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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.

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.

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2263 Poster Board II-240 Objective There is compelling evidence that increased levels of Tissue-factor (TF) and TF-bearing microparticles (MPs) as well as P-selectin and its receptor P-selectin glycoprotein ligand 1 (PSGL-1) play an important role in disease progression, angiogenesis and cancer-related coagulopathy in solid and hematologic malignancies. We therefore hypothesized that numbers of circulating TF- and PSGL-1 bearing MPs might have substantial influence on outcome in allogeneic stem cell transplantation (alloSCT). Patients and Methods Blood samples were obtained from 33 patients with different hematologic diseases (AML=19, ALL=5, CML/MPS=2, CLL/lymphoma=2, myeloma=2, aplastic anemia=2) during the course of alloSCT at distinct time points: at admission (“start”), during conditioning therapy before anti-thymocyte globulin (ATG) infusion (“pre-ATG”), on day 3 of ATG infusion (“during-ATG”), before transplantation (“pre-Tx”), 1 h after transplantation (“post-Tx), on day 1 after transplantation (“d+1post-Tx”) and on day 1 after neutrophil engraftment (“engraftment”). Conditioning regimens included either standard therapies (n=17), FLAMSA (n=11) or other (n=5). GvHD prophylaxis consisted of either CsA/MTX/ATG (n=19), CsA/MMF/ATG (n=11) or CsA/Steroids (n=3). Typical patient and transplant risk factors were included in statistical analysis. Platelet-free plasma (PFP) was obtained by three-step centrifugation, snap-frozen in liquid nitrogen and stored at -80°C. After thawing, 50μl of PFP were resuspended in Annexin V binding or control buffer and labeled with 1μl Annxin V-Cy5 and 0.5μg of CD142-FITC (TF), CD162-FITC (PSGL-1) or isotype control. In flow cytometry MPs were gated by size (<1μm) and Annexin V positivity and Trucount tubes® were used for MP enumeration. Results Mean follow-up time was 759 (10-1305) days. 17 (51.5%) patients died, 9 due to TRM (27.3%), and 12 (36.4%) had recurrence or progression during follow-up. Median overall survival (OS) was 815 days, median disease-free or progression survival (DFS/PFS) 440 (10-1272) days and median time to progression (TTP) 365.5 (130-1183) days. MP counts are shown in table 1. There was a significant reduction (p<0.05) for Annexin V- and PSGL-1-MPs from “start” to “pre-Tx” and an increase after transplantation while TF-MPs had significant higher numbers at “d+1post-Tx” compared to “start” values. Univariate Cox analysis identified TF-MPs at “start” (p=0.011, HR=1.006 per 1 MP/μl) and “pre-ATG” (p=0.004, HR=1.011) to be significantly associated with OS. When corrected for known risk factors, TF-MPs “pre-ATG” remained the only independent predictive parameter for OS in multivariate Cox analysis s(p=0.036, HR 1.022). A cut-off value was determined for stratification of patients by calculating the AUC of a ROC–curve (p=0.030, AUC 0.762). Thus, Patients with TF-MP values >140/μl showed a significantly shorter survival time (p=0.007; mean OS: 469.4±160.0 vs. 1038.5±110.3 days). Same was done for DFS/PFS and again TF-MP values >140/μl were predictive of worse DFS/PFS (p=0.020, HR=3.61) in multivariate Cox regression with a shortened DFS/PFS (p=0.013; mean: 350.9±113.8 vs. 852.5±129.6 days). These findings were caused by an increased treatment-related mortality (TRM) with a cumulative incidence at 1 year of 55.5% vs. 13.3% for corresponding TF-MP groups (p=0.029). TRM was caused by infection (n=4), VOD (n=2) or other reasons (n=3). There was no significant association with relapse or acute GvHD. Furthermore, we found PSGL-1-MP counts below 250/μl “post-Tx” (and on “engraftment”) to be a significant predictor of relapse/progression (p=0.044, HR=4.91) with a cumulative incidence at 3 years of 81% (95%-CI: 61.9-100) vs. 11.1% (95%-CI: 1.8-70.5) and a mean TTP of 602±106.1 vs. 1140±104.5 days (p=0.027). Conclusions TF-bearing MPs before and during conditioning therapy are prognostic markers of OS and DFS/PFS due to increased TRM while PSGL-1 bearing MPs after transplantation and at engraftment predict relapse/progression and TTP. Course of MP counts suggests increased TF-MP release by monocytes, endothelial and stromal cell while significant differences in PSGL-1-MPs might rather be graft-related. Disclosures: Trummer: CSL Behring: Research Funding.