Platelet Contamination in Plasma: a Possible Source of Biologic Mediators Linked to Transfusion-Related Acute Lung Injury.
Authors: Bercovitz Rachel S. , Khan Samina Y. , Kelher Marguerite R. , Ball James B. , Berry Travis H. , Land Kevin J. , Silliman Christopher C. 
Published: October 2019

Abstract
Abstract 2109
Poster Board II-86
Introduction: Transfusion of plasma is associated with the highest risk of transfusion related acute lung injury (TRALI)-related death. We hypothesize that the current preparation regimen of plasma derived from whole blood has led to significant platelet contamination of plasma, and these platelets contribute to the high rates of TRALI in recipients of plasma. Materials and Methods: Ten units of whole blood were drawn from volunteer donors (5 females and 5 males). All units were centrifuged at 5,316g for 6 minutes to achieve acellular centrifugal effect (ACE) of 4.61×107 in accordance with the blood center's operating procedure for the primary separation of red cells from plasma. We divided each unit into 4 equal aliquots and compared 4 preparation regimens to evaluate the effect of freezing and cell depletion centrifugation (CDC) on platelets and associated factors: not frozen/no CDC, frozen/no CDC, not frozen/CDC, and frozen/CDC. CDC samples were centrifuged at an additional 12,500g for 7 minutes at 4°C to achieve cell depletion prior to freezing, and frozen samples were prepared according to the American Association of Blood Banks (AABB) standard for the preparation of frozen plasma. For each of the four preparation regimens we measured platelet levels using a Beckman Coulter Counter (model: AcT 5Diff AL). We also measured levels of four biologic mediators associated with platelets using commercially available ELISA kits: adenosine diphosphate (ADP), which is found in dense bodies; soluble CD40 ligand (sCD40L), which is found on the surface of platelets; vascular endothelial growth factor (VEGF) and platelet factor 4 (PF4), both found in the alpha granules. Results: We found significant platelet contamination (p<0.05) in the samples that did not undergo CDC with a mean platelet count of 38.8×103/μL (SEM ± 3.0, n=20) versus CDC samples which had a mean platelet count of 1.8×103/μL (SEM ± 0.9, n=20). Samples which had undergone both freezing and CDC had significantly lower levels of VEGF, sCD40L, and PF4 (p<0.05) than samples that had not undergone either process (Table 1). Conclusions: In our study, there was a direct correlation between the concentration of platelet-derived factors and platelet counts in plasma samples. Freezing plasma did not result in a significant decrease in platelet count, but was associated with a decrease in sCD40L. Performing the cell depletion centrifugation resulted in significant decreases in platelet count as well as sCD40L, VEGF, and PF4 levels. Finding a mechanism to reduce platelet contamination of frozen plasma may mitigate TRALI risk by decreasing the levels of biologic mediators, such as sCD40L, implicated in some cases of TRALI.
Preparation sCD40L (ng/mL) VEGF (pg/mL) PF4 (ng/mL) ADP (nmol/μL) Not Frozen, No CDC 0.930 ± 0.138 † 0.087 ± 0.006 51.160 ± 2.942 0.065 ± 0.006 Not Frozen, CDC 0.230 ± 0.106 *† 0.072 ± 0.008 28.820 ± 3.810 *† 0.064 ± 0.006 Frozen, No CDC 0.388 ± 0.104 * 0.083 ± 0.006 47.310 ± 1.719 0.069 ± 0.007 Frozen, CDC 0.127 ± 0.108 *† 0.059 ± 0.006 *† 34.260 ± 3.561 *† 0.069 ± 0.009 Table 1. The data is expressed as the mean ± SEM in pg/ml or ng/ml of the measured biologic mediators from 10 units of plasma each prepared in 4 different ways; * = p<0.05 significance when compared to the not frozen, not CDC sample; † = p<0.05 significance when compared to the frozen, not CDC sample.

Disclosures:
No relevant conflicts of interest to declare.