J. B. Schriner1, A. Mankame3, B. S. Gill4, C. S. Cox2, S. D. Olson2, C. E. Wade1 1University Of Texas Health Science Center At Houston, Center For Translational Injury Research, Houston, TX, USA 2University Of Texas Health Science Center At Houston, Pediatric Surgery, Houston, TX, USA 3University Of Texas Health Science Center At Houston, McGovern Medical School, Houston, TX, USA 4University Of Texas Health Science Center At Houston, Department Of Surgery, Houston, TX, USA
Introduction:
Anticoagulation (AC) of blood products for laboratory evaluation and blood banking relies on chelation of calcium to prevent thrombus formation. Different AC impacts platelet count, volume, and shape, but there is relatively little research regarding the effect of AC on platelet activation. Citrate phosphate dextrose (CPD) solution, the most common AC for blood product storage in the US, uses trisodium citrate to chelate calcium.
We used flow cytometry (FC), thromboelastography (TEG), and a proprietary clot contraction assay (zFlex) to measure platelet activation and clot formation in CPD vs trisodium citrate (TC, standard blue top tube). Donor samples were collected into both TC and CPD. We hypothesized that there would be a difference in platelet activation between the AC.
Methods:
Healthy donors had not taken antiplatelet medications in the prior 14 days. Samples were anticoagulated with 0.105M TC to a final concentration of 11.7mM and CPD to a final concentration of 10.2mM (standard 1:9 dilution). For FC, samples (n=5) underwent soft spin to isolate plasma. Platelets were activated with thrombin receptor activating peptide-6 (TRAP) and compared to nonactivated platelets. Samples were stained with anti-CD42B-FITC (BP1bα: platelet marker, constitutively expressed), anti-CD62P-APC (p-selectin), and anti-CD63-APC-Cy7 (Tspan30: both are markers of activation).
Whole blood (WB) was used for TEG and zFlex (n=7). WB was activated with kaolin and run on a TEG 5000. WB was recalcified to a final concentration of 6mM for zFlex use. Data were analyzed using paired t-test, all p-values are two-tailed.
Results:
The mean fluorescence intensity (MFI) of CD62P in the baseline TC group was 3,419±604 while the baseline MFI in the CPD group was 3,237±615(p=0.29). The MFI of CD62P in TC TRAP was 52,483±12,154 while the MFI in CPD TRAP was 65,814±9,939(p=0.007). The trends in baseline and activated MFI for CD63 were similar. TEG results demonstrated mean R time of 3.9±1.2 minutes for TC vs 8.7±2.6 minutes for CPD(p<0.001). K time TC 1.6±0.4 minutes vs 3.2±2.1 minutes for CPD(p=0.01). TC MA 60±3.7mm while CPD MA was 59.9±5.7mm(p=0.95). LY30 TC 1.3±1% vs 0.4±0.4%(p=0.0013) while LY60 TC was 4.7±2.2% vs 2.7±1.9%(p<0.001). zFlex showed max clot contraction force of 5012±727µN for TC vs 4310±851µN for CPD(p=0.19).
Conclusion:
Platelet activation is dependent on AC choice, though maximum clot strength was mostly unaffected by AC choice. CPD increased the presentation of p-selectin on platelets after activation. While CPD WB took twice as long to generate a clot, CPD was able to maintain clot strength at 30 and 60 minutes better than TC. Further study includes transfusion in animal models and kinetic thrombin assays to better understand the effect of AC on platelet activation response, global clotting parameters, and coagulopathies.