Platelet transfusion improves clot formation and platelet function in severely thrombocytopenic haematology patients.

Prophylactic platelet (PLT) transfusion is a common practice in severely thrombocytopenic patients that reduces mortality, but responses to platelet transfusions are variable and difficult to predict in individual patients. In this prospective study, we evaluated the outcome of PLT transfusions in 40 patients with haematological malignancies, linking corrected count increment (CCI) to clot formation and agonist-induced platelet activation after transfusion. The CCI was highly variable between patients and 34% showed no response (1-h CCI < 7,5). Short time since the last PLT transfusion and extended storage time of the PLT product were linked to poor transfusion response, while patient sex, C-reactive protein or the number of chemotherapy cycles prior to transfusion did not influence transfusion outcome. High CCI and good PLT responsiveness to agonist stimulation predicted efficient clot formation in rotational thromboelastometry, but transfusion did not restore poor PLT function in patients to the level of healthy controls. Our study provides new insights into factors affecting PLT transfusion outcome in haematology patients with severe thrombocytopenia, and suggests that the thrombocytopenic environment, or disease-associated factors, may hamper platelet responsiveness.

Cryopreservation of buffy coat derived platelets: Paired in vitro characterization using uncontrolled versus controlled freezing rate protocols.

BACKGROUND: Cryopreserved platelets show a reduced recovery and viability after freezing and thawing including several ultrastructural and phenotypic deteriorations compared with liquid-stored platelets. It is suggested that using Controlled-Rate Freezing (CRF) can reduce variability and optimize the functionality profile for cells. The objective of the study is to compare cellular, metabolic, phenotypic and functional effects on platelets after cryopreservation using different freezing rate protocols. STUDY DESIGN AND METHODS: To evaluate the possible effects of different freezing rate protocols a two-experimental study comparing diverse combinations was tested with a pool and split design. Uncontrolled freezing of platelets in materials with different thermal conductivity (metal vs cardboard) was evaluated in experiment 1. Experiment 2 evaluated uncontrolled vs a controlled-rate freezing protocol in metal boxes. All variables were assessed pre and post cryopreservation. RESULTS: Directly after thawing, no major differences in platelet recovery, LDH, ATP, Δψ, CD62P, CD42b, platelet endothelial cell adhesion molecule and sCD40L were seen between units frozen with different thermal conductivity for temperature. In contrast, we observed signs of increased activation after freezing using the CRF protocol, reflected by increased cell surface expression of CD62P, PAC-1 binding and increased concentration of LDH. Agonist induced expression of a conformational epitope on the GPIIb/IIIa complex and contribution to blood coagulation in an experimental rotational thromboelastometry setup were not statistically different between the groups. CONCLUSION: The use of a uncontrolled freezing rate protocol is feasible, creating a platelet product comparable to using a controlled rate freezing equipment during cryopreservation of platelets.

Platelet function analysed by ROTEM platelet in cardiac surgery after cardiopulmonary bypass and platelet transfusion.

OBJECTIVES: The aim of this study was to investigate whether ROTEM platelet can provide additional information to the traditional ROTEM analysis to guide treatment with platelet transfusions in cardiac surgery and to identify factors triggering platelet administration. BACKGROUND: Platelets play a crucial role in coagulation and haemostasis after cardiac surgery. Excessive bleeding after cardiopulmonary bypass usually requires transfusions of blood products, including platelets. The ROTEM platelet is a novel point-of-care analysis for whole blood. MATERIALS AND METHODS: We included 23 patients scheduled for complex cardiac surgery. ROTEM (in-tem, ex-tem), ROTEM platelet (ARA-tem, ADP-tem and TRAP-tem) and platelet count were analysed before induction of anaesthesia (T0), after cardiopulmonary bypass and protamine reversal (T1) and after platelet transfusion (T2, n = 10). RESULTS: ROTEM and ROTEM platelet tests were all significantly reduced between T0 and T1. ROTEM parameters improved significantly after platelet transfusion. Regarding ROTEM platelet, only TRAP-tem increased between T1 and T2 (P = .008). Factors triggering platelet transfusion were long duration of surgery and time on cardiopulmonary bypass. CONCLUSION: ROTEM platelet with thrombin activation, TRAP-tem, improved significantly, indicating that platelet transfusion may reverse cardiopulmonary bypass-induced platelet dysfunction. Further studies are needed to evaluate whether TRAP-tem can be a valuable analysis regarding indications for transfusion of platelets after extensive cardiac surgery.

Cryopreservation of buffy coat-derived platelet concentrates photochemically treated with amotosalen and UVA light.

BACKGROUND: Cryopreserved platelets (CPPs) are considered a promising approach for extended platelet storage, bridging inventory shortages of conventionally stored platelets. It is unknown if platelet concentrates exposed to photochemical treatment (PCT) with amotosalen and ultraviolet A (UVA) light, to inactivate pathogens, are suitable for freezing. The objective of this study was to analyze potential effects of PCT on CPPs as compared with untreated CPPs. STUDY DESIGN AND METHODS: A total of 12 PCT-treated and 12 untreated platelet units from buffy coats were cryopreserved at -80°C in 5% dimethyl sulfoxide. CPPs of both types were rapidly thawed at 37°C and resuspended in 200 mL fresh plasma. In vitro properties were analyzed prefreezing, postfreezing and thawing, and on Day 1 after thawing. RESULTS: Directly after thawing, no major differences in platelet content, lactase hydrogenase, adenosine triphosphate, mitochondrial membrane potential, CD62P, CD42b, and platelet endothelial cell adhesion molecule were seen between PCT-CPPs and conventional CPPs. Agonist-induced PAC-1 expression and contribution of CPPs to blood coagulation in an experimental rotational thromboelastometry setup were also similar between the groups. On Day 1 after thawing, the CPPs of both types performed less well. The PCT-CPPs tended to be more affected by the freezing process than the conventional CPPs. CONCLUSIONS: PCT-CPPs appeared slightly more susceptible to lesion effects by freezing than conventional CPPs, in particular in assays on Day 1 after thawing, but these differences were small relative to the dramatic effects of the freezing process itself.

Whole blood coagulation assays ROTEM and T-TAS to monitor dabigatran treatment.

BACKGROUND: A rapid and reliable assessment of the dabigatran effect is desirable in dabigatran treated patients with uncontrolled bleeding or before acute surgery. OBJECTIVE: To evaluate how the viscoelastic point-of-care test Rotational thromboelastometry (ROTEM) and Total Thrombus-formation system (T-TAS), which studies thrombus formation under flowing conditions, correlate with dabigatran concentrations in patients with atrial fibrillation (AF). METHOD: ROTEM using the reagents In-tem, Ex-tem, Fib-tem or low tissue factor concentration (TF), and T-TAS with the AR-chip (shear rate 600s-1, representing flow in large arteries) were investigated in whole blood samples. Plasma concentrations were determined by mass spectrometry (LC-MS/MS) at trough and post-dose in 30 patients on dabigatran 150mg BID. RESULTS: Median plasma dabigatran concentrations at trough were 86ng/mL (29-150) and post-dose (2.8h after ingestion) 175ng/mL (67-490). The ROTEM clotting time (CT) correlated strongly with dabigatran concentrations when activated with the reagents Ex-tem (r=0.92, p<0.01) and Fib-tem (r=0.93, p<0.01), while with In-tem and low TF the correlation was weaker (r=0.72 and r=0.36, p<0.01). There were significant but weaker correlations also between dabigatran concentrations and T-TAS variables (r-values 0.39-0.41, p<0.01), aPTT (r=0.70, p<0.01) and PT-INR (r=0.43, p<0.01) respectively. CONCLUSIONS: ROTEM Ex-tem and Fib-tem CT shows a strong correlation with dabigatran concentrations in real-life AF-patients, and results are obtained within minutes. This could make ROTEM useful in acute situations. T-TAS detect differences in hemostasis caused by dabigatran, but the relationships to plasma concentrations of dabigatran are weaker than for ROTEM CT with the settings used in this study.

Haemostasis monitored in stored red blood cells, plasma and platelet concentrates in the proportion of 4 :  4 :  1 diluted with crystalloids and colloids.

The aim of this in-vitro study was to evaluate haemostasis analysed with thromboelastometry and blood gas and blood count variables, in stored blood components and the effects after dilution with Ringer[Combining Acute Accent]s acetate, albumin and hydroxyethyl starch (HES). Aliquots from stored red blood cells, plasma and platelet concentrates were mixed in the proportion of 4 : 4 : 1 and analysed with rotational thromboelastometry (ROTEM), blood count [haemoglobin (Hb), haematocrit, platelet count] and blood gas (pH, calcium, sodium, potassium, glucose levels). The blood mix was thereafter diluted 20 and 33% with Ringer's acetate, albumin or HES. The stored blood component mix in a ratio of 4 : 4 : 1 had a low pH (7.11 ±â€Š0.03, mean ±â€Šstandard deviation), nonmeasurable calcium level, and high concentrations of sodium, potassium and glucose but ROTEM curves within normal range after recalcification. With Ringer's acetate dilution, the ROTEM variables changed almost linearly with increasing dilution volume. When albumin was used in the 33% dilution, the clot firmness of the fibrin clot (FibTEM) was further reduced, and with HES dilution, there was a pronounced impairment. The stored blood mix had a low pH and calcium level, both of which might have a significant influence on the coagulation process but normal ROTEM curves after recalcification. Dilution with Ringer's acetate and albumin resulted in moderate deterioration, while dilution with HES showed severely impaired haemostasis.

Constructing a population-based research database from routine maternal screening records: a resource for studying alloimmunization in pregnant women.

BACKGROUND: Although screening for maternal red blood cell antibodies during pregnancy is a standard procedure, the prevalence and clinical consequences of non-anti-D immunization are poorly understood. The objective was to create a national database of maternal antibody screening results that can be linked with population health registers to create a research resource for investigating these issues. STUDY DESIGN AND METHODS: Each birth in the Swedish Medical Birth Register was uniquely identified and linked to the text stored in routine maternal antibody screening records in the time window from 9 months prior to 2 weeks after the delivery date. These text records were subjected to a computerized search for specific antibodies using regular expressions. To illustrate the research potential of the resulting database, selected antibody prevalence rates are presented as tables and figures, and the complete data (from more than 60 specific antibodies) presented as online moving graphical displays. RESULTS: More than one million (1,191,761) births with valid screening information from 1982-2002 constitute the study population. Computerized coverage of screening increased steadily over time and varied by region as electronic records were adopted. To ensure data quality, we restricted analysis to birth records in areas and years with a sustained coverage of at least 80%, representing 920,903 births from 572,626 mothers in 17 of the 24 counties in Sweden. During the study period, non-anti-D and anti-D antibodies occurred in 76.8/10,000 and 14.1/10,000 pregnancies respectively, with marked differences between specific antibodies over time. CONCLUSION: This work demonstrates the feasibility of creating a nationally representative research database from the routine maternal antibody screening records from an extended calendar period. By linkage with population registers of maternal and child health, such data are a valuable resource for addressing important clinical questions, such as the etiological significance of non-anti-D antibodies.

Fetal hemolytic anemia and intrauterine death caused by anti-M immunization.

BACKGROUND: Antibodies with anti-M specificity are detected in 10 percent of pregnant women with a positive antibody screen, but anti-M is only rarely associated with hemolytic anemia in the fetus. STUDY DESIGN AND METHODS: This study reports on three pregnancies in one family that all resulted in severe fetal anemia. The first fetus died in utero with hydrops fetalis during the 20th gestational week and the second child was delivered after 28 weeks of gestation with hydrops fetalis and a hemoglobin level of 16 g per L whereas the third affected child was treated with intrauterine red cell (RBC) transfusions before delivery at 28 weeks of gestation. RESULTS: The direct antiglobulin test was negative but anti-M in a low titer was detected through the three pregnancies, and its clinical relevance, which initially was uncertain, was confirmed by pronounced in vivo hemolysis in maternal blood of chromate ((51)Cr)-labeled M+ RBCs and normal survival of (51)Cr labeled M- RBCs. CONCLUSION: It is concluded that anti-M immunization in a few cases may cause severe fetal hemolytic anemia and intrauterine death. It remains to be elucidated why a normally clinically insignificant antibody is this aggressive in a small proportion of cases. Because the condition is treatable, anti-M must be considered as a possible cause of fetal anemia and intrauterine death.

Characterization of red cell autoantibodies in consecutive DAT-positive patients with relation to in vivo haemolysis.

The clinical symptoms of patients with autoimmune haemolytic anaemia (AIHA) may range from no symptoms at all to severe haemolysis with life-threatening anaemia. The aim of the present study was to characterize the autoantibodies serologically and to evaluate their activity in vitro in monocyte monolayer assay (MMA) in consecutive direct antiglobulin test (DAT)-positive patients from a haematological department. We also aimed to evaluate the monocyte production of cytokines in vitro and relate all findings to in vivo haemolysis. Twenty-nine patients with positive DAT were included in the study and clinical characteristics were documented. The patients were divided into three groups based on laboratory parameters, severe haemolysis, moderate haemolysis and no sign of haemolysis. Severe haemolysis was related to a strongly positive DAT and positive results in MMA. When eluates were analysed in MMA, a positive result was associated with a higher concentration in the supernatants of interleukin (IL)-8 after 6 h incubation. We conclude that MMA activity and in vitro cytokine production may reflect the in vivo activity of autoantibodies. This may be of importance in understanding the mechanisms of haemolysis and in predicting the harmfulness of antibodies, especially when blood transfusions are needed.