Joyce Curvers

Effects of skin disinfection method, deviation bag, and bacterial screening on clinical safety of platelet transfusions in the Netherlands

BACKGROUND:  Bacterial contamination of blood products is a great hazard for development of fatal transfusion reactions. Bacterial screening of platelet concentrates (PC) by aerobic and anaerobic culturing (BacT/ALERT, bioMérieux) was introduced in the Netherlands in October 2001.

Shedding of procoagulant microparticles from unstimulated platelets by integrin-mediated destabilization of actin cytoskeleton

Platelet activation by potent, Ca2+‐mobilizing agonists results in shedding of microparticles that are active in coagulation. Here we show that platelets under storage produce procoagulant microparticles in the absence of agonist. Microparticle formation by resting platelets results from αIIbβ3 signaling to destabilization of the actin cytoskeleton in the absence of calpain activation. Integrin‐mediated spreading of platelets over fibrinogen similarly results in microparticle formation. After transfusion of stored platelet preparations to thrombocytopenic patients, the microparticles contribute to coagulant activity in vivo.

Effects of storage‐induced platelet microparticles on the initiation and propagation phase of blood coagulation

Platelets shed microparticles, which support haemostasis via adherence to the damaged vasculature and by promoting blood coagulation. We investigated mechanisms through which storage‐induced microparticles might support blood coagulation. Flow cytometry was used to determine microparticle number, cellular origin and surface expression of tissue factor (TF), procoagulant phosphatidylserine (PtdSer) and glycoprotein (GP) Ib‐α. The influence of microparticles on initiation and propagation of coagulation were examined in activated factor X (factor Xa; FXa) and thrombin generation assays and compared with that of synthetic phospholipids. About 75% of microparticles were platelet derived and their number significantly increased during storage of platelet concentrates. About 10% of the microparticles expressed functionally active TF, as measured in a FXa generation assay. However, TF‐driven thrombin generation was only found in plasma in which tissue factor pathway inhibitor (TFPI) was neutralised, suggesting that microparticle‐associated TF in platelet concentrates is of minor importance. Furthermore, 60% of all microparticles expressed PtdSer. In comparison with synthetic procoagulant phospholipids, the maximal rate of thrombin formation in TF‐activated plasma was 15‐fold higher when platelet‐free plasma was titrated with microparticles. This difference could be attributed to the ability of microparticles to propagate thrombin generation by thrombin‐activated FXI. Collectively, our findings indicate a role of microparticles in supporting haemostasis by enhancement of the propagation phase of blood coagulation.

Acquired APC resistance and oral contraceptives : differences between two functional tests

Resistance to activated protein C (APC) is often associated with a mutation in factor V (factor VLeiden). Individuals without factor VLeiden who exhibit a response in functional APC‐resistance tests similar to that of carriers of factor VLeiden are considered to be acquired APC resistant. This phenomenon is particularly observed in women using oral contraceptives (OC).

In vitro comparison of platelet storage in plasma and in four platelet additive solutions, and the effect of pathogen reduction: a proposal for an in vitro rating system.

Background  The introduction of platelet (PLT) additive solutions (PASs) and pathogen reduction (PR) technologies possibly allow extension of PLT shelf life. It was our aim to compare in vitro quality of leucocyte‐reduced PLT concentrates (PCs) stored in various PASs, including PR, with those in plasma during 8 days of storage. The study was performed in four blood centres where each tested four conditions.

Decreased responsiveness and development of activation markers of PLTs stored in plasma

BACKGROUND:  Circulating PLTs have a low activation state and high responsiveness, which ensures adequate hemostatic activity at sites of vessel wall damage. PLTs collected for transfusion purposes preferably have retained these properties to restore impaired hemostasis with thrombocytopenia.

Platelet ADP response deteriorates in synthetic storage media

BACKGROUND:  During storage under blood bank conditions, platelets (PLTs) are known to secrete ADP. PLT stimulation by ADP results in refractoriness to restimulation, making this response one of the most unstable PLT reactions. The goal of this study was to evaluate the ADP‐induced responses of PLTs stored in full plasma or in plasma and additive solution (AS).

Increased thrombin generation and fibrinogen level after therapeutic plasma transfusion: relation to bleeding.

In a clinical setting, fresh frozen plasma (FFP) is transfused to diluted patients with complicated surgery or trauma, as guided by prolonged conventional coagulation times or low fibrinogen levels. However, the limited sensitivity of these coagulation tests may restrict their use in measuring the effect of transfusion and hence predicting the risk of perioperative bleeding. We used the more sensitive, calibrated automated thrombogram (CAT) method to evaluate the result of therapeutic FFP transfusion to 51 patients with dilutional coagulopathy. Thrombin generation was measured in pre- and post-transfusion plasma samples in the presence of either platelets or phospholipids. For all patients, the transfusion led to higher plasma coagulation factor levels, a shortened activated partial thromboplastin time, and a significant increase in thrombin generation (peak height and endogenous thrombin potential). Interestingly, thrombin generation parameters and fibrinogen levels were higher in post-transfusion plasmas from patients who stopped bleeding (n = 32) than for patients with ongoing bleeding (n = 19). Plasmas from 15 of the 19 patients with ongoing bleeding were markedly low in either thrombin generation or fibrinogen level. We conclude that the thrombin generation method detects improved haemostatic activity after plasma transfusion. Furthermore, the data suggest that thrombin generation and fibrinogen are independent determinants of the risk of perioperative bleeding in this patient group.

Flow cytometric measurement of CD62P (P-selectin) expression on platelets : a multicenter optimization and standardization effort

BACKGROUND: One of the variables to determine the quality of platelets (PLTs) in vitro is measurement of CD62P expression. Different protocols are in use, however, making comparison of results virtually impossible. It was our aim to develop a uniform CD62P protocol that would yield comparable results in various laboratories.

Novel methodology for assessment of prophylactic platelet transfusion therapy by measuring increased thrombus formation and thrombin generation.

Currently, patients developing severe thrombocytopenia during chemotherapy treatment are prophylactically transfused with platelets. We developed two platelet function tests to report the improved haemostasis in the transfused patients, which were capable of detecting aberrant responsiveness of the platelets after transfusion. First, in a whole‐blood flow test, platelet adhesion and thrombus formation were determined under high‐shear flow conditions. Second, the procoagulant function of platelets was assayed in platelet‐rich plasma by measurement of thrombin generation. Experimental conditions were established, where flow‐induced adhesion and thrombin generation test parameters increased semi‐linearly with the platelet concentration, and informed on the activation properties of platelets. The transfusion effects were evaluated for 38 thrombocytopenic patients, who were transfused with platelets stored in plasma or in synthetic medium (platelet additive solution II). In most but not all patients, transfusion resulted in increased adhesion and thrombus formation, as well as in improved platelet‐dependent coagulation. Taken together, the increase in platelet count after transfusion explained 57% of the overall improvement in platelet function. In acute graft‐versus‐host disease, thrombus formation was normal, while platelet‐dependent coagulation was higher than expected. We conclude that assessment of flow‐induced adhesion and thrombin generation in acquired thrombocytopenia adequately determines the improved haemostatic activity by transfused platelets.