Effects of blood components and whole blood in a model of severe trauma-induced coagulopathy

Abstract

Introduction:

Plasma resuscitation ameliorates hyperfibrinolysis (HF) and trauma-induced coagulopathy (TIC). However, the use of other blood components to reduce HF has not been evaluated. Therefore, our aim was to determine the effect of individual blood components and whole blood (WB), on an in vitro model of severe HF/TIC.

Methods:

A “TIC” solution was made with 1:1 dilution of WB with saline and exacerbated with tissue plasminogen activator (tPA). Components were added in proportions equivalent to the thromboelastography (TEG) based goal-directed resuscitation used at our institution. Whole blood was added at proportions equal to what has been transfused in injured patients. Samples (n=9) underwent citrated native and tPA-challenge (75ng/ml) TEG with analysis of R-time, angle, MA and LY30. Statistical analyses were completed employing the non-parametric Kruskal-Wallis and Dunn’s multiple comparisons tests.

Results:

Compared to control, TIC solution had a decrease in clot strength (MA 41mm vs 51.5mm, p<0.01). The addition of tPA resulted in a severe coagulopathy (MA 24.5mm vs 41mm and LY30 52.8% vs 2.4%, p<0.03 for all). The addition of 4U of WB improved clot strength compared to TIC+tPA (p=0.03). No individual blood component resulted in improved fibrinolysis (p>0.7). Cryoprecipitate improved R-time (7.5 vs 11.9 min, p<0.01), angle (56.8 vs 30.2degrees) and MA (49mm vs 36.25mm), while platelets improved MA (44mm vs 36.25mm) compared to TIC+tPA (p<0.03 for all).

Conclusion:

No single blood component or volume of whole blood led to attenuation of tPA-mediated fibrinolysis in an in vitro model of TIC. Cryoprecipitate was the most effective at improving coagulation function.

Introduction:

Trauma-induced coagulopathy (TIC) is characterized by deficiencies in thrombin generation, low fibrinogen concentration or impaired function, decreased platelet function, and excessive or absent fibrinolysis.^1–6 The most lethal form of TIC is hyperfibrinolysis (HF), which manifests as excessive clot breakdown.^{7, 8} Resuscitation strategies have been proposed to attenuate HF. Plasma-first resuscitation has been shown to reduce HF in trauma patients, while pre-hospital resuscitation with crystalloid has been shown to exacerbate HF.^7,9,10

A number of studies have evaluated the use of in-hospital ratio-based resuscitation protocols for the treatment of hemorrhage in the injured patient and their effects on patient outcomes.^11–15 More recently, the military experience with the implementation of whole blood transfusion protocols has stimulated a renewed interest in the use and development of civilian whole blood massive transfusion protocols to treat hemorrhage and combat TIC.^16–18 These studies have stimulated the development of transfusion protocols including indications for transfusion of whole blood or individual blood component therapies. While these studies have focused on plasma or whole blood transfusion, the effects of other individual blood components on HF and TIC are limited.

Ratio-based massive transfusion protocols and thrombelastography (TEG)-based massive transfusion protocols, which rely on TEG measurements of time to clot initiation, clot propagation, clot strength, and fibrinolysis to guide resuscitation with blood products, are the primary modalities used in urban trauma centers to guide resuscitation in the injured patient.^{12,14,15,19–22} At our institution, a massive transfusion protocol is initiated with a transfusion ratio of 1:2 (plasma: red blood cells) and then transitioned to TEG-guided metrics for subsequent blood product transfusions. We transfuse plasma for prolonged time of clot initiation, cryoprecipitate for impaired dynamics of clot formation, and platelets for reduced clot strength.^12,19

While significant clinical work has been done to evaluate ratio-based, TEG-based, and whole blood massive transfusion protocols, the specific effects of these individual components on viscoelastic coagulation characteristics in a model of TIC has not been fully evaluated. Specifically, the effects of individual components on reducing the incidence of HF has not been evaluated. Most in vitro models that test blood components do not use proportional volumes of these blood products and instead use large ratios compared to the patient’s blood volume.^9,23,24 Therefore, the purpose of this study was to evaluate the effect of individual blood components and whole blood, using a clinically relevant proportion, in an in vitro model of severe TIC associated with HF.

Methods

Materials

Human single-chain tissue plasminogen activator (tPA) was purchased from Molecular Innovations (Novi, MI) and underwent dilution with 5% bovine serum albumin in phosphate-buffered solution (PBS) followed by separation into aliquots and storage at −80°C. As previously described, tPA was thawed on the day of experiment and added to a TIC solution to a desired concentration of 75ng/ml.⁹ This has been shown to produce a reliable and reproducible fibrinolysis profile. ^9,23,24 AB blood type fresh frozen plasma (FFP), AB blood type cryoprecipitate, and apheresis platelets were obtained from Vitalant (Denver, CO). Normal saline for intravenous infusion was purchased from Baxter International (Deerfield, IL).

Healthy volunteer blood collection

After obtaining informed consent, blood samples were collected from nine healthy volunteers between December 2017 and January 2018 in three 3.3-ml buffered sodium citrate (3.2%) tubes (Vacutainer, Becton-Dickingson, Franklin Lakes, NJ) under the Colorado Multiple Institutional Review Board (COMIRB) protocol number 10–0477. Of the volunteers, 6 of the 9 were men, they were aged 27–34 years, and no volunteers were pregnant or taking any medications at the time of blood draw.

Estimated blood volume and estimated in vitro component volume

In order to administer proportional doses of blood components and whole blood, we first determined the estimated blood volume of a typical patient at our institution. Based on our trauma activation protocol (TAP) database, there were 1067 patients that met trauma activation criteria between April 2014 and August 2019 at the Ernest E. Moore Shock Trauma Center at Denver Health Medical Center. Of these, body weight was obtained for 980 patients. The median body weight was 79.4 kg (IQR:70–90.7 kg). For simplicity this was rounded to 80 kg. While weight-based estimates of blood volumes vary, we estimated an 80 kg patient had a blood volume of 6L.²⁵ Further, volume of blood components varies based on country, institution, and provider of blood components. We therefore used estimated volumes of blood components that fell within described volumes in the literature and were in line with what we saw at our institution. The estimated volume of blood components included one unit of plasma being 200 mL, one unit (10 bag) of cryoprecipitate equal to 250 mL, one unit of apheresis platelets 300 mL, and one unit of whole blood 500 mL in volume.^26–29 We then projected these volumes of individual components and WB relative to a 6L estimated blood volume to our model which had a total volume of 500 μL.

Preparation of blood components and whole blood

One unit of AB (Rh-) fresh frozen plasma (FFP) was thawed and separated into 1000 uL aliquots followed by flash freeze with liquid nitrogen. FFP was subsequently thawed on the day of experiment for addition to TIC solution. One unit of AB (Rh-) cryoprecipitate was thawed and separated into 500 μL aliquots followed by flash freeze in liquid nitrogen. Cryoprecipitate was subsequently thawed on the day of experiment for addition to TIC solution. Apheresis platelets were collected on the day of the experiment. The final concentration of apheresis platelets was >100 k/μL for all apheresis samples used in these experiments. Apheresis platelets were stored at 4°C until addition to the TIC solution. We used the blood collected from the healthy volunteer as the whole blood (WB) administered to the TIC solution.

Thrombelastography

A schematic representation of control and experimental groups is shown in Figure 1. Non-diluted whole blood was used as a control (WB only). In order to establish an in vitro model of TIC, a combination of WB and saline (1:1) was created to establish a “TIC” solution (TIC), simulating the dilutional coagulopathy and acidosis that occurs during hemorrhagic shock and resuscitation with prehospital crystalloid. We then added tPA to simulate the HF phenotype (TIC+tPA), which was treated with addition of individual components or WB. At our institution, 2 units of plasma are transfused for prolonged clot initiation, 10 bags of cryoprecipitate are transfused for impaired dynamics of clot formation, and 1 unit of platelets transfused for reduced clot strength.¹⁹ Components were added in proportions equivalent to the TEG-based goal-directed resuscitation used at our institution with plasma (2U), cryoprecipitate (one 10 bag) and apheresis platelets (1U) projected on a final volume of 500 μL. A recent study evaluating transfusion of WB found that 1–4U of WB was safe²⁹ and we subsequently used these proportions to evaluate effects of WB administration on our model of severe TIC. Samples (n=9) underwent citrated native and tPA-challenge (75ng/ml) TEG as previously described using the TEG 5000 Thrombelastograph Hemostasis Analyzer (Haemonetics, Niles, IL).⁹ Blood components or WB were added to these solutions. Based on the calculated weight of our TAP patients, and the volume of specific blood components, these blood components were added in the appropriate ratio to a TIC solution in a centrifuge tube with a final volume of 500 μL. TEG properties including speed of clot initiation (R-time), rate of clot formation (angle), maximum clot strength (maximum amplitude, MA), and percent lysis at 30 minutes (LY30) were analyzed.

Figure 1:

Schematic representation of control groups and experimental groups. TIC = trauma induced coagulopathy, tPA = tissue plasminogen activator, and WB = whole blood

Statistics

TEG values are reported as median with interquartile ranges. Statistical analysis was done using SPSS version 24 (IBM) and GraphPad Prism version 7.0a (GraphPad Software, Inc; La Jolla, CA). TEG values R-time, angle, MA, and LY30 had a skewed, non-normal distribution. Differences across groups were determined using a non-parametric Mann-Whitney test or the Kruskal-Wallis test and Dunn’s multiple comparisons test. Statistical significance was set at p<0.05.

Results:

Creation of a severe TIC

A TIC solution was created with a 1:1 dilution of WB and normal saline. Compared to control, the TIC solution without tPA had similar R-time (13.3 IQR 10.1–18.4 vs 12.6 IQR 9.2–15.3 min, p=0.53), similar angle (32.7 IQR 28.9–47.9 vs 33.3 IQR 32.6–44.0 degrees, p=0.78), diminished MA (51.5 IQR 45.3–55.3 vs 41 IQR 40.0–45.0 mm, p<0.01), and lower LY30 (2.4 IQR 1.2–4.5 vs 0.8 IQR 0.5–1.1 percent, p=0.01). The addition of tPA to a TIC solution worsened coagulopathy as measured by viscoelastic coagulation parameters. TIC+tPA had a similar R-time and angle compared to the control and TIC without tPA solution (p>0.99), but had a significantly reduced MA (24.5 IQR 13.5–37 mm, p<0.03) and increased LY30 (52.8 IQR 44.6–68.25 percent, p<0.03) compared to control and TIC without tPA solutions.

Whole blood transfusion effects on severe TIC

When added to the TIC+tPA solution, whole blood improved MA only if the equivalent of 4U of WB was administered. Table 1 shows the TEG parameters of TIC+tPA and TIC+tPA with the addition of 1U, 2U, or 4U of whole blood. There was no change in R-time or angle. There was no reduction in LY30 with increasing number of WB units added to the TIC+tPA solution.

Table 1:

TEG parameters of the TIC+tPA solution as well as TIC+tPA with addition of 1U, 2U, or 4U of whole blood. There was no change in the coagulation parameters when increasing units of WB are given except for 4U of WB, which shows a significant increase in clot strength (MA).

	R-Time (sec)	Angle (degrees)	MA (mm)	LY30 (%)
TIC+tPA	11.9 (10.6–15.2)	30.2 (24.9–43.7)	24.5 (13.5–36.3)	52.8 (44.6–68.3)
1U WB	9.8 (9.7–14.4	40.8 (25–43.9)	26 (19.5–37.3)	60.8 (44.3–63.2)
2U WB	12.1 (9.7–12.9)	38.6 (33.5–45.8)	33 (26.8–41.8)	45.2 (32.3–59.3)
4U WB	12.1 (9.6–15)	38.4 (32.3–45.2)	39 (32.5–49.5)*	40.4 (25.4–49.4)

^*P=0.027

Individual blood component effects on severe TIC

Based on the transfusion protocol at our institution, 2 units of FFP are administered for a prolonged R-time, one 10 bag of cryoprecipitate is administered for a reduced angle, and 1 unit of platelets is administered for a decreased MA. R-time was enhanced with the addition of the equivalent of 1 bag of cryoprecipitate (7.5 IQR 6.9–9.25 min, p<0.01), while the addition of plasma (10.8 IQR 9.45–14.1 min, p>0.99) and platelets (9.7 IQR 9.2–11.65 min, p=0.19) did not improve R-time when added to a TIC+tPA solution (11.9 IQR 10.55–15.15 min) (Figure 2). R-time was unchanged from control (13.3 IQR 10.1–18.4 min, p<0.01), but cryoprecipitate added to a TIC+tPA solution led to enhanced angle compared to control. Plasma (41 IQR 34.55–51.5 degrees, p=0.58) and platelets (43.1 IQR 31.2–52.05 degrees, p=0.72) did not lead to an increased angle compared to TIC+tPA solution (30.2 IQR 24.85–43.7 degrees). However, the addition of the equivalent of 1 bag of cryoprecipitate led to a significant improvement in angle (56.8 IQR 52.55–60.75 degrees, p<0.01)(Figure 3). Further, the angle was unchanged from control (32.7 IQR 28.85–47.9 degrees, p>0.99) when plasma or platelets were added to a TIC+tPA solution. In contrast, cryoprecipitate led to an angle greater than that of control when added to TIC+tPA (p<0.01). Compared to TIC+tPA (24.5 IQR 13.5–36.25 mm), MA increased with the addition of platelets (44 IQR 34.5–49 mm, p=0.02) and cryoprecipitate (49 IQR 47–54 mm, p<0.01). Further, compared to whole blood control (51.5 IQR 45.25–55.25 mm), platelets and cryoprecipitate resulted in near normal MA (p>0.3 for both). The addition of plasma did not increase MA (31.5 IQR 25–37 mm, p>0.99) (Figure 4). Plasma (56.6 IQR 47.15–59.1 percent, p>0.7), platelets (48.2 IQR 33.45–57.9 percent,, p>0.7), nor cryoprecipitate (47.3 IQR 43.55–61.05 percent, p>0.7) reduced tPA mediate fibrinolysis in a TIC+tPA solution (52.8 IQR 44.6–68.25 percent, p>0.7) (Figure 5).

Figure 2:

The addition of cryoprecipitate to a TIC+tPA solution led to enhanced clot initiation. The addition of plasma or platelets had a similar clot initiation compared to a TIC+tPA solution. Cryoprecipitate also is shown to have superior clot initiation compared to whole blood control *p<0.01 compared to TIC+tPA, †p<0.01 compared to control

Figure 3:

The addition of cryoprecipitate to a TIC+tPA solution led to enhanced dynamics of clot formation. Angle was enhanced compared to whole blood control. The addition of plasma or platelets had a similar angle compared to a TIC+tPA solution. *p<0.01 compared to TIC+tPA, †p<0.01 compared to control

Figure 4:

The addition of cryoprecipitate and platelets to a TIC+tPA solution led to enhanced clot strength. The addition of plasma had a clot strength compared to a TIC+tPA solution. *p<0.03 compared to TIC+tPA, †p<0.01 compared to control

Figure 5:

Compared to a TIC+tPA solution, there was not a difference in fibrinolysis when plasma, platelets, nor cryoprecipitate were added. *p<0.01 compared to control

Discussion:

A 1:1 dilution of normal saline and whole blood, with the addition of tPA, created a model of TIC with reduced clot strength and hyperfibrinolysis. While lower doses of WB (1U and 2U) did not improve the coagulopathy seen with the TIC+tPA solution, addition of the equivalent of 4U of WB improved clot strength compared to a TIC+tPA solution. Clinical proportions of platelets improved clot strength, while cryoprecipitate led to enhanced clot initiation, dynamics of clot formation, and clot strength. Neither whole blood, plasma, platelets, nor cryoprecipitate resulted in reduction of tPA mediated fibrinolysis in a model of severe TIC. This model of TIC is the first to employ clinically relevant proportions of individual blood products in the in vitro setting to evaluate their effect on hemostatic potential using TEG. In the TEG-based resuscitation protocol at our institution, plasma is administered to correct deficiencies in clot formation, cryoprecipitate is administered to treat fibrinogen deficiencies, platelets are transfused to treat reduced clot strength, and antifibrinolytics (such as tranexamic acid) are given to treat hyperfibrinolysis.^12,19,22 In our model, plasma did not improve any parameter of coagulation based on TEG. In vitro, animal, and clinical studies have evaluated the use of plasma-first resuscitation, specifically to reverse or reduce the effects of fibrinolysis.^9,10,30 In our study however, we did not see an improvement in coagulation measurements when the equivalent of 2U of FFP was administered to our TIC+tPA solution. Compared to two previous studies,^9,10 we developed a greater degree of coagulopathy, particularly hyperfibrinolysis, and used much smaller proportions of plasma in our in vitro model. Our study illustrates that the equivalent of a single unit of apheresis platelets, in severe TIC, can improve clot strength. Platelet activity correlates with clot strength.³¹ However, saline has been shown to exacerbate platelet dysfunction.^32–34 The addition of functional platelets may therefore compensate for the impaired clot strength seen in the TIC+tPA solution.

Cryoprecipitate had the greatest efficacy in improving TEG-based coagulation parameters. Clot initiation, dynamics of clot formation, and clot strength were all improved with addition of a single unit of cryoprecipitate. Cryoprecipitate is most commonly transfused to treat disorders of fibrinogen depletion. The high concentration of coagulation factors such as von Willebrand factor and factor XIII, likely contributes to its ability to reduce clot initiation time.²⁷ Fibrin usually contributes to 20% of clot strength, explaining how administration of this blood product would increase TEG MA.³⁵ Further, in renal failure patients, it is postulated that clot strength is maintained, even in settings of platelet dysfunction, by the increase in fibrinogen concentration that is seen in this population.³⁶ In patients receiving a massive transfusion (defined as >4 units of PRBC in the first hour), over 40% of patients had TEG abnormalities that indicated a need for cryoprecipitate transfusion.³⁷ With cryoprecipitate acting as the most efficacious single blood product that effects TEG-based metrics of hemostasis, earlier transfusion of cryoprecipitate may be indicated. However, studies designed to determine the effects of early cryoprecipitate would need to be pursued to better understand the risks and benefits of an early cryoprecipitate transfusion strategy.

Recently, the military has had the most robust experience with the use of whole blood for massive transfusion during the conflicts in Iraq and Afghanistan.^16,17 These studies have illustrated the benefit of WB transfusion in injured patients. Since then, work has been done to translate this to the civilian population. Recently, protocols have been established for the use of WB transfusions at civilian centers.^18,29 However, recent studies have illustrated that WB massive transfusion alone does not adequately correct the coagulopathy seen in the injured patient.¹⁸ These patients may require supplemental blood products to achieve normal clotting properties in addition to the WB that is transfused¹⁸. A study by Kornblith et al. showed that whole blood exhibits greater hemostatic properties than did either 1:1:1 or 1:1:2 ratios of blood components.³⁸ In particular, this study showed that WB had increased maximum clot firmness (a measure of cloth strength) compared to mixes of 1:1:1 or 1:1:2 ratios of blood products and components.³⁸ While WB did not lead to significant improvement in clot initiation or angle in our study, the equivalent of 4 units of WB in volume led to improvement in clot strength when added to an in vitro model of severe TIC. Lower doses of WB however, did not lead to improvement in TEG-based coagulation parameters. However, it is likely that correction using 4 units of WB was primarily due to reversal of a hemodiluted state in our model. Whole blood also may exert effects through proteomic, metabolomic, or effects of the endothelium, but these are unable to be measured or quantified in this in vitro model.

There are limitations to this study. While increased tPA activity has been shown in the plasma of trauma patients,^39,40 there are undoubtedly other abnormalities (metabolic or proteomic) that effect the fibrinolysis profile that were not included in the development of this model of severe TIC. The tPA added in vitro does not completely replicate the coagulopathic changes seen in injured patients. However, the use of exogenous tPA to an in vitro model produces a reliable fibrinolysis profile that can be modulated to study the effects of interventions, specifically addition of WB or blood component therapy. In an attempt to use proportional volumes of blood products and whole blood, estimates of the volumes of blood products needed to be made and ultimately translated to equivalent volumes in a 500 μL in vitro model. While these estimates were informed through literature review and discussion with our institutional blood bank, volumes and even concentrations of coagulation factors (such as fibrinogen) may vary from institution to institution and even country to country. Furthermore, this model does not include effects of the endothelium. Recent studies have illustrated the development of endotheliopathy following injury and a higher degree of endothelial damage is associated with hypocoaguability and hyperfibrinolysis.⁴¹ While this study was not designed to evaluate the endothelium, future studies should focus on effects of blood and blood components and maintaining a functional endothelium or reducing endotheliopathy following trauma. Finally, these experiments were performed with the blood of healthy volunteers and therefore do not fully reflect the spectrum of metabolic and proteomic abnormalities observed in the severely injured trauma patient.

This study illustrates the effects of WB and single blood components, at proportional doses, on TEG-based coagulation parameters in a model of severe trauma induced coagulopathy. Clot strength was the parameter that was able to be improved upon by the greatest number of products while cryoprecipitate was the most efficacious at enhancing multiple coagulation parameters and should be considered early in transfusion of a severely coagulopathic and injured patient. No single component or amount of WB was able to improve fibrinolysis. Further studies should focus on metabolites, proteins, and the endothelium as targets to reduce fibrinolysis in injured patients.