Abstract

Background. The hemostatic activity of plasma is due to thrombin generation (TG) provided by the interrelated activation of platelets and the plasma coagulation system. The global capacity for plasma TG can be assessed in the presence of procoagulant phospholipids (PL) as a substitute for activated platelets initiation of the reaction by tissue factor (TF). Photochemical treatment (PCT) of plasma with amotosalen and UVA light (INTERCEPT Blood System, Cerus Europe, Leusden, The Netherlands) retains adequate levels of procoagulant and antithrombotic factors according to European standards and is clinically effective. We have assessed the impact of PCT on TG using the calibrated automated thrombogram (CAT).

Methods. Plasma (650mL) was collected by apheresis from 90 donors (MCS+, Haemonetics, Braintree, MA). Plasma was held at 20°–24°C before PCT and flash frozen at −80°C within 8 hrs of collection. Plasma units of 200mL were stored at −30°C for up to 1 year. TG was continuously measured in platelet poor plasma with a multiwell-plate reader using CAT and reagents (Thrombinoscope BV, Biodis, Signes, France). 20 μL PPP- reagent Low or High (1 or 20 pM TF and 4μM PL) were added at 37°C to triplicates of 80 μL PPP. TG measures were started by addition of 20 μL thrombin fluorescent substrate Z-GGR-AMC (2.5 mM) and CaCl2 (17 mM). Fluorescence accumulation from cleaved AMC was measured at 390 nm and 460 nm wavelengths and converted into nanomolar thrombin using a calibrator. The area under the curve corresponds to the total potential amount of endogenous thrombin formed (ETP) and the height of the peak to the maximum concentration of thrombin (MT).

Results. Plasma from 3 A, 1 AB and 3 O donors (Exp.1, Table) were tested for FVIII: before PCT, 1.08 ± 0.33 U/mL and after, 0.63 ± 0.20 U/mL (p < 0.01). TG was compared before and after PCT (Exp.1) at TF 1pM (control: ETP 1,906 ± 129 nM.min and MT 183–327 nM) and TF 20pM (control: ETP 2,026 ± 151 nM.min and MT 416–474 nM). Low TF (1pM) was further used to discriminate the effect of PCT on TG. In experiment 2, 12 donors (6 O, 1 AB and 5 A) tested after at day T0, before PCT (ETP 1,597 ± 205 nM.min) and after (ETP 1,680 ± 254 nM.min) were not different. Results were similar after day T30 storage (PCT: ETP 1,703 ± 174 nM.min). Results were identical for QC (Exp.3) of 12 pools of PCT of 6 units (3O and 3A donors): mean FVIII (0.69 ± 0.10 U/mL) and ETP (1,704 ± 93 nM.min).

Conclusions. Traditional standards have focused on FVIII levels, but TG, not modified by PCT of apheresis plasma, is of far greater relevance as it measures the efficacy of the entire hemostatic system, regardless of FVIII.

ETP and MT in control and PCT plasma

Experiment NbPlasma (n)F VIII (U/mL)TF (pM)ETP (nM.min)MT (nM)
Exp.1. Control (6) 1.08 ± 0.33 Low 1 1,906 ± 129 183–327 
Exp.1. PCT (6) 0.63 ± 0.20 Low 1 1,683 ± 172 134–223 
Exp.1. Control (6) 1.08 ± 0.33 High 20 2,026 ± 151 416–474 
Exp.1. PCT (6) 0.63 ± 0.20 High 20 1947 ± 151 383 ± 440 
Exp.2. T0 day Control (12) 0.82 ± 0.17 Low 1 1,597 ± 205 84–273 
Exp.2. T0 day PCT (12) 0.56 ± 0.13 Low 1 1,680 ± 254 94–233 
Exp.2. T30 days Control (12) 0.78 ± 0.17 Low 1 1,530 ± 128  
Exp.2. T30 days PCT (12) 0.51 ± 0.09 Low 1 1,703± 174  
Exp.3.QC PCT (12.6=72) 0.69 ± 0.10 Low 1 1,704 ± 93 162–226 
Experiment NbPlasma (n)F VIII (U/mL)TF (pM)ETP (nM.min)MT (nM)
Exp.1. Control (6) 1.08 ± 0.33 Low 1 1,906 ± 129 183–327 
Exp.1. PCT (6) 0.63 ± 0.20 Low 1 1,683 ± 172 134–223 
Exp.1. Control (6) 1.08 ± 0.33 High 20 2,026 ± 151 416–474 
Exp.1. PCT (6) 0.63 ± 0.20 High 20 1947 ± 151 383 ± 440 
Exp.2. T0 day Control (12) 0.82 ± 0.17 Low 1 1,597 ± 205 84–273 
Exp.2. T0 day PCT (12) 0.56 ± 0.13 Low 1 1,680 ± 254 94–233 
Exp.2. T30 days Control (12) 0.78 ± 0.17 Low 1 1,530 ± 128  
Exp.2. T30 days PCT (12) 0.51 ± 0.09 Low 1 1,703± 174  
Exp.3.QC PCT (12.6=72) 0.69 ± 0.10 Low 1 1,704 ± 93 162–226 

Author notes

Disclosure:Research Funding: JP Cazenave has received research funding from Cerus. Honoraria Information: JP Cazenave has received honoraria for service on advisory boards to Cerus. Membership Information: JP Cazenave has served on Cerus scientific advisory boards.