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. Author manuscript; available in PMC: 2019 Nov 19.
Published in final edited form as: Transfus Med. 2019 Aug 5;29(5):351–357. doi: 10.1111/tme.12622

Effects of whole blood leukoreduction on platelet function and hemostatic parameters

M C Morris 1, R Veile 1, L A Friend 1, D Oh 2,3, T A Pritts 1, W C Dorlac 4, P C Spinella 5, M D Goodman 1
PMCID: PMC6863083  NIHMSID: NIHMS1059346  PMID: 31382318

SUMMARY

Aims/Objectives:

The aim of this study was to evaluate the hemostatic consequences of whole blood leukoreduction (LR).

Background:

Whole blood is being used for trauma resuscitation in the military, and an increasing number of civilian trauma centres across the nation. The benefits of LR, such as decreased infectious and transfusion-related complications, are well established, but the effects on hemostatic parameters remain a concern.

Methods:

Twenty-four units of whole blood were assigned to one of the four groups: non-leukoreduced (NLR), leukoreduced at 1 h and a height of 33 in. (LR-1), leukoreduced at 4 h and a height of 33 in. (LR-4(33)), or leukoreduced at 4 h and a height of 28 in. (LR-4(28)). Viscoelastic parameters, platelet aggregation, cell counts, physiological parameters and thrombin potential were evaluated immediately before and after LR, and on days 1, 7, 14 and 21 following LR.

Results:

The viscoelastic parameters and thrombin generation potential were unchanged between the groups. Platelet aggregation was reduced in the LR-1 group compared with NLR after 7 days. The LR-4(28) group also showed a trend of reduced platelet aggregation compared with NLR. Aggregation in LR-4(33) was similar to NLR throughout the storage time. Physiological and electrolyte changes over the whole blood storage period were not affected by LR.

Conclusion:

Our study shows that whole blood can be LR at 4 h after collection and a height of 33 in. while maintaining platelet count and without altering platelet function and hemostatic performance.

Keywords: coagulopathy, haemorrhage, resuscitation, whole blood, shock, trauma

Injury remains the fourth leading cause of overall mortality in the United States. Haemorrhage and haemorrhagic shock are the leading potentially preventable causes of trauma-related mortality (Sauaia et al., 1995). Trauma-induced coagulopathy is observed in up to 28% of injured patients (MacLeod et al., 2003). The need for massive transfusion has been estimated to be approximately 12–15% in the adult trauma population who requires blood (Nunez et al., 2009; Cotton et al., 2010). Haemorrhagic shock, massive tissue injury and coagulopathy have been associated with the need for massive transfusion and increased mortality (McLaughlin et al., 2008; Niles et al., 2008; Spinella et al., 2008).

Increasing recognition of trauma-induced coagulopathy has contributed to the transition back to blood-first resuscitation strategies. The use of whole blood, component therapy and crystalloid in trauma resuscitation has varied over the past century (Hess & Thomas, 2003). Because of concerns regarding resource utilization, whole blood fractionation became popular and component therapy became the primary form of resuscitation in the latter part of the 20th century (Ho et al., 2005). However, limited use of whole blood persisted in the military (Repine et al., 2006). Recent studies of combat-related injuries have demonstrated the ability of fresh whole blood to increase survival in traumatic haemorrhage. Whole blood resuscitation has also been shown to reduce total transfusion volumes in selected civilian populations (Cotton et al., 2013).

Logistically, it is difficult to implement fresh whole blood transfusion protocols in civilian environments (Spinella, 2008). The risks associated with fresh whole blood, including virus transmission and transfusion reactions, have also remained a concern (Spinella et al., 2007). Fresh whole blood is typically rapidly transfused and is unable to go through real-time transfusion-transmitted disease testing. By contrast, cold-stored whole blood is approved by the Food and Drug Administration (FDA), and there is growing interest in implementation of low-titre group O whole blood (LTOWB) in the civilian setting (Yazer et al., 2018).

Leukoreduction (LR) is thought to be an additional step that could improve safety in the use of whole blood. LR is able to reduce human leukocyte antigen (HLA) alloimmunization, febrile reactions and viral transmission. However, whole blood platelet function has been shown to decrease over time during storage (Pidcoke et al., 2013). The effects on platelet quantity and mass, function and hemostatic performance following LR using an FDA-approved platelet-sparing filter at various heights are unknown. We aimed to examine the effect of in vitro LR on whole blood hemostatic properties. We additionally sought to examine the effects of timing and height of LR on whole blood platelet and coagulation performance.

MATERIALS AND METHODS

Grouping, LR, storage and sampling

Fresh whole blood from male donors was purchased from Hoxworth Blood Center. Donation of 500 mL of whole blood acquired by the standard blood donation protocol was transferred to an FDA-approved IMUFLEX WB-SP blood bag system with an integrated whole blood leukocyte reduction platelet-sparing filter (Terumo BCT, Lake Zurich, CO, USA). Each IMUFLEX WB-SP bag set contains 63 mL of citrate phosphate dextrose (CPD) anticoagulant and SAG-M (saline adenine glucose mannitol) additive solution. The whole blood units were transported within an hour of donation to the laboratory and remained untreated and at room temperature until LR. The units were then randomly assigned to one of the four different groups: non-leukoreduced (NLR), leukoreduced at 1 h at the standard height (33 in.) (LR-1(33)), leukoreduced at 4 h at the standard manufacturer-recommended height (LR-4(33)) or leukoreduced at 4 h at a lower height (28 in.) (LR-4(28)). The units were leukoreduced according to the manufacturer’s instructions and then stored at 4 °C without agitation. This variability in height was hypothesized to slow the LR process and possibly allow for better retention of platelets or decreased platelet activation. The height is measured from the bottom of the blood bag to the table that the post-filtration collection bag is placed on. A total of 20 mL samples were taken from each unit on the day of collection (day 0) before and after LR, and days 1, 7, 14 and 21 after filtration. Prior to sampling, the units were gently mixed to ensure adequate mixing and homogeneous samples.

Complete blood count and arterial blood gas analysis

A complete blood count for each unit was obtained at each timepoint utilizing a Coulter AcT 10 Hematology Analyzer (Beckman Coulter, Brea, CA, USA). An arterial blood gas sample was analysed at each timepoint utilizing VetScan iSTAT (Abaxis, Union City, CA, USA). Prothrombin time evaluation was attempted, but did not generate a result using the iSTAT at any timepoint possibly because of an interaction of the storage solution with the iSTAT cartridge.

Thromboelastometry

Viscoelastic coagulation testing was performed using the rotational thromboelastometry (ROTEM) delta WB analyzer (ROTEM, TEM Systems Inc., Durham, NC, USA) following the manufacturer’s instructions. A total of 300 μL of whole blood was aliquoted into each test cup. Coagulation was initiated for EXTEM and FIBTEM testing with 20 μL of thrombo-plastin. FIBTEM testing was performed with the addition of cytochalasin D for platelet inhibition. The temperature was maintained at 37 °C, and the samples were allowed to run through 60 min after the maximal clot firmness (MCF) was reached, assessing the clotting time (CT), clot formation time (CFT), alpha angle and clot lysis. Platelet contribution to clot strength (%MCFPlatelet) was calculated by the equation: (EXTEMMCF – FIBTEMMCF)/EXTEMMCF, similar to the methods previously described (Midura et al., 2015).

Platelet function

Platelet aggregation was measured by using a multiplate impedance aggregometer by Roche Diagnostics (Risch-Rotkreuz, Switzerland) according to the manufacturer’s instructions. Four different agonists were used including adenosine diphosphate (ADP), arachidonic acid (ASPi), collagen (COL) and thrombin receptor agonist peptide (TRAP). The test was performed by incubating 300 μL of whole blood with 300 μL of 0·9% sodium chloride for 3 min. After incubation, 20 μL of the respective agonist was added (6·5 μmol L−1 ADP, 0·5 mmol L−1 ASPi, 3·2 μg mL−1 collagen or 32 μmol L−1 TRAP). Platelet aggregation velocity, total platelet aggregation (AU) and the area under the curve (AUC) were measured.

Thrombin generation

Thrombin generation was evaluated using a calibrated automated thrombinoscope (Diagnostica Stago, Parsippany, NJ, USA) according to the manufacturer’s instructions. The platelet poor plasma (PPP) and platelet-rich plasma (PRP) were processed from each whole blood sample immediately prior to analysis. For PPP, whole blood was centrifuged at 2000 g for 10 min at room temperature. The plasma was then collected and centrifuged again at 10000 g × 1000 for another 10 min. The PPP was removed from above the platelet pellets and was aliquoted for testing. For PRP, whole blood was centrifuged at 260g for 6 min. The plasma was collected and centrifuged for another 6 min at 640g to ensure buffy coat removal.

For the analysis of PPP, samples were aliquoted into the test well and 20 μL of each chosen reagent was added [PPP low, PPP high and microparticles (MP)]. For PRP, the sample to be tested was aliquoted into the test well and 20 μL of the PRP reagent was added. The samples were incubated at 37 °C for 10 min and 20 μL of the FluCa reagent was added. Each sample was run in triplicate as recommended by the manufacturer, assessing the endogenous thrombin potential (ETP), time to peak (ttPeak), peak thrombin generation and thrombin generation velocities.

Serum markers of platelet function and activation

Whole blood was placed in serum separator tubes (BD Bioscience, San Diego, CA, USA) and centrifuged at 1000g for 10 min, and the resulting serum was collected. Serum levels of fibrinogen, CD40L, P-selectin and platelet factor-4 (PF4) were measured by ELISA according to the manufacturer’s protocols (Abcam, Cambridge, MA, USA). Thromboxane A2 (TXA2) was also assessed by ELISA according to the manufacturer’s protocol (Antibody Research Corporation, St. Peters, MO, USA). The lower limits of detections were as follows: fibrinogen 1·56 ng mL−1, CD40L 8·23 pg mL−1, P selectin 0·041 ng mL−1, PF4 0·04 ng mL−1 and TXA2 62·5 pg mL−1.

Statistical analysis

Prism 6 (GraphPad Software, La Jolla, CA, USA) was used for all statistical analyses. All data are presented as the median (interquartile range). The Kruskal–Wallis test was used to compare continuous nonparametric data between the different groups. Analysis was performed to compare non-LR vs LR at 1 h (33 in.) vs LR at 4 h (33 in.) vs LR at 4 h (28 in.) at day 0 pre-LR and post-LR, day 1, day 7, day 14 and day 21. A P value of <0·05 was considered significant.

RESULTS

Effect of LR on complete blood count

The percentage of platelet recovery following LR is shown in Table 1. Importantly, platelet recovery was similar between all leukoreduced groups. The platelet count was similar before and after LR. In the NLR group, platelet count was decreased by day 1 from baseline and also decreased from day 1 to day 14. In the LR-1(33) and LR-4(28) groups, platelet count was decreased by day 7 from baseline. However, the platelet count in the LR-4(33) group did not change over time. Prior to LR, the NLR group had a higher platelet count than the LR-4(28) group, and after LR, the LR-4(33) group had a higher platelet count than the LR-4(28) group. There were no other differences between the groups at any timepoint (Fig. 1a). The white blood cell (WBC) count remained higher on day 1 for the NLR group than for the LR-4(28) group. The WBC count was maintained in the NLR blood but was not measurable in the LR groups (Fig. 1b). No other significant differences were found between the groups. Haemoglobin was similar between groups at all timepoints (data not shown).

Table 1.

Average platelet and red blood cell (RBC) counts recovery percentage following leukoreduction in each group

Platelet recovery (%) RBC recovery (%)
LR at 1 h (33 in.) 69·5 92·7
LR at 4 h (33 in.) 90·1 94·9
LR at 4 h (28 in.) 73·7 97·4
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Fig. 1.

Fig. 1.

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Average (a) platelet count and (b) white blood cell (WBC) count in each group over the study period.

Effect of LR on arterial blood gas and electrolyte analysis

The pH and potassium levels were not significantly different between the groups at any timepoint (Fig. 2a,b). However, the pH was significantly decreased in each group by day 21 when compared to pre-LR, and potassium was increased by day 1 compared to pre-LR. Base excess also did not demonstrate any significant differences between the groups, but did show a negative trend over time (Fig. 2c). Sodium level did not have any differences between groups, but did significantly decrease in each group by day 7 (data not shown).

Fig. 2.

Fig. 2.

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Average (a) pH, (b) base excess (BE) or (c) potassium (K) level in each group over the study period.

Effect of LR on platelet aggregation

In the LR-1 group, platelet aggregation was reduced at days 1 (ADP, COL, TRAP), 7 (COL, TRAP, ASPi), 14 (all agonists) and 21 (all agonists) compared to NLR. In the LR-4(28) group, platelet aggregation was reduced at days 1 (ADP, TRAP, ASPi), 7 (TRAP), 14 (COL) and 21 (ADP, COL, TRAP) compared to NLR. In the LR-4(33) group, platelet aggregation was similar to NLR. The AUC in the LR-4(28) group was also significantly reduced at day 21 compared to pre-LR in all four agonist groups, whereas only the TRAP-induced AUC was reduced for the LR-4(33) group at day 21 compared to pre-LR. The NLR group was not decreased on day 21 compared to baseline for any of the four agonists (Fig. 3a–d).

Fig. 3.

Fig. 3.

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Platelet aggregation shown by the area under curve (AUC) in each group when stimulated with (a) adenosine diphosphate (ADP), (b) thrombin receptor agonist peptide (TRAP), (c) arachidonic acid (ASPi) or (d) collagen (COL) over the study period.

Effect of LR on viscoelastic parameters

There were no differences in EXTEM CT, CFT, alpha angle and MCF between the groups at any timepoint. The EXTEM MCF was reduced in all groups from day 0 to day 21 (Fig. 4a). In addition, the FIBTEM CT, MCF and alpha were similar between all groups. Therefore, there were no notable differences in stored whole blood clot initiation, formation, or strength after LR. In addition, the platelet contribution to clot (%MCF-Platelet) did not change from day 0 to day 21 (Fig. 4b).

Fig. 4.

Fig. 4.

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Maximal clot firmness (MCF) of the (a) extrinsic coagulation cascade (EXTEM) and (b) platelets (Plts) in each group over the study period. (c) Microparticle (MP)-associated endogenous thrombin potential (ETP) in each group over the study period.

Effect of LR on thrombin generation potential

The median ETP in both high and low tissue factor experiments showed no difference between the groups. In the PRP and MP experiments, the median ETP was also similar between the groups. However, the median ETP in the MP experiment was increased from day 0 to day 21 in all groups except the LR-4(28) group (Fig. 4c). In all four experiments, the Peak, ttPeak and velocities of thrombin generation were similar between all groups. Therefore, there were no notable differences in thrombin generation following LR.

Effect of LR on platelet factors and degradation products

Serum PF4 levels significantly decreased over time in all groups (Fig. 5a). The PF4 levels were significantly higher in the LR-4(28) than in NLR at days 14 and 21. Thromboxane A2 and fibrinogen levels were similar between all groups. Fibrinogen levels were also unchanged from day 0 to day 21 in all groups. Although serum P-selectin and CD40L levels increased over time by day 21, there were no differences demonstrated following LR (Fig. 5b,c).

Fig. 5.

Fig. 5.

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Enzyme-linked immunosorbent assay (ELISA) concentrations in each group over the study period for (a) platelet factor 4 (PF4), (b) P selectin and (c) CD40 ligand (CD40L). * indicates P < 0·05 and – indicates P < 0·05 for each respective symbol.

DISCUSSION

This study evaluated hemostatic and platelet function parameters over a 21-day period following the platelet-sparing LR of the donated whole blood. Although whole blood LR outcomes have been previously published, this is the first study evaluating LR at different heights and times following donation. The group that underwent LR at 28 in. was affected by the reduced height, and the currently recommended height is likely optimal. However, our study did demonstrate that blood leukoreduced at 1 h following collection had a lower platelet aggregation than NLR blood, whereas LR at 4 h did not reduce the platelet function.

LR at a lower height was performed to slow filtration and evaluate whether this change would allow for enhanced platelet recovery and reduced filtration-induced platelet activation. LR at 28 in. consistently showed decreased platelet aggregation compared to the NLR group. This is a novel finding and suggests that the currently recommended height for this platelet-sparing LR filter may yield optimal results. However, the ability to maintain hemostatic function in the LR-4(33) group is in contrast to previously published data demonstrating impaired coagulation in leukoreduced whole blood. Siletz et al. (2017) demonstrated delayed clot initiation, clot progression and maximal clot formation in leukoreduced blood over a 30-day time period. However, a non-platelet-sparing filter was used and may account for the differences in clotting. Another study demonstrated significant reduction in viscoelastic parameters and thrombin potential in leukoreduced whole blood compared to NLR blood (Remy et al., 2018). The current results, therefore, suggest that the discrepancy from previous data may be because of the type of filter used for LR or the technique of platelet-sparing LR. Therefore, filtration at the appropriate height and time from collection with a platelet-sparing filter may allow for preserved hemostatic function of stored whole blood.

Similarly, another interesting finding was the decreased platelet aggregation in the LR-1 group. Our hypothesis is that disturbing the platelets a second time within a short interval following blood donation may induce additional activation and thereby a reduction of remaining platelet aggregability. Remy et al. (2018) showed a similar decrease in platelet aggregation in leukoreduced whole blood over a 15-day period and LR in that study occurred at 1 h following collection. Other studies have also shown similar findings. Pidcoke et al. (2013) demonstrated a similar decrease in platelet aggregation, but showed attenuation of this effect by using refrigeration. These results have also been corroborated by other studies (Jobes et al., 2011; Sivertsen et al., 2018). Jobes et al. demonstrated decreased aggregation in LR blood when stimulated with collagen and ristocetin, but not with ADP and epinephrine (Jobes et al., 2011). In addition, a study evaluating forced filtration at higher pressures demonstrated decreased aggregation by day 10 of storage (Sivertsen et al., 2018). These previous studies also showed a similar decrease in pH and sodium as well as an increase in potassium over time, which is similar to the current results.

Previous studies have evaluated platelet aggregation of apheresis platelets following storage. Reddoch et al. showed that cold storage produced better platelet aggregation than platelets stored at room temperature. They also demonstrated that TRAP-induced platelet aggregation decreased over time, regardless of the storage technique (Reddoch et al., 2014). When stimulated with ADP or collagen, only the platelets stored at room temperature appeared to decrease aggregability over time. In another study by Getz et al., the authors demonstrated reduced platelet aggregation over a 15-day study period following stimulation with TRAP or collagen (Getz et al., 2016). These studies demonstrate that platelet aggregation may decrease over time, regardless of LR but may be mildly maintained by cold storage. However, these platelet aggregation tests were not created to simulate platelet aggregation in a trauma patient in haemorrhagic shock and are limited by the absence of endothelial and flow dynamics contribution to clotting. Therefore, the applicability of platelet aggregation alone compared to whole blood hemostatic function and viscoelastic coagulability following LR is unknown.

This study also demonstrates successful LR using the IMUFLEX WB-SP filter as evaluated by the general haematological and physiological properties of whole blood over time. We demonstrated successful decreases in WBC count following LR and a difference in WBC count compared to NLR blood, validating the efficacy of the LR filter. It is also important to note that LR did not affect haemoglobin concentration, and platelet recovery was not affected by the timing or height during LR. Upon blood gas analysis, we demonstrated that the height of filtration did not have any effect on pH, base deficit, sodium or potassium levels after filtration or during subsequent storage. All values changed over time, but this was a consistent finding throughout all groups. These findings are novel and demonstrate the physiological similarities, regardless of filtration height and post-donation timing used for LR.

The viscoelastic properties observed in EXTEM and ROTEM did not show any differences between groups. Although the EXTEM MCF was reduced over the time period, it was decreased in both LR and NLR groups. The EXTEM results are likely unchanged because this test is also dependent on coagulation factors, which have been shown to be preserved and effective for over 21 days (Nilsson et al., 1983). In addition, fibrinogen and thrombin generation were also preserved over time following LR. This is compared to a previous study that showed ETP was reduced in low tissue factor experiments in the LR groups compared with NLR blood. However, in the high tissue factor experiments, the ETP was similar between the NLR and LR groups (Siletz et al., 2017; Remy et al., 2018). This preservation of thrombin generation supports the notion that the decreased platelet aggregation observed may not be clinically relevant since thrombin generation is also reliant on platelet activation.

The limitations of this study are the in vitro experimental conditions and lack of direct clinical implementation. Whole blood LR in the trauma population remains a controversial topic. The benefits of LR are well studied (Kim et al., 2016), but it is unclear if the benefits outweigh the risks in a trauma patient with haemorrhagic shock. Any clinical significance of this in vitro study would need to be confirmed by clinical studies assessing hemostatic properties following the transfusion of whole blood units. The hemostatic parameters in this study are limited by the lack of endothelial contribution and flow dynamics to hemostasis. This study focused only on a single LR filter, so the results may not be generalizable to all LR filters, timing and techniques. Finally, for homogeneity of blood samples in this study, only blood from male donors was analysed and the variability of gender or blood type may need further consideration in future studies as not all whole blood may age or function similarly following storage.

In conclusion, this in vitro analysis of whole blood LR demonstrates that NLR and LR whole blood have similar thrombin generation potential, viscoelastic parameters and amount of platelet degradation products. Platelet aggregation was significantly decreased in the group that was leukoreduced at 1 h or at a lower height than recommended by the manufacturer. Therefore, this study suggests that whole blood LR at 4 h from a height of 33 in. is safe and does not have any significant deleterious effects on platelet function and hemostatic properties compared to NLR blood.

ACKNOWLEDGMENTS

M. C. M. and M. D. G. contributed to the design of the study. M. C. M., R. V. and L. A. F. contributed to acquisition of data. M. C. M., D. O., T. A. P., W. C. D., P. C. S. and M. D. G. contributed to data analysis. M. C. M. and M. G. D. drafted the article. All authors contributed to critical review of the article and have agreed to the final version of the article.

Footnotes

CONFLICT OF INTEREST

The authors have no competing interests.

Publisher's Disclaimer: DISCLAIMER

The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Air Force, the Department of Defense, or the U.S. Government.

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