The efficacy of WB to treat or prevent coagulopathic bleeding has traditionally been based on minimal time from donation to administration, hence the term “fresh” WB. The question of how long WB can be stored and retain normal coagulation and PLT function properties from an integrated functional perspective has received little attention. The primary objective of our study was to investigate the functional coagulation and PLT function properties of WB units stored in CPDA-1 anticoagulant at 4°C over a period of 31 days, which closely approximates the industry standard shelf life of 35 days. As an individual component of coagulation, PLTs retain normal responsiveness to agonists for 7 to 21 days. However, integrated coagulation function as determined by TEG remains normal up to 14 days of refrigerated storage.
The integrated function of coagulation proteins and PLTs was measured by TEG. TEG has been used to diagnose abnormal coagulation states in surgical and other settings of blood loss and replacement.16-18
A normal TEG has been shown to have a high negative predictive value for inadequate hemostasis due to coagulopathy.16,19-22
We postulated that TEG measurements of stored blood would be analogous to clinical use in detecting abnormalities or, if within the range of normal, would suggest an intact coagulation state. The R value in TEG is the time required for initial fibrin formation; R values in TEG remained unchanged throughout the duration of our study.
Significant departure of other TEG variables from adult normal values did not occur until Day 14. Both K and alpha variables quantify the rate of clotting; K is mostly influenced by the fibrinogen level, whereas alpha reflects PLT activity in stimulating fibrin polymerization to a greater extent. MA measures the maximum strength (amplitude) of the blood clot and correlates with the aggregation function of PLT contribution to clot formation. The significant departure of TEG alpha and MA values from baseline indicates that PLT contribution to clot development begins to decline at 14 days.
The LTA results imply that normal agonist-induced PLT function (reaching >60% light transmittance)23
is preserved for at least 17 days, with the exception of collagen-induced aggregation, which was reduced beginning on Day 7. However, there was no concomitant effect on alpha or MA on Day 7; both remained unchanged until Day 14. Also notable is spontaneous agglutination before the addition of agonists, which progressively increases. While agglutination increases from Day 4, no TEG change is observed until Day 14, suggesting no significant impairment of PLT contribution to clotting. Refrigeration of PLT concentrates at 4°C has been known to cause changes in PLT structure and function. Cold-induced morphologic change on the PLT membrane leads to clearance of PLTs from the circulation as early as 1 to 2 days after transfusion.24,25
However, it is important to note that the reduced duration of transfused PLTs in the circulation is entirely separable from the ability to contribute to the blood clotting process.24,26
While it is true that in vivo survivability of refrigerated PLTs is compromised when compared to room temperature–stored PLTs, their aggregation functionality is, in fact, arguably better. Several studies have shown that refrigerated PLTs perform better than room temperature–stored PLTs in aggregation assays.27-29
A clinical trial in pediatric cardiac surgical patients comparing PLT aggregation test results up to 3 hours after transfusion concluded that PLTs of refrigerated WB demonstrated better functionality compared to standard PLT concentrates and was associated with reduced bleeding and transfusion requirements.7
A review of the literature reveals that most coagulation proteins are not significantly depleted during 4°C storage. Factor (F)II, FVII, FIX, FX, FXII, and FXIII were found to remain above the lower limit of normal when stored in WB at 4°C through 35 days.11
Furthermore, fibrinogen and plasminogen activity did not change. FV and FVIII are known to be the most labile coagulation factors exhibiting greater reduced concentration during storage. Even so, results from previous studies suggest that FVIII activity remains above bleeding threshold (30%) for 14 to 42 days and FV (10%) for up to 28 days.11,30-33
In our study TEG R value remained normal up to Day 31, suggesting that although likely reduced, the protein concentrations are functionally adequate.
Biochemical changes in WB units during storage are collectively termed as the storage lesion. Progressive reductions in pH, ATP, and 2,3-diphosphoglycerate acid and an increase in plasma hemoglobin and potassium have been documented.15
Cell count and metabolic assay data of our study demonstrated signs of storage lesions as early as Day 4. Levels of potassium and lactate were elevated, whereas blood pH, WBC, and PLT counts gradually declined. Interestingly, the onset of these metabolic alterations did not result in changes in TEG variables or PLT aggregation. CPDA-1 solution and the storage time of samples of WB units may have affected results of some laboratory tests. Blood samples obtained by venipuncture from patients for CBC (PLT count) are collected in ethylenediaminetetraacetate anticoagulant because PLTs tend to progressively clump in citrate, resulting in lower than true counts. Thus, our observations of declining PLT count may be related to clumping in citrate anticoagulant. Similarly, we believe that PLT clumping was responsible for the failure (“flow obstruction error”) of the PFA-100 assay. Standard laboratory practice for PFA-100 dictates that the assay must be performed within 4 hours after the blood sample is collected from a patient in acitrated tube. We documented the presence of clumped PLTs using standard light microscopy. The results of TEG and LTA on stored blood are not yet known to predict clinical efficacy. However, a comparison of in vitro PLT function with an in vivo animal model of hemostatic efficacy suggests that in vivo function may be satisfactory despite in vitro test changes.16
While the fractionation of WB is clearly efficient and advantageous for the majority who require only a component(s), there are specific applications where patients may be better served by using WB. The use of nonrefrigerated WB versus components in cardiac surgery demonstrated qualitative and quantitative laboratory evidence of improved coagulation profile in infants34
and increased PLT count and function in adults.35
Transfusion of freshly drawn WB in combat casualties compared to components was associated with improved survival.2
An observational study of refrigerated WB (stored 8-28 days; mean, 14 days) demonstrated no evidence of coagulopathic bleeding until 20+ units were transfused.5
Prospective randomized trials demonstrated decreased blood loss after pediatric cardiac surgery when refrigerated WB (48 hr) compared to components is used during and immediately after surgery.6,7
Secondary outcomes in those studies include improved PLT function,7
lower inotropic score, fewer donor exposures, less time on ventilator support, and shorter hospital length of stay.6
Similarly, stored WB versus components during liver transplantation resulted in reduced donor exposures.36
Unfortunately, there are no clinical studies addressing the effect of refrigerated storage between 48 hours6,7
and 8 to 20 days.5
A significant limitation in improving the utilization of WB is its availability, which is directly related to current restrictive guidelines for use, shelf life, and difficulty in obtaining WB from blood centers. Establishing a longer duration of hemostatic quality (shelf life) would increase availability and utility. This would be particularly beneficial in locations where donation and refrigeration are available but component separation is not, such as in military or disaster relief scenarios and remote locations.37
Other certain elective surgical patients who require large-volume blood replacement such as pediatric craniofacial reconstruction, liver transplant, or emergency major vascular surgery and trauma patients would also benefit.37-39
Attempts to determine optimum ratios of components for clinical efficacy in large-volume transfusion have not surprisingly concluded that WB should be replicated, that is, should be administered in a 1:1:1 ratio of RBCs:fresh-frozen plasma (FFP):PLTs.38,40
The continuous immediate availability of a “transfusion package” (5 units of RBCs, 5 units of thawed FFP, 2 units of pooled PLTs) for unexpected massively bleeding patients reduced transfusion requirements and increased survival significantly.38
Substituting WB in such a program would be more efficient resulting in fewer donor exposures and less wastage of outdated plasma and PLTs. We believe that there is sufficient direct and indirect evidence from this and other studies to warrant clinical trials of refrigerated WB stored longer than 48 hours for selected patient populations.