- Email: [email protected]
C0340 An automated progressive assay of antithrombin
S182
Abstracts / Thrombosis Research 130 (2012) S100–S202
well as arachidonic acid (AA) was measured. LD EPI and ADP aggregation was considered exaggerated if greater than 15%, a cutoff suggested by the manufacturer. PLAG results were correlated with clinical presentation, blood smear platelet morphology and PFA100 closure time (CT). Exclusion criteria included history of bleeding with no evidence of thromboemboli (i.e. wrong assay ordered). Results: 126 studies were included in the study. A thromboembolic event was documented in 104 of 126 (83%) patients, 76 (73%) of which showed a decreased threshold for aggregation with LD EPI (“hyperaggregable platelets”). Of the 76 patients with abnormal aggregation, 46 (61%) experienced venous, 28 (37%) experienced arterial and 2 (3%) patients experienced both venous and arterial thromboemboli. 10 patients without thromboemboli, but with coronary artery disease, orthopedic surgery, atrial fibrillation or obesity, also had a decreased threshold for aggregation with LD EPI. Antiplatelet therapy was documented in 40 of 86 (47%) patients with exaggerated LD EPI aggregation, 34 of which showed aspirin effect as AA inhibition. Significant spontaneous aggregation was not observed. Platelet morphology was unremarkable. A shortened PFA100 ADP CT was seen in only 23 of 126 patients with no difference between hyperaggregable and non-hyperaggregable studies. Comment:
Results: We first detected agglutination by measuring absorbance increase at 405 nm and 671 nm, and chose 405 nm for the assay because the calibration curve slope was steeper. Over 10 runs, the calibration curves had median R2 value of 0.999 (range 0.996 – 1.000) with a linear-linear plot. Within run precision for ATAL at normal AT level (107 IU/dL) tested 10 times, was good, the coefficient of variation (CV) was 0.96% and for a sample with a low AT level (33 IU/dL) CV was 2.5%. Despite the reconstituted latex being stable for at least 4 weeks at 4 °C, we corrected results utilising a frozen (−80 °C) pooled plasma that was tested in each run. Over 13 runs, a normal sample (AT 103 IU/dL) showed a CV of 2.2% and a low ATAL sample (66 IU/dL) showed a CV of 5.3%. Eighty-two samples tested by ATAE and ATAL showed median AT antigen levels of 85 IU/dl and 87 IU/dL, respectively, the results correlated well (r=0.96) and results were not significantly different (P=0.23, by Wilcoxon matched-pairs signed-ranks test). Comment: The automated latex-based AT assay performs well and is now in routine use in our laboratory. We have achieved a significant saving in hands-on time, and improved turnaround time, we can assay 10 specimens in approximately 30 minutes.
1. A distinct “hyperaggregable” PLAG pattern (i.e. decreased threshold for aggregation with LD EPI) was identified in 73% of patients with thromboemboli and may indicate an additional risk factor for arterial or venous thrombosis. 2. PLAG may also be useful to identify patients on aspirin who show persistent aggregation to LD EPI, suggesting they may benefit from a higher aspirin dose and/or alternative antiplatelet agent. 3. A shortened PFA100 ADP CT is not clinically significant.
C0340 An automated progressive assay of antithrombin
doi:10.1016/j.thromres.2012.08.211
C0339 An automated antithrombin antigen assay on instrumentation laboratory ACL top analyser Peter Cooper1, Kieron Hickey1, Fiona Coath2, Mike Makris2 Royal Hallamshire Hospital, Coagulation Laboratory, Department of Haematology - Glossop Road, Sheffield, UK; 2Royal Hallamshire Hospital, Haematology - Glossop Road, Sheffield, UK 1
Background: Antithrombin (AT) deficiency is characterised as type I when activity and antigen levels are similarly reduced, and type II deficiency when activity level is disproportionately reduced compared to antigen. Type IIRS has defective AT interaction with substrate, type IIHBS shows defective AT binding to heparin, and type IIPE has more than one defect. Subjects with type IIHBS usually have a benign thrombotic phenotype. We sought to establish an automated AT antigen assay to replace our precise but labour intensive ELISA assay. Methods: We developed an automated latex-based AT antigen assay (ATAL) utilising reagents from the (manual) Liatest ATIII antigen assay kit (Stago, Thiele, UK). The assay was adapted for the ACL TOP auto analyser (Instrumentation Laboratory, Warrington, UK). Sample dilution in saline was mixed with latex particles coated with polyclonal antibody to AT, then agglutination was recorded as increase in optical density (405nM) and used to determine AT antigen level. We compared the performance of the automated ATAL assay with our established in-house ELISA assay (ATAE) that utilises polyclonal antibodies from Dako (High Wycombe, UK).
doi:10.1016/j.thromres.2012.08.212
Peter Cooper1, Kieron Hickey1, Fiona Coath2, Mike Makris2 Royal Hallamshire Hospital, Coagulation Laboratory, Department of Haematology - Glossop Road, Sheffield, UK; 2Royal Hallamshire Hospital, Haematology - Glossop Road, Sheffield, UK 1
Background: Type II antithrombin (AT) deficiency is classified as type IIRS with defective AT interaction with substrate, type IIHBS with defective AT binding to heparin and type IIPE which shows more than one defect. Subjects with type IIHBS have impaired AT binding to heparin and normal progressive AT activity (AT activity in absence of heparin) and usually have a benign thrombotic phenotype. We have developed an automated chromogenic progressive AT assay (PATA) for the ACL TOP analyser (Instrumentation Laboratory, Warrington, UK) that detects inhibition of bovine thrombin by patient plasma dilution in the absence of heparin. Methods: In the PATA assay, sample dilution is mixed with Pefabloc FG (Hart Biologicals Ltd., Hartlepool, UK) to stop clot formation during a 30 minute incubation with bovine thrombin (Thromboclotin; Sysmex, Milton Keynes, UK). Residual thrombin is then detected by adding Chromozyme TH (Roche Diagnostics, Burgess Hill, UK) and measuring increase of optical density (405 nM). A calibration curve was created, and as AT depleted plasma (AT antigen b 1 IU/dL) returned a PATA value of 25 U/dL, a normal pooled plasma was diluted in AT deficient plasma to generate an algorithm to help correct for the non-AT inhibition. Corrected PATA (CPATA) was compared to a routine bovine thrombin based AT assay that incorporates heparin (Berichrom, Siemens) (AT3C). Results: A sample with normal AT3C level had within-assay coefficient of variation (CV) of 1.1%, and a sample with low AT3C had a CV of 1.6%; inter-assay CV was 3.5% and 9%, respectively. A CPATA reference range was assessed by testing 58 healthy donors, and was 89-108 U/dL. Thirteen patients with phenotypic type I AT deficiency had reduced CPATA level. Three patients with type IIRS deficiency (AT Glasgow) had clearly abnormal CPATA . In type IIHBS, one patient with AT Geneva (AT3C 62 IU/dL) had CPATA of 113 U/dL and two patients with borderline AT3C (AT Basel) had normal CPATA. Excluding type II deficiency, the correlation of AT3C with CPATA was good, r = 0.9528 (n = 71).
Abstracts / Thrombosis Research 130 (2012) S100–S202
Comment: We recommend that the progressive AT assay is performed on all patients shown to have type II antithrombin deficiency, to help differentiate between type IIHBS and other type II AT defects. doi:10.1016/j.thromres.2012.08.213
C0351 In vitro response of thrombin generation (CAT) and fibrin formation (ROTEM) with enoxaparin, dabigatran and rivaroxaban Michel Meyer Samama, Céline Guinet, Lena Le Flem Biomnis R&D94200 Ivry sur Seine FRANCE - 78 avenue de Verdun, France Background: Thromboelastometry (TEM) has the advantage of studying whole blood (WB) or platelet rich plasma (PRP), in contrast to thrombin generation test (TGT) which cannot be performed in WB. The aim is to investigate the response in ROTEM® delta and TGT to enoxaparin, dabigatran and rivaroxaban and determine if these different methods show a dose/response relationship. A correlation between ROTEM (WB or PRP) and TGT parameters is also investigated. Methods: Citrated WB was obtained from 18 healthy volunteers from a blood bank and tested in ROTEM with increasing concentrations between 0 and 0.5 μg/mL for dabigatran and rivaroxaban, and 0 and 0.5 anti-Xa IU/mL for enoxaparin. After spinning the WB, the PRP obtained (without adjusting platelet count) has been tested in ROTEM and TGT with the 3 anticoagulants studied. TEM and TGT were triggered with tissue factor: 0.36 and 1 pM final respectively. Statistical analysis has been performed with XLSTAT software. Results: Parameters which show the best correlation to the anticoagulant doses are CT for ROTEM and LT for TGT with a Spearman rhô N 0.9. The 3 drugs influence the other parameters in a weak dose-dependent manner, except for ROTEM amplitude and TGT ETP which are not modified. In general, CT in WB is more sensitive than in PRP. The concentrations needed to prolong the ROTEM CT 2 fold (Tx2) are 0.27 IU/mL, 0.06 and 0.20 μg/mL in WB versus 0.38 IU/mL, 0.10 and 0.18 μg/mL in PRP for enoxaparin, dabigatran and rivaroxaban respectively. TGT LT is less sensitive with Tx2 at 0.50 IU/mL, 0.15 and 0.30 μg/mL for enoxaparin, dabigatran and rivaroxaban respectively. The statistical analysis show a good correlation between WB and PRP in ROTEM CT, and between ROTEM CT and TGT LT with a Spearman rhôN 0.9. Comment: TGT and ROTEM are influenced by enoxaparin, dabigatran and rivaroxaban concentrations in a significant dose/response relationship for time parameters. The different methods studied could be used for the evaluation of the anticoagulation level. ROTEM CT was found more sensitive than TGT LT for the 3 anticoagulants. According to these in vitro results, it is likely that both tests may be used as a functional test in treated patients. doi:10.1016/j.thromres.2012.08.214
C0236 Pre-analytical conditions of centrifugation and freezing but not temperature affect MP levels Virtudes Vila1, Silvia Navarro1, Verónica Sánchez-López2, Remedios Otero2, Vicenta Martínez-Sales1 1 Hospital Universitario La FeCentro de Investigación - Avda, Campanar 21, Valencia-46009, Spain; 2Hospital Universitario Virgen del RocioHUVR-IBIS.CIBERES - Avda, Manuel Siurot s/n - 41013 - Sevilla, Spain
S183
Background: Pre-analytical steps used in analysis of circulating microparticles (MP) may cause variability in the results; however there are not well established recommendations. We have analysed the levels of MP and their procoagulant activity in fresh and frozen plasma, obtained at different speeds of centrifugation and temperature to assess their impact. Methods: Blood samples from 24 healthy individuals obtained on trisodium citrate (0.109 M) were analyzed. MP-rich plasmas (MPRP) were prepared by a single centrifugation at 1500 ×g 30 min or by double centrifugation at 2500 ×g 10 min, at 4 °C and 20 °C. MP-free plasmas (MPFP) were obtained by centrifugation at 25000 ×g 120 min. Samples were analyzed in plasma fresh and frozen (−80 °C)-thawed. The annexinV-positive MP (0.5-1 μm) were quantified by flow cytometry (EPICS XL-MCL, Beckman-Coulter) using standard beads (Megamix, BioCytex).The procoagulant activity of plasma MP-mediated was analyzed by the thrombin generation assay without exogenous TF or phospholipids added (TG) (CAT, Thrombinoscope BV) and by the procoagulant phospholipids-dependent clotting time assay (PPL) (STAProcoagPPL, Stago). MPRP and MPFP samples were analyzed. Results: MPRP analysis showed no differences temperature-dependent (4 °C vs 20 °C). However, it was observed significant differences dependent on centrifugation and freezing. Fresh plasma had lower MP (428±409 vs 2668 ±1911/μl, p b 0.001) and TG (20 ±15 vs 65 ±51 nM, pb 0.01) and higher PPL (97 ±13 vs 49±15 s, pb 0.001) than frozenthawed plasma. The double centrifugation (2500xg) compared with simple centrifugation (1500xg) caused decreased number of MP (fresh: 294±241 vs 555±496/μl, pb 0.05; frozen: 1676 ±1250 vs 3167 ±1980/ μl, p b 0.001) and TG (fresh: 16 ± 12 vs 24 ± 15 nM, p b 0.05; frozen: 42 ± 27 vs 87 ± 58 nM, p b 0.001) and increased PPL (fresh: 102 ± 14 vs 92± 10 s, P b 0.01; frozen: 58 ± 15 vs 41 ± 9 s, P b 0.001). In MPFP samples it was found a reduction in annexinV + MP of 95% and a concomitant reduction in TG of 94%. The correlation study showed a significant association of number of MP with PPL (r= −0.77, .p b 0.001) and TG (r= 0.69, p b 0.001). There was also correlation between TG and PPL (r= −0.65, p b 0.001). Comment: The pre-analytical conditions of centrifugation and freezing, but not temperature influence the evaluation of MP. The freezing conditions of plasma increases both the number of MP as well as their procoagulant activity. The MP level may determine the procoagulant capacity of plasma. FIS-PI- 080124. doi:10.1016/j.thromres.2012.08.215
C0280 Correlation between annexinV-FITC and Annexin V-V450 positive microparticles in two high sensitivity cytometers Verónica Sánchez-López1, Virtudes Vila2, Vicenta Martinez-Salas2, Emilia Martín-Rodriguez1, Ana Montes-Worboys1, Elena Arellano1, Teresa Elias1, Remedios Otero1 1 Hospital Universitario Virgen del Rocio HUVR-IBIS - Avda, Manuel Siurot s/n - 41013 - Sevilla, Spain; 2Hospital Universitario La Fe, Centro de Investigación - Avda, Campanar 21, Valencia-46009, Spain Background: Circulating microparticles are biomarkers in coagulation, inflammation and cancer. Flow cytometry allows enumeration and characterization of MP cellular origin. MP production implies the externalization of phosphatidilserine of cell membrane and staining with annexinV. Our aim was to compare fluorescein isothiocyanate labeled Annexin V-MP positive (AnnV-FITC) or by AnnexinV-V450MP positive (AnnV-V450) in two high sensitivity cytometers. Methods: Samples from 20 patients suffering venous thromboembolism were drawn from the antecubital vein punction with a light tourniquet. Patients provided written informed consent. Blood was
Abstracts / Thrombosis Research 130 (2012) S100–S202
well as arachidonic acid (AA) was measured. LD EPI and ADP aggregation was considered exaggerated if greater than 15%, a cutoff suggested by the manufacturer. PLAG results were correlated with clinical presentation, blood smear platelet morphology and PFA100 closure time (CT). Exclusion criteria included history of bleeding with no evidence of thromboemboli (i.e. wrong assay ordered). Results: 126 studies were included in the study. A thromboembolic event was documented in 104 of 126 (83%) patients, 76 (73%) of which showed a decreased threshold for aggregation with LD EPI (“hyperaggregable platelets”). Of the 76 patients with abnormal aggregation, 46 (61%) experienced venous, 28 (37%) experienced arterial and 2 (3%) patients experienced both venous and arterial thromboemboli. 10 patients without thromboemboli, but with coronary artery disease, orthopedic surgery, atrial fibrillation or obesity, also had a decreased threshold for aggregation with LD EPI. Antiplatelet therapy was documented in 40 of 86 (47%) patients with exaggerated LD EPI aggregation, 34 of which showed aspirin effect as AA inhibition. Significant spontaneous aggregation was not observed. Platelet morphology was unremarkable. A shortened PFA100 ADP CT was seen in only 23 of 126 patients with no difference between hyperaggregable and non-hyperaggregable studies. Comment:
Results: We first detected agglutination by measuring absorbance increase at 405 nm and 671 nm, and chose 405 nm for the assay because the calibration curve slope was steeper. Over 10 runs, the calibration curves had median R2 value of 0.999 (range 0.996 – 1.000) with a linear-linear plot. Within run precision for ATAL at normal AT level (107 IU/dL) tested 10 times, was good, the coefficient of variation (CV) was 0.96% and for a sample with a low AT level (33 IU/dL) CV was 2.5%. Despite the reconstituted latex being stable for at least 4 weeks at 4 °C, we corrected results utilising a frozen (−80 °C) pooled plasma that was tested in each run. Over 13 runs, a normal sample (AT 103 IU/dL) showed a CV of 2.2% and a low ATAL sample (66 IU/dL) showed a CV of 5.3%. Eighty-two samples tested by ATAE and ATAL showed median AT antigen levels of 85 IU/dl and 87 IU/dL, respectively, the results correlated well (r=0.96) and results were not significantly different (P=0.23, by Wilcoxon matched-pairs signed-ranks test). Comment: The automated latex-based AT assay performs well and is now in routine use in our laboratory. We have achieved a significant saving in hands-on time, and improved turnaround time, we can assay 10 specimens in approximately 30 minutes.
1. A distinct “hyperaggregable” PLAG pattern (i.e. decreased threshold for aggregation with LD EPI) was identified in 73% of patients with thromboemboli and may indicate an additional risk factor for arterial or venous thrombosis. 2. PLAG may also be useful to identify patients on aspirin who show persistent aggregation to LD EPI, suggesting they may benefit from a higher aspirin dose and/or alternative antiplatelet agent. 3. A shortened PFA100 ADP CT is not clinically significant.
C0340 An automated progressive assay of antithrombin
doi:10.1016/j.thromres.2012.08.211
C0339 An automated antithrombin antigen assay on instrumentation laboratory ACL top analyser Peter Cooper1, Kieron Hickey1, Fiona Coath2, Mike Makris2 Royal Hallamshire Hospital, Coagulation Laboratory, Department of Haematology - Glossop Road, Sheffield, UK; 2Royal Hallamshire Hospital, Haematology - Glossop Road, Sheffield, UK 1
Background: Antithrombin (AT) deficiency is characterised as type I when activity and antigen levels are similarly reduced, and type II deficiency when activity level is disproportionately reduced compared to antigen. Type IIRS has defective AT interaction with substrate, type IIHBS shows defective AT binding to heparin, and type IIPE has more than one defect. Subjects with type IIHBS usually have a benign thrombotic phenotype. We sought to establish an automated AT antigen assay to replace our precise but labour intensive ELISA assay. Methods: We developed an automated latex-based AT antigen assay (ATAL) utilising reagents from the (manual) Liatest ATIII antigen assay kit (Stago, Thiele, UK). The assay was adapted for the ACL TOP auto analyser (Instrumentation Laboratory, Warrington, UK). Sample dilution in saline was mixed with latex particles coated with polyclonal antibody to AT, then agglutination was recorded as increase in optical density (405nM) and used to determine AT antigen level. We compared the performance of the automated ATAL assay with our established in-house ELISA assay (ATAE) that utilises polyclonal antibodies from Dako (High Wycombe, UK).
doi:10.1016/j.thromres.2012.08.212
Peter Cooper1, Kieron Hickey1, Fiona Coath2, Mike Makris2 Royal Hallamshire Hospital, Coagulation Laboratory, Department of Haematology - Glossop Road, Sheffield, UK; 2Royal Hallamshire Hospital, Haematology - Glossop Road, Sheffield, UK 1
Background: Type II antithrombin (AT) deficiency is classified as type IIRS with defective AT interaction with substrate, type IIHBS with defective AT binding to heparin and type IIPE which shows more than one defect. Subjects with type IIHBS have impaired AT binding to heparin and normal progressive AT activity (AT activity in absence of heparin) and usually have a benign thrombotic phenotype. We have developed an automated chromogenic progressive AT assay (PATA) for the ACL TOP analyser (Instrumentation Laboratory, Warrington, UK) that detects inhibition of bovine thrombin by patient plasma dilution in the absence of heparin. Methods: In the PATA assay, sample dilution is mixed with Pefabloc FG (Hart Biologicals Ltd., Hartlepool, UK) to stop clot formation during a 30 minute incubation with bovine thrombin (Thromboclotin; Sysmex, Milton Keynes, UK). Residual thrombin is then detected by adding Chromozyme TH (Roche Diagnostics, Burgess Hill, UK) and measuring increase of optical density (405 nM). A calibration curve was created, and as AT depleted plasma (AT antigen b 1 IU/dL) returned a PATA value of 25 U/dL, a normal pooled plasma was diluted in AT deficient plasma to generate an algorithm to help correct for the non-AT inhibition. Corrected PATA (CPATA) was compared to a routine bovine thrombin based AT assay that incorporates heparin (Berichrom, Siemens) (AT3C). Results: A sample with normal AT3C level had within-assay coefficient of variation (CV) of 1.1%, and a sample with low AT3C had a CV of 1.6%; inter-assay CV was 3.5% and 9%, respectively. A CPATA reference range was assessed by testing 58 healthy donors, and was 89-108 U/dL. Thirteen patients with phenotypic type I AT deficiency had reduced CPATA level. Three patients with type IIRS deficiency (AT Glasgow) had clearly abnormal CPATA . In type IIHBS, one patient with AT Geneva (AT3C 62 IU/dL) had CPATA of 113 U/dL and two patients with borderline AT3C (AT Basel) had normal CPATA. Excluding type II deficiency, the correlation of AT3C with CPATA was good, r = 0.9528 (n = 71).
Abstracts / Thrombosis Research 130 (2012) S100–S202
Comment: We recommend that the progressive AT assay is performed on all patients shown to have type II antithrombin deficiency, to help differentiate between type IIHBS and other type II AT defects. doi:10.1016/j.thromres.2012.08.213
C0351 In vitro response of thrombin generation (CAT) and fibrin formation (ROTEM) with enoxaparin, dabigatran and rivaroxaban Michel Meyer Samama, Céline Guinet, Lena Le Flem Biomnis R&D94200 Ivry sur Seine FRANCE - 78 avenue de Verdun, France Background: Thromboelastometry (TEM) has the advantage of studying whole blood (WB) or platelet rich plasma (PRP), in contrast to thrombin generation test (TGT) which cannot be performed in WB. The aim is to investigate the response in ROTEM® delta and TGT to enoxaparin, dabigatran and rivaroxaban and determine if these different methods show a dose/response relationship. A correlation between ROTEM (WB or PRP) and TGT parameters is also investigated. Methods: Citrated WB was obtained from 18 healthy volunteers from a blood bank and tested in ROTEM with increasing concentrations between 0 and 0.5 μg/mL for dabigatran and rivaroxaban, and 0 and 0.5 anti-Xa IU/mL for enoxaparin. After spinning the WB, the PRP obtained (without adjusting platelet count) has been tested in ROTEM and TGT with the 3 anticoagulants studied. TEM and TGT were triggered with tissue factor: 0.36 and 1 pM final respectively. Statistical analysis has been performed with XLSTAT software. Results: Parameters which show the best correlation to the anticoagulant doses are CT for ROTEM and LT for TGT with a Spearman rhô N 0.9. The 3 drugs influence the other parameters in a weak dose-dependent manner, except for ROTEM amplitude and TGT ETP which are not modified. In general, CT in WB is more sensitive than in PRP. The concentrations needed to prolong the ROTEM CT 2 fold (Tx2) are 0.27 IU/mL, 0.06 and 0.20 μg/mL in WB versus 0.38 IU/mL, 0.10 and 0.18 μg/mL in PRP for enoxaparin, dabigatran and rivaroxaban respectively. TGT LT is less sensitive with Tx2 at 0.50 IU/mL, 0.15 and 0.30 μg/mL for enoxaparin, dabigatran and rivaroxaban respectively. The statistical analysis show a good correlation between WB and PRP in ROTEM CT, and between ROTEM CT and TGT LT with a Spearman rhôN 0.9. Comment: TGT and ROTEM are influenced by enoxaparin, dabigatran and rivaroxaban concentrations in a significant dose/response relationship for time parameters. The different methods studied could be used for the evaluation of the anticoagulation level. ROTEM CT was found more sensitive than TGT LT for the 3 anticoagulants. According to these in vitro results, it is likely that both tests may be used as a functional test in treated patients. doi:10.1016/j.thromres.2012.08.214
C0236 Pre-analytical conditions of centrifugation and freezing but not temperature affect MP levels Virtudes Vila1, Silvia Navarro1, Verónica Sánchez-López2, Remedios Otero2, Vicenta Martínez-Sales1 1 Hospital Universitario La FeCentro de Investigación - Avda, Campanar 21, Valencia-46009, Spain; 2Hospital Universitario Virgen del RocioHUVR-IBIS.CIBERES - Avda, Manuel Siurot s/n - 41013 - Sevilla, Spain
S183
Background: Pre-analytical steps used in analysis of circulating microparticles (MP) may cause variability in the results; however there are not well established recommendations. We have analysed the levels of MP and their procoagulant activity in fresh and frozen plasma, obtained at different speeds of centrifugation and temperature to assess their impact. Methods: Blood samples from 24 healthy individuals obtained on trisodium citrate (0.109 M) were analyzed. MP-rich plasmas (MPRP) were prepared by a single centrifugation at 1500 ×g 30 min or by double centrifugation at 2500 ×g 10 min, at 4 °C and 20 °C. MP-free plasmas (MPFP) were obtained by centrifugation at 25000 ×g 120 min. Samples were analyzed in plasma fresh and frozen (−80 °C)-thawed. The annexinV-positive MP (0.5-1 μm) were quantified by flow cytometry (EPICS XL-MCL, Beckman-Coulter) using standard beads (Megamix, BioCytex).The procoagulant activity of plasma MP-mediated was analyzed by the thrombin generation assay without exogenous TF or phospholipids added (TG) (CAT, Thrombinoscope BV) and by the procoagulant phospholipids-dependent clotting time assay (PPL) (STAProcoagPPL, Stago). MPRP and MPFP samples were analyzed. Results: MPRP analysis showed no differences temperature-dependent (4 °C vs 20 °C). However, it was observed significant differences dependent on centrifugation and freezing. Fresh plasma had lower MP (428±409 vs 2668 ±1911/μl, p b 0.001) and TG (20 ±15 vs 65 ±51 nM, pb 0.01) and higher PPL (97 ±13 vs 49±15 s, pb 0.001) than frozenthawed plasma. The double centrifugation (2500xg) compared with simple centrifugation (1500xg) caused decreased number of MP (fresh: 294±241 vs 555±496/μl, pb 0.05; frozen: 1676 ±1250 vs 3167 ±1980/ μl, p b 0.001) and TG (fresh: 16 ± 12 vs 24 ± 15 nM, p b 0.05; frozen: 42 ± 27 vs 87 ± 58 nM, p b 0.001) and increased PPL (fresh: 102 ± 14 vs 92± 10 s, P b 0.01; frozen: 58 ± 15 vs 41 ± 9 s, P b 0.001). In MPFP samples it was found a reduction in annexinV + MP of 95% and a concomitant reduction in TG of 94%. The correlation study showed a significant association of number of MP with PPL (r= −0.77, .p b 0.001) and TG (r= 0.69, p b 0.001). There was also correlation between TG and PPL (r= −0.65, p b 0.001). Comment: The pre-analytical conditions of centrifugation and freezing, but not temperature influence the evaluation of MP. The freezing conditions of plasma increases both the number of MP as well as their procoagulant activity. The MP level may determine the procoagulant capacity of plasma. FIS-PI- 080124. doi:10.1016/j.thromres.2012.08.215
C0280 Correlation between annexinV-FITC and Annexin V-V450 positive microparticles in two high sensitivity cytometers Verónica Sánchez-López1, Virtudes Vila2, Vicenta Martinez-Salas2, Emilia Martín-Rodriguez1, Ana Montes-Worboys1, Elena Arellano1, Teresa Elias1, Remedios Otero1 1 Hospital Universitario Virgen del Rocio HUVR-IBIS - Avda, Manuel Siurot s/n - 41013 - Sevilla, Spain; 2Hospital Universitario La Fe, Centro de Investigación - Avda, Campanar 21, Valencia-46009, Spain Background: Circulating microparticles are biomarkers in coagulation, inflammation and cancer. Flow cytometry allows enumeration and characterization of MP cellular origin. MP production implies the externalization of phosphatidilserine of cell membrane and staining with annexinV. Our aim was to compare fluorescein isothiocyanate labeled Annexin V-MP positive (AnnV-FITC) or by AnnexinV-V450MP positive (AnnV-V450) in two high sensitivity cytometers. Methods: Samples from 20 patients suffering venous thromboembolism were drawn from the antecubital vein punction with a light tourniquet. Patients provided written informed consent. Blood was