EAST 2014 PLENARY PAPER
Never-frozen liquid plasma blocks endothelial permeability as effectively as thawed fresh frozen plasma Yanna Cao, MD, Anahita Dua, MD, MS, MBA, Nena Matijevic, PhD, Yao-Wei Wang, MD, Shibani Pati, MD, PhD, Charles E. Wade, PhD, Tien C. Ko, MD, and John B. Holcomb, MD, Houston, Texas
BACKGROUND: Thawed fresh frozen plasma (TP) is a preferred plasma product for resuscitation but can only be used for up to 5 days after thawing. Never-frozen, liquid plasma (LQP) is approved for up to 26 days when stored at 1-C to 6-C. We have previously shown that TP repairs tumor necrosis factor > (TNF->)Yinduced permeability in human endothelial cells (ECs). We hypothesized that stored LQP repairs permeability as effectively as TP. METHODS: Three single-donor LQP units were pooled. Aliquots were frozen, and samples were thawed on Day 0 (TP0) then refrigerated for 5 days (TP5). The remaining LQP was kept refrigerated for 28 days, and aliquots were analyzed every 7 days. The EC monolayer was stimulated with TNF-> (10 ng/mL), inducing permeability, followed by a treatment with TP0, TP5, or LQP aged 0, 7, 14, 21, and 28 days. Permeability was measured by leakage of fluorescein isothiocyanateYdextran through the EC monolayer. Hemostatic profiles of samples were evaluated by thrombogram and thromboelastogram. Statistical analysis was performed using two-way analysis of variance, with p G 0.05 deemed significant. RESULTS: TNF-> increased permeability of the EC monolayer twofold compared with medium control. There was a significant decrease in permeability at 0, 7, 14, 21, and 28 days when LQP was used to treat TNF->Yinduced EC monolayers (p G 0.001). LQP was as effective as TP0 and TP5 at reducing permeability. Stored LQP retained the capacity to generate thrombin and form a clot. CONCLUSION: LQP corrected TNF->Yinduced EC permeability and preserved hemostatic potential after 28 days of storage, similar to TP stored for 5 days. The significant logistical benefit (fivefold) of prolonged LQP storage improves the immediate availability of plasma as a primary resuscitative fluid for bleeding patients. (J Trauma Acute Care Surg. 2014;77: 28Y33. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: Liquid plasma; FFP; endothelial permeability; thawed plasma; resuscitation.
H
emorrhage is a leading cause of preventable death, accounting for up to 40% of trauma-related mortality. Of these deaths, more than 50% occur within the first 24 hours, and current management of severe bleeding involves early plasma administration.1Y3 Preclinical studies have demonstrated the ability of plasma to promote clot generation and decrease endothelial permeability.4,5 Several large multicenter studies have shown that plasma is of benefit in hemorrhaging trauma patients and early, balanced administration in conjunction with packed red blood cells is associated with both decreased transfusion requirements and mortality.6Y10 Unfortunately, 1 U of fresh frozen plasma (FFP) takes appropriately 45 minutes to thaw, limiting its immediate availability in rapidly bleeding patients. Submitted: December 1, 2013, Revised: January 28, 2014, Accepted: January 29, 2014. From the Center for Translational Injury Research (CeTIR) (Y.C., A.D., M.M., Y.-W.W., C.E.W.,T.C.K., J.B.H.), Department of Surgery, University of TexasHouston, Houston, Texas; and Blood Systems Research Institute (S.P.), Department of Laboratory Medicine and Surgery, University of California-San Francisco, San Francisco, California. *Y.C. and A.D. contributed equally to this study. This study was presented at the 27th Eastern Association for the Surgery of Trauma Annual Scientific Assembly, January 14Y18, 2014, in Naples, Florida. Address for reprints: Anahita Dua, MD, MS, Center for Translational Injury Research (CeTIR), Department of Surgery, University of Houston-Texas, 6431 Fannin St, Houston, TX 77030; email: [email protected]. DOI: 10.1097/TA.0000000000000276
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Data to support the beneficial effect of early, balanced plasma administration in patients that require substantial transfusion are accumulating, resulting in the increasing use of thawed plasma (TP) as a primary resuscitative fluid.6,11,12 TP is an AABB (formerly known as the American Association of Blood Banks)Yapproved product and a common form of plasma administered. TP is the thawed form of FFP and can be refrigerated at 1-C to 6-C for 5 days. Bleeding patients benefit from early plasma administration.6Y10 Recently, TP has been placed in the emergency department and prehospital (ambulance, helicopter) environment for immediate transfusion in hemorrhaging patients. While TP is logistically superior to FFP, the 5-day shelf life continues to present supply- and cost-related issues. Maintaining a constant supply of TP for emergency situations can result in a substantial wastage of product if no patients requiring plasma present within the 5-day window. This is an issue for all 1,590 US trauma centers but especially for the 664 Level II and Level III trauma centers that may not have the patient volume to avoid wastage of this 5-day product. This issue is even more acute for the remaining 4,134 nontrauma hospitals that occasionally see significantly injured or bleeding patients.13 A potential solution for this logistical issue is the use of the AABB-approved product liquid plasma (LQP). LQP is separated from whole blood within 8 hours of blood collection and is approved for use for up to 26 days when stored at 1-C to 6-C. It is never frozen (or thawed) and has been shown to have a superior hemostatic profile in vitro J Trauma Acute Care Surg Volume 77, Number 1
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when compared with TP.4 A unit of FFP costs approximately $65, while a unit of LQP costs $75. The proposed mechanisms of action of TP, aside from improved hemostasis, include modulation of endothelial function, resulting in both a decrease in inflammation and inhibition of vascular endothelial cell (EC) permeability.5,14 The effect of LQP on EC monolayer permeability is unknown. The purpose of this study was to evaluate whether LQP decreases EC monolayer permeability as effectively as TP.
PATIENTS AND METHODS This in vitro study was conducted by inducing permeability of a human pulmonary endothelial monolayer with the use of TNF->. The leaky monolayer was then treated with TP Day 0 (TP0), TP Day 5 (TP5), as well as LQP aged Day 0, 7, 14, 21, and 28. Permeability was then measured to determine the impact of different plasma storage types on the monolayer.
Plasma Three single-donor plasma units from healthy blood donors (Type A, two females and one male) were obtained from the Gulf Coast Regional Blood Center (Houston, Texas). FFP served as positive control. The LQP samples were pooled and refrigerated at 1-C to 6-C for 28 days. Aliquots of the LQP were taken using sterile connections on Day 0 (LQP-0), Day 7 (LQP-7), Day 14 (LQP-14), and Day 28 (LQP-28) for analysis. TP samples were acquired from the same pooled plasma sample; aliquots were taken on Day 0 from the pooled LQP and frozen at j80-C. To create TP0, a frozen plasma aliquot was thawed in a 37-C water bath as per standard AABB operating procedures on the day of testing. To create TP5, a frozen aliquot was thawed and stored at 1-C to 6-C for 5 days. TP0 and TP5 were analyzed concurrently with LQP at each time points 7, 14, 21, and 28 days for comparison of permeability profiles.
Primary Cells First passage human lung microvascular ECs termed pulmonary ECs (PECs) were purchased from Lonza Inc. (Allendale, NJ). The PECs were cultured in endothelial growth medium (EGM-2 BulletKit) at 37-C in a humidified incubator containing 5% CO2 and used at Passage 4 for all experiments.
EC Permeability An in vitro Vascular Permeability Assay kit including collagen precoated 24-well plate was purchased from EMD Millipore (Billerica, MA). The PECs were seeded at 10,000 cells per insert and cultured for 48 hours, allowing cell attachment, adhesion, and monolayer formation. TNF-> was applied to EC monolayers of PECs to induce permeability. Cells were treated with TNF-> (10 ng/mL) for 20 hours to 24 hours, followed by LQP or TP treatment at 10% for 1 hour at 37-C. The fluorescein isothiocyanate (FITC)Y conjugated dextran solution was then added to the insert at 1:20 dilution and incubated at room temperature for 60 minutes. Aliquots (100 KL) from the bottom well were taken and transferred to the wells of a 96-well microplate (BD Bio, Bedford, MA). Fluorescence signals were read using a fluorimeter (Molecular Devices, Sunnyville, CA) at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
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To investigate whether LQP storage altered its inhibitory effects on EC permeability in vitro, LQP was examined using the permeability assay every 7 days (time point, 0, 7, 14, 21, and 28 days) in comparison with its donor-matched TP0 and TP5.
Hemostatic Potential Hemostatic potential of LQP-7, TP0, and TP5 was evaluated by measuring thrombin generation using the Calibrated Automated Thrombogram (CAT, Thrombinoscope, Maastricht, the Netherlands). A parameter of CAT is endogenous thrombin potential (ETP) (nMminute) where the area under the curve represents thrombin generation and decay in time. CAT objectively measures thrombin generation potential of a sample, which is directly correlated with clot formation. Thrombelastography (TEG 5000 Thrombelastograph Analyzer, Haemoscope, Niles, IL) was used to evaluate the kinetics of plasma clot formation, strength, and stability. Clot formation was triggered with recombinant tissue factor (Recombiplastin, Instrumentation Laboratory, Lexington, MA), with final concentration of 3 pM. Under these conditions, obtained TEG tracings represent fibrin clot generated from plasma clotting factors/inhibitors and residual platelets but without red blood cells or leukocytes given that their presence in plasma products is negligible.4,15
Statistical Analysis For in vitro permeability assays, data were analyzed using one-way analysis of variance for single time point studies and two-way analysis of variance for time course studies. When significant, an appropriate multiple comparison Holm-Sidak method was applied. Data are presented as mean T SEM with a p G 0.05 deemed significant.
RESULTS Hemostatic Potential To compare the hemostatic capabilities between plasma groups, TP0, TP5, and LQP-7 (stored for 7 days) were evaluated using CAT and TEG. The hemostatic profiles were not significantly different between the groups (Table 1). To test for differences in endothelial permeability in the experimental assay, the effect of five groups on TNF->Yinduced endothelium were evaluated: medium alone (negative control), seven-donor pooled independent FFP (positive control), TP0, TP5, LQP (Days 0, 7, 14, 21, or 28).
TNF-> Induces Compromise of the PEC Monolayer Which Is Inhibited by the Treatment With TP and LQP In our in vitro assays, we used TNF-> to induce permeability in the EC monolayers. Qualitative analysis in Figure 1 reveals that TNF-> treatment of the EC monolayer creates TABLE 1. ETP and TEG Maximum Amplitude Values Comparing TP0, TP5, and LQP-7 ETP, nMminute TEG maximum amplitude, mm
TP0
TP5
LQP-7
1,537 31
1,585 32
1,574 T 37 32.5 T 3
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Figure 1. Human PEC (hPEC) monolayer on the inserts. Following the permeability assay for LQP, cell monolayers on the inserts were stained, and representative images were captured with original magnification of 200. Qualitative analysis reveals that TNF-> treatment of the monolayer creates gaps between adjacent ECs, which is consistent with compromise of the EC monolayer and increased paracellular permeability. Treatment with all plasma groups (seven-pooled positive control FFP, TP0, TP5, LQP-28) decreases the gaps between the endothelial monolayers, indicating repair or decreased EC permeability.
intercellular gaps between adjacent ECs, which is consistent with compromise of the EC monolayer and increased paracellular permeability. Treatment with all plasma groups (FFP positive control, TP0, TP5, LQP-28) decreased these gaps between the endothelial monolayers, indicating repair or decreased EC permeability (Fig. 1).
LQP Inhibits PEC Permeability Similar to FFP To examine the potential of LQP to inhibit EC permeability in vitro, TNF-> was applied to EC monolayers of PECs to induce paracellular permeability. EC monolayers were treated with LQP or an equivalent amount of FFP (pooled from seven donors). We used the positive control, pooled seven-donor FFP sample as the comparison group for this experiment because TP0 and TP5 same-donor plasma were unavailable on LQP-0 when this experiment was conducted. Medium alone was added as a negative control. TNF-> induces a two fold increase in permeability compared with medium control (Fig. 2). LQP significantly inhibited TNF-alpha-induced permeability.
LQP Blocks Endothelial Permeability as Effectively as TP To compare the effects of LQP on endothelial permeability in vitro with TP, LQP-7 (stored 7 days) and donormatched TP0 and TP5 were assayed simultaneously. As shown in Figure 3, LQP-7 blocked endothelial permeability as efficiently as TP0 and TP5. LQP was as effective at blocking permeability as TP0 and TP5. The seven-donor pooled FFP sample was again included as a positive control and demonstrated no significant differences between TP or LQP. 30
Storage of LQP for 28 Days Does Not Alter the Protective Effects on Endothelial Permeability To compare the different groups tested over the time course of the experiment (28 days), all groups and data were plotted on the same graph. Figure 4 depicts that LQP retained its ability to decrease endothelial permeability as effectively as TP0 and TP5 regardless of age (Day 0Y28). Partial data of LQP-0 and LQP-7 from Figures 2 and 3 were included for a summarized presentation of the entire time course. There was a significant decrease in EC permeability at all LQP age points (0, 7, 14, 21, and 28 days) when compared with the TNF>Ystimulated EC monolayer (p G 0.001). LQP Day 0, 7, 14, 21, and 28 inhibited EC permeability as effectively as TP0 and TP5 (Fig. 4). This effectiveness of LQP on endothelial permeability was not altered with storage for up to 28 days.
DISCUSSION As early plasma becomes more widely accepted as a primary resuscitative fluid for hemorrhaging patients, the logistics of storage and delivery of this product have become increasingly important. With approximately 2 million units transfused annually, the most commonly administered form of plasma in the United States is FFP.16 As FFP requires 45 minutes to thaw before administration, it has limited utility in rapidly bleeding patients. For the prehospital setting (ambulances and helicopters) as well as the emergency department, this means plasma must be thawed and refrigerated (TP) to be available for immediate administration. This strategy may result in significant cost and product waste. A potential solution to this logistical issue is the use of LQP. LQP is never frozen or thawed and approved for use for up * 2014 Lippincott Williams & Wilkins
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Figure 2. Liquid plasma blocks permeability of EC monolayers in vitro. hPECs were seeded on inserts for 48 hours, and the EC monolayers were formed. The cell monolayers were stimulated by TNF-> (10 ng/mL) for 20 hours to 24 hours, medium alone as control. LQP-0 was applied to the inserts for 1 hour, equal amount of medium was added to the medium or TNF-> alone groups, and FFP served as positive control. Permeability of FITC-dextran added on the cell monolayer was measured as fluorescence signal at excitation 485 nm and emission 535 nm. Data are expressed as mean T SEM. n = 4 inserts per group. *p G 0.05 compared with medium control, #p G 0.05 compared with TNF-> treatment.
to 26 days. This gives trauma centers ample time to use the blood product while ensuring that transfusion-ready plasma is available for immediate resuscitation of bleeding patients. Preparation and storage of plasma products vary significantly.17 The impact of plasma preparations on hemostatic potential and endothelial permeability has been studied in a limited capacity. Matijevic et al.4 found that LQP had a better capacity to generate thrombin and form clot when compared with TP in an experiment that evaluated hemostatic potential of these two plasma preparations. Even at Day 26, LQP was comparable with TP in its ability to form clot and generate thrombin, with the majority of LQP clotting factors retaining their initial activity level.4 In vitro studies comparing different preparations of plasma report no significant differences on endothelial permeability models. Wataha et al.14 compared spray-dried plasma and TP to determine the impact on permeability and inflammation in in vitro studies. Both spraydried plasma and TP equivalently decreased vascular EC permeability, EC adherens junction breakdown, and endothelial white blood cell binding.14 The in vivo protective effects of thawed FFP administration as compared with lactated Ringer’s solution on lung endothelial permeability induced by hemorrhagic shock and trauma in a rodent model of injury have been
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described.18,19 FFP was found to partially restore the endothelial glycocalyx, which attenuated lung injury, thereby decreasing pulmonary edema.18,19 Our study found that LQP, regardless of age for up to 28 days, decreased TNF->Yinduced endothelial permeability as effectively as TP. There are numerous clinical studies that demonstrate the proposed advantage of early plasma administration. However, no randomized control trials comparing different types of plasma preparations (LQP, spray-dried, freeze-dried, TP, FFP, etc.) exist. A large cohort study inclusive of 84,986 patients compared the mortality rates in patients administered with TP (n = 212,541 U administered) to those transfused LQP (142,169 U administered) and found no overall difference between groups.17 LQP transfusions were divided into categories based on the age of the LQP product (1Y3 days, 4Y14 days, and Q15 days). Interestingly, there was no significant difference in patient outcome (mortality) associated with the duration of storage.17 Another study by Young et al.20 described their institutional experience with two different massive transfusion protocols (MTPs) for traumatically injured patients.20 The study reported that LQP and TP were used interchangeably; one institutional location used LQP as part of the MTP algorithm, and the other incorporated TP into their MTP.20 There were no details regarding differences in patient outcomes in this study, but the authors did comment that within the University Health System Consortium, inclusive of 339 institutions (academic and affiliated), TP was available for early transfusion in only 60% of the institutions.20 This likely reflects the logistical issues associated with TP (thawing time, waste associated with
Figure 3. LQP (Day 7) inhibits endothelial permeability as effectively as TP. hPECs were seeded on inserts for 48 hours to establish EC monolayers. The cell monolayers were stimulated by TNF-> (10 ng/mL) for 20 hours to 24 hours with medium alone as control. LQP-7, the donor-matched TP0, or TP5 were applied to the inserts for 1 hour, and equal amount of medium was added to the medium or TNF-> alone groups. FFP served as positive control. Permeability of FITC-dextran added on the cell monolayer on inserts to the bottom wells was measured as fluorescence signal at excitation 485 nm and emission 535 nm. Data are expressed as mean T SEM. n = 4 inserts per group. *p G 0.05 compared with medium control, #p G 0.05 compared with TNF-> treatment.
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LIMITATIONS
Figure 4. The effects of LQP on endothelial permeability were not altered during storage. hPECs were seeded on inserts for 48 hours, and the EC monolayers were formed. The cell monolayers were stimulated by TNF-> (10 ng/mL) for 20 hours to 24 hours, medium alone as control. LQPs and their donor-matched TPs were applied to the inserts for 1 hour, and equal amount of medium was added to TNF-> alone groups. Permeability of FITC-dextran added on the cell monolayer was measured as fluorescence signal at excitation 485 nm and emission 535 nm. Data are expressed as mean T SEM of fold changes compared with TNF-> treatment. n = 4 inserts per group. Partial data of LQP-0 and LQP-7 from Figures 2 and 3 are included for the summarized presentation purpose of this entire time course of LQPs. The same FFP was included in all time point assays as positive control. *p G 0.05 as comparison of TNF-> + plasma treatment with TNF-> treatment alone. NS, not significant between LQP and TP and throughout all LQP time points.
expiration).20 In the LQP group during a 6-month period, only five LQP units became outdated, while in the TP cohort, 17 U expired.20,21 Given the present study and earlier research demonstrating clinical equivalency of TP and LQP, our institution has replaced TP with LQP on helicopters, decreasing the logistical burden on the blood bank and helicopter crews. In the past, unused TP had to be exchanged from the helicopter on Day 3, but by placing LQP on the helicopters, we have increased our time to plasma exchange to Day 12. During the last 2 years, we have wasted only 1.9% of all plasma placed on our helicopters. While many high-volume centers have instituted TP as part of their resuscitative paradigms, the 5-day expiration date on TP makes it difficult for smaller centers to implement early plasma use, given the perceived potential for waste and associated increased cost. In actual practice, most centers have seen a decrease in waste and cost when a TP program is implemented.20Y22 This waste and its associated cost may be even further reduced with the implementation of an LQP program. Data from our endothelial permeability and coagulation studies in conjunction with the retrospective clinical data of Norda et al.17 demonstrate that LQP, regardless of the duration of storage up to 28 days, is as good as or superior to TP.4,17 LQP improves the immediate and 28-day availability of plasma as a primary resuscitative fluid for bleeding patients. 32
A limitation of this study is the unclear clinical significance of specifically regulating endothelial permeability on outcomes in trauma patients. We hypothesize that limiting EC permeability in injured patients will significantly impact endothelial dysfunction, inflammation, and dysregulated coagulation after hemorrhagic shock and trauma, resulting in both decreased edema and end-organ damage. Further clinical studies are required to determine whether EC permeability can be used as a measure of quality and function of plasma-based therapeutics and whether modulating EC permeability impacts outcomes in trauma. Another limitation of this study was that plasma was collected from only three donors and pooled. Because our study design focused on comparing TP and LQP from the same pooled donor sample, we were unable to compare same-donor TP0 or TP5 to LQP-0. Because the TP samples came from thawing the frozen aliquots of plasma from the same pooled LQP donor bag, we did not have a TP sample that had been previously frozen to generate a TP0 or TP5 sample on LQP-0.
CONCLUSION Plasma is becoming an increasingly accepted primary resuscitative fluid for rapidly bleeding patients; therefore, demand for a plasma product that can be stored long term, in a form readily available for immediate transfusion, is increasing. The current AABB-approved answer to this logistical issue is the use of LQP. Previous work by our group and the current study found that LQP preserves hemostatic potential as effectively as TP.4 This study found that LQP corrects TNF->Yinduced EC permeability after 28 days of storage, similar to TP stored for up to 5 days. Our previous and current data combined with the clinical study by Norda et al. show that LQP is comparable with TP in its ability to correct hemostatic profiles and decrease EC monolayer permeability.4,17 LQP can be considered for use in trauma patients requiring immediate plasma resuscitation. AUTHORSHIP Y.C. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision. A.D. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision. N.M. contributed to the study design, data collection, data analysis, data interpretation, writing, and critical revision. Y.-W.W. contributed to the study design, data collection, data analysis, data interpretation, writing, and critical revision. S.P. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision. C.E.W. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision. T.C.K. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision. J.B.H. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision.
DISCLOSURE The authors declare no conflicts of interest.
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REFERENCES 1. Holcomb JB. Optimal use of blood products in severely injured trauma patients. Hematology Am Soc Hematol Educ Program. 2010;2010:465Y469. 2. Johansson PI, Stensballe J. Effect of haemostatic control resuscitation on mortality in massively bleeding patients: a before and after study. Vox Sang. 2009;96(2):111Y118. 3. Holcomb JB, Wade CE, Michalek JE, Chisholm GB, Zarzabal LA, Schreiber MA, Gonzalez EA, Pomper GJ, Perkins JG, et al. Increased plasma and platelet to red blood cell ratios improves outcome in 466 massively transfused civilian trauma patients. Ann Surg. 2008;248(3):447Y458. 4. Matijevic N, Wang YW, Cotton BA, Hartwell E, Barbeau JM, Wade CE, Holcomb JB. Better hemostatic profiles of never-frozen liquid plasma compared with thawed fresh frozen plasma. J Trauma Acute Care Surg. 2013;74(1):84Y90; discussion 90Y91. 5. Pati S, Matijevic N, Doursout MF, Ko T, Cao Y, Deng X, Kozar RA, Hartwell E, Conyers J, Holcomb JB. Protective effects of fresh frozen plasma on vascular endothelial permeability, coagulation, and resuscitation after hemorrhagic shock are time dependent and diminish between days 0 and 5 after thaw. J Trauma. 2010;69(Suppl 1):S55YS63. 6. Holcomb JB, Fox EE, Wade CE; PROMMTT Study Group. The PRospective Observational Multicenter Major Trauma Transfusion (PROMMTT) study. J Trauma Acute Care Surg. 2013;75(1 Suppl 1):S1YS2. 7. Peiniger S, Nienaber U, Lefering R, Braun M, Wafaisade A, Wutzler S, Borgmann M, Spinella PC, Maegele M; Trauma Registry of the Deutsche Gesellschaft fu¨r Unfallchirurgie. Balanced massive transfusion ratios in multiple injury patients with traumatic brain injury. Crit Care. 2011;15(1):R68. 8. Dente CJ, Shaz BH, Nicholas JM, Harris RS, Wyrzykowski AD, Patel S, Shah A, Vercruysse GA, Feliciano DV, Rozycki GS, et al. Improvements in early mortality and coagulopathy are sustained better in patients with blunt trauma after institution of a massive transfusion protocol in a civilian level I trauma center. J Trauma. 2009;66(6):1616Y1624. 9. del Junco DJ, Holcomb JB, Fox EE, Brasel KJ, Phelan HA, Bulger EM, Schreiber MA, Muskat P, Alarcon LH, Cohen MJ, et al.; PROMMTT Study Group. Resuscitate early with plasma and platelets or balance blood products gradually: findings from the PROMMTT study. J Trauma Acute Care Surg. 2013;75(1 Suppl 1):S24YS30. 10. Brakenridge SC, Phelan HA, Henley SS, Golden RM, Kashner TM, Eastman AE, Sperry JL, Harbrecht BG, Moore EE, et al.; Inflammation and the Host Response to Injury Investigators. Early blood product and crystalloid volume resuscitation: risk association with multiple organ dysfunction after severe blunt traumatic injury. J Trauma. 2011;71(2):299Y305. 11. Radwan ZA, Bai Y, Matijevic N, del Junco DJ, McCarthy JJ, Wade CE, Holcomb JB, Cotton BA. An emergency department thawed plasma protocol for severely injured patients. JAMA Surg. 2013;148(2):170Y175. 12. Kim BD, Zielinski MD, Jenkins DH, Schiller HJ, Berns KS, Zietlow SP. The effects of prehospital plasma on patients with injury: a prehospital plasma resuscitation. J Trauma Acute Care Surg. 2012;73(2 Suppl 1):S49YS53. 13. American Hospital Association. Fast Fact US Hospitals. Available at: http://www.aha.org/research/rc/stat-studies/fast-facts.shtml. Accessed December 1, 2013. 14. Wataha K, Menge T, Deng X, Shah A, Bode A, Holcomb JB, Potter D, Kozar R, Spinella PC, Pati S. Spray-dried plasma and fresh frozen plasma modulate permeability and inflammation in vitro in vascular endothelial cells. Transfusion. 2013;53(Suppl 1):80SY90S. 15. Matijevic N, Wang YW, Kostousov V, Wade CE, Vijayan KV, Holcomb JB. Decline in platelet microparticles contributes to reduced hemostatic potential of stored plasma. Thromb Res. 2011;128(1):35Y41.
16. Arya RC, Wander G, Gupta P. Blood component therapy: which, when and how much. J Anaesthesiol Clin Pharmacol. 2011;27(2):278Y284. 17. Norda R, Andersson TM, Edgren G, Nyren O, Reilly M. The impact of plasma preparations and their storage time on short-term posttransfusion mortality: a population-based study using the Scandinavian Donation and Transfusion database. J Trauma Acute Care Surg. 2012;72(4):954Y960. 18. Kozar RA, Peng Z, Zhang R, Holcomb JB, Pati S, Park P, Ko TC, Paredes A. Plasma restoration of endothelial glycocalyx in a rodent model of hemorrhagic shock. Anesth Analg. 2011;112(6):1289Y1295. 19. Peng Z, Pati S, Potter D, Brown R, Holcomb JB, Grill R, Wataha K, Park PW, Xue H, Kozar RA. Fresh frozen plasma lessens pulmonary endothelial inflammation and hyperpermeability after hemorrhagic shock and is associated with loss of syndecan 1. Shock. 2013;40(3):195Y202. 20. Young PP, Cotton BA, Goodnough LT. Massive transfusion protocols for patients with substantial hemorrhage. Transfus Med Rev. 2011;25(4):293Y303. 21. Goodnough LT, Daniels K, Wong AE, Viele M, Fontaine MF, Butwick AJ. How we treat: transfusion medicine support of obstetric services. Transfusion. 2011;51(12):2540Y2548. 22. Wehrli G, Taylor NE, Haines AL, Brady TW, Mintz PD. Instituting a thawed plasma procedure: it just makes sense and saves cents. Transfusion. 2009;49(12):2625Y2630.
EDITORIAL CRITIQUE The Houston group has added to a developing body of bench and clinical literature suggesting similarities of hemostatic and endothelial-reactive effects of liquid plasma compared to thawed plasma. Given that liquid plasma is far superior to thawed plasma from a logistical standpoint (since it has a 26-day lifespan as opposed to thawed plasma’s 5 days), if similarities in effectiveness can be demonstrated it would be powerful evidence to move toward more widespread use of this underutilized resource. This important work goes a long way toward accomplishing this goal. As with most research, this study also raises as many questions as it answers. Chief among these is the finding that all plasma preparations lowered the ‘‘leakiness’’ of the endothelial cell matrices well below the baseline value of untreated cells. This is interesting since, while I would expect to see all plasma preparations attenuate TNF’s actions, one would not expect them to overcorrect so zealously. The significance of this finding remains unknown. Additionally, the authors’ group is also on the forefront of the role of the endothelial glycocalyx in the response to resuscitation after trauma. I would encourage them to pursue liquid plasma’s actions in this realm. In conclusion, Dr. Dua and her colleagues are to be commended for this important contribution to the advancement of the care of the injured patient. Herb A. Phelan, MD, MSCS Division of Burns/Trauma/Critical Care UT-Southwestern Medical Center Dallas, Texas
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Never-frozen liquid plasma blocks endothelial permeability as effectively as thawed fresh frozen plasma Yanna Cao, MD, Anahita Dua, MD, MS, MBA, Nena Matijevic, PhD, Yao-Wei Wang, MD, Shibani Pati, MD, PhD, Charles E. Wade, PhD, Tien C. Ko, MD, and John B. Holcomb, MD, Houston, Texas
BACKGROUND: Thawed fresh frozen plasma (TP) is a preferred plasma product for resuscitation but can only be used for up to 5 days after thawing. Never-frozen, liquid plasma (LQP) is approved for up to 26 days when stored at 1-C to 6-C. We have previously shown that TP repairs tumor necrosis factor > (TNF->)Yinduced permeability in human endothelial cells (ECs). We hypothesized that stored LQP repairs permeability as effectively as TP. METHODS: Three single-donor LQP units were pooled. Aliquots were frozen, and samples were thawed on Day 0 (TP0) then refrigerated for 5 days (TP5). The remaining LQP was kept refrigerated for 28 days, and aliquots were analyzed every 7 days. The EC monolayer was stimulated with TNF-> (10 ng/mL), inducing permeability, followed by a treatment with TP0, TP5, or LQP aged 0, 7, 14, 21, and 28 days. Permeability was measured by leakage of fluorescein isothiocyanateYdextran through the EC monolayer. Hemostatic profiles of samples were evaluated by thrombogram and thromboelastogram. Statistical analysis was performed using two-way analysis of variance, with p G 0.05 deemed significant. RESULTS: TNF-> increased permeability of the EC monolayer twofold compared with medium control. There was a significant decrease in permeability at 0, 7, 14, 21, and 28 days when LQP was used to treat TNF->Yinduced EC monolayers (p G 0.001). LQP was as effective as TP0 and TP5 at reducing permeability. Stored LQP retained the capacity to generate thrombin and form a clot. CONCLUSION: LQP corrected TNF->Yinduced EC permeability and preserved hemostatic potential after 28 days of storage, similar to TP stored for 5 days. The significant logistical benefit (fivefold) of prolonged LQP storage improves the immediate availability of plasma as a primary resuscitative fluid for bleeding patients. (J Trauma Acute Care Surg. 2014;77: 28Y33. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: Liquid plasma; FFP; endothelial permeability; thawed plasma; resuscitation.
H
emorrhage is a leading cause of preventable death, accounting for up to 40% of trauma-related mortality. Of these deaths, more than 50% occur within the first 24 hours, and current management of severe bleeding involves early plasma administration.1Y3 Preclinical studies have demonstrated the ability of plasma to promote clot generation and decrease endothelial permeability.4,5 Several large multicenter studies have shown that plasma is of benefit in hemorrhaging trauma patients and early, balanced administration in conjunction with packed red blood cells is associated with both decreased transfusion requirements and mortality.6Y10 Unfortunately, 1 U of fresh frozen plasma (FFP) takes appropriately 45 minutes to thaw, limiting its immediate availability in rapidly bleeding patients. Submitted: December 1, 2013, Revised: January 28, 2014, Accepted: January 29, 2014. From the Center for Translational Injury Research (CeTIR) (Y.C., A.D., M.M., Y.-W.W., C.E.W.,T.C.K., J.B.H.), Department of Surgery, University of TexasHouston, Houston, Texas; and Blood Systems Research Institute (S.P.), Department of Laboratory Medicine and Surgery, University of California-San Francisco, San Francisco, California. *Y.C. and A.D. contributed equally to this study. This study was presented at the 27th Eastern Association for the Surgery of Trauma Annual Scientific Assembly, January 14Y18, 2014, in Naples, Florida. Address for reprints: Anahita Dua, MD, MS, Center for Translational Injury Research (CeTIR), Department of Surgery, University of Houston-Texas, 6431 Fannin St, Houston, TX 77030; email: [email protected]. DOI: 10.1097/TA.0000000000000276
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Data to support the beneficial effect of early, balanced plasma administration in patients that require substantial transfusion are accumulating, resulting in the increasing use of thawed plasma (TP) as a primary resuscitative fluid.6,11,12 TP is an AABB (formerly known as the American Association of Blood Banks)Yapproved product and a common form of plasma administered. TP is the thawed form of FFP and can be refrigerated at 1-C to 6-C for 5 days. Bleeding patients benefit from early plasma administration.6Y10 Recently, TP has been placed in the emergency department and prehospital (ambulance, helicopter) environment for immediate transfusion in hemorrhaging patients. While TP is logistically superior to FFP, the 5-day shelf life continues to present supply- and cost-related issues. Maintaining a constant supply of TP for emergency situations can result in a substantial wastage of product if no patients requiring plasma present within the 5-day window. This is an issue for all 1,590 US trauma centers but especially for the 664 Level II and Level III trauma centers that may not have the patient volume to avoid wastage of this 5-day product. This issue is even more acute for the remaining 4,134 nontrauma hospitals that occasionally see significantly injured or bleeding patients.13 A potential solution for this logistical issue is the use of the AABB-approved product liquid plasma (LQP). LQP is separated from whole blood within 8 hours of blood collection and is approved for use for up to 26 days when stored at 1-C to 6-C. It is never frozen (or thawed) and has been shown to have a superior hemostatic profile in vitro J Trauma Acute Care Surg Volume 77, Number 1
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when compared with TP.4 A unit of FFP costs approximately $65, while a unit of LQP costs $75. The proposed mechanisms of action of TP, aside from improved hemostasis, include modulation of endothelial function, resulting in both a decrease in inflammation and inhibition of vascular endothelial cell (EC) permeability.5,14 The effect of LQP on EC monolayer permeability is unknown. The purpose of this study was to evaluate whether LQP decreases EC monolayer permeability as effectively as TP.
PATIENTS AND METHODS This in vitro study was conducted by inducing permeability of a human pulmonary endothelial monolayer with the use of TNF->. The leaky monolayer was then treated with TP Day 0 (TP0), TP Day 5 (TP5), as well as LQP aged Day 0, 7, 14, 21, and 28. Permeability was then measured to determine the impact of different plasma storage types on the monolayer.
Plasma Three single-donor plasma units from healthy blood donors (Type A, two females and one male) were obtained from the Gulf Coast Regional Blood Center (Houston, Texas). FFP served as positive control. The LQP samples were pooled and refrigerated at 1-C to 6-C for 28 days. Aliquots of the LQP were taken using sterile connections on Day 0 (LQP-0), Day 7 (LQP-7), Day 14 (LQP-14), and Day 28 (LQP-28) for analysis. TP samples were acquired from the same pooled plasma sample; aliquots were taken on Day 0 from the pooled LQP and frozen at j80-C. To create TP0, a frozen plasma aliquot was thawed in a 37-C water bath as per standard AABB operating procedures on the day of testing. To create TP5, a frozen aliquot was thawed and stored at 1-C to 6-C for 5 days. TP0 and TP5 were analyzed concurrently with LQP at each time points 7, 14, 21, and 28 days for comparison of permeability profiles.
Primary Cells First passage human lung microvascular ECs termed pulmonary ECs (PECs) were purchased from Lonza Inc. (Allendale, NJ). The PECs were cultured in endothelial growth medium (EGM-2 BulletKit) at 37-C in a humidified incubator containing 5% CO2 and used at Passage 4 for all experiments.
EC Permeability An in vitro Vascular Permeability Assay kit including collagen precoated 24-well plate was purchased from EMD Millipore (Billerica, MA). The PECs were seeded at 10,000 cells per insert and cultured for 48 hours, allowing cell attachment, adhesion, and monolayer formation. TNF-> was applied to EC monolayers of PECs to induce permeability. Cells were treated with TNF-> (10 ng/mL) for 20 hours to 24 hours, followed by LQP or TP treatment at 10% for 1 hour at 37-C. The fluorescein isothiocyanate (FITC)Y conjugated dextran solution was then added to the insert at 1:20 dilution and incubated at room temperature for 60 minutes. Aliquots (100 KL) from the bottom well were taken and transferred to the wells of a 96-well microplate (BD Bio, Bedford, MA). Fluorescence signals were read using a fluorimeter (Molecular Devices, Sunnyville, CA) at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
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To investigate whether LQP storage altered its inhibitory effects on EC permeability in vitro, LQP was examined using the permeability assay every 7 days (time point, 0, 7, 14, 21, and 28 days) in comparison with its donor-matched TP0 and TP5.
Hemostatic Potential Hemostatic potential of LQP-7, TP0, and TP5 was evaluated by measuring thrombin generation using the Calibrated Automated Thrombogram (CAT, Thrombinoscope, Maastricht, the Netherlands). A parameter of CAT is endogenous thrombin potential (ETP) (nMminute) where the area under the curve represents thrombin generation and decay in time. CAT objectively measures thrombin generation potential of a sample, which is directly correlated with clot formation. Thrombelastography (TEG 5000 Thrombelastograph Analyzer, Haemoscope, Niles, IL) was used to evaluate the kinetics of plasma clot formation, strength, and stability. Clot formation was triggered with recombinant tissue factor (Recombiplastin, Instrumentation Laboratory, Lexington, MA), with final concentration of 3 pM. Under these conditions, obtained TEG tracings represent fibrin clot generated from plasma clotting factors/inhibitors and residual platelets but without red blood cells or leukocytes given that their presence in plasma products is negligible.4,15
Statistical Analysis For in vitro permeability assays, data were analyzed using one-way analysis of variance for single time point studies and two-way analysis of variance for time course studies. When significant, an appropriate multiple comparison Holm-Sidak method was applied. Data are presented as mean T SEM with a p G 0.05 deemed significant.
RESULTS Hemostatic Potential To compare the hemostatic capabilities between plasma groups, TP0, TP5, and LQP-7 (stored for 7 days) were evaluated using CAT and TEG. The hemostatic profiles were not significantly different between the groups (Table 1). To test for differences in endothelial permeability in the experimental assay, the effect of five groups on TNF->Yinduced endothelium were evaluated: medium alone (negative control), seven-donor pooled independent FFP (positive control), TP0, TP5, LQP (Days 0, 7, 14, 21, or 28).
TNF-> Induces Compromise of the PEC Monolayer Which Is Inhibited by the Treatment With TP and LQP In our in vitro assays, we used TNF-> to induce permeability in the EC monolayers. Qualitative analysis in Figure 1 reveals that TNF-> treatment of the EC monolayer creates TABLE 1. ETP and TEG Maximum Amplitude Values Comparing TP0, TP5, and LQP-7 ETP, nMminute TEG maximum amplitude, mm
TP0
TP5
LQP-7
1,537 31
1,585 32
1,574 T 37 32.5 T 3
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Figure 1. Human PEC (hPEC) monolayer on the inserts. Following the permeability assay for LQP, cell monolayers on the inserts were stained, and representative images were captured with original magnification of 200. Qualitative analysis reveals that TNF-> treatment of the monolayer creates gaps between adjacent ECs, which is consistent with compromise of the EC monolayer and increased paracellular permeability. Treatment with all plasma groups (seven-pooled positive control FFP, TP0, TP5, LQP-28) decreases the gaps between the endothelial monolayers, indicating repair or decreased EC permeability.
intercellular gaps between adjacent ECs, which is consistent with compromise of the EC monolayer and increased paracellular permeability. Treatment with all plasma groups (FFP positive control, TP0, TP5, LQP-28) decreased these gaps between the endothelial monolayers, indicating repair or decreased EC permeability (Fig. 1).
LQP Inhibits PEC Permeability Similar to FFP To examine the potential of LQP to inhibit EC permeability in vitro, TNF-> was applied to EC monolayers of PECs to induce paracellular permeability. EC monolayers were treated with LQP or an equivalent amount of FFP (pooled from seven donors). We used the positive control, pooled seven-donor FFP sample as the comparison group for this experiment because TP0 and TP5 same-donor plasma were unavailable on LQP-0 when this experiment was conducted. Medium alone was added as a negative control. TNF-> induces a two fold increase in permeability compared with medium control (Fig. 2). LQP significantly inhibited TNF-alpha-induced permeability.
LQP Blocks Endothelial Permeability as Effectively as TP To compare the effects of LQP on endothelial permeability in vitro with TP, LQP-7 (stored 7 days) and donormatched TP0 and TP5 were assayed simultaneously. As shown in Figure 3, LQP-7 blocked endothelial permeability as efficiently as TP0 and TP5. LQP was as effective at blocking permeability as TP0 and TP5. The seven-donor pooled FFP sample was again included as a positive control and demonstrated no significant differences between TP or LQP. 30
Storage of LQP for 28 Days Does Not Alter the Protective Effects on Endothelial Permeability To compare the different groups tested over the time course of the experiment (28 days), all groups and data were plotted on the same graph. Figure 4 depicts that LQP retained its ability to decrease endothelial permeability as effectively as TP0 and TP5 regardless of age (Day 0Y28). Partial data of LQP-0 and LQP-7 from Figures 2 and 3 were included for a summarized presentation of the entire time course. There was a significant decrease in EC permeability at all LQP age points (0, 7, 14, 21, and 28 days) when compared with the TNF>Ystimulated EC monolayer (p G 0.001). LQP Day 0, 7, 14, 21, and 28 inhibited EC permeability as effectively as TP0 and TP5 (Fig. 4). This effectiveness of LQP on endothelial permeability was not altered with storage for up to 28 days.
DISCUSSION As early plasma becomes more widely accepted as a primary resuscitative fluid for hemorrhaging patients, the logistics of storage and delivery of this product have become increasingly important. With approximately 2 million units transfused annually, the most commonly administered form of plasma in the United States is FFP.16 As FFP requires 45 minutes to thaw before administration, it has limited utility in rapidly bleeding patients. For the prehospital setting (ambulances and helicopters) as well as the emergency department, this means plasma must be thawed and refrigerated (TP) to be available for immediate administration. This strategy may result in significant cost and product waste. A potential solution to this logistical issue is the use of LQP. LQP is never frozen or thawed and approved for use for up * 2014 Lippincott Williams & Wilkins
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Figure 2. Liquid plasma blocks permeability of EC monolayers in vitro. hPECs were seeded on inserts for 48 hours, and the EC monolayers were formed. The cell monolayers were stimulated by TNF-> (10 ng/mL) for 20 hours to 24 hours, medium alone as control. LQP-0 was applied to the inserts for 1 hour, equal amount of medium was added to the medium or TNF-> alone groups, and FFP served as positive control. Permeability of FITC-dextran added on the cell monolayer was measured as fluorescence signal at excitation 485 nm and emission 535 nm. Data are expressed as mean T SEM. n = 4 inserts per group. *p G 0.05 compared with medium control, #p G 0.05 compared with TNF-> treatment.
to 26 days. This gives trauma centers ample time to use the blood product while ensuring that transfusion-ready plasma is available for immediate resuscitation of bleeding patients. Preparation and storage of plasma products vary significantly.17 The impact of plasma preparations on hemostatic potential and endothelial permeability has been studied in a limited capacity. Matijevic et al.4 found that LQP had a better capacity to generate thrombin and form clot when compared with TP in an experiment that evaluated hemostatic potential of these two plasma preparations. Even at Day 26, LQP was comparable with TP in its ability to form clot and generate thrombin, with the majority of LQP clotting factors retaining their initial activity level.4 In vitro studies comparing different preparations of plasma report no significant differences on endothelial permeability models. Wataha et al.14 compared spray-dried plasma and TP to determine the impact on permeability and inflammation in in vitro studies. Both spraydried plasma and TP equivalently decreased vascular EC permeability, EC adherens junction breakdown, and endothelial white blood cell binding.14 The in vivo protective effects of thawed FFP administration as compared with lactated Ringer’s solution on lung endothelial permeability induced by hemorrhagic shock and trauma in a rodent model of injury have been
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described.18,19 FFP was found to partially restore the endothelial glycocalyx, which attenuated lung injury, thereby decreasing pulmonary edema.18,19 Our study found that LQP, regardless of age for up to 28 days, decreased TNF->Yinduced endothelial permeability as effectively as TP. There are numerous clinical studies that demonstrate the proposed advantage of early plasma administration. However, no randomized control trials comparing different types of plasma preparations (LQP, spray-dried, freeze-dried, TP, FFP, etc.) exist. A large cohort study inclusive of 84,986 patients compared the mortality rates in patients administered with TP (n = 212,541 U administered) to those transfused LQP (142,169 U administered) and found no overall difference between groups.17 LQP transfusions were divided into categories based on the age of the LQP product (1Y3 days, 4Y14 days, and Q15 days). Interestingly, there was no significant difference in patient outcome (mortality) associated with the duration of storage.17 Another study by Young et al.20 described their institutional experience with two different massive transfusion protocols (MTPs) for traumatically injured patients.20 The study reported that LQP and TP were used interchangeably; one institutional location used LQP as part of the MTP algorithm, and the other incorporated TP into their MTP.20 There were no details regarding differences in patient outcomes in this study, but the authors did comment that within the University Health System Consortium, inclusive of 339 institutions (academic and affiliated), TP was available for early transfusion in only 60% of the institutions.20 This likely reflects the logistical issues associated with TP (thawing time, waste associated with
Figure 3. LQP (Day 7) inhibits endothelial permeability as effectively as TP. hPECs were seeded on inserts for 48 hours to establish EC monolayers. The cell monolayers were stimulated by TNF-> (10 ng/mL) for 20 hours to 24 hours with medium alone as control. LQP-7, the donor-matched TP0, or TP5 were applied to the inserts for 1 hour, and equal amount of medium was added to the medium or TNF-> alone groups. FFP served as positive control. Permeability of FITC-dextran added on the cell monolayer on inserts to the bottom wells was measured as fluorescence signal at excitation 485 nm and emission 535 nm. Data are expressed as mean T SEM. n = 4 inserts per group. *p G 0.05 compared with medium control, #p G 0.05 compared with TNF-> treatment.
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LIMITATIONS
Figure 4. The effects of LQP on endothelial permeability were not altered during storage. hPECs were seeded on inserts for 48 hours, and the EC monolayers were formed. The cell monolayers were stimulated by TNF-> (10 ng/mL) for 20 hours to 24 hours, medium alone as control. LQPs and their donor-matched TPs were applied to the inserts for 1 hour, and equal amount of medium was added to TNF-> alone groups. Permeability of FITC-dextran added on the cell monolayer was measured as fluorescence signal at excitation 485 nm and emission 535 nm. Data are expressed as mean T SEM of fold changes compared with TNF-> treatment. n = 4 inserts per group. Partial data of LQP-0 and LQP-7 from Figures 2 and 3 are included for the summarized presentation purpose of this entire time course of LQPs. The same FFP was included in all time point assays as positive control. *p G 0.05 as comparison of TNF-> + plasma treatment with TNF-> treatment alone. NS, not significant between LQP and TP and throughout all LQP time points.
expiration).20 In the LQP group during a 6-month period, only five LQP units became outdated, while in the TP cohort, 17 U expired.20,21 Given the present study and earlier research demonstrating clinical equivalency of TP and LQP, our institution has replaced TP with LQP on helicopters, decreasing the logistical burden on the blood bank and helicopter crews. In the past, unused TP had to be exchanged from the helicopter on Day 3, but by placing LQP on the helicopters, we have increased our time to plasma exchange to Day 12. During the last 2 years, we have wasted only 1.9% of all plasma placed on our helicopters. While many high-volume centers have instituted TP as part of their resuscitative paradigms, the 5-day expiration date on TP makes it difficult for smaller centers to implement early plasma use, given the perceived potential for waste and associated increased cost. In actual practice, most centers have seen a decrease in waste and cost when a TP program is implemented.20Y22 This waste and its associated cost may be even further reduced with the implementation of an LQP program. Data from our endothelial permeability and coagulation studies in conjunction with the retrospective clinical data of Norda et al.17 demonstrate that LQP, regardless of the duration of storage up to 28 days, is as good as or superior to TP.4,17 LQP improves the immediate and 28-day availability of plasma as a primary resuscitative fluid for bleeding patients. 32
A limitation of this study is the unclear clinical significance of specifically regulating endothelial permeability on outcomes in trauma patients. We hypothesize that limiting EC permeability in injured patients will significantly impact endothelial dysfunction, inflammation, and dysregulated coagulation after hemorrhagic shock and trauma, resulting in both decreased edema and end-organ damage. Further clinical studies are required to determine whether EC permeability can be used as a measure of quality and function of plasma-based therapeutics and whether modulating EC permeability impacts outcomes in trauma. Another limitation of this study was that plasma was collected from only three donors and pooled. Because our study design focused on comparing TP and LQP from the same pooled donor sample, we were unable to compare same-donor TP0 or TP5 to LQP-0. Because the TP samples came from thawing the frozen aliquots of plasma from the same pooled LQP donor bag, we did not have a TP sample that had been previously frozen to generate a TP0 or TP5 sample on LQP-0.
CONCLUSION Plasma is becoming an increasingly accepted primary resuscitative fluid for rapidly bleeding patients; therefore, demand for a plasma product that can be stored long term, in a form readily available for immediate transfusion, is increasing. The current AABB-approved answer to this logistical issue is the use of LQP. Previous work by our group and the current study found that LQP preserves hemostatic potential as effectively as TP.4 This study found that LQP corrects TNF->Yinduced EC permeability after 28 days of storage, similar to TP stored for up to 5 days. Our previous and current data combined with the clinical study by Norda et al. show that LQP is comparable with TP in its ability to correct hemostatic profiles and decrease EC monolayer permeability.4,17 LQP can be considered for use in trauma patients requiring immediate plasma resuscitation. AUTHORSHIP Y.C. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision. A.D. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision. N.M. contributed to the study design, data collection, data analysis, data interpretation, writing, and critical revision. Y.-W.W. contributed to the study design, data collection, data analysis, data interpretation, writing, and critical revision. S.P. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision. C.E.W. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision. T.C.K. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision. J.B.H. contributed to the literature search, study design, data collection, data analysis, data interpretation, writing, and critical revision.
DISCLOSURE The authors declare no conflicts of interest.
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REFERENCES 1. Holcomb JB. Optimal use of blood products in severely injured trauma patients. Hematology Am Soc Hematol Educ Program. 2010;2010:465Y469. 2. Johansson PI, Stensballe J. Effect of haemostatic control resuscitation on mortality in massively bleeding patients: a before and after study. Vox Sang. 2009;96(2):111Y118. 3. Holcomb JB, Wade CE, Michalek JE, Chisholm GB, Zarzabal LA, Schreiber MA, Gonzalez EA, Pomper GJ, Perkins JG, et al. Increased plasma and platelet to red blood cell ratios improves outcome in 466 massively transfused civilian trauma patients. Ann Surg. 2008;248(3):447Y458. 4. Matijevic N, Wang YW, Cotton BA, Hartwell E, Barbeau JM, Wade CE, Holcomb JB. Better hemostatic profiles of never-frozen liquid plasma compared with thawed fresh frozen plasma. J Trauma Acute Care Surg. 2013;74(1):84Y90; discussion 90Y91. 5. Pati S, Matijevic N, Doursout MF, Ko T, Cao Y, Deng X, Kozar RA, Hartwell E, Conyers J, Holcomb JB. Protective effects of fresh frozen plasma on vascular endothelial permeability, coagulation, and resuscitation after hemorrhagic shock are time dependent and diminish between days 0 and 5 after thaw. J Trauma. 2010;69(Suppl 1):S55YS63. 6. Holcomb JB, Fox EE, Wade CE; PROMMTT Study Group. The PRospective Observational Multicenter Major Trauma Transfusion (PROMMTT) study. J Trauma Acute Care Surg. 2013;75(1 Suppl 1):S1YS2. 7. Peiniger S, Nienaber U, Lefering R, Braun M, Wafaisade A, Wutzler S, Borgmann M, Spinella PC, Maegele M; Trauma Registry of the Deutsche Gesellschaft fu¨r Unfallchirurgie. Balanced massive transfusion ratios in multiple injury patients with traumatic brain injury. Crit Care. 2011;15(1):R68. 8. Dente CJ, Shaz BH, Nicholas JM, Harris RS, Wyrzykowski AD, Patel S, Shah A, Vercruysse GA, Feliciano DV, Rozycki GS, et al. Improvements in early mortality and coagulopathy are sustained better in patients with blunt trauma after institution of a massive transfusion protocol in a civilian level I trauma center. J Trauma. 2009;66(6):1616Y1624. 9. del Junco DJ, Holcomb JB, Fox EE, Brasel KJ, Phelan HA, Bulger EM, Schreiber MA, Muskat P, Alarcon LH, Cohen MJ, et al.; PROMMTT Study Group. Resuscitate early with plasma and platelets or balance blood products gradually: findings from the PROMMTT study. J Trauma Acute Care Surg. 2013;75(1 Suppl 1):S24YS30. 10. Brakenridge SC, Phelan HA, Henley SS, Golden RM, Kashner TM, Eastman AE, Sperry JL, Harbrecht BG, Moore EE, et al.; Inflammation and the Host Response to Injury Investigators. Early blood product and crystalloid volume resuscitation: risk association with multiple organ dysfunction after severe blunt traumatic injury. J Trauma. 2011;71(2):299Y305. 11. Radwan ZA, Bai Y, Matijevic N, del Junco DJ, McCarthy JJ, Wade CE, Holcomb JB, Cotton BA. An emergency department thawed plasma protocol for severely injured patients. JAMA Surg. 2013;148(2):170Y175. 12. Kim BD, Zielinski MD, Jenkins DH, Schiller HJ, Berns KS, Zietlow SP. The effects of prehospital plasma on patients with injury: a prehospital plasma resuscitation. J Trauma Acute Care Surg. 2012;73(2 Suppl 1):S49YS53. 13. American Hospital Association. Fast Fact US Hospitals. Available at: http://www.aha.org/research/rc/stat-studies/fast-facts.shtml. Accessed December 1, 2013. 14. Wataha K, Menge T, Deng X, Shah A, Bode A, Holcomb JB, Potter D, Kozar R, Spinella PC, Pati S. Spray-dried plasma and fresh frozen plasma modulate permeability and inflammation in vitro in vascular endothelial cells. Transfusion. 2013;53(Suppl 1):80SY90S. 15. Matijevic N, Wang YW, Kostousov V, Wade CE, Vijayan KV, Holcomb JB. Decline in platelet microparticles contributes to reduced hemostatic potential of stored plasma. Thromb Res. 2011;128(1):35Y41.
16. Arya RC, Wander G, Gupta P. Blood component therapy: which, when and how much. J Anaesthesiol Clin Pharmacol. 2011;27(2):278Y284. 17. Norda R, Andersson TM, Edgren G, Nyren O, Reilly M. The impact of plasma preparations and their storage time on short-term posttransfusion mortality: a population-based study using the Scandinavian Donation and Transfusion database. J Trauma Acute Care Surg. 2012;72(4):954Y960. 18. Kozar RA, Peng Z, Zhang R, Holcomb JB, Pati S, Park P, Ko TC, Paredes A. Plasma restoration of endothelial glycocalyx in a rodent model of hemorrhagic shock. Anesth Analg. 2011;112(6):1289Y1295. 19. Peng Z, Pati S, Potter D, Brown R, Holcomb JB, Grill R, Wataha K, Park PW, Xue H, Kozar RA. Fresh frozen plasma lessens pulmonary endothelial inflammation and hyperpermeability after hemorrhagic shock and is associated with loss of syndecan 1. Shock. 2013;40(3):195Y202. 20. Young PP, Cotton BA, Goodnough LT. Massive transfusion protocols for patients with substantial hemorrhage. Transfus Med Rev. 2011;25(4):293Y303. 21. Goodnough LT, Daniels K, Wong AE, Viele M, Fontaine MF, Butwick AJ. How we treat: transfusion medicine support of obstetric services. Transfusion. 2011;51(12):2540Y2548. 22. Wehrli G, Taylor NE, Haines AL, Brady TW, Mintz PD. Instituting a thawed plasma procedure: it just makes sense and saves cents. Transfusion. 2009;49(12):2625Y2630.
EDITORIAL CRITIQUE The Houston group has added to a developing body of bench and clinical literature suggesting similarities of hemostatic and endothelial-reactive effects of liquid plasma compared to thawed plasma. Given that liquid plasma is far superior to thawed plasma from a logistical standpoint (since it has a 26-day lifespan as opposed to thawed plasma’s 5 days), if similarities in effectiveness can be demonstrated it would be powerful evidence to move toward more widespread use of this underutilized resource. This important work goes a long way toward accomplishing this goal. As with most research, this study also raises as many questions as it answers. Chief among these is the finding that all plasma preparations lowered the ‘‘leakiness’’ of the endothelial cell matrices well below the baseline value of untreated cells. This is interesting since, while I would expect to see all plasma preparations attenuate TNF’s actions, one would not expect them to overcorrect so zealously. The significance of this finding remains unknown. Additionally, the authors’ group is also on the forefront of the role of the endothelial glycocalyx in the response to resuscitation after trauma. I would encourage them to pursue liquid plasma’s actions in this realm. In conclusion, Dr. Dua and her colleagues are to be commended for this important contribution to the advancement of the care of the injured patient. Herb A. Phelan, MD, MSCS Division of Burns/Trauma/Critical Care UT-Southwestern Medical Center Dallas, Texas
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