Comparison of the Quantra QPlus and ROTEM Goal-Directed Transfusion Protocols in Cardiothoracic Surgery Patients: A Prospective Observational Study.

OBJECTIVES: To compare the designed treatment protocols for the Quantra QPlus and rotational thromboelastometry (ROTEM) with regard to transfusion advice. DESIGN: Prospective observational study. SETTING: Maastricht University Medical Center, The Netherlands. PARTICIPANTS: Adults with elective cardiopulmonary bypass surgery with a ROTEM test. INTERVENTIONS: ROTEM tests were performed postoperatively for standard monitoring of coagulation status and clinical decision making. Simultaneously, a concurrent sample was analyzed for the Quantra QPlus. MEASUREMENTS AND MAIN RESULTS: A total of 100 samples were analyzed using both the ROTEM and Quantra QPlus. Agreement between the transfusion advice for the ROTEM and Quantra QPlus protocols were compared using Cohen κ values for i.a. fibrinogen, platelet concentrates, and fresh frozen plasma (FFP). The agreement between ROTEM and Quantra QPlus was poor for overall transfusion (0.174) and fibrinogen transfusion (0.300). The agreement of cutoff values for fibrinogen clot stiffness for the Quantra QPlus and EXTEM A10 for the ROTEM was poor (0.160). The fibrinogen clot stiffness and FIBTEM A10 had a moderate agreement (0.731). A Cohen κ could not be calculated for the agreement of protamine, thrombocytes, FFP or cutoff values for these transfusions since frequencies included zero in these cases. The Quantra QPlus transfusion protocol advises transfusion in many non-bleeders, adjustments appear to be necessary. In a small group of cases in which clinically relevant blood loss was observed, the Quantra QPlus advised administration of transfusion products, whereas the ROTEM tests did not. CONCLUSION: ROTEM-guided and Quantra-guided transfusion did not correspond in this patient group, and agreement was moderate at best. Specificity and sensitivity for transfusion within protocols were heterogeneous between the methods. More clinical research in high-bleeding risk populations is needed to determine the clinical impact of the different protocols.

Proactive monitoring of drug-drug interactions between direct oral anticoagulants and small-molecule inhibitors in patients with non-small cell lung cancer.

BACKGROUND: Small-molecule inhibitors (SMIs) have revolutionised the treatment of non-small cell lung cancer (NSCLC). However, SMI-induced drug-drug interactions (DDIs) with frequently co-administered direct oral anticoagulants (DOACs), increase thromboembolic and bleeding risks. This study investigated and proactively managed the consequences of DOAC-SMI DDIs. METHODS: This prospective, observational study enrolled patients with NSCLC concomitantly using a DOAC and SMI. The primary outcome was the proportion of patients with DOAC plasma trough (Ctrough) and peak (Cpeak) concentrations outside expected ranges. Secondary outcomes included DOAC treatment modifications, incidence of bleeding and thromboembolic events and feasibility evaluation of pharmacokinetically guided DOAC dosing. RESULTS: Thirty-three patients were analysed. Thirty-nine percent (13/33) had DOAC Ctrough and/or Cpeak were outside the expected ranges in 39% (13/33). In 71% (5/7) of patients with DOAC concentrations quantified before and during concurrent SMI use, DOAC Ctrough and/or Cpeak increased or decreased >50% upon SMI initiation. In all patients in whom treatment modifications were deemed necessary, DOAC concentrations were adjusted to within the expected ranges. CONCLUSION: Proactive monitoring showed that a substantial proportion of patients had DOAC concentrations outside the expected ranges. DOAC concentrations were successfully normalised after treatment modifications. These results highlight the importance of proactive monitoring of DOAC-SMI DDIs to improve treatment in patients with NSCLC.

Antibodies against Platelet Glycoproteins in Clinically Suspected VITT Patients.

Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare but severe complication following COVID-19 vaccination, marked by thrombocytopenia and thrombosis. Analogous to heparin-induced thrombocytopenia (HIT), VITT shares similarities in anti-platelet factor 4 (PF4) IgG-mediated platelet activation via the FcγRIIa. To investigate the involvement of platelet-antibodies in VITT, we analyzed the presence of platelet-antibodies directed against glycoproteins (GP)IIb/IIIa, GPV and GPIb/IX in the serum of 232 clinically suspected VITT patients determined based on (suspicion of) occurrence of thrombocytopenia and/or thrombosis in relation to COVID-19 vaccination. We found that 19% of clinically suspected VITT patients tested positive for anti-platelet GPs: 39%, 32% and 86% patients tested positive for GPIIb/IIIa, GPV and GPIb/IX, respectively. No HIT-like VITT patients (with thrombocytopenia and thrombosis) tested positive for platelet-antibodies. Therefore, it seems unlikely that platelet-antibodies play a role in HIT-like anti-PF4-mediated VITT. Platelet-antibodies were predominantly associated with the occurrence of thrombocytopenia. We found no association between the type of vaccination (adenoviral vector vaccine versus mRNA vaccine) or different vaccines (ChAdOx1 nCoV-19, Ad26.COV2.S, mRNA-1273, BTN162b2) and the development of platelet-antibodies. It is essential to conduct more research on the pathophysiology of VITT, to improve diagnostic approaches and identify preventive and therapeutic strategies.

Restrictive versus liberal fluid administration strategy (REFILL study) in postpartum hemorrhage and its effects on thromboelastometry (ROTEM®) values: a randomized, controlled trial.

OBJECTIVE: Current obstetric guidelines for postpartum hemorrhage (PPH) vary in fluid resuscitation management. This study aimed to evaluate the effect of fluid management on coagulation parameters in early PPH. METHODS: We performed a multicenter, randomized trial. Women who had 500 mL of blood loss in the third stage of labor were randomized to receive a restrictive fluid administration strategy or a liberal fluid administration strategy. A rotational thromboelastometry panel was performed in 72 patients. We evaluated within-group and between-group differences in the EXTEM clotting time (CT), EXTEM amplitude at 10 minutes (A10), INTEM CT, and FIBTEM A10. We also evaluated the mean fibrinogen concentration, activated partial thromboplastin time, and partial thromboplastin time in the total study population (n = 249). RESULTS: There were no significant differences in hemostatic parameters between the groups after correction for baseline values. CONCLUSIONS: In women with PPH <1500 mL, there is no clinically relevant effect of a restrictive or liberal fluid administration strategy on thromboelastometric hemostatic and regular coagulation parameters.

Role of SHP2 (PTPN11) in glycoprotein VI-dependent thrombus formation: Improved platelet responsiveness by the allosteric drug SHP099 in Noonan syndrome patients.

INTRODUCTION: The protein tyrosine phosphatase SHP2 (PTPN11) is a negative regulator of glycoprotein VI (GPVI)-induced platelet signal under certain conditions. Clinical trials with derivatives of the allosteric drug SHP099, inhibiting SHP2, are ongoing as potential therapy for solid cancers. Gain-of-function mutations of the PTPN11 gene are observed in part of the patients with the Noonan syndrome, associated with a mild bleeding disorder. Assessment of the effects of SHP2 inhibition in platelets from controls and Noonan syndrome patients. MATERIALS AND METHODS: Washed human platelets were incubated with SHP099 and stimulated with collagen-related peptide (CRP) for stirred aggregation and flow cytometric measurements. Whole-blood microfluidics assays using a dosed collagen and tissue factor coating were performed to assess shear-dependent thrombus and fibrin formation. Effects on clot formation were evaluated by thromboelastometry. RESULTS: Pharmacological inhibition of SHP2 did not alter GPVI-dependent platelet aggregation under stirring, but it enhanced integrin αIIbß3 activation in response to CRP. Using whole-blood microfluidics, SHP099 increased the thrombus buildup on collagen surfaces. In the presence of tissue factor and coagulation, SHP099 increased thrombus size and reduced time to fibrin formation. Blood from PTPN11-mutated Noonan syndrome patients, with low platelet responsiveness, after ex vivo treatment with SHP099 showed a normalized platelet function. In thromboelastometry, SHP2 inhibition tended to increase tissue factor-induced blood clotting profiles with tranexamic acid, preventing fibrinolysis. CONCLUSION: Pharmacological inhibition of SHP2 by the allosteric drug SHP099 enhances GPVI-induced platelet activation under shear conditions with a potential to improve platelet functions of Noonan syndrome patients.

Serial thrombin generation and exploration of alternative anticoagulants in critically ill COVID-19 patients: Observations from Maastricht Intensive Care COVID Cohort.

Background: COVID-19 associated coagulopathy (CAC) is associated with an increase in thromboembolic events. Current guidelines recommend prophylactic heparins in the management of CAC. However, the efficacy of this strategy in the intensive care population remains uncertain. Objective: We aimed to measure thrombin generation (TG) to assess CAC in intensive care unit (ICU) patients receiving thromboprophylaxis with low molecular weight heparin (LMWH) or unfractionated heparin (UFH). In addition, we performed statistical modeling to link TG parameters to patient characteristics and clinical parameters. Lastly, we studied the potency of different anticoagulants as an alternative to LMWH treatment in ex vivo COVID-19 plasma. Patients/Methods: We included 33 patients with confirmed COVID-19 admitted at the ICU. TG was measured at least twice over the course of 6 weeks after admission. Thrombin generation parameters peak height and endogenous thrombin potential (ETP) were compared to healthy controls. Results were subsequently correlated with a patient characteristics and laboratory measurements. In vitro spiking in TG with rivaroxaban, dabigatran, argatroban and orgaran was performed and compared to LMWH. Results: Anti-Xa levels of all patients remained within the therapeutic range throughout follow-up. At baseline, the mean (SE) endogenous thrombin potential (ETP) was 1,727 (170) nM min and 1,620 (460) nM min for ellagic acid (EA) and tissue factor (TF), respectively. In line with this we found a mean (SE) peak height of 353 (45) nM and 264 (96) nM for EA and TF. Although fluctuating across the weeks of follow-up, TG parameters remained elevated despite thromboprophylaxis. In vitro comparison of LMWHs and direct thrombin inhibitors (e.g., agratroban, dabigatran) revealed a higher efficacy in reducing coagulation potential for direct thrombin inhibition in both ellagic acid (EA) and tissue factor (TF) triggered TG. Conclusion: In a sub-group of mechanically ventilated, critically ill COVID-19 patients, despite apparent adequate anti-coagulation doses evaluated by anti-Xa levels, thrombin generation potential remained high during ICU admission independent of age, sex, body mass index, APACHE II score, cardiovascular disease, and smoking status. These observations could, only partially, be explained by (anti)coagulation and thrombosis, inflammation, and multi-organ failure. Our in vitro data suggested that direct thrombin inhibition compared with LMWH might offer an alternate, more effective anticoagulant strategy in COVID-19.

Assessment of need for hemostatic evaluation in patients taking valproic acid: A retrospective cross-sectional study.

INTRODUCTION: Valproic acid (VPA) is a frequently prescribed anti-epileptic drug. Since its introduction side effects on hemostasis are reported. However, studies show conflicting results, and the clinical relevance is questioned. We aimed to determine the coagulopathies induced by VPA in patients who undergo high-risk surgery. The study results warrant attention to this issue, which might contribute to reducing bleeding complications in future patients. METHODS: Between January 2012 and August 2020, 73 consecutive patients using VPA were retrospectively included. Extensive laboratory hemostatic assessment (including platelet function tests) was performed before elective high-risk surgery. Patient characteristics, details of VPA treatment, and laboratory results were extracted from medical records. RESULTS: 46.6% of the patients using VPA (n = 73) showed coagulopathy. Mainly, platelet function disorder was found (36.4%). Thrombocytopenia was seen in 9.6% of the patients. Data suggested that the incidence of coagulopathies was almost twice as high in children as compared to adults and hypofibrinogenemia was only demonstrated in children. No association was found between the incidence of coagulopathies and VPA dosage (mg/kg/day). CONCLUSION: A considerable number of patients using VPA were diagnosed with coagulopathy, especially platelet function disorder. Further prospective studies are needed to confirm the need for comprehensive laboratory testing before elective high-risk surgery in these patients.

Antithrombotic prophylaxis for surgery-associated venous thromboembolism risk in patients with inherited platelet disorders. The SPATA-DVT Study.

Major surgery is associated with an increased risk of venous thromboembolism (VTE), thus the application of mechanical or pharmacologic prophylaxis is recommended. The incidence of VTE in patients with inherited platelet disorders (IPD) undergoing surgical procedures is unknown and no information on the current use and safety of thromboprophylaxis, particularly of low-molecular-weight-heparin in these patients is available. Here we explored the approach to thromboprophylaxis and thrombotic outcomes in IPD patients undergoing surgery at VTE-risk participating in the multicenter SPATA study. We evaluated 210 surgical procedures carried out in 155 patients with well-defined forms of IPD (VTE-risk: 31% high, 28.6% intermediate, 25.2% low, 15.2% very low). The use of thromboprophylaxis was low (23.3% of procedures), with higher prevalence in orthopedic and gynecological surgeries, and was related to VTE-risk. The most frequently employed thromboprophylaxis was mechanical and appeared to be effective, as no patients developed thrombosis, including patients belonging to the highest VTE-risk classes. Low-molecular-weight-heparin use was low (10.5%) and it did not influence the incidence of post-surgical bleeding or of antihemorrhagic prohemostatic interventions use. Two thromboembolic events were registered, both occurring after high VTE-risk procedures in patients who did not receive thromboprophylaxis (4.7%). Our findings suggest that VTE incidence is low in patients with IPD undergoing surgery at VTE-risk and that it is predicted by the Caprini score. Mechanical thromboprophylaxis may be of benefit in patients with IPD undergoing invasive procedures at VTE-risk and low-molecular-weight-heparin should be considered for major surgery.

Fibrinolysis in patients with chemotherapy-induced thrombocytopenia and the effect of platelet transfusion.

Essentials Bleeding in chemotherapy induced thrombocytopenia (CIT) might be influenced by hyperfibrinolysis. t-PA-thromboelastography is a fast and reliable assay for hyperfibrinolysis in CIT patients. Clots of CIT patients are more susceptible to t-PA induced lysis compared to healthy individuals. Besides platelets, other factors are likely to influence clot lysis in CIT patients. BACKGROUND: Bleeding events in chemotherapy-induced thrombocytopenic (CIT) patients with similar platelet counts might be influenced by changes in clot lysis potential. OBJECTIVES: To investigate, in an observational study, thromboelastographic lysis parameters, alterations in clot strength and susceptibility to clot lysis in CIT patients. To identify factors associated with fibrinolytic profiles, and to evaluate the effects of platelet transfusions. METHODS: Independent determinants of tissue-type plasminogen activator (t-PA)-ROTEM lysis parameters were identified with multivariable linear regression. Clot formation, strength and lysis parameters were compared with the results of healthy individuals. Characteristics of CIT patients with and without hyperfibrinolytic profiles were compared. t-PA-ROTEM results before, 1 hour after and 24 hours after platelet transfusion were compared. RESULTS: A total of 72 consecutive CIT patients were included. t-PA-ROTEM lysis parameters correlated with changes in fibrinolytic proteins. Clot formation time was longer, maximum clot firmness was weaker and lysis times were shorter than in healthy individuals. CIT patients had low plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor levels, and 40% showed hyperfibrinolytic profiles. Platelet transfusions resulted in less hyperfibrinolytic profiles in many, but not all CIT patients. Patients without hyperfibrinolytic profiles had higher fibrinogen, factor VIII and α2 -antiplasmin levels. CONCLUSIONS: t-PA-ROTEM can be used as a fast and reliable assay to detect hyperfibrinolytic profiles in CIT patients. CIT patients have weaker clots, which are more susceptible to clot lysis, than healthy individuals. Besides platelets, other factors are likely to influence clot susceptibility to fibrinolysis in CIT patients. The impact of a hyperfibrinolytic t-PA-ROTEM profile on bleeding remains to be investigated.

Platelet Aggregation in Healthy Participants is Not Affected by Smoking, Drinking Coffee, Consuming a High-Fat Meal, or Performing Physical Exercise.

Platelet aggregation can be measured using optical aggregation (light transmission aggregometry, LTA) as well as by impedance (Multiplate analyzer). The LTA (the gold standard method) can be influenced by many preanalytical variables. Several guidelines differ in recommendations for the duration patients should refrain from smoking, coffee, fatty meals, and physical exercise prior to blood collection for performing platelet function tests. In this pilot study, the influence of smoking, coffee, high-fat meal, or physical exercise on platelet aggregation was investigated to improve patient friendliness and laboratory logistics in platelet function diagnostics. Standardized blood collection was performed when participants were fasting and after each parameter (n=5 per group). As a control for diurnal fluctuations, participants (n=6) were fasting during both blood collections. Platelet aggregation was executed using standardized methods for LTA and Multiplate analyzer. Statistical analysis of the results using Wilcoxon signed-rank test did not show any significant differences in platelet aggregation in healthy participants under different preanalytical variables. Therefore, these variables are not expected to adversely affect testing, which can avoid canceling tests for those patients who inevitably did.

Shifts of Transfusion Demand in Cardiac Surgery After Implementation of Rotational Thromboelastometry-Guided Transfusion Protocols: Analysis of the HEROES-CS (HEmostasis Registry of patiEntS in Cardiac Surgery) Observational, Prospective Open Cohort Database.

OBJECTIVES: Rotational thromboelastometry (ROTEM)-guided transfusion algorithms in cardiac surgery have been proven to be successful in reducing blood loss in randomized controlled trials. Using an institutional hemostasis registry of patients in cardiac surgery (HEROES-CS), the authors hypothesized that the use of ROTEM-guided transfusion algorithms would save blood products and overall costs in cardiac surgery in every day practice. DESIGN: Observational, prospective open cohort database. SETTING: Single-center academic hospital. PARTICIPANTS: Cardiac surgery patients. INTERVENTIONS: Implementation of ROTEM-guided bleeding management. MEASUREMENTS AND MAIN RESULTS: A classical-guided algorithm and a ROTEM-guided algorithm were used for patient blood management in 2 cohorts. Primary outcome was the use and amount of blood products and hemostatic medication. Secondary outcomes were amount of rethoracotomies, length of stay, and 30-day mortality. Finally, costs and savings were calculated. The classical-guided cohort comprised 204 patients, and ROTEM-guided cohort comprised 151 patients. Baseline characteristics showed excellent similarities after propensity score matching of 202 patients. Blood loss was lower after ROTEM guidance (p < 0.001). Absolute risk reduction was 17% for red blood cells (p = 0.024), 12% for fresh frozen plasma (p = 0.019), and 4% for thrombocyte concentrates (p = 0.582). More tranexamic acid was given, but not more fibrinogen concentrate, while desmopressin was given less often. Hospital length of stay was reduced by an overall median of 2 and a mean of 4 days (p < 0.001). Mortality and rethoracotomy rates were not affected. Potential savings were about €4,800 ($5,630) per patient. CONCLUSIONS: Implementation of a ROTEM-guided transfusion algorithm in cardiac surgery patients reduced the use of blood products and hemostatic medication, hereby saving costs. Reductions in mortality and rethoracotomy rates could not be found.

Impaired mitochondrial activity explains platelet dysfunction in thrombocytopenic cancer patients undergoing chemotherapy.

Severe thrombocytopenia (≤50×109 platelets/L) due to hematological malignancy and intensive chemotherapy is associated with an increased risk of clinically significant bleeding. Since the bleeding risk is not linked to the platelet count only, other hemostatic factors must be involved. We studied platelet function in 77 patients with acute leukemia, multiple myeloma or malignant lymphoma, who experienced chemotherapy-induced thrombocytopenia. Platelets from all patients - independent of disease or treatment type - were to a variable extent compromised in Ca2+ flux, integrin a ß activation and P-selectin expression when stimulated with a panelIIbof3 agonists. The patients' platelets were also impaired in spreading on fibrinogen. Whereas the Ca2+ store content was unaffected, the patients' platelets showed ongoing phosphatidylserine exposure, which was not due to apoptotic caspase activity. Interestingly, mitochondrial function was markedly reduced in platelets from a representative subset of patients, as evidenced by a low mitochondrial membrane potential (P<0.001) and low oxygen consumption (P<0.05), while the mitochondrial content was normal. Moreover, the mitochondrial impairments coincided with elevated levels of reactive oxygen species (Spearman's rho=-0.459, P=0.012). Markedly, the impairment of platelet function only appeared after two days of chemotherapy, suggesting origination in the megakaryocytes. In patients with bone marrow recovery, platelet function improved. In conclusion, our findings disclose defective receptor signaling related to impaired mitochondrial bioenergetics, independent of apoptosis, in platelets from cancer patients treated with chemotherapy, explaining the low hemostatic potential of these patients.

Restrictive Versus Massive Fluid Resuscitation Strategy (REFILL study), influence on blood loss and hemostatic parameters in obstetric hemorrhage: study protocol for a randomized controlled trial.

BACKGROUND: Postpartum hemorrhage (PPH) is associated with maternal morbidity and mortality and has an increasing incidence in high-resource countries, despite dissemination of guidelines, introduction of skills training, and correction for risk factors. Current guidelines advise the administration, as fluid resuscitation, of almost twice the amount of blood lost. This advice is not evidence-based and could potentially harm patients. METHODS: All women attending the outpatient clinic who are eligible will be informed of the study; oral and written informed consent will be obtained. Where there is more than 500 ml blood loss and ongoing bleeding, patients will be randomized to care as usual, fluid resuscitation with 1.5-2 times the amount of blood loss or fluid resuscitation with 0.75-1.0 times the blood loss. Blood loss will be assessed by weighing all draping. A blood sample, for determining hemoglobin concentration, hematocrit, thrombocyte concentration, and conventional coagulation parameters will be taken at the start of the study, after 60 min, and 12-18 h after delivery. In a subgroup of women, additional thromboelastometric parameters will be obtained. DISCUSSION: Our hypothesis is that massive fluid administration might lead to a progression of bleeding due to secondary coagulation disorders. In non-pregnant individuals with massive blood loss, restrictive fluid management has been shown to prevent a progression to dilution coagulopathy. These data, however, cannot be extrapolated to women in labor. Our objective is to compare both resuscitation protocols in women with early, mild PPH (blood loss 500-750 ml) and ongoing bleeding, taking as primary outcome measure the progression to severe PPH (blood loss > 1000 ml). TRIAL REGISTRATION: Netherlands Trial Register, NTR 3789 . Registered on 11 January 2013.

Detecting clinically relevant rivaroxaban or dabigatran levels by routine coagulation tests or thromboelastography in a cohort of patients with atrial fibrillation.

BACKGROUND: Traditional coagulation tests are included in emergency guidelines for management of patients on direct oral anticoagulants (DOACs) who experience acute bleeding or require surgery. We determined the ability of traditional coagulation tests and fast whole blood thromboelastography (ROTEM®) to screen for anticoagulation activity of dabigatran and rivaroxaban as low as 30 ng/mL. METHODS: One hundred eighty-four citrated blood samples (75 dabigatran, 109 rivaroxaban) were collected from patients with non-valvular atrial fibrillation (NVAF), to perform screening tests from different manufacturers, (diluted, D) PT, aPTT, TT and ROTEM®. The activity of DOACs was quantitatively determined by clot detection assays: Hemoclot DTT and DiXaI test (Biophen), on CS2100 (Siemens). The clotting time (CT) of INTEM and EXTEM ROTEM® (Werfen) were used as test parameters. RESULTS: Dabigatran, ≥ 30 ng/mL, was accurately detected by five coagulation tests: APTT Actin FSL (93%), PT Neoplastin (93%), APTT Cephascreen, Thromboclotin, and Thrombin (all 100%), but not by PT Innovin (49%). CT-EXTEM (91%) was sufficiently sensitive, but not CT-INTEM (52%). APTT Cephascreen and Thrombin showed good linearity (R2 = 0.71,R2 = 0.72). For the other tests linearity was moderate to poor. Rivaroxaban was accurately detected by PT Neoplastin (98%) and less so by APTT Cephascreen (85%). In addition, rivaroxaban was also accurately detected by CT-INTEM (96%). PT Neoplastin showed good linearity (R2 = 0.81), all other tests had moderate to poor linearity. CONCLUSION: In patients with NVAF, the ability of routine coagulation tests to detect the presence of significant levels of DOACs is test and reagent dependent. CT-INTEM and CT-EXTEM may be fast whole blood alternatives. TRIAL REGISTRATION: The Institutional Review Board of the MUMC approved this study (December 2011, project number 114069).

Routine haemostasis testing before transplanted kidney biopsy: a cohort study.

Kidney biopsy can result in bleeding complications. Prebiopsy testing using bleeding time (BT) is controversial. New whole blood haemostasis tests, such as platelet function analyser-100 (PFA-100) and multiple electrode aggregometry (MEA), might perform better. We postulated that PFA-100 would be suitable to replace BT prebiopsy. In 154 patients, transplanted kidney biopsies were performed after measurement of bleeding time, PFA-100, MEA and mean platelet volume (MPV). Bleeding outcome (haemoglobin (Hb) drop, haematuria (±bladder catheterization), ultrasound finding of a bleeding, need for (non)surgical intervention and/or transfusion) after the biopsy was correlated to each test. Male-female ratio was 2:1. 50% had a surveillance biopsy at either three or 12 months. Around 17% (had) used acetylsalicylic acid (ASA) prebiopsy. Of 17 bleeding events, one subject needed a transfusion. Most bleeding events were Hb reductions over 1 mmol/l and all resolved uneventful. BT, PFA-100, MEA and MPV did not predict a bleeding outcome; prior ASA use however could (odds ratio 3.19; 95%-CI 1.06 to 9.61). Diagnostic performance data and Bland-Altman analysis showed that BT could not be substituted by PFA-100. ASA use was the best determinant of bleeding after kidney biopsy. Routine haemostasis testing prebiopsy has no added value.

Bleeding risk of surgery and its prevention in patients with inherited platelet disorders.

Excessive bleeding at surgery is a feared complication in patients with inherited platelet disorders. However, very few studies have evaluated the frequency of surgical bleeding in these hemorrhagic disorders. We performed a worldwide, multicentric, retrospective study to assess the bleeding complications of surgery, the preventive and therapeutic approaches adopted, and their efficacy in patients with inherited platelet disorders: the Surgery in Platelet disorders And Therapeutic Approach (SPATA) study. We rated the outcome of 829 surgical procedures carried out in 423 patients with well-defined forms of inherited platelet disorders: 238 inherited platelet function disorders and 185 inherited platelet number disorders. Frequency of surgical bleeding was high in patients with inherited platelet disorders (19.7%), with a significantly higher bleeding incidence in inherited platelet function disorders (24.8%) than in inherited platelet number disorders (13.4%). The frequency of bleeding varied according to the type of inherited platelet disorder, with biallelic Bernard Soulier syndrome having the highest occurrence (44.4%). Frequency of bleeding was predicted by a pre-operative World Health Organization bleeding score of 2 or higher. Some types of surgery were associated with a higher bleeding incidence, like cardiovascular and urological surgery. The use of pre-operative pro-hemostatic treatments was associated with a lower bleeding frequency in patients with inherited platelet function disorders but not in inherited platelet number disorders. Desmopressin, alone or with antifibrinolytic agents, was the preventive treatment associated with the lowest bleedings. Platelet transfusions were used more frequently in patients at higher bleeding risk. Surgical bleeding risk in inherited platelet disorders is substantial, especially in inherited platelet function disorders, and bleeding history, type of disorder, type of surgery and female sex are associated with higher bleeding frequency. Prophylactic pre-operative pro-hemostatic treatments appear to be required and are associated with a lower bleeding incidence.

Application of an optimized flow cytometry-based quantification of Platelet Activation (PACT): Monitoring platelet activation in platelet concentrates.

BACKGROUND: Previous studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage. STUDY DESIGN AND METHODS: The optimized PACT was used to monitor 5 PCs during 10 days of storage. In brief, optimized PACT uses a ready-to-use reaction mix, which is stable at -20°C. When needed, a test strip is thawed and platelet activation is initiated by mixing PC with PACT. PACT was based on the following agonists: adenosine diphosphate (ADP), collagen-related peptide (CRP) and thrombin receptor-activating peptide (TRAP-6). Platelet activation was measured as P-selectin expression. Light transmission aggregometry (LTA) was performed as a reference. RESULTS: Both PACT and LTA showed platelet function decline during 10-day storage after stimulation with ADP and collagen/CRP; furthermore, PACT showed decreasing TRAP-induced activation. Major differences between the two tests are that PACT is able to measure the status of platelets in the absence of agonists, and it can differentiate between the number of activated platelets and the amount of activation, whereas LTA only measures aggregation in response to an agonist. Also, PACT is more time-efficient compared to LTA and allows high-throughput analysis. CONCLUSION: PACT is an optimized platelet function test that can be used to monitor the activation of PCs. PACT has the same accuracy as LTA with regard to monitoring PCs, but it is superior to both LTA and conventional flow cytometry based tests with regard to labor-, time- and cost efficiency.

The use of regression analysis in determining reference intervals for low hematocrit and thrombocyte count in multiple electrode aggregometry and platelet function analyzer 100 testing of platelet function.

Low platelet counts and hematocrit levels hinder whole blood point-of-care testing of platelet function. Thus far, no reference ranges for MEA (multiple electrode aggregometry) and PFA-100 (platelet function analyzer 100) devices exist for low ranges. Through dilution methods of volunteer whole blood, platelet function at low ranges of platelet count and hematocrit levels was assessed on MEA for four agonists and for PFA-100 in two cartridges. Using (multiple) regression analysis, 95% reference intervals were computed for these low ranges. Low platelet counts affected MEA in a positive correlation (all agonists showed r2 ≥ 0.75) and PFA-100 in an inverse correlation (closure times were prolonged with lower platelet counts). Lowered hematocrit did not affect MEA testing, except for arachidonic acid activation (ASPI), which showed a weak positive correlation (r2 = 0.14). Closure time on PFA-100 testing was inversely correlated with hematocrit for both cartridges. Regression analysis revealed different 95% reference intervals in comparison with originally established intervals for both MEA and PFA-100 in low platelet or hematocrit conditions. Multiple regression analysis of ASPI and both tests on the PFA-100 for combined low platelet and hematocrit conditions revealed that only PFA-100 testing should be adjusted for both thrombocytopenia and anemia. 95% reference intervals were calculated using multiple regression analysis. However, coefficients of determination of PFA-100 were poor, and some variance remained unexplained. Thus, in this pilot study using (multiple) regression analysis, we could establish reference intervals of platelet function in anemia and thrombocytopenia conditions on PFA-100 and in thrombocytopenia conditions on MEA.