Chandigarh, India. In this prospective observational study, severe ITP (newly diagnosed) and without active bleeding (WHO bleeding grade <2) were included. All these patient were clinically evaluated, and blood samples were collected as per unit protocol for ITP including Sonoclot analysis. For Sonoclot analysis, fresh blood samples were drawn in citrated vacutainer and analyzed by Sonoclot coagulation and platelet function analyzer made by Sienco, Inc, model SCP1 (USA). The sonoclot signature assessment gives activated clotting time (ACT), clot rate (R1) and platelet function (PF). The samples for sonoclot analysis were monthly drawn for six months in patients who continued to have severe ITP.Results: A total of 50 patients with severe ITP were included. Twenty patients were not included in the final analysis as their platelet remained above 30,000/μL during follow up. The remaining 30 patients were divided into two groups based on normal platelet function (PF) (>1.5) and low PF (<1.5) as measured by the Sonoclot analyzer. The normal PF group (n= 17) had only one patient who had clinically significant bleeding (WHO grade > 2), while low PF group had four patients with clinically significant bleeding (Figure 1). Though the statistical significance level could not be achieved, likely due to a small cohort of patients, the results look promising and shows the potential of sonoclot to give valuable input regarding the risk of bleeding in severe ITP.Conclusion: The patients with poor platelet function as measured by ‘Sonoclot coagulation and platelet function analyzer’ had more bleeding episodes as compared to patients with normal platelet function. Therefore, Sonoclot may work as a point of care investigation to predict the risk of bleeding in severe ITP patients. This will help the clinician in being more conservative in the management of severe ITP patients with low risk of bleeding and avoid unnecessary therapy. We conclude that the use of Sonoclot during follow-up in severe ITP patients has prognostic significance. DisclosuresNo relevant conflicts of interest to declare.

Initial Evaluation of Adolescent Females Hospitalized with Heavy Menstrual Bleeding

Background: Heavy menstrual bleeding (HMB) in adolescents can be severe and life-threatening. Up to 30% of young women who are hospitalized with anemia due to HMB have a bleeding disorder. Guidelines from the American College of Obstetrics and Gynecology (ACOG) and the National Heart, Lung, and Blood Institute (NHLBI) recommend evaluation for bleeding disorders in such patients. ACOG recommendations include testing for von Willebrand disease (VWD) and specify that consultation with a hematologist may help in interpreting results. NHLBI recommends testing for vWD be done in conjunction with a hematologist. As von Willebrand factor is an acute phase reactant, testing when patients are severely anemic and bleeding may not provide accurate results. ACOG guidelines do not include testing for platelet function disorders (PFD), though PFD may be as prevalent as VWD in females with HMB. Early and accurate diagnosis of bleeding disorders is important for health and quality of life, yet limited data exists on the diagnostic evaluation for bleeding disorders in adolescent females hospitalized for HMB.Objectives: To evaluate the diagnostic evaluation of bleeding disorders in adolescent females hospitalized for HMB.Methods: A retrospective, single center chart review of female patients aged 9-21 years hospitalized for HMB and anemia at a tertiary care children's hospital from January 1, 2000 until December 31, 2017 was done. HMB was defined as menses ≥7 days in length, use of 8 or more pads or tampons per day during menses, pictorial bleeding assessment chart (PBAC) score greater than 100, or symptomatic anemia. Patients were identified from our Hemophilia Treatment Center (HTC) registry, review of patients seen at a comprehensive clinic staffed by pediatric hematologists and gynecologists for adolescent females with HMB and bleeding disorders, and by an Electronic Medical Record (EMR) query of admission and discharge diagnoses of HMB and anemia. Data obtained included clinical features, diagnostic evaluation, and laboratory results.Results: 118 patients hospitalized for HMB and anemia were included. Inpatient Hematology consult or outpatient referral occurred in 68 (58%) of the patients; 60/68 (88%) had a bleeding disorder evaluation completed. 34 patients had a hematologic disorder. PFD was the most common (15/34; 44%) followed by VWD (9/34; 26%). 42% (50/118) of the patients did not have a Hematology consult or outpatient referral (Table 1). While hospitalized for HMB and anemia, 29 of the 50 patients had testing for vWD performed and only 4/29 (14%) had testing repeated as an outpatient once hemoglobin normalized. No patients tested for VWD while inpatient had results consistent with the diagnosis. Platelet function testing was performed in 10/50 patients using the platelet function analyzer (PFA-100) in 8 patients and platelet aggregometry in 2 patients.Conclusions: Despite national guidelines and the presence of known risk factors such as HMB since menarche and HMB causing severe anemia, the hematology service was not involved in the diagnostic process for a significant number of adolescent females. In these patients, testing often occurred while patients were hospitalized and was not repeated. Testing for platelet function disorders occurred infrequently and mainly consisted of the PFA-100 which lacks sensitivity and specificity. When patients were evaluated by Hematology and tested for bleeding disorders, a large proportion had a bleeding disorder, of which PFD were most common. This study demonstrates the need for standardization of the evaluation of adolescent females hospitalized for HMB. Guidelines should be updated to include testing for PFD. Hematologists should be involved when females are hospitalized for HMB and anemia. [Display omitted] DisclosuresCarpenter:Genentech Incorporated: Membership on an entity's Board of Directors or advisory committees; Nationwide Children's Hospital: Speakers Bureau; Bayer: Honoraria; Kedrion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk Pharmaceuticals, Inc: Consultancy; HEMA Biologics: Consultancy; American Academy of Pediatrics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Consultancy; National Hemophilia Foundation (Impact Education): Speakers Bureau; Kane County State's Attorney: Consultancy; CSL Behring: Speakers Bureau; 4th Judicial District Attorney's Office- Colorado: Consultancy; Kedrion Biopharmaceuticals: Consultancy.

Flow Cytometric Analysis of Platelet Function in Patients on Antiplatelet Therapy and Suspected Thrombocytopathy

Background: Light transmission aggregometry (LTA) is still the most used test for the identification and diagnosis of platelet function defects (PFD). Disadvantages of LTA include its laborious nature, the large volumes of blood required and the relative insensitivity to small changes in platelet function. We investigated if the flow cytometry-based whole blood platelet activation test (WB-PACT) correlates with LTA or could be used as a complimentary test.Methods: WB-PACT quantifies αIIbβ3 activation and P-selectin expression in response to 3 agonists and VWF binding to platelets in response to ristocetin. In total, 212 patients (51 on dual antiplatelet therapy (acetylsalicylic acid and ADP receptor antagonist), 161 suspected for bleeding diathesis) were tested. For WB-PACT parameters, the 2.5th, 10th and 25th percentiles were determined in 56 healthy donors to score the patient samples. For LTA, maximal aggregation, disaggregation and prolongation of the lagtime in response to 4 agonists and ristocetin was scored. Patients with at least one parameter lower than the 10th percentile measured with WB-PACT in healthy donors or at least one value deviating from the normal range measured with LTA were diagnosed with PFD. A bleeding score (BS) was calculated with the ISTH/SSC bleeding assessment tool (Rodeghiero et al., JTH, 2010).Results: A moderate correlation between LTA versus WB-PACT was found with a R of 0.60 (Figure 1). In total, 95 patients were diagnosed with PFD by both tests (κ=0.40, Table 1) and BS were recorded for 28/95 patients. Of these 28 patients, 25% had an elevated BS (adapted according to gender and age: >5 in women, >3 in men and >2 in children) and 36% had a BS>3. BS were recorded for 30/38 and 22/24 patients who were diagnosed with PFD according to WB-PACT only and LTA only, respectively. Interestingly, 27% of patients with PFD according to WB-PACT had an elevated BS and 40% had a BS>3. For LTA, only 13% of patients with PFD had an elevated BS and 13% had a BS>3.Conclusions: Flow cytometric analysis of platelet function by WB-PACT gives additional value to LTA to determine PFD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Multiple Myeloma Treatment Is Associated with Enhanced Platelet Reactivity

Multiple myeloma (MM) is associated with elevated levels of thrombotic disease. Poor mobility due to bone disease and older age are contributory factors but a satisfactory molecular or cellular explanation for this has not been established. Furthermore, the treatment of newly-diagnosed or relapsed MM patients with immunomodulatory drugs increases thrombosis risk. In this study, we therefore sought to determine whether platelet function and/or haemostasis is altered during the clinical progression from the benign monoclonal gammopathy of underdetermined significance (MGUS) to the smouldering myeloma (SM, untreated) state, and to establish the impact of lenalidomide therapy in MM patients on platelet reactivity.Patients (MGUS (N=10), SM (N=10) MM (N=4), healthy volunteer/control (N=10)) were recruited at the myeloma clinics at the Royal Berkshire and Oxford University Hospital Trusts. Single blood samples were obtained from controls, MGUS and smoldering myeloma participants. For patients undergoing therapy (lenalidomide, dexamethasone and anti-coagulant or anti-platelet therapy), samples were taken at predose Cy1, Cy1 D14, Cy1 D28 (a week free from lenalidomide administration), and Cy4 D14. All blood samples were taken less than 12 hours from Lenalidomide dosing, and analysis performed within 4 hours. High throughput platelet function analysis was performed on platelet-rich plasma and washed platelets to measure platelet aggregation, secretion and fibrinogen binding in response collagen, collagen-related peptide (a collagen receptor GPVI-selective ligand), ADP, thrombin receptor activatory peptide (TRAP), the thromboxane mimetic U46619 and epinephrine, and full concentration-response profiles obtained for each. The levels of key platelet receptors including GPVI, CD61, CD41b, CD36 and CD31, were also measured by flow cytometry and remained unaltered between groups. In comparison with control samples, platelet functional responses (maximal response, maximal gain and EC50) from MGUS and smoldering myeloma patients were unaltered (EC50: ADP 1µM, collagen-related peptide (CRP-XL) 0.1µg/ml, U46619 1µM, TRAP 1.6µM, epinephrine 1.3µM). Similarly circulating levels of platelet-bound fibrinogen and P-selectin exposure were unaltered (control: 709±194 AU, MGUS: 581±129 AU, SM: 655±148 AU 9 (Mean±SD)). In MM patients during therapy, the levels of these platelet responses (aggregation, fibrinogen binding and P-selectin exposure - a marker for a-granule secretion) were increased 2-4 fold. In most patients this was notable at week 2 and had declined approximately 50% by week 4 of cycle 1 but remained above predose levels, and remained elevated to a similar level in drug cycle 4.The study of further patients (including Bortezomib treated patients as controls) will be required in order to validate these findings which suggest that (1) baseline platelet function is not substantially compromised in MM and SM, (2) lenalidomide and other immunomodulatory drugs may be able to modulate platelets in patients causing increased platelet reactivity, and (3) effects noted are lenalidomide-dependent and not related to inflammatory state secondary to disease response. The effects of exposure of platelets to lenalidomide in vitro in the presence or absence of dexamethasone and a proteasome inhibitor are being tested to explore potential direct effects of the drugs on circulating platelets. Results from these experiments will also be presented. DisclosuresGibbins:Celgene Corporation: Research Funding. Rana:Celgene Corporation: Research Funding. Khan:Celgene Corporation: Research Funding. Shapiro:Freeline Therapeutics Ltd: Consultancy. Grech:Novartis: Other: sponsorship for scientific meetings. Ramasamy:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Chronic Lymphocytic Leukaemia Is Associated with a Broad Platelet-Function Defect Which Is Exacerbated By Ibrutinib and Acalabrutinib

INTRODUCTIONBleeding from thrombocytopathy is a common complication of advanced chronic lymphocytic leukaemia (CLL). In addition to disease-related thrombocytopenia, the presence of the CLL clone and/or therapeutic interventions may further impair platelet function. In particular, the BTK inhibitors ibrutinib and acalabrutinib are known to inhibit platelet glycoprotein VI (GPVI)-mediated platelet aggregation. We compared platelet function and markers of GPVI activation between untreated CLL patients, ibrutinib-treated CLL patients and healthy controls, and studied the in vitro effects of ibrutinib and acalabrutinib on clinically utilised platelet function assays to assess their impact on GPVI-mediated as well as non-GPVI-mediated platelet activation pathways.METHODSBlood samples from 17 healthy volunteers and 8 untreated CLL patients were spiked with vehicle or comparable plasma concentrations of ibrutinib (0.3µM, 1.0µM) and acalabrutinib (1.8µM, 6.0µM) attainable during the treatment of CLL. Additional samples were obtained from 5 CLL patients undergoing ibrutinib treatment. Platelet function was evaluated using whole blood multiple electrode aggregometry (MEA - Multiplate®) and light transmission aggregometry (LTA - AggRAM®) in response to varying concentrations of aggregation-inducing reagents (collagen, CRP-XL, ADP, TRAP, ristocetin, arachidonic acid, and adrenaline). Shear-induced platelet adhesion was assessed using PFA-100®. Soluble GPVI plasma levels were assessed by ELISA.RESULTSIn the absence of treatment, CLL patients exhibited significant platelet defects on whole-blood platelet function analyses in response to various agonists including ADP, ristocetin, TRAP and collagen (MEA) and prolongation of PFA-100® collagen/epinephrine closure time. This impairment was not replicated in assays using platelet-rich plasma (LTA). Ibrutinib-treated CLL patients demonstrated an additive impairment of platelet function, especially in regards to collagen-mediated activation by MEA or PFA-100®. There was no significant difference in soluble GPVI levels between normal, untreated or ibrutinib treated CLL patients.Addition of clinically-attainable concentrations of ibrutinib and acalabrutinib in vitro produced similar concentration-dependent inhibition of platelet function in healthy controls, with inhibition of aggregation evident in response to various agonists including collagen, CRP-XL, ristocetin and ADP but not arachidonic acid or TRAP. Ibrutinib also impaired aggregation in response to epinephrine, and caused selective prolongation of the PFA-100® collagen/epinephrine closure time, an effect not observed with acalabrutinib. MEA appears more sensitive and reproducible than LTA to describe the various inhibitory effects on platelet aggregation. Similar concentration-dependent inhibition of platelet function was observed by adding ibrutinib and acalabrutinib in vitro to blood samples from untreated CLL patients.CONCLUSIONSCLL is associated with a broad platelet function defect, which can be exacerbated by BTK inhibitors. Acalabrutinib induces a platelet function defect similar but less potent to that observed with ibrutinib, with the exception of shear-induced platelet adhesion (PFA-100®) which was only abnormal with ibrutinib. Routine platelet function assays are capable of quantifying BTK inhibitor-induced platelet dysfunction in CLL patients, with the most sensitive and reproducible measure being collagen-induced aggregation by MEA. There was no evidence for BTK-dependent platelet GPVI cleavage. Whole-blood platelet function assays may have utility in managing CLL patients presenting with bleeding or requiring urgent surgery during therapy with BTK inhibitors. DisclosuresMcGregor:Pfizer: Other: Conference travel support; Bristol-Myers Squibb: Other: Conference travel support; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria. Baker:CSL Behring: Research Funding; Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support; Roche: Other: Conference travel support; Novo Nordisk: Other: Conference travel support.

Comparative evaluation of Plateletworks, Multiplate analyzer and Platelet function analyzer-200 in cardiology patients.

The objective of this study was to comparatively evaluate three commercial whole-blood platelet function analyzer systems: Platelet Function Analyzer-200 (PFA; Siemens Canada, Mississauga, Ontario, Canada), Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), and Plateletworks Combo-25 kit (PLW; Helena Laboratories, Beaumont, TX, USA). Venipuncture was performed on 160 patients who visited a department of cardiology. Pairwise agreement among the three platelet function assays was assessed using Cohen's kappa coefficient and percent agreement within the reference limit. Kappa values with the same agonists were poor between PFA-collagen (COL; agonist)/adenosine diphosphate (ADP) and MP-ADP (-0.147), PFA-COL/ADP and PLW-ADP (0.089), MP-ADP and PLW-ADP (0.039), PFA-COL/ADP and MP-COL (-0.039), and between PFA-COL/ADP and PLW-COL (-0.067). Nonetheless, kappa values for the same assay principle with a different agonist were slightly higher between PFA-COL/ADP and PFA-COL/EPI (0.352), MP-ADP and MP-COL (0.235), and between PLW-ADP and PLW-COL (0.247). The range of percent agreement values was 38.7% to 73.8%. Therefore, various measurements of platelet function by more than one method were needed to obtain a reliable interpretation of platelet function considering low kappa coefficient and modest percent agreement rates among 3 different platelet function tests.

Frequency of development of aspirin resistance in the early postoperative period and inadequate inhibition of thromboxane A2 production after coronary artery bypass surgery

This study aims to investigate the frequency of the development of aspirin resistance, whether or not this resistance was reversible, and to evaluate the efficiency of the mechanism of incomplete inhibition of thromboxane A2 in development of aspirin resistance in the early postoperative period in patients who had undergone coronary artery bypass grafting. Eighty patients (55 males, 25 females; mean age 63.1±9.2 years; range 51 to 75 years) who underwent coronary artery bypass grafting between February 2009 and March 2010 at our clinic were prospectively evaluated. Venous blood samples were collected from all patients and evaluated by a platelet function analyzer in the preoperative period and on postoperative days 7 and 15. Aspirin resistance diagnosis was defined as collagen-epinephrine closure time less than 186 seconds. The urine levels of 11-dehidro thromboxane B2 were also measured on postoperative day one. Aspirin resistance was found in 23 patients (28.75%) in the preoperative period, in 31 patients (38.75%) on the postoperative seventh day and in 25 patients (31.25%) on the postoperative 15th day. The urine levels of 11-dehidro thromboxane B2 in patients with aspirin resistance on the postoperative seventh day were significantly higher than those in patients without aspirin resistance (p<0.001). The mean aortic cross-clamping time (p=0.003) and cardiopulmonary bypass time (p=0.029) in the patients with aspirin resistance on the postoperative seventh day were significantly higher than those in patients without aspirin resistance. The results of this study suggest that aspirin resistance develops within the first seven days after coronary artery bypass grafting and is highly reversible, and that the mechanism of inadequate inhibition of thromboxane A2 by aspirin has a role in the development of aspirin resistance in the early postoperative period.