Background. There is compelling evidence that blood-borne tissue factor that is predominantly found on circulating microparticles (MPs) plays an important role in both, cancer biology and organ and stem-cell transplantation (SCT). Therefore, we hypothesized that numbers of tissue factor bearing MPs might be associated with complications and outcome in allogeneic SCT (allo-SCT). Materials and Methods. In a prospective study, we enumerated total, platelet, endothelial, and tissue factor bearing MPs in plasma samples obtained from up to 60 patients with hematologic diseases at different time-points during the course of allo-SCT by flow cytometry. Patient- and transplant-related risk factors were included in statistical analysis. Results. Mean follow-up time was 968 days (0–1981 days). Thirty-four (56.7%) patients died, 17 due to transplant-related mortality (28.3%). High numbers of tissue factor positive MPs more than 500/&mgr;L before conditioning were predictive for shorter overall survival (P=0.017, hazard ratio=4.5) in multivariate analysis. This was mainly caused by an increase in transplant-related mortality (P=0.010, hazard ratio=11.0) with cumulative incidences at 1 year of 68.8% compared with patients with lower values (20.1%; P=0.002). Conclusions. Tissue factor bearing MPs might be useful biomarkers for risk stratification in allo-SCT patients and further studies should investigate their origin, functional properties, and optimal cut-off values.

The aetiology of anti-factor VIII (FVIII) autoantibody formation in acquired haemophilia remains unknown. We hypothesised that encounter of antigenically different, allogeneic FVIII may challenge inhibitor formation after presentation on MHC class II. Eighteen consecutive cases with acquired haemophilia were enrolled (nine females, nine males). A control group comprised 50 male and 50 female healthy blood donors. The coding region of the FVIII gene and the HLA-DRB1 genotype were studied. The presentation of foreign FVIII variants on the patient's MHC class II alleles was predicted using SYFPEITHI algorithm. A rare FVIII variant (E2004K) was found in one patient with acquired haemophilia after massive transfusion; the 2004 K allele was predicted to be presented on the patient's HLA-DRB1*0101. Moreover, distribution of a polymorphism (D1241E) was significantly skewed comparing patients and controls. Three of three patients with transfusion-associated disease carried 1241D in homozygous or hemizygous form and were predicted to present 1241E (foreign), but not 1241D (self), on their HLA-DRB1*0301. Therefore, encounter of 1241E may result in the presentation of a new T cell epitope in these patients. The same conditions were not found in any patient with acquired haemophilia of other causes. The expected frequency in the general Caucasoid population undergoing transfusion is 3% to 4%. In conclusion, encounter of variant allogeneic FVIII presented on a suitable MHC background could be a risk factor for inhibitor formation.

Consent regarding the correct processing and storage of blood microparticles is lacking and different protocols for the freeze-thaw cycle exist. Therefore, three different thawing procedures were evaluated regarding their influence on recovery and composition of microparticles. Microparticles were prepared by TRAP-6 or A23187 stimulation of platelet-rich plasma from smokers and nonsmokers (n = 8), from an endothelial cell line or directly obtained from platelet-free plasma of septic patients (n = 5). After snap-freezing in liquid nitrogen platelet-free samples were thawed at 37 degrees, at room temperature or on ice and staining of microparticles was carried out with Annexin V-Cy5 as well as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Microparticle concentrations were determined by means of Trucount tubes. Recovery of platelet microparticles was significantly reduced when samples were thawed on ice (P = 0.001 for all antigens) compared with the two other techniques (P = 0.6 for 37 degrees and P = 0.7 for room temperature, respectively) whereas microparticles of endothelial origin appeared to be less influenced. There was a strong trend towards altered microparticle composition as microparticle counts detected by CD41 staining showed a stronger decrease on ice than Annexin V enumeration (P = 0.07). For microparticle detection thawing of snap-frozen, platelet-free plasma samples should be carried out at room temperature or at 37 degrees C in a water bath but not on ice.

Background: The characterisation and quantification of cell-derived microparticles (MPs) using flow cytometry are often complicated by a tow staining intensity and a non-discrete signal pattern of many cell surface antigens. Fluorescence-labelled isotype controls (ICs) are commonly used to set limits for the discrimination of antigen positive vs. negative events.Objectives: The influence of different ICs on the characterisation and quantification of MPs was studied. Antigen negative MPs stained with an antibody of interest were evaluated as an alternative control.Methods: MPs were prepared from platelets, endothelial cell Lines and leucemic cell lines and stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) Labelled antibodies or isotype controls.Results are given as the mean fluorescence intensity (MFI) or percentage of "false-positive" events above a fluorescence intensity >1. Results: Using identical instrument settings, seven different ICs (FITC-conjugates N = 3, PE-conjugatesN=4) resulted in a wide range of MFI and percentage of positive events with a mean coefficient of variation (CV) of 0.77. Instead, NMPs showed less variability with a mean CV of 0.50 and allowed a reliable and reproducible quantification of MPs when set as controls with <2% false-positive events above an FI >1. As a result, the expression of certain antigens (e.g. CD62P) was lower compared to previous reports in the literature.Conclusions: Diversity in the staining intensity of isotype controls is a potential source of error in the characterisation and quantification of MPs by flow cytometry. The use of antigen negative MPs to adjust instrument settings is suggested. (C) 2008 Elsevier Ltd. ALL rights reserved.

Acquired von Willebrand's syndrome (AVWS) is a rare but probably underdiagnosed hemorrhagic disorder often associated with hematological or cardiovalvular disorders. Diagnostic workup remains challenging, particularly in patients with normal or increased von Willebrand factor antigen (Ag) and ristocetin cofactor (RCo). Here, we present a retrospective single-center study of 35 patients diagnosed with AVWS based on (i) a history of recent onset of bleeding, (ii) a negative family history of von Willebrand's disease, and (iii) abnormal plasma VWF multimers. AVWS was associated with monoclonal gammopathy (n=11), cardiovalvular disorders (n=16), or other conditions (n=8) including myeloproliferative and autoimmune disorders. The PFA-100® screening test was inconclusive due to anemia (hematocrit <30 %) or thrombocytopenia (<100/nl) in 10 patients (29 %); prolonged closure times were observed using collagen/epinephrine and collagen/adrenalin in 20 of 25 (80 %) and 18 of 25 (72 %) patients, respectively. Factor VIII:C was reduced below 50 IU/dl in 7 of 35 patients (20 %). VWF Ag and RCo were reduced below 50 IU/dl in 8 patients (23 %). VWF Ag and RCo were normal or increased in all patients with cardiovalvular disease and in four of eleven patients with gammopathy. Median VWF Ag was higher in cardiovalvular disease (median 202 IU/dl, range 90 to 608) compared to gammopathy (median 31 IU/dl, range 8 to 468, p<0.02 by Mann Whitney U test). Of 27 patients with normal or increased VWF Ag and RCo, 12 (44 %) had a reduced collagen binding activity (CBA) or CBA to Ag ratio <0.7; 10 (37 %) had a borderline CBA ratio between 0.7 and 0.8; 5 (19 %) had a normal CBA >0.8. A normal or increased VWF Ag together with a CBA ratio >0.7 was observed both in patients with cardiovalvular disease (n=9), gammopathy (n=2) and other disorders (n=4). However, in all patients the largest VWF multimers were decreased (n=14) or absent (n=21). In conclusion, no single test was sufficient to detect all cases of AVWS. The PFA-100® test is of limited use in this population because of its limitation in anemia or thrombocytopenia and because of its low sensitivity. A significant number of patients present with a normal or increased VWF Ag and RCo as well as a CBA ratio >0.7 emphasizing the importance of multimer analysis in all patients with suspected AVWS.