Comparison of in vitro responses to fresh whole blood and reconstituted whole blood after collagen stimulation

Abstract

Background: In patients who have large bleeds, there is a tendency to transfuse more plasma and platelets than recommended in earlier guidelines, and accordingly many hospitals now provide "transfusion packages" with an intended red cell:platelet:plasma ratio of 1:1:1. The purpose of this study was to investigate in vitro functions of transfusion packs compared with fresh whole blood.

Material and methods: "Reconstituted whole blood" was prepared with the same ratio of red cells, platelets and plasma as used in local transfusion packages. The aggregation and thrombin-antithrombin complex formation responses to collagen stimulation of this reconstituted whole blood were compared with those of fresh whole blood. The storage time of red cells and platelets was varied in a systematic manner, giving nine different compositions of reconstituted whole blood that simulated transfusion packs.

Results: The responses varied significantly between whole blood and reconstituted whole blood -and between the reconstituted whole blood of different compositions. A significant decrease (p<0.005) in collagen-induced platelet count reduction was seen with increasing platelet and red blood cell age. Thrombin-antithrombin complex formation peaked in studies with platelets stored for 5 days. The red cells stored for the longest time induced the greatest thrombin-antithrombin complex formation. Fresh whole blood gives more consistent responses, and the aggregation response to collagen is stronger than in reconstituted whole blood.

Discussion: Our results indicate that in vitro responses of reconstituted whole blood vary substantially according to how long the red cells and platelets are stored for. As the responses obtained by testing whole blood are more consistent and usually stronger, the alternative use fresh whole blood in special conditions should not be excluded without further consideration.

Figure 1

The effect of collagen (10 μg/mL) (Chrono-log Corporation) stimulation on CPCR in reconstituted whole blood and fresh whole blood. A matrix system (Table I) was set up to provide nine variations of reconstituted whole blood (varying storage age of red cells and platelets), whilst the fresh whole blood was stored for a maximum of 4 hours. The figure shows the results for platelets stored for 1 day (A), 3 days (B), and 5 days (C). The samples were stimulated for a minimum of 5 minutes prior to analysis. All groups show a decrease in CPCR after stimulation with collagen. A significant decrease in platelet response to agonist was observed in control samples compared with stimulated samples (p<0.05, Wilcoxon’s test for paired samples). The results are presented as CPCR of control samples and stimulated samples. A statistically significant decrease of CPCR was found between fresh whole blood and all groups of reconstituted whole blood (p<0.05, Wilcoxon’s test for paired samples). P-values are indicated above the graphs.

Figure 2

The determination of CPCR in reconstituted whole blood and fresh whole blood after collagen (10 μg/mL) (Chrono-log Corporation) stimulation. The figure shows the results for red blood cells stored for 0–4 days (A), 12–16 days (B), and 26–35 days (C). The samples were stimulated for a minimum of 5 minutes prior to analysis. All groups show a decrease in CPCR after stimulation with collagen. A significant decrease in platelet response to agonist was observed in control samples compared with stimulated samples (p<0.05, Wilcoxon’s test for paired samples). The results are presented as CPCR for control samples and stimulated samples. A statistically significant decrease of CPCR was found between whole blood and all groups of reconstituted whole blood (p<0.05, Wilcoxon’s test for paired samples). P-values are indicated above the graphs.

Figure 3

Determination of thrombin generation in reconstituted whole blood and fresh whole blood after collagen stimulation (10 μg/mL) (Chrono-log Corporation). The figure shows the results for platelets stored for 1 day (A), 3 days (B), and 5 days (C). All results are presented as TAT complex levels (pg/mL). Significant differences were found between whole blood and groups B, D, E, F, G, and I (p<0.05). The Wilcoxon’s test for paired samples was used for statistical comparisons, and p-values are shown above the graphs.

Figure 4

The effect of collagen (10 μg/mL) (Chrono-log Corporation) stimulation on thrombin generation in reconstituted whole blood and fresh whole blood. The figure shows the results for red blood cells stored for 0–4 days (A), 12–16 days (B), and 26–35 days (C). All results are presented as TAT complex levels (pg/mL). Significant differences were found between whole blood and groups B, D, E, F, G, and I (p<0.05). Wilcoxon’s test for paired samples was used for statistical comparisons, and p-values are shown above the graphs.