Rick Wetzels

Rapid and correct prediction of thrombocytopenia and hypofibrinogenemia with rotational thromboelastometry in cardiac surgery.

OBJECTIVES In the present study, the authors have investigated whether rotational thromboelastometry (ROTEM) could predict thrombocytopenia and hypofibrinogenemia in cardiac surgery using the clot amplitude after 5 minutes (A5). Another parameter, PLTEM, in which the contribution of fibrinogen is eliminated by subtracting a fibrin-specific ROTEM test (FIBTEM) from an extrinsically-activated ROTEM test (EXTEM), was investigated. Furthermore, the turnaround time of ROTEM was compared to conventional laboratory tests. DESIGN Prospective cohort study. SETTING Single academic medical center. PARTICIPANTS Ninety-seven patients undergoing cardiac surgery between July 2011 until August 2012. INTERVENTIONS The correlations between EXTEM/FIBTEM A5, A10, and maximal clot formation (MCF), EXTEM/PLTEM (A5/A10, and MCF) and platelet count, and FIBTEM (A5/A10, and MCF) and fibrinogen were evaluated using the Pearsons correlation coefficient and receiver-operating characteristic curves. Turnaround times of ROTEM tests and conventional laboratory tests were assessed in the central laboratory. MEASUREMENTS AND MAIN RESULTS EXTEM A5 and FIBTEM A5 showed an excellent correlation with A10 (R:0.99/1.00) and MCF (R:0.97/0.99). The correlation between EXTEM A5 and platelet count (R:0.74) was comparable with the correlation of A10 (R:0.73) and MCF (R:0.70) with platelet count. FIBTEM A5 predicted fibrinogen levels (R:0.87) as well as A10 (R:0.86) and MCF (R:0.87). PLTEM A5 (R:0.85) correlated better with platelet count than EXTEM A5 (R:0.74; p = 0.04) and showed significantly better area under the curve values than EXTEM for predicting thrombocytopenia (A5 p = 0.012, A10 p = 0.019). Turnaround time for ROTEM tests, 12 minutes, was comparable with emergency requests for platelet count, 13 minutes, and shorter than emergency requests for fibrinogen levels, 37 minutes. CONCLUSIONS Implementation of PLTEM and FIBTEM A5 in ROTEM-guided transfusion protocols may improve transfusion management.

Gradual increase in thrombogenicity of juvenile platelets formed upon offset of prasugrel medication

In patients with acute coronary syndrome, dual antiplatelet therapy with aspirin and a P2Y12 inhibitor like prasugrel is prescribed for one year. Here, we investigated how the hemostatic function of platelets recovers after discontinuation of prasugrel treatment. Therefore, 16 patients who suffered from ST-elevation myocardial infarction were investigated. Patients were treated with aspirin (100 mg/day, long-term) and stopped taking prasugrel (10 mg/day) after one year. Blood was collected at the last day of prasugrel intake and at 1, 2, 5, 12 and 30 days later. Platelet function in response to ADP was normalized between five and 30 days after treatment cessation and in vitro addition of the reversible P2Y12 receptor antagonist ticagrelor fully suppressed the regained activation response. Discontinuation of prasugrel resulted in the formation of an emerging subpopulation of ADP-responsive platelets, exhibiting high expression of active integrin αIIbβ3. Two different mRNA probes, thiazole orange and the novel 5′Cy5-oligo-dT probe revealed that this subpopulation consisted of juvenile platelets, which progressively contributed to platelet aggregation and thrombus formation under flow. During offset, juvenile platelets were overall more reactive than older platelets. Interestingly, the responsiveness of both juvenile and older platelets increased in time, pointing towards a residual inhibitory effect of prasugrel on the megakaryocyte level. In conclusion, the gradual increase in thrombogenicity after cessation of prasugrel treatment is due to the increased activity of juvenile platelets.

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

Abstract 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.

Screening for platelet function disorders with Multiplate and platelet function analyzer

Abstract Light transmission aggregation (LTA) is the gold standard for the diagnosis of platelet function disorders (PFDs), but it is time-consuming and limited to specialized laboratories. Whole-blood impedance aggregometry (Multiplate) and platelet function analyzer (PFA) may be used as rapid screening tools to exclude PFDs. The aim of this study is to assess the diagnostic performance of Multiplate and PFA for PFDs, as detected by LTA.Data from preoperative patients, patients referred to the hematologist for bleeding evaluation, and patients with a diagnosed bleeding disorder were used. PFDs were defined as ≥2 abnormal LTA curves. Diagnostic performance of Multiplate and PFA for detecting PFDs was expressed as sensitivity and specificity. The ability of Multiplate agonists and PFA kits to detect corresponding LTA curve abnormalities was expressed as area under the receiver operating characteristic curve. Prevalence of PFDs was 16/335 (4.8%) in preoperative patients, 10/54 (18.5%) in referred patients, and 3/25 (12%) in patients with a diagnosed bleeding disorder. In preoperative and referred patients, the sensitivity of Multiplate and PFA for detecting mild PFDs varied between 0% and 40% and AUCs for detecting corresponding LTA curve abnormalities were close to 0.50. In patients with a diagnosed bleeding disorder, both assays could detect Glanzmann thrombasthenia (GT) with sensitivity of 100% and AUCs of 0.70–1.00. Multiplate and PFA cannot discriminate between preoperative and referred patients with and without mild PFDs, meaning that they cannot be used as screening tests to rule out mild PFDs in these populations. Both Multiplate and PFA can detect GT in previously diagnosed patients.

Increased coagulation and fibrinolytic potential of solvent-detergent plasma: a comparative study between Omniplasma and fresh frozen plasma.

In this study, differences in levels of proteins involved in coagulation and fibrinolysis were compared between fresh frozen (quarantine plasma) and Omniplasma. Furthermore, thawing conditions and plasma stability after thawing were studied.

Early platelet recovery following cardiac surgery with cardiopulmonary bypass

Abstract Coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) is frequently associated with low platelet count (PC) and disturbed platelet function (PF). While PC is easy to measure, PF is more difficult to assess. Moreover, the time-related platelet dysfunction and recovery after CPB is not fully elucidated. Platelet dysfunction could lead to bleeding but also to coronary graft failure. Laboratory tests could provide more insights into PF after CABG. The aim of the current study was to investigate the time-related PF induced by CPB. Blood samples of 20 patients with a preoperative PC of more than 250 × 109/L were collected before incision, after weaning from CPB, and 24 h postoperative. Platelet contribution to coagulation was quantified by PLTEM (calculated by means of EXTEM and FIBTEM results). PF was assessed by multiple electrode impedance aggregometry (MEIA) in whole blood and by light transmission aggregometry (LTA) in platelet-rich plasma after stimulation with arachidonic acid (AA), adenosine diphosphate, collagen, and thrombin-receptor-activating peptide. LTA and MEIA analysis demonstrated significant platelet dysfunction after CPB, with partial recovery within 24 h after surgery. AA-induced platelet aggregation increased to higher levels within 24 h after surgery compared to baseline values as measured by LTA. PLTEM maximum clot firmness remained unchanged throughout the study. Correlation analyses revealed that MEIA and rotational thromboelastometry (ROTEM), but not LTA, were dependent on PC and hematocrit. No correlations were found between LTA, MEIA, ROTEM, PC, and clinical outcome parameters. Our results demonstrate a reversible platelet dysfunction recovering within 24 h after CPB. Interestingly, AA-induced platelet aggregation increases to higher levels during the first 24 h postoperatively, which might be important for early initiation of antiplatelet therapy after CABG. MEIA as POC test is able to detect platelet dysfunction during cardiac surgery with a PC of ≥150 × 109/L.

OC-08 - Multiple functional defects in platelets from thrombocytopenic cancer patients undergoing chemotherapy.

INTRODUCTION Severe thrombocytopenia (≤50×10(9) platelets/L) is often the consequence of hematological malignancies and intensive chemotherapy. The risk of clinically significant bleeding is increased in these patients, despite the use of prophylactic platelet transfusions. The fact that there is no clear correlation between the platelet count and the risk of hemorrhage, suggests that there are other contributing factors. The contribution of impairments in platelet and coagulant function remains poorly understood. AIM In patients with chemotherapy-induced thrombocytopenia due to hematological malignancies, we evaluate platelet and coagulant functions and determine the effects of platelet transfusion. Ultimately, we can identify specific hemostatic factors that aid in the prediction of bleeding. MATERIALS AND METHODS In total 58 patients were included and blood was collected before and, if indicated (≤10×10(9) platelets/L), 1 hour after transfusion with platelet concentrate. Platelet function was assessed using flow cytometry by determining: 1) integrin αIIbβ3 activation (PAC-1 antibody), 2) P-selectin expression (anti-P-selectin antibody), 3) phosphatidylserine exposure (Annexin-V) and 4) intracellular calcium (Fluo-4 AM). Factor levels were determined in plasma. Thrombus and fibrin formation was assessed by perfusion of whole blood over a collagen-tissue factor surface at a shear rate of 1,000 s-1. RESULTS Platelets from the thrombocytopenic patients before transfusion showed markedly reduced integrin αIIbβ3 activation and P-selectin expression in response to thrombin, collagen-related peptide and ADP, compared to healthy donor platelets. Also, agonist-induced intracellular calcium fluxes were greatly reduced. However, calcium fluxes with thapsigargin, a SERCA pump inhibitor, were similar in patient and control platelets, suggesting a normal calcium store content in the patient platelets. Furthermore, phosphatidylserine exposure was increased in unstimulated patient platelets compared to control platelets (8.2 vs. 1.8%, p<0.0001). Coagulation factor levels were within the normal range, with the exception of von Willebrand factor and fibrinogen levels, which were elevated. Platelet transfusion partly recovered the platelet integrin αIIbβ3 activation and P-selectin expression induced by agonists. Platelet deposition (6.7 vs. 1.7%, p<0.0001) and fibrin formation (7.6 vs. 0.9%, p=0.0005) under flow conditions were substantially improved after platelet transfusion. CONCLUSIONS Platelets from cancer patients undergoing chemotherapy appear to display impaired functional responses to activating stimuli. Platelet transfusion partly restores these functional defects, resulting in improved thrombus and fibrin formation.

Preoperative screening for bleeding disorders: A comprehensive laboratory assessment of clinical practice

Essentials Patients with bleeding disorders are at risk of operative bleeding, but screening for these disorders is challenging. Patients with and without bleeding symptoms on a guideline‐based screening questionnaire were included and hemostatically phenotyped. The questionnaire could not differentiate between patients with and without hemostatic abnormalities. The discriminative power of the PT, aPTT, TT, Euglobulin lysis time, PFA, and the ISTH‐BAT was also limited.

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 β 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.