- Email: [email protected]
Impact of blood products on platelet function in patients with traumatic injuries: a translational study
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Available online at www.sciencedirect.com
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Impact of blood products on platelet function in patients with traumatic injuries: a translation study
Hanne Hee Henriksen, MD,a,b,1 Alexandra G. Grand,a,1 Sandra Viggers,b Lisa A. Baer,a S. Solbeck,b Bryan A. Cotton,a N. Matijevic,a Sisse R. Ostrowski,b Jakob Stensballe,b,c Erin E. Fox,a Tzu-An Chen,a John B. Holcomb,a Pa¨r I. Johansson,a,b Jessica C. Cardenas,a a, Q2Q3 and Charles E. Wade, PhD *
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a
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Center for Translational Injury Research (CeTIR) and Department of Surgery, University of Texas Health Science Center, Houston, Texas b Section for Transfusion Medicine, Capital Region Blood Bank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark c Department of Anesthesia, Centre of Head and Orthopedics, Rigshospitalet, Copenhagen University Hospital, Denmark
article info
abstract
Article history:
Background: Reductions in platelet (PLT) count and function are associated with poor
Received 11 October 2016
outcomes in trauma patients. We proposed to determine if patients expected to receive
Received in revised form
blood products have a decrease in PLT function higher than expected based on the
22 December 2016
reduction in PLT count, and if the reduction in function could be associated with the donor
Accepted 16 February 2017
plasma/supernatant received.
Available online xxx
Methods: PLT count and function were measured on admission to the emergency department and intensive care unit in severely injured patients expected to receive a transfusion.
Keywords:
PLT function was measured by Multiplate aggregometry in response to five agonists.
Blood
Function was corrected for alterations in count. In vitro studies were conducted in the blood
Platelet
of normal subjects to assess the effect of dilutions with AB donor plasma on PLT function.
Patients
Results: Forty-six patients were enrolled, with 87% requiring a transfusion. Median Injury
Trauma
Severity Score was 23 (13.29) and mortality 15%. PLT count and function were decreased
Translation
from emergency department to intensive care unit admission by 25% and 58%, respec-
Study
tively. Decreases in function persisted after adjustment for count. Patients requiring large volumes of blood products had reductions in function that were disproportionately greater. Reductions in PLT function were greatest after transfusion of PLTs. In in vitro studies with a 30% dilution by autologous plasma caused a relational reduction in function, whereas allogenic plasma resulted in greater decreases that were highly variable between donors. Conclusions: Within hours of injury a decrease in both PLT count and function occurs, that is aggravated with the administration of blood products, with transfusion of PLTs showing
* Corresponding author. University of Texas Health Science Center at Houston, 6431 Fannin St., MSB 5.204, Houston, TX 77030. Tel.: þ1 713-500-6818; fax: þ1 713-500-0685. E-mail address: [email protected] (C.E. Wade). 1 These authors contributed equally to this study. 0022-4804/$ e see front matter ª 2017 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jss.2017.02.037
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the greatest effect. The effect on PLT function of allogenic transfused plasma appears to be
131 highly donor related. 132 133 134 135 136 Introduction 137 138 Platelet (PLT) count and function are highly associated with 139 morbidity and mortality of the patient with traumatic 140 injuries.1-5 A low PLT count at admission has been associated 141 142 with as a decreased rate of survival and a reduction in PLT 143 function. Decreases in PLT function are associated with higher 144 incidence of acute coagulopathy of trauma higher ventilation 145 requirements, inflammation/infection, traumatic brain 146 injury, and mortality.1-5 In addition, a high degree of 147 variability exists in PLT function adjusted for PLT count in 148 patients with severe traumatic injuries who have been 149 transfused large volumes of blood products. 150 In vitro, the use of blood products is suggested to decrease 151 PLT function and to vary as to how long the red cells and PLTs 152 153 Q6 were stored.6 In addition, the supernate (plasma) from stored 154 red blood cells (RBCs) inhibits PLT function.7 Evaluation of 155 in vitro PLT function in reconstituted whole blood variants 156 suggest that additives or components in packed RBCs or fresh 157 frozen plasma (FFP) depress PLT function. Thus, the age of 158 blood products and constituents of the products appear to 159 affect PLT function. 160 For these reasons, we developed this study to test the 161 hypothesis that patients expected to receive large amounts of 162 blood products would have a higher than expected decrease in 163 PLT function based on PLT count. A second hypothesis is that 164 165 the reduction in PLT function is associated with the donor 166 plasma/supernatant received in stored blood products. 167 168 169 Methods 170 171 172 Human subjects 173 174 This prospective observational study was conducted at 175 Memorial Hermann Hospital Texas Medical Center, a Level I 176 trauma center, and The University of Texas Health Science 177 Center at Houston (UTHealth). Prior approval was obtained 178 from the UTHealth Institutional Review Board (HSC-GEN-12179 0059). Adult admitted patients were eligible for inclusion in 180 this study if they met the hospital criteria for the highest level 181 182 of trauma team activation. This was a sampling of conve183 nience based on when research laboratory staff were available 184 to obtain and process samples. Patients were excluded if they 185 were aged <16 y, pregnant, prisoners, enrolled in other 186 studies, or declined to give consent. There were 813 patients 187 screened for enrollment in studies over the 8-mo period. The 188 majority were excluded as they were not expected to receive a 189 transfusion. Many of those expected by staff to receive a 190 transfusion of blood products were enrolled in ongoing 191 randomized trials.8 Patients from whom an initial blood draw 192 could not be obtained were also excluded from enrollment. 193 194 Consent was obtained from the patient or a legally authorized 195 representative within 72 h of admission. A waiver of consent
ª 2017 Published by Elsevier Inc.
was obtained from the Institutional Review Board for those patients discharged or who died within 24 h. In the remaining cases in which consent could not be obtained, the patient was excluded from the study, and their blood samples were destroyed. Blood samples were also collected from consented healthy subjects to serve as controls and for the in vitro experiments under a separate protocol (HSC-MS-09-0314).
Clinical studies On hospital admission, 20 mL of blood was obtained. Blood was transferred into vacutainer tubes containing 3.2% citrate and inverted to ensure proper anticoagulation. If the patient was expected by the research staff to receive a transfusion of blood products in the course of their initial care, a second sample was obtained on admission to the intensive care unit (ICU). Patient demographics, vital signs, standard laboratory values, mechanisms, and severity of injuries were collected at the time of admission. Blood products transfused were identified as RBCs, FFP, and PLTs. A unit of PLTs was defined as six packs. The age of PLTs transfused was also recorded.
In vitro studies Twenty milliliters of blood were obtained from control subjects who were not on PLT inhibitors or had an infection. Serial dilutions with saline and autologous plasma were performed to assess the effect of the dilution on PLT function in blood. Subject autologous FFP was prepared by snap freezing a plasma sample in liquid nitrogen then thawing it in a 37 water bath. We have previously demonstrated this procedure to decrease the hemostatic function of plasma.9,10 Additional dilution studies with AB plasma purchased from the local blood bank were also performed. The donor FFP was collected in citrate phosphate dextrose anticoagulant and frozen before using following standard blood banking procedures.
PLT function Investigations on whole blood PLTs function were performed 30 min after blood draw by trained personnel on-call. The analyses were made on Multiplate (Verum Diagnostica GmbH, Munich, Germany) using the multiple electrode aggregometry technology that has a turnaround time of 10 min per test period.11-13 Multiplate has five different test cells that allow simultaneous measurement of five different agonists to interrogate specific functional pathways11-13; each cell has two sets of 3-mm silver-coated copper electrode, a Tefloncoated stirring magnet, and requires 300 mL of whole citrate blood. PLTs were activated through five different agonist: adenosine diphosphate (ADP; 20 mL 6.5 mM, receptor pathway P2Y12), collagen (COL; 20 mL 3.2 mg/mL, GPIa/IIa, and GPVI receptors pathways), thrombin receptor-activating peptide-6 (TRAP; 20 mL 32 mM, PAR receptor pathway), arachidonic acid
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henriksen et al coagulation and platelet function in trauma
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(AA; 20 mL 0.5 mM, COX pathway) and Ristocetin (RISTO; 50 mL 0.8 mg/mL, GPIba receptor pathway). These reagents activated the PLT to adhere and aggregate on the electrode. The result of aggregation is increasing resistance, measured between the two sensor electrodes, continuously recorded as area under the curve (AUC) during the 6-min measuring time.
Statistical analysis All statistical analyses were conducted using STATA (12.1; College Station, TX). Nonparametric tests, including KruskaleWallis and Chi-square tests, were used to assess differences over time. Parametric tests (t-test and analysis of variance) were used where appropriate. To determine the effect of PLT transfusion analysis of covariance was used with the emergency department (ED) parameter being treated as a confounder. Statistical significance was set at the 5% level for a two-sided test.
Results Patients Forty-six patients, expected to subsequently require a transfusion, had blood samples obtained on arrival to the ED and on admission to the ICU. Of these patients, 40 (87%) subsequently required transfusion of a blood product in the next 24 h. The patients were severely injured based on admission demographics and laboratory values (Table 1). The median time
Table 1 e Patient demographics, n [ 46. Variable Age (y)
39 18.3
Height (cm)
175 10.6
Weight (kg)
83 21.2
3
between hospital admission and ICU admission was 202 (interquartile range [IQR] 139e310) min. There was a significant reduction in hemoglobin concentration comparing the ED sample, 12.6 2.07 to 11.6 1.94 g/dL (P < 0.001) to the ICU sample. PLT count was also reduced from 249 69.4 to 185 64.9 (P < 0.001). The percent reductions were 7 11.5% and 24 19.6% for hemoglobin and PLT count, respectively. There were also reductions over time in all measures of PLT function (Fig. 1A). To account for differences in PLT count, PLT function was divided by count.14,15 When corrected for the PLT count, the decrease in PLT function from the ED sample to the ICU sample persisted (Fig. 1B). Thus, decreases in PLT function were disproportional to the reductions in PLT count. The decreases in PLT function after correction for PLT count were weakly associated with the volume of blood products infused (r values from 0.24 to 0.41). For those subjects receiving blood products, the median number of total units transfused was 8 (IQR 4e17). Comparing PLT function between patients receiving no blood products and those administered 1-8 or >8 units of any blood product demonstrated that patients administered more than 8 units of blood products over 24 h had greater reductions in PLT function when adjusted for PLT count for all agonists except TRAP and AA (Table 2). Of these patients, 35% (n ¼ 16) were administered PLTs as well as other blood products. These patients received a median of 1 (IQR 1e2) unit of PLTs with a median age of 4 (IQR 3e4.5) d. This raises the issue of the influence of PLT transfusion on function as storage decreases PLT function.16 Comparison of patients administered PLTs and those not receiving PLTs demonstrated similar counts on admission to the ED and ICU (Table 3). PLT function adjusted for count was not different between groups on admission to the ED. In the ICU, while both groups had decreases in PLT function, patients administered PLTs had a significantly greater reductions in function when activated by ADP, COL, and RISTO (Table 3).
In vitro studies
Male (%)
78
Assay performance
Blunt (%)
76
As repeated samples were evaluated, the variability of PLT function was determined in blood from seven subjects as well as with varying dilutions of up to 30% with autologous fresh plasma or autologous FFP. For all agonist, there was a strong relationship between repeated measures (r values: ADP 0.95, COL 0.94, TRAP 0.71, AA 0.94, RISTO 0.94: n ¼ 21, all P < 0.001).
SBP (mm Hg)
108 34.1
DBP (mm Hg)
68 20.4
HR (beats/min) SBP (%) 90 mm Hg
108 26.2 41
GCS
3 (3, 15)
pH
7.23 0.113
BE
7 4.6
ISS
23 (13, 29)
Mortality (%)
15
24-h blood products (units) RBC
3 (1, 4.75)
Plasma
3 (1, 6.75)
PLTs
0 (0, 1)
DBP ¼ diastolic blood pressure; HR ¼ heart rate; ISS ¼ Injury Severity Score; SBP ¼ systolic blood pressure. Values are mean standard deviation or median (interquartile range) were appropriate.
Serial dilutions For three subjects with a mean PLT count of 221 27,103/mL (standard error of the mean [SEM]), blood samples were serially diluted with autologous fresh plasma or saline up to 40%. The magnitude of the decrease in PLT function was associated with the degree of dilution irrespective of the diluent. For example, a 30% dilution with saline resulted in a 29 2% decrease in ADP and a 24 9% reduction with autologous fresh plasma.
Single donor response To determine the response to allogenic plasma, the response to a 30% dilution with FFP from a single donor was evaluated
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Figure e PLT function (AUC) for the various activators with and without adjustment for PLT count (AUC/PLT count).
compared with autologous FFP in eight subjects. Compared with dilution with autologous FFP, the allogenic donor FFP decreased PLT activation with ADP by 13 4.3%, for COL by 13 3.0%, and 16 3.6% with AA (Table 4). The response to TRAP was not significantly altered while there was a 30 11% increase in PLT activation with RISTO. The coefficients of variation for autologous and donor FFP were similar for all agonist indicative of similar individual subject variation.
Multiple donor responses The response to five different donors was assessed in four subjects. Blood from a subject was evaluated after a 30% dilution with each of the five donors to assess the variation in response to different donors. Responses to donor plasma at a 30% dilution were compared with the subjects autologous FFP (Table 5). There was a high degree of variability between donors in responses to each agonist. The coefficient of variation of the means between donors for ADP was 49%, 72% for COL, 60% with TRAP, 31% for AA, and 230% for RISTO.
These high levels of variance are indicative of variable responses to donors.
Discussion Patients with severe traumatic injuries have a significant acute decrease in circulating PLT count.2,17-21 This reduction in PLT count has been associated with decreases in functional measures of hemostasis and markers of inflammation.3,22-25 However, these associations have been weak. With the advent of readily available measures of PLT function, clinical data suggest these measures provide insights into patient outcomes. We observed that PLT function was reduced in patients receiving large amounts of blood products and in some patients the reduction in function exceeded the decrease in PLT count.24 In the present study, it was determined that patients with traumatic injuries have a decrease in PLT function that persists after adjusting PLT count. In
Table 2 e PLT function adjusted for PLT count on admission to the ED and ICU in those patients transfused no blood products and those receiving PLT compared with those administered PLTs. No transfusion (n ¼ 6) a
ED Total blood products (units)
1-8 units (n ¼ 22)
ICU
ED
0
b
>8 units (n ¼ 18)
ICU
ED
4 (2.25, 6)
P value c
ICU 17 (11, 27)
PLT (count 103)ac**bc**
250 78.2
228 64.7
254 65.8
201 70.3
243 74.2
150 38.4
G ¼ 0.001; ED < 0.001
ADP (AUC/count 103)bc*
0.26 0.143
0.16 0.068
0.22 0.093
0.15 0.063
0.30 0.164
0.10 0.573
G ¼ 0.01; ED ¼ 0.354
COL (AUC/count 103)bc*
0.21 0.121
0.12 0.063
0.18 0.110
0.12 0.064
0.24 0.176
0.07 0.050
G ¼ 0.014; ED ¼ 0.152
TRAP (AUC/count 103)
0.48 0.175
0.40 0.117
0.46 0.179
0.37 0.108
0.51 0.231
0.30 0.169
G ¼ 0.107; ED ¼ 0.031
AA (AUC/count 103)
0.22 0.067
0.20 0.065
0.19 0.096
0.17 0.077
0.021 0.127
0.14 0.157
G ¼ 0.417; ED ¼ 0.273
RISTO (AUC/count 103)bc**
0.22 0.121
0.10 0.072
0.17 0.137
0.15 0.128
0.15 0.097
0.07 0.104
G ¼ 0.007; ED ¼ 0.001
Values are median (interquartile range). *P < 0.05; **P < 0.01; ***P < 0.001; G ¼ group effect: No transfusion (a) versus 1-8 units (b) versus >8 units (c); ED ¼ ED parameter confounder effect.
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Table 3 e PLT function adjusted for PLT count on admission to the ED and ICU in those patients transfused no PLT compared with those administered PLTs. No platelets (n ¼ 30) ED 3
ICU
Received platelets (n ¼ 16) ED
P-value
ICU
PLT (count 10 )
253 68.3
190 53.2
241 74.9
173 86.1
G ¼ 0.371; ED < 0.001
ADP (AUC/count 103)**
0.22 0.108
0.15 0.062
0.31 0.172
0.10 0.054
G ¼ 0.001; ED ¼ 0.282
COL (AUC/count 103)**
0.18 0.111
0.11 0.064
0.26 0.188
0.07 0.043
G ¼ 0.002; ED ¼ 0.078
TRAP (AUC/count 103)
0.46 0.185
0.37 0.134
0.54 0.227
0.31 0.153
G ¼ 0.066; ED ¼ 0.028
AA (AUC/count 103)***
0.19 0.090
0.17 0.072
0.21 0.139
0.09 0.078
G < 0.001; ED ¼ 0.029
0.18 0.131
0.12 0.119
0.15 0.100
0.06 0.078
G ¼ 0.048; ED ¼ 0.001
3 *
RISTO (AUC/count 10 )
Values are mean SD. *P < 0.05; **P < 0.01; ***P < 0.001; G ¼ group effect: No PLT versus PLT; ED: ED parameter confounder effect.
addition, the reduction in PLT function was greater in patients receiving large volumes (>8 units) of blood products, specifically in those requiring PLT transfusions. In the present study, those patients receiving large volumes of blood products, specifically PLT transfusions, showed a further reduction in PLT count with a disproportionate decrease in function. Others have noted a reduction in PLT function in patients with traumatic injuries and reported that the primary cause was the decrease in PLT count. Jacoby et al. reported a 30% decrease over 24 h in PLT count and significant reductions in PLT function in response to COL and ADP activation of 32 and 29%, respectively. Wohlauer et al. found after major trauma patients presented with PLT dysfunction, determined by ADP and AA activation being decreased using a thromboelastography-based PLT function assays.2 These reductions were observed in the presence of normal PLT counts and before substantial fluids or blood were administered. Kutcher et al. found a 35% decrease in PLT count within 6 h after admission.3 PLT function assessed by Multiplate with ADP, COL, TRAP, or AA was reduced and was weakly associated with PLT count (r ¼ 0.42 to 0.51). The authors attributed the reduction in PLT function to the decrease in count. Solomon et al. reported a reduction in PLT count of 15% on admission in nonsurvivors compared with survivors, and a proportional decrease in PLT function in response to ADP and TRAP activation of 14% and 15%,
respectively.26 The findings of the present study are unique as they demonstrate a reduction in PLT function that persisted after accounting for the decrease in PLT count. Furthermore, while the amount of blood products administered in previous studies was reported, the volume was not accounted for. Windeløv et al. reported a significant reduction in PLT aggregation to stimulation with TRAP and COL in trauma patients receiving massive transfusion compared with patients not receiving one.27 Stored PLTs have consistently been shown to have progressive reductions in function over time.28-30 Ponschab et al. assessed PLT function over 5 d of storage by Multiplate.16 PLT aggregation in response to ADP, COL, and AA were all reduced by 40%-50%. Transfusion of stored PLTs compared with fresh PLTs has been demonstrated to result in a reduced improvement in aggregation.26,31-33 In addition, overtime stored PLTs become activated and take days to recover their function posttransfusion. Recently, Pidcoke et al. evaluated pools of transfused products in patients undergoing burn excision.14 The PLT function of the transfused pools was abnormally low even after correction of PLT count. More importantly, the transfused PLTs were functionally inferior to the native PLTs. In the present study, the median age of PLTs administered was 4 d. Therefore, while PLT count was sustained in patients receiving a transfusion, the administered PLTs may have had a low function. The lower function of
Table 4 e Comparison of dilution by 30% with fresh autologous plasma, autologous snap frozen, and thawed plasma (FFP) or allogenic FFP from a single donor on PLT activation wit in eight subjects. Fresh autologous plasma
Autologous FFP
Allogenic FFP
Fresh versus autologous
Autologous versus allogenic
P value
P value
ADP (AUC)
67 7.1
62 5.5
53 4.9
0.32
0.018
COL (AUC)
44 3.5
45 3.6
39 3.5
0.53
0.005
114 5.3
105 5.5
104 3.7
0.03
0.576
AA (AUC)
56 3.7
55 4.4
46 4.4
0.71
0.004
RISTO (AUC)
70 6.0
64 5.3
80 4.3
0.12
0.015
TRAP (AUC)
Values are the AUC presented as mean SEM.
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651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715
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Table 5 e Comparison of donors: in response to a 30% dilution, the percent reduction from autologous FFP in the response to five different donors. Q10
Donor 1
Donor 2
Donor 3
Donor 4
Donor 5
ADP (% reduction)
39 6.1
29 3.5
21 2.3
21 11.4
8 5.8
COL (% reduction)
29 5.11
6 5.5
8 5.0
9 8.1
11 1.9
TRAP (% reduction)
32 4.7
23 8.9
16 2.4
12 6.8
5 2.3
AA (% reduction)
27 7.6
12 3.6
21 4.8
23 14.5
15 4.2
RISTO (% reduction)
4 10.6
3 14.3
49 15.6
6 16.1
1 13.4
Presented is the percentage reduction in PLT function for each donor allogenic FFP. Values are the AUC presented as mean SEM.
stored PLTs thus in part could account for the decrease in aggregation observed in those patients receiving a PLT transfusion. These findings bolster the argument advocating for the early administration of normal functioning PLTs to patients requiring large volumes of blood products. In addition, in vitro studies have demonstrated reductions in PLT function with exposure to blood products. Matheu and McFaul assessed PLT aggregation response to collagen after incubation of PLTs with supernates from leukoreduced and nonleukoreduced stored RBCs.7 After incubation with 42 d of storage nonleukoreduced RBC supernatant, PLT aggregation was decreased 31% compared with day 0. Leukoreduced supernatant had no effect. Nepstad et al. compared PLT function in response to collagen stimulation in the presence of fresh whole blood or reconstituted whole blood. Reconstituted whole blood was the ratio of RBCs, plasma, and PLTs used in their transfusion packets. PLT function was reduced with exposure to reconstituted whole blood. The decrease in function was further aggravated if older RBCs were used. Ponschab et al. evaluated PLT function by Multiplate aggregometry in reconstituted whole blood of various product types.16 Leukoreduced whole blood with apheresis PLTs added was contrasted with mixtures of RBCs and apheresis PLTs with FFP or solvent detergent plasma. On day 0, there were no differences between groups in PLT count. Compared with leukoreduced whole blood with PLTs added, reconstituted whole bloodereduced PLT aggregation for all activators with a further reduction noted in some with FFP compared with solvent detergent plasma. Rahbar et al. identified a reduction in PLT count and function over 24 h in patients with active bleeding requiring emergent use of uncross-matched blood in the ED.22 Patients subsequently received modified whole blood with PLTs or reconstituted blood. While there were no differences in PLT counts between groups over time, PLT aggregation to all agonist was generally higher in those patients receiving leukoreduced whole blood with PLTs. These studies demonstrated a reduction in PLT function because of exposure to component blood products possible due to the additives in RBCs and plasma or cellular aggregates due to processing and storage. In the present study, dilution with fresh autologous plasma or saline resulted in proportional decreases in PLT function. Caballo et al. diluted whole blood by 30% with Ringer’s lactate and reported a 30% reduction in PLT interactions.34 Hanke et al. demonstrated a dose response between PLT count and function with dilutions when assessed using Multiplate aggregometry.35 Using blood from control subjects, they
prepared samples with varying PLT counts and found PLT function to decrease with counts below the normal range (<150,000). At counts of 100,000 and 50,000, functional results decreased by 18% and 37%, respectively. Of note, they reported a large interindividual variation in these responses. Thus, reductions of PLT count by dilution or dose results in proportional decreases in function. It should be noted, however, that reduction in PLT count below <150,000 significantly influence PLT function as evaluated by Multiplate and, hence, discriminating between a reduction in PLT functionality caused by low count versus impaired intrinsic function is difficult36 and is a limitation of the present study. In the present study, a comparison of autologous FFP with a single allogenic donor FFP demonstrated differences in all activators except TRAP. Of note, the variance of the response between subjects to the single donor was similar. In contrast, the response to FFP from five different donors was highly variable. For example, dilution with plasma from donor 1 resulted in a 39% reduction in activity to ADP and 4% increase with RISTO in contrast to the 8% and 48% reductions to ADP and RISTO, respectively, for donor 5 plasma. In our evaluation of clinical subjects, while there were reductions from the ED to the ICU, the magnitude of the responses were highly variable between patients and weakly associated with the amount of blood product administered. This inconsistency would appear to be in part related to the variance within the allogenic donor FFP. Among and between the various plasma products, inconsistencies have been reported in factor activity, functional measures of coagulation, residual cell and micro-particle distributions, as well as actions on endothelial permeability.9,10 Between FFP collections factor, activity has been reported to vary by as much as 100% between donors. Functional measures of coagulation also show a wide variation. This may in part due to the variation in the volume of blood collected on a set volume of anticoagulant preservatives, as well as residual cells and microparticles have being increased as a result of the plasma being retained for a longer period of time with the blood cells. FFP may be refrigerated for 5 d before use, modifying all these characteristics. Finally, there are innate differences between donors. The observed variations of the responses of PLT function to various donor plasmas are another demonstration of the lack of consistency in plasma products obtained from single donors. Limitations of the present study include the small number of subjects limiting broad applicability of the findings. However, the samples were collected at similar periods in the
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course of care, up on admission to the ED and ICU, in contrast to larger studies where sampling was based on time only, but at varying points in their course of care. Another limitation is the absence of adequate information as to prehospital medications, specifically anti-PLT drugs. We reviewed clinical records and were unable to find documentation of preadmission anti-PLT medications. Use of blood products in the treatment of the patient with severe injuries and shock has evolved over the past 50 y. In response to the administration of blood products, the physiology of the patient often improves as does the hemostatic status. With the transfusion of blood products, the hemostatic potential of the patient who is hypocoagulable improves based on measures of clotting, viscoelastometry, and thrombin generation in spite of a decrease in PLT count and function.3,22,37 The present study identifies, in patients with traumatic injuries, a reduction of both PLT count and function between admission to the ED and the ICU, which is further aggravated with the administration of the plasma/supernatant of blood products, specifically with stored PLTs. The disproportionate reduction in PLT function, in relation to count, may in part be because of the administration of allogenic blood products.
Acknowledgment
7. 8.
9.
10.
11.
12.
13.
14.
15.
C.E.W., H.H.H., A.G.G., L.A.B., J.C.C., and S.R.O. designed the study; C.E.W., H.H.H., A.G.G., L.A.B., J.C.C., S.V., S.S., N.M., B.A.C., J.B.H., and S.R.O. collected the data; C.E.W., H.H.H., A.G.G., L.A.B., J.C.C., S.R.O., J.B.H., P.I.J., and T.A.C. analyzed the data; All authors drafted, edited, revised, and approved the final version of the article.
16.
17.
Disclosure 18.
There are no conflicts of interest to report. 19.
references 20. 1. Solomon C, Traintinger S, Ziegler B, et al. Platelet function following trauma. A multiple electrode aggregometry study. Thromb Haemost. 2011;106:322e330. 2. Wohlauer MV, Moore EE, Thomas S, et al. Early platelet dysfunction: an unrecognized role in the acute coagulopathy of trauma. J Am Coll Surg. 2012;214:739e746. 3. Kutcher ME, Redick BJ, McCreery RC, et al. Characterization of platelet dysfunction after trauma. J Trauma Acute Care Surg. 2012;73:13e19. 4. Nystrup KB, Windelov NA, Thomsen AB, Johansson PI. Reduced clot strength upon admission, evaluated by thrombelastography (TEG), in trauma patients is independently associated with increased 30-day mortality. Scand J Trauma Resusc Emerg Med. 2011;19:52. 5. Ramsey MT, Fabian TC, Shahan CP, et al. A prospective study of platelet function in trauma patients. J Trauma Acute Care Surg. 2016;80:726e732. 6. Nepstad I, Reikvam H, Strandenes G, et al. Comparison of in vitro responses to fresh whole blood and reconstituted
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whole blood after collagen stimulation. Blood Transfus. 2014;12:50e55. Matheu FA, McFaul SJ. Supernates from stored red blood cells inhibit platelet aggregation. Transfusion. 2010;50:1196e1202. Holcomb JB, Tilley BC, Baraniuk S, et al. Transfusion of plasma, platelets, and red blood cells in a 1:1:1 vs a 1:1:2 ratio and mortality in patients with severe trauma: the PROPPR randomized clinical trial. JAMA. 2015;313:471e482. Matijevic N, Kostousov V, Wang YW, et al. Multiple levels of degradation diminish hemostatic potential of thawed plasma. J Trauma. 2011;70:71e79. discussion 79e80. Matijevic N, Wang YW, Kostousov V, et al. Decline in platelet microparticles contributes to reduced hemostatic potential of stored plasma. Thromb Res. 2011;128:35e41. Paniccia R, Antonucci E, Maggini N, et al. Assessment of platelet function on whole blood by multiple electrode aggregometry in high-risk patients with coronary artery disease receiving antiplatelet therapy. Am J Clin Pathol. 2009;131:834e842. Peerschke EI, Castellone DD, Stroobants AK, Francis J. Reference range determination for whole-blood platelet aggregation using the Multiplate analyzer. Am J Clin Pathol. 2014;142:647e656. Rubak P, Villadsen K, Hvas AM. Reference intervals for platelet aggregation assessed by multiple electrode platelet aggregometry. Thromb Res. 2012;130:420e423. Pidcoke HF, Isbell CL, Herzig MC, et al. Acute blood loss during burn and soft tissue excisions: an observational study of blood product resuscitation practices and focused review. J Trauma Acute Care Surg. 2015;78:S39eS47. Wurtz M, Hvas AM, Kristensen SD, Grove EL. Platelet aggregation is dependent on platelet count in patients with coronary artery disease. Thromb Res. 2012;129:56e61. Ponschab M, Schlimp CJ, Zipperle J, et al. Platelet function in reconstituted whole blood variants: an observational study over 5 days of storage time. J Trauma Acute Care Surg. 2015;79:797e804. Brown LM, Call MS, Margaret Knudson M, et al. A normal platelet count may not be enough: the impact of admission platelet count on mortality and transfusion in severely injured trauma patients. J Trauma. 2011;71:S337eS342. Harr JN, Moore EE, Chin TL, et al. Platelets are dominant contributors to hypercoagulability after injury. J Trauma Acute Care Surg. 2013;74:756e762. discussion 762e755. Stansbury LG, Hess AS, Thompson K, et al. The clinical significance of platelet counts in the first 24 hours after severe injury. Transfusion. 2013;53:783e789. Bowbrick VA, Mikhailidis DP, Stansby G. Influence of platelet count and activity on thromboelastography parameters. Platelets. 2003;14:219e224. Kornblith LZ, Kutcher ME, Redick BJ, et al. Fibrinogen and platelet contributions to clot formation: implications for trauma resuscitation and thromboprophylaxis. J Trauma Acute Care Surg. 2014;76:255e256. discussion 262e253. Rahbar E, Cardenas JC, Matijevic N, et al. Trauma, time, and transfusions: a longitudinal analysis of coagulation markers in severely injured trauma patients receiving modified whole blood or component blood products. Shock. 2015;44:417e425. Plotkin AJ, Wade CE, Jenkins DH, et al. A reduction in clot formation rate and strength assessed by thrombelastography is indicative of transfusion requirements in patients with penetrating injuries. J Trauma. 2008;64:S64eS68. Jacoby RC, Owings JT, Holmes J, et al. Platelet activation and function after trauma. J Trauma. 2001;51:639e647. Nekludov M, Bellander BM, Blomback M, Wallen HN. Platelet dysfunction in patients with severe traumatic brain injury. J Neurotrauma. 2007;24:1699e1706.
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26. Mohr R, Martinowitz U, Lavee J, et al. The hemostatic effect of transfusing fresh whole blood versus platelet concentrates after cardiac operations. J Thorac Cardiovasc Surg. 1988;96:530e534. 27. Windelov NA, Sorensen AM, Perner A, et al. Platelet aggregation following trauma: a prospective study. Blood Coagul Fibrinolysis. 2014;25:67e73. 28. Akay OM, Gunduz E, Basyigit H, Gulbas Z. Platelet function testing during 5-day storage of single and random donor plateletpheresis. Transfus Apher Sci. 2007;36:285e289. 29. Shrivastava M. The platelet storage lesion. Transfus Apher Sci. 2009;41:105e113. 30. Ostrowski SR, Bochsen L, Windelov NA, et al. Hemostatic function of buffy coat platelets in additive solution treated with pathogen reduction technology. Transfusion. 2011;51:344e356. 31. Rosenfeld BA, Herfel B, Faraday N, Fuller A, Braine H. Effects of storage time on quantitative and qualitative platelet function after transfusion. Anesthesiology. 1995;83:1167e1172. 32. Owens M, Holme S, Heaton A, Sawyer S, Cardinali S. Posttransfusion recovery of function of 5-day stored platelet concentrates. Br J Haematol. 1992;80:539e544.
33. Rinder HM, Murphy M, Mitchell JG, et al. Progressive platelet activation with storage: evidence for shortened survival of activated platelets after transfusion. Transfusion. 1991;31:409e414. 34. Caballo C, Escolar G, Diaz-Ricart M, et al. Impact of experimental haemodilution on platelet function, thrombin generation and clot firmness: effects of different coagulation factor concentrates. Blood Transfus. 2013;11:391e399. 35. Hanke AA, Roberg K, Monaca E, et al. Impact of platelet count on results obtained from multiple electrode platelet aggregometry (Multiplate). Eur J Med Res. 2010;15:214e219. 36. Stissing T, Dridi NP, Ostrowski SR, Bochsen L, Johansson PI. The influence of low platelet count on whole blood aggregometry assessed by Multiplate. Clin Appl Thromb Hemost. 2011;17:E211eE217. 37. Holcomb JB, Wade CE, Michalek JE, et al. Increased plasma and platelet to red blood cell ratios improves outcome in 466 massively transfused civilian trauma patients. Ann Surg. 2008;248:447e458.
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Available online at www.sciencedirect.com
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Impact of blood products on platelet function in patients with traumatic injuries: a translation study
Hanne Hee Henriksen, MD,a,b,1 Alexandra G. Grand,a,1 Sandra Viggers,b Lisa A. Baer,a S. Solbeck,b Bryan A. Cotton,a N. Matijevic,a Sisse R. Ostrowski,b Jakob Stensballe,b,c Erin E. Fox,a Tzu-An Chen,a John B. Holcomb,a Pa¨r I. Johansson,a,b Jessica C. Cardenas,a a, Q2Q3 and Charles E. Wade, PhD *
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a
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Center for Translational Injury Research (CeTIR) and Department of Surgery, University of Texas Health Science Center, Houston, Texas b Section for Transfusion Medicine, Capital Region Blood Bank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark c Department of Anesthesia, Centre of Head and Orthopedics, Rigshospitalet, Copenhagen University Hospital, Denmark
article info
abstract
Article history:
Background: Reductions in platelet (PLT) count and function are associated with poor
Received 11 October 2016
outcomes in trauma patients. We proposed to determine if patients expected to receive
Received in revised form
blood products have a decrease in PLT function higher than expected based on the
22 December 2016
reduction in PLT count, and if the reduction in function could be associated with the donor
Accepted 16 February 2017
plasma/supernatant received.
Available online xxx
Methods: PLT count and function were measured on admission to the emergency department and intensive care unit in severely injured patients expected to receive a transfusion.
Keywords:
PLT function was measured by Multiplate aggregometry in response to five agonists.
Blood
Function was corrected for alterations in count. In vitro studies were conducted in the blood
Platelet
of normal subjects to assess the effect of dilutions with AB donor plasma on PLT function.
Patients
Results: Forty-six patients were enrolled, with 87% requiring a transfusion. Median Injury
Trauma
Severity Score was 23 (13.29) and mortality 15%. PLT count and function were decreased
Translation
from emergency department to intensive care unit admission by 25% and 58%, respec-
Study
tively. Decreases in function persisted after adjustment for count. Patients requiring large volumes of blood products had reductions in function that were disproportionately greater. Reductions in PLT function were greatest after transfusion of PLTs. In in vitro studies with a 30% dilution by autologous plasma caused a relational reduction in function, whereas allogenic plasma resulted in greater decreases that were highly variable between donors. Conclusions: Within hours of injury a decrease in both PLT count and function occurs, that is aggravated with the administration of blood products, with transfusion of PLTs showing
* Corresponding author. University of Texas Health Science Center at Houston, 6431 Fannin St., MSB 5.204, Houston, TX 77030. Tel.: þ1 713-500-6818; fax: þ1 713-500-0685. E-mail address: [email protected] (C.E. Wade). 1 These authors contributed equally to this study. 0022-4804/$ e see front matter ª 2017 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jss.2017.02.037
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the greatest effect. The effect on PLT function of allogenic transfused plasma appears to be
131 highly donor related. 132 133 134 135 136 Introduction 137 138 Platelet (PLT) count and function are highly associated with 139 morbidity and mortality of the patient with traumatic 140 injuries.1-5 A low PLT count at admission has been associated 141 142 with as a decreased rate of survival and a reduction in PLT 143 function. Decreases in PLT function are associated with higher 144 incidence of acute coagulopathy of trauma higher ventilation 145 requirements, inflammation/infection, traumatic brain 146 injury, and mortality.1-5 In addition, a high degree of 147 variability exists in PLT function adjusted for PLT count in 148 patients with severe traumatic injuries who have been 149 transfused large volumes of blood products. 150 In vitro, the use of blood products is suggested to decrease 151 PLT function and to vary as to how long the red cells and PLTs 152 153 Q6 were stored.6 In addition, the supernate (plasma) from stored 154 red blood cells (RBCs) inhibits PLT function.7 Evaluation of 155 in vitro PLT function in reconstituted whole blood variants 156 suggest that additives or components in packed RBCs or fresh 157 frozen plasma (FFP) depress PLT function. Thus, the age of 158 blood products and constituents of the products appear to 159 affect PLT function. 160 For these reasons, we developed this study to test the 161 hypothesis that patients expected to receive large amounts of 162 blood products would have a higher than expected decrease in 163 PLT function based on PLT count. A second hypothesis is that 164 165 the reduction in PLT function is associated with the donor 166 plasma/supernatant received in stored blood products. 167 168 169 Methods 170 171 172 Human subjects 173 174 This prospective observational study was conducted at 175 Memorial Hermann Hospital Texas Medical Center, a Level I 176 trauma center, and The University of Texas Health Science 177 Center at Houston (UTHealth). Prior approval was obtained 178 from the UTHealth Institutional Review Board (HSC-GEN-12179 0059). Adult admitted patients were eligible for inclusion in 180 this study if they met the hospital criteria for the highest level 181 182 of trauma team activation. This was a sampling of conve183 nience based on when research laboratory staff were available 184 to obtain and process samples. Patients were excluded if they 185 were aged <16 y, pregnant, prisoners, enrolled in other 186 studies, or declined to give consent. There were 813 patients 187 screened for enrollment in studies over the 8-mo period. The 188 majority were excluded as they were not expected to receive a 189 transfusion. Many of those expected by staff to receive a 190 transfusion of blood products were enrolled in ongoing 191 randomized trials.8 Patients from whom an initial blood draw 192 could not be obtained were also excluded from enrollment. 193 194 Consent was obtained from the patient or a legally authorized 195 representative within 72 h of admission. A waiver of consent
ª 2017 Published by Elsevier Inc.
was obtained from the Institutional Review Board for those patients discharged or who died within 24 h. In the remaining cases in which consent could not be obtained, the patient was excluded from the study, and their blood samples were destroyed. Blood samples were also collected from consented healthy subjects to serve as controls and for the in vitro experiments under a separate protocol (HSC-MS-09-0314).
Clinical studies On hospital admission, 20 mL of blood was obtained. Blood was transferred into vacutainer tubes containing 3.2% citrate and inverted to ensure proper anticoagulation. If the patient was expected by the research staff to receive a transfusion of blood products in the course of their initial care, a second sample was obtained on admission to the intensive care unit (ICU). Patient demographics, vital signs, standard laboratory values, mechanisms, and severity of injuries were collected at the time of admission. Blood products transfused were identified as RBCs, FFP, and PLTs. A unit of PLTs was defined as six packs. The age of PLTs transfused was also recorded.
In vitro studies Twenty milliliters of blood were obtained from control subjects who were not on PLT inhibitors or had an infection. Serial dilutions with saline and autologous plasma were performed to assess the effect of the dilution on PLT function in blood. Subject autologous FFP was prepared by snap freezing a plasma sample in liquid nitrogen then thawing it in a 37 water bath. We have previously demonstrated this procedure to decrease the hemostatic function of plasma.9,10 Additional dilution studies with AB plasma purchased from the local blood bank were also performed. The donor FFP was collected in citrate phosphate dextrose anticoagulant and frozen before using following standard blood banking procedures.
PLT function Investigations on whole blood PLTs function were performed 30 min after blood draw by trained personnel on-call. The analyses were made on Multiplate (Verum Diagnostica GmbH, Munich, Germany) using the multiple electrode aggregometry technology that has a turnaround time of 10 min per test period.11-13 Multiplate has five different test cells that allow simultaneous measurement of five different agonists to interrogate specific functional pathways11-13; each cell has two sets of 3-mm silver-coated copper electrode, a Tefloncoated stirring magnet, and requires 300 mL of whole citrate blood. PLTs were activated through five different agonist: adenosine diphosphate (ADP; 20 mL 6.5 mM, receptor pathway P2Y12), collagen (COL; 20 mL 3.2 mg/mL, GPIa/IIa, and GPVI receptors pathways), thrombin receptor-activating peptide-6 (TRAP; 20 mL 32 mM, PAR receptor pathway), arachidonic acid
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henriksen et al coagulation and platelet function in trauma
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(AA; 20 mL 0.5 mM, COX pathway) and Ristocetin (RISTO; 50 mL 0.8 mg/mL, GPIba receptor pathway). These reagents activated the PLT to adhere and aggregate on the electrode. The result of aggregation is increasing resistance, measured between the two sensor electrodes, continuously recorded as area under the curve (AUC) during the 6-min measuring time.
Statistical analysis All statistical analyses were conducted using STATA (12.1; College Station, TX). Nonparametric tests, including KruskaleWallis and Chi-square tests, were used to assess differences over time. Parametric tests (t-test and analysis of variance) were used where appropriate. To determine the effect of PLT transfusion analysis of covariance was used with the emergency department (ED) parameter being treated as a confounder. Statistical significance was set at the 5% level for a two-sided test.
Results Patients Forty-six patients, expected to subsequently require a transfusion, had blood samples obtained on arrival to the ED and on admission to the ICU. Of these patients, 40 (87%) subsequently required transfusion of a blood product in the next 24 h. The patients were severely injured based on admission demographics and laboratory values (Table 1). The median time
Table 1 e Patient demographics, n [ 46. Variable Age (y)
39 18.3
Height (cm)
175 10.6
Weight (kg)
83 21.2
3
between hospital admission and ICU admission was 202 (interquartile range [IQR] 139e310) min. There was a significant reduction in hemoglobin concentration comparing the ED sample, 12.6 2.07 to 11.6 1.94 g/dL (P < 0.001) to the ICU sample. PLT count was also reduced from 249 69.4 to 185 64.9 (P < 0.001). The percent reductions were 7 11.5% and 24 19.6% for hemoglobin and PLT count, respectively. There were also reductions over time in all measures of PLT function (Fig. 1A). To account for differences in PLT count, PLT function was divided by count.14,15 When corrected for the PLT count, the decrease in PLT function from the ED sample to the ICU sample persisted (Fig. 1B). Thus, decreases in PLT function were disproportional to the reductions in PLT count. The decreases in PLT function after correction for PLT count were weakly associated with the volume of blood products infused (r values from 0.24 to 0.41). For those subjects receiving blood products, the median number of total units transfused was 8 (IQR 4e17). Comparing PLT function between patients receiving no blood products and those administered 1-8 or >8 units of any blood product demonstrated that patients administered more than 8 units of blood products over 24 h had greater reductions in PLT function when adjusted for PLT count for all agonists except TRAP and AA (Table 2). Of these patients, 35% (n ¼ 16) were administered PLTs as well as other blood products. These patients received a median of 1 (IQR 1e2) unit of PLTs with a median age of 4 (IQR 3e4.5) d. This raises the issue of the influence of PLT transfusion on function as storage decreases PLT function.16 Comparison of patients administered PLTs and those not receiving PLTs demonstrated similar counts on admission to the ED and ICU (Table 3). PLT function adjusted for count was not different between groups on admission to the ED. In the ICU, while both groups had decreases in PLT function, patients administered PLTs had a significantly greater reductions in function when activated by ADP, COL, and RISTO (Table 3).
In vitro studies
Male (%)
78
Assay performance
Blunt (%)
76
As repeated samples were evaluated, the variability of PLT function was determined in blood from seven subjects as well as with varying dilutions of up to 30% with autologous fresh plasma or autologous FFP. For all agonist, there was a strong relationship between repeated measures (r values: ADP 0.95, COL 0.94, TRAP 0.71, AA 0.94, RISTO 0.94: n ¼ 21, all P < 0.001).
SBP (mm Hg)
108 34.1
DBP (mm Hg)
68 20.4
HR (beats/min) SBP (%) 90 mm Hg
108 26.2 41
GCS
3 (3, 15)
pH
7.23 0.113
BE
7 4.6
ISS
23 (13, 29)
Mortality (%)
15
24-h blood products (units) RBC
3 (1, 4.75)
Plasma
3 (1, 6.75)
PLTs
0 (0, 1)
DBP ¼ diastolic blood pressure; HR ¼ heart rate; ISS ¼ Injury Severity Score; SBP ¼ systolic blood pressure. Values are mean standard deviation or median (interquartile range) were appropriate.
Serial dilutions For three subjects with a mean PLT count of 221 27,103/mL (standard error of the mean [SEM]), blood samples were serially diluted with autologous fresh plasma or saline up to 40%. The magnitude of the decrease in PLT function was associated with the degree of dilution irrespective of the diluent. For example, a 30% dilution with saline resulted in a 29 2% decrease in ADP and a 24 9% reduction with autologous fresh plasma.
Single donor response To determine the response to allogenic plasma, the response to a 30% dilution with FFP from a single donor was evaluated
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4
391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 Q9 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455
j o u r n a l o f s u r g i c a l r e s e a r c h - 2 0 1 7 ( - ) 1 e8
Figure e PLT function (AUC) for the various activators with and without adjustment for PLT count (AUC/PLT count).
compared with autologous FFP in eight subjects. Compared with dilution with autologous FFP, the allogenic donor FFP decreased PLT activation with ADP by 13 4.3%, for COL by 13 3.0%, and 16 3.6% with AA (Table 4). The response to TRAP was not significantly altered while there was a 30 11% increase in PLT activation with RISTO. The coefficients of variation for autologous and donor FFP were similar for all agonist indicative of similar individual subject variation.
Multiple donor responses The response to five different donors was assessed in four subjects. Blood from a subject was evaluated after a 30% dilution with each of the five donors to assess the variation in response to different donors. Responses to donor plasma at a 30% dilution were compared with the subjects autologous FFP (Table 5). There was a high degree of variability between donors in responses to each agonist. The coefficient of variation of the means between donors for ADP was 49%, 72% for COL, 60% with TRAP, 31% for AA, and 230% for RISTO.
These high levels of variance are indicative of variable responses to donors.
Discussion Patients with severe traumatic injuries have a significant acute decrease in circulating PLT count.2,17-21 This reduction in PLT count has been associated with decreases in functional measures of hemostasis and markers of inflammation.3,22-25 However, these associations have been weak. With the advent of readily available measures of PLT function, clinical data suggest these measures provide insights into patient outcomes. We observed that PLT function was reduced in patients receiving large amounts of blood products and in some patients the reduction in function exceeded the decrease in PLT count.24 In the present study, it was determined that patients with traumatic injuries have a decrease in PLT function that persists after adjusting PLT count. In
Table 2 e PLT function adjusted for PLT count on admission to the ED and ICU in those patients transfused no blood products and those receiving PLT compared with those administered PLTs. No transfusion (n ¼ 6) a
ED Total blood products (units)
1-8 units (n ¼ 22)
ICU
ED
0
b
>8 units (n ¼ 18)
ICU
ED
4 (2.25, 6)
P value c
ICU 17 (11, 27)
PLT (count 103)ac**bc**
250 78.2
228 64.7
254 65.8
201 70.3
243 74.2
150 38.4
G ¼ 0.001; ED < 0.001
ADP (AUC/count 103)bc*
0.26 0.143
0.16 0.068
0.22 0.093
0.15 0.063
0.30 0.164
0.10 0.573
G ¼ 0.01; ED ¼ 0.354
COL (AUC/count 103)bc*
0.21 0.121
0.12 0.063
0.18 0.110
0.12 0.064
0.24 0.176
0.07 0.050
G ¼ 0.014; ED ¼ 0.152
TRAP (AUC/count 103)
0.48 0.175
0.40 0.117
0.46 0.179
0.37 0.108
0.51 0.231
0.30 0.169
G ¼ 0.107; ED ¼ 0.031
AA (AUC/count 103)
0.22 0.067
0.20 0.065
0.19 0.096
0.17 0.077
0.021 0.127
0.14 0.157
G ¼ 0.417; ED ¼ 0.273
RISTO (AUC/count 103)bc**
0.22 0.121
0.10 0.072
0.17 0.137
0.15 0.128
0.15 0.097
0.07 0.104
G ¼ 0.007; ED ¼ 0.001
Values are median (interquartile range). *P < 0.05; **P < 0.01; ***P < 0.001; G ¼ group effect: No transfusion (a) versus 1-8 units (b) versus >8 units (c); ED ¼ ED parameter confounder effect.
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Q11
456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520
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henriksen et al coagulation and platelet function in trauma
521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585
Table 3 e PLT function adjusted for PLT count on admission to the ED and ICU in those patients transfused no PLT compared with those administered PLTs. No platelets (n ¼ 30) ED 3
ICU
Received platelets (n ¼ 16) ED
P-value
ICU
PLT (count 10 )
253 68.3
190 53.2
241 74.9
173 86.1
G ¼ 0.371; ED < 0.001
ADP (AUC/count 103)**
0.22 0.108
0.15 0.062
0.31 0.172
0.10 0.054
G ¼ 0.001; ED ¼ 0.282
COL (AUC/count 103)**
0.18 0.111
0.11 0.064
0.26 0.188
0.07 0.043
G ¼ 0.002; ED ¼ 0.078
TRAP (AUC/count 103)
0.46 0.185
0.37 0.134
0.54 0.227
0.31 0.153
G ¼ 0.066; ED ¼ 0.028
AA (AUC/count 103)***
0.19 0.090
0.17 0.072
0.21 0.139
0.09 0.078
G < 0.001; ED ¼ 0.029
0.18 0.131
0.12 0.119
0.15 0.100
0.06 0.078
G ¼ 0.048; ED ¼ 0.001
3 *
RISTO (AUC/count 10 )
Values are mean SD. *P < 0.05; **P < 0.01; ***P < 0.001; G ¼ group effect: No PLT versus PLT; ED: ED parameter confounder effect.
addition, the reduction in PLT function was greater in patients receiving large volumes (>8 units) of blood products, specifically in those requiring PLT transfusions. In the present study, those patients receiving large volumes of blood products, specifically PLT transfusions, showed a further reduction in PLT count with a disproportionate decrease in function. Others have noted a reduction in PLT function in patients with traumatic injuries and reported that the primary cause was the decrease in PLT count. Jacoby et al. reported a 30% decrease over 24 h in PLT count and significant reductions in PLT function in response to COL and ADP activation of 32 and 29%, respectively. Wohlauer et al. found after major trauma patients presented with PLT dysfunction, determined by ADP and AA activation being decreased using a thromboelastography-based PLT function assays.2 These reductions were observed in the presence of normal PLT counts and before substantial fluids or blood were administered. Kutcher et al. found a 35% decrease in PLT count within 6 h after admission.3 PLT function assessed by Multiplate with ADP, COL, TRAP, or AA was reduced and was weakly associated with PLT count (r ¼ 0.42 to 0.51). The authors attributed the reduction in PLT function to the decrease in count. Solomon et al. reported a reduction in PLT count of 15% on admission in nonsurvivors compared with survivors, and a proportional decrease in PLT function in response to ADP and TRAP activation of 14% and 15%,
respectively.26 The findings of the present study are unique as they demonstrate a reduction in PLT function that persisted after accounting for the decrease in PLT count. Furthermore, while the amount of blood products administered in previous studies was reported, the volume was not accounted for. Windeløv et al. reported a significant reduction in PLT aggregation to stimulation with TRAP and COL in trauma patients receiving massive transfusion compared with patients not receiving one.27 Stored PLTs have consistently been shown to have progressive reductions in function over time.28-30 Ponschab et al. assessed PLT function over 5 d of storage by Multiplate.16 PLT aggregation in response to ADP, COL, and AA were all reduced by 40%-50%. Transfusion of stored PLTs compared with fresh PLTs has been demonstrated to result in a reduced improvement in aggregation.26,31-33 In addition, overtime stored PLTs become activated and take days to recover their function posttransfusion. Recently, Pidcoke et al. evaluated pools of transfused products in patients undergoing burn excision.14 The PLT function of the transfused pools was abnormally low even after correction of PLT count. More importantly, the transfused PLTs were functionally inferior to the native PLTs. In the present study, the median age of PLTs administered was 4 d. Therefore, while PLT count was sustained in patients receiving a transfusion, the administered PLTs may have had a low function. The lower function of
Table 4 e Comparison of dilution by 30% with fresh autologous plasma, autologous snap frozen, and thawed plasma (FFP) or allogenic FFP from a single donor on PLT activation wit in eight subjects. Fresh autologous plasma
Autologous FFP
Allogenic FFP
Fresh versus autologous
Autologous versus allogenic
P value
P value
ADP (AUC)
67 7.1
62 5.5
53 4.9
0.32
0.018
COL (AUC)
44 3.5
45 3.6
39 3.5
0.53
0.005
114 5.3
105 5.5
104 3.7
0.03
0.576
AA (AUC)
56 3.7
55 4.4
46 4.4
0.71
0.004
RISTO (AUC)
70 6.0
64 5.3
80 4.3
0.12
0.015
TRAP (AUC)
Values are the AUC presented as mean SEM.
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6
651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715
j o u r n a l o f s u r g i c a l r e s e a r c h - 2 0 1 7 ( - ) 1 e8
Table 5 e Comparison of donors: in response to a 30% dilution, the percent reduction from autologous FFP in the response to five different donors. Q10
Donor 1
Donor 2
Donor 3
Donor 4
Donor 5
ADP (% reduction)
39 6.1
29 3.5
21 2.3
21 11.4
8 5.8
COL (% reduction)
29 5.11
6 5.5
8 5.0
9 8.1
11 1.9
TRAP (% reduction)
32 4.7
23 8.9
16 2.4
12 6.8
5 2.3
AA (% reduction)
27 7.6
12 3.6
21 4.8
23 14.5
15 4.2
RISTO (% reduction)
4 10.6
3 14.3
49 15.6
6 16.1
1 13.4
Presented is the percentage reduction in PLT function for each donor allogenic FFP. Values are the AUC presented as mean SEM.
stored PLTs thus in part could account for the decrease in aggregation observed in those patients receiving a PLT transfusion. These findings bolster the argument advocating for the early administration of normal functioning PLTs to patients requiring large volumes of blood products. In addition, in vitro studies have demonstrated reductions in PLT function with exposure to blood products. Matheu and McFaul assessed PLT aggregation response to collagen after incubation of PLTs with supernates from leukoreduced and nonleukoreduced stored RBCs.7 After incubation with 42 d of storage nonleukoreduced RBC supernatant, PLT aggregation was decreased 31% compared with day 0. Leukoreduced supernatant had no effect. Nepstad et al. compared PLT function in response to collagen stimulation in the presence of fresh whole blood or reconstituted whole blood. Reconstituted whole blood was the ratio of RBCs, plasma, and PLTs used in their transfusion packets. PLT function was reduced with exposure to reconstituted whole blood. The decrease in function was further aggravated if older RBCs were used. Ponschab et al. evaluated PLT function by Multiplate aggregometry in reconstituted whole blood of various product types.16 Leukoreduced whole blood with apheresis PLTs added was contrasted with mixtures of RBCs and apheresis PLTs with FFP or solvent detergent plasma. On day 0, there were no differences between groups in PLT count. Compared with leukoreduced whole blood with PLTs added, reconstituted whole bloodereduced PLT aggregation for all activators with a further reduction noted in some with FFP compared with solvent detergent plasma. Rahbar et al. identified a reduction in PLT count and function over 24 h in patients with active bleeding requiring emergent use of uncross-matched blood in the ED.22 Patients subsequently received modified whole blood with PLTs or reconstituted blood. While there were no differences in PLT counts between groups over time, PLT aggregation to all agonist was generally higher in those patients receiving leukoreduced whole blood with PLTs. These studies demonstrated a reduction in PLT function because of exposure to component blood products possible due to the additives in RBCs and plasma or cellular aggregates due to processing and storage. In the present study, dilution with fresh autologous plasma or saline resulted in proportional decreases in PLT function. Caballo et al. diluted whole blood by 30% with Ringer’s lactate and reported a 30% reduction in PLT interactions.34 Hanke et al. demonstrated a dose response between PLT count and function with dilutions when assessed using Multiplate aggregometry.35 Using blood from control subjects, they
prepared samples with varying PLT counts and found PLT function to decrease with counts below the normal range (<150,000). At counts of 100,000 and 50,000, functional results decreased by 18% and 37%, respectively. Of note, they reported a large interindividual variation in these responses. Thus, reductions of PLT count by dilution or dose results in proportional decreases in function. It should be noted, however, that reduction in PLT count below <150,000 significantly influence PLT function as evaluated by Multiplate and, hence, discriminating between a reduction in PLT functionality caused by low count versus impaired intrinsic function is difficult36 and is a limitation of the present study. In the present study, a comparison of autologous FFP with a single allogenic donor FFP demonstrated differences in all activators except TRAP. Of note, the variance of the response between subjects to the single donor was similar. In contrast, the response to FFP from five different donors was highly variable. For example, dilution with plasma from donor 1 resulted in a 39% reduction in activity to ADP and 4% increase with RISTO in contrast to the 8% and 48% reductions to ADP and RISTO, respectively, for donor 5 plasma. In our evaluation of clinical subjects, while there were reductions from the ED to the ICU, the magnitude of the responses were highly variable between patients and weakly associated with the amount of blood product administered. This inconsistency would appear to be in part related to the variance within the allogenic donor FFP. Among and between the various plasma products, inconsistencies have been reported in factor activity, functional measures of coagulation, residual cell and micro-particle distributions, as well as actions on endothelial permeability.9,10 Between FFP collections factor, activity has been reported to vary by as much as 100% between donors. Functional measures of coagulation also show a wide variation. This may in part due to the variation in the volume of blood collected on a set volume of anticoagulant preservatives, as well as residual cells and microparticles have being increased as a result of the plasma being retained for a longer period of time with the blood cells. FFP may be refrigerated for 5 d before use, modifying all these characteristics. Finally, there are innate differences between donors. The observed variations of the responses of PLT function to various donor plasmas are another demonstration of the lack of consistency in plasma products obtained from single donors. Limitations of the present study include the small number of subjects limiting broad applicability of the findings. However, the samples were collected at similar periods in the
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henriksen et al coagulation and platelet function in trauma
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course of care, up on admission to the ED and ICU, in contrast to larger studies where sampling was based on time only, but at varying points in their course of care. Another limitation is the absence of adequate information as to prehospital medications, specifically anti-PLT drugs. We reviewed clinical records and were unable to find documentation of preadmission anti-PLT medications. Use of blood products in the treatment of the patient with severe injuries and shock has evolved over the past 50 y. In response to the administration of blood products, the physiology of the patient often improves as does the hemostatic status. With the transfusion of blood products, the hemostatic potential of the patient who is hypocoagulable improves based on measures of clotting, viscoelastometry, and thrombin generation in spite of a decrease in PLT count and function.3,22,37 The present study identifies, in patients with traumatic injuries, a reduction of both PLT count and function between admission to the ED and the ICU, which is further aggravated with the administration of the plasma/supernatant of blood products, specifically with stored PLTs. The disproportionate reduction in PLT function, in relation to count, may in part be because of the administration of allogenic blood products.
Acknowledgment
7. 8.
9.
10.
11.
12.
13.
14.
15.
C.E.W., H.H.H., A.G.G., L.A.B., J.C.C., and S.R.O. designed the study; C.E.W., H.H.H., A.G.G., L.A.B., J.C.C., S.V., S.S., N.M., B.A.C., J.B.H., and S.R.O. collected the data; C.E.W., H.H.H., A.G.G., L.A.B., J.C.C., S.R.O., J.B.H., P.I.J., and T.A.C. analyzed the data; All authors drafted, edited, revised, and approved the final version of the article.
16.
17.
Disclosure 18.
There are no conflicts of interest to report. 19.
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33. Rinder HM, Murphy M, Mitchell JG, et al. Progressive platelet activation with storage: evidence for shortened survival of activated platelets after transfusion. Transfusion. 1991;31:409e414. 34. Caballo C, Escolar G, Diaz-Ricart M, et al. Impact of experimental haemodilution on platelet function, thrombin generation and clot firmness: effects of different coagulation factor concentrates. Blood Transfus. 2013;11:391e399. 35. Hanke AA, Roberg K, Monaca E, et al. Impact of platelet count on results obtained from multiple electrode platelet aggregometry (Multiplate). Eur J Med Res. 2010;15:214e219. 36. Stissing T, Dridi NP, Ostrowski SR, Bochsen L, Johansson PI. The influence of low platelet count on whole blood aggregometry assessed by Multiplate. Clin Appl Thromb Hemost. 2011;17:E211eE217. 37. Holcomb JB, Wade CE, Michalek JE, et al. Increased plasma and platelet to red blood cell ratios improves outcome in 466 massively transfused civilian trauma patients. Ann Surg. 2008;248:447e458.
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