Summary.The need for quality control of leucoreduction of blood products has led to the development of various methods to count low levels of residual leucocytes. We compared five platforms side-by-side: the Nageotte haemocytometer and four based on fluorescent staining of nuclei: two flowcytometers (Beckman Coulter, BD Biosciences) with methods based on counting beads, a volumetric flow cytometer (Partec) and the microvolumic fluorimeter imagn2000 (BD Biosciences), all according to their manufacturers' recommended methods. Analysis of doublefiltered red cell concentrates (RCCs) and platelet concentrates (PCs), spiked with various numbers of leucocytes, revealed good linearity for all methods over the range of 1´6±32´7 leucocytes/ml, all with r 2 . 0´99. At the rejection level of leucocyte-reduced blood components, i.e. 1 Â 10 6 per unit corresponding with approximately 3´3 leucocytes/ml, the Nageotte haemocytometer had low accuracy (0% for RCCs, 56% for PCs), and was relatively imprecise [coefficient of variance (CV) of 34% and 30% respectively]. The Partec flow cytometer gave good results for RCCs (accuracy 67%, CV 22%), but not for PCs (accuracy 0%, CV 25%). The imagn2000 had an accuracy of 44% for RCCs and 89% for PCs, but the precision was variable (CV 32% for RCCs, 15% for PCs). The best results were obtained with the Beckman Coulter (RCCs: accuracy 86%, CV 13%, PCs: accuracy 67%, CV 16%), and BD Biosciences platforms (RCCs: accuracy 100%, CV 10%; PCs: accuracy 89%, CV 11%). We conclude that, at the rejection level of 1 Â 10 6 leucocytes per unit, the widely used Nageotte haemocytometer performs poorly in terms of inaccuracy and imprecision, and that both counting-bead-based, flow cytometric methods performed best.
In the present study we have investigated whether the collagen receptor a2b1 (GPIa-IIa; GP, glycoprotein) regulates protein tyrosine phosphorylation in platelets directly through activation of tyrosine kinases or indirectly through modification of the response to GPVI. The interaction of collagen with a2b1 was inhibited in two distinct ways, using the metalloprotease jararhagin, which cleaves the b1 subunit, or the antibody P1E6 which competes with binding of collagen to the integrin. The two inhibitors caused a shift to the right in the collagen concentration response curves for protein tyrosine phosphorylation and platelet activation consistent with a causal relationship between the two events. There was no change in the overall pattern of tyrosine phosphorylation in response to high concentrations of collagen in the presence of a2b1 blockade demonstrating that the integrin is not required for this event. In contrast, jararhagin and P1E6 had a small, almost negligible inhibitory effect against responses to the GPVI-selective agonist collagen-related peptide (CRP) and the G protein-coupled receptor agonist thrombin. Crosslinking of a2b1 in solution or by adhesion to a monolayer using a variety of antibodies to either subunit of the integrin did not induce detectable protein tyrosine phosphorylation in whole cell lysates. The snake venom toxin trimucytin-stimulated a similar pattern of tyrosine phosphorylation to that induced by crosslinking of GPVI which was maintained in the presence of jararhagin. Trimucytin may therefore induce activation via GPVI rather than a2b1 as previously thought. These observations show that the integrin a2b1 is not required for regulation of tyrosine phosphorylation by collagen.Keywords: collagen; GPIa-IIa; GPVI; platelets; tyrosine phosphorylation.When the subendothelium lining is damaged, platelets adhere to newly exposed collagen fibres and undergo activation leading to thromboxane A 2 formation, aminophospholipid exposure, granule secretion and platelet aggregation. Several surface glycoproteins have been implicated as collagen receptors, including glycoprotein (GP) IV (also known as CD36), GPVI, a recently cloned protein of 65 kDa protein, a 85/90 kDa protein and the integrin a2b1. GPVI and a2b1 are believed to play pivotal roles in the interaction of platelets with collagen as patients deficient in the expression of either protein have impaired collagen±platelet interactions and mild bleeding disorders. The consensus is that the integrin a2b1 plays a critical role in adhesion, whilst GPVI is essential for activation. There is evidence for minor roles of other receptors in these responses (reviewed in [1±3]).GPVI is associated with the Fc receptor g-chain (FcR g-chain) on the platelet surface [4,5], and crosslinking leads to phosphorylation of the FcR g-chain on a sequence known as an immunoreceptor tyrosine-based activation motif (ITAM) [6], probably by a Src kinase [7,8]. This enables binding and activation of the tyrosine kinase Syk, starting a chain of events that leads to t...
The WBC-reduced PCs conformed to specifications. These WBC-reduced PCs could be stored at least 5 days with maintenance of pH, and they gave sufficient increments after transfusion to patients.
SummaryBased on in vitro studies, thrombin-activatable fibrinolysis inhibitor (TAFI) has been hypothesized as a link between coagulation and fibrinolysis, but the physiological role of TAFI in vivo has not yet been established. To anticipate on the availability of genetically modified mouse models, we studied the endogenous expression of TAFI in mice. Functional TAFI was found in mouse plasma. TAFI mRNA was only detectable in the liver, showing a hepatocyte-specific expression with a pericentral lobular distribution pattern. The murine TAFI cDNA was cloned and sequenced. The deduced amino acid sequence revealed that murine TAFI is highly identical to human TAFI. The murine cDNA was stably expressed and the activated recombinant protein was functionally active; it converted the substrate hippuryl-arginine, and prolonged the clot lysis time of TAFI depleted plasma. We conclude that mice have functional TAFI in plasma, which is highly similar to human TAFI. Therefore, genetically modified mice may provide useful models to study the role of TAFI in vivo. Abbreviations: TAFI, thrombin-activatable fibrinolysis inhibitor; CPI, carboxypeptidase inhibitor from potato tubers; CPN, carboxypeptidase N; t-PA, tissue-type plasminogen activator; PPACK, H-D-Phe-Pro-Arg-chloromethylketone.
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