Novel use of XSTAT 30 for mitigation of lethal non-compressible torso hemorrhage in swine.

BACKGROUND: Management of Non-Compressible Torso Hemorrhage (NCTH) consists primarily of aortic occlusion which has significant adverse outcomes, including ischemia-reperfusion injury, in prolonged field care paradigms. One promising avenue for treatment is through use of RevMedx XSTAT 30™ (an FDA approved sponge-based dressing utilized for extremity wounds). We hypothesized that XSTAT 30™ would effectively mitigate NCTH during a prolonged pre-hospital period with correctable metabolic and physiologic derangements. METHODS AND
FINDINGS: Twenty-four male swine (53±2kg) were anesthetized, underwent line placement, and splenectomy. Animals then underwent laparoscopic transection of 70% of the left lobe of the liver with hemorrhage for a period of 10min. They were randomized into three groups: No intevention (CON), XSTAT 30™-Free Pellets (FP), and XSTAT 30™-Bagged Pellets (BP). Animals were observed for a pre-hospital period of 180min. At 180min, animals underwent damage control surgery (DCS), balanced blood product resuscitation and removal of pellets followed by an ICU period of 5 hours. Postoperative fluoroscopy was performed to identify remaining pellets or bags. Baseline physiologic and injury characteristics were similar. Survival rates were significantly higher in FP and BP (p<0.01) vs CON. DCS was significantly longer in FP in comparison to BP (p = 0.001). Two animals in the FP group had pellets discovered on fluoroscopy following DCS. There was no significant difference in blood product or pressor requirements between groups. End-ICU lactates trended to baseline in both FP and BP groups.
CONCLUSIONS: While these results are promising, further study will be required to better understand the role for XSTAT in the management of NCTH.

Introduction

Traumatic non-compressible torso hemorrhage (NCTH) is the most common cause of potentially survivable deaths in both civilian and military trauma [1, 2]. Current treatment for junctional and torso hemorrhage consist of balanced blood product resuscitation, resuscitative thoracotomy with aortic cross clamping and placement of endovascular balloon occlusion of the aorta when logistically feasible [3, 4]. However, in austere environments, there may be a decreased availability of blood products. Additionally, increasing transport times limit the usefulness of extended Zone 1 aortic occlusion due to the high risk of ischemia reperfusion injury [5, 6]. Hemostatic gauzes, such as QuikClot Combat Gauze, have been used to manage junctional and extremity hemorrhage, however are not compatible with injury patterns that include abdominal hemorrhage [7].

XSTAT 30™, an FDA-approved sponge based hemostatic dressing that is used for the management of extremity bleeding, has also shown promise in the control of non-compressible junctional hemorrhage [8]. The device is composed of an applicator filled with highly compressed sponges that expand in the presence of a liquid. The individual compressed sponges (pellets) that make up the XSTAT 30™ have radio-opaque markers for identification and have most recently been refined for use in severe uterine hemorrhage where sponges are deployed within a radio-opaque bag [9]. Through these developments, XSTAT 30™ may have a broader application for pre-hospital intra-abdominal hemorrhage control.

The purpose of this study was to determine the efficacy of XSTAT 30™ in pre-hospital management of NCTH in a swine model. We hypothesized the following: H10 XSTAT 30™ would not elicit a survival benefit in an animal model of NCTH compared to Hextend resuscitation alone as evidenced by pre-hospital survival and intra-cavitary shed blood; H20 physiologic derangements incurred following pre-hospital application of XSTAT 30™ would not be amenable to correction by modern damage control therapies as evidence by significant disturbances in metabolic parameters to include but not limited to: serum lactate, pH, base excess and bicarbonate at the end of intensive care observation.

Materials and methods

Oregon Health and Science University’s Institutional Animal Care and Use Committee reviewed and approved this protocol prior to the initiation of the study. Telazol 8mg/kg IM was used for sedation. Inhaled Isoflurane was used for anesthesia. Euthanasia solution 1mL/10lb was used for euthanasia. Experiments were carried out in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care at Oregon Health and Science University, Portland, Oregon. Animals were used in accordance with The Guide for the Care and Use of Laboratory Animals. A priori power analysis was performed which demonstrated an estimated 60% survival difference detected with n = 8 animals.

Twenty-four male Yorkshire swine (53±2kg) from a single source vendor (Oak Hill Genetics Inc, Ewing, IL, USA) were anesthetized with Telazol (Zoetis, Persippany-Troy Hills, NJ, USA) IM injection and maintained with Isoflurane after oro-tracheal intubation for the entirety of the experiment until euthanasia. No animals regained consciousness after initial sedation. Following intubation, the animals were given buprenorphine (IM) injection for intra-operative analgesia. Vascular access was achieved by Seldinger technique and included right external jugular pulmonary artery catheter, left carotid artery catheter, two left external jugular venous catheters, and femoral arterial and venous catheters. Cut-down was performed for right carotid artery access for carotid flow probe monitor placement. Physiologic telemetry was monitored, including the following: EKG, invasive arterial pressures, cardiac output, systemic vascular resistance, EtCO2, SvO2, VO2, tissue oxygenation by near-infrared spectroscopy (StO2), and carotid flow.

The investigators implemented a previously described swine model of NCTH as follows [3, 10]: Laparotomy and devascularization of the spleen (which was left in the anatomical position) were performed to eliminate auto-transfusion and create a standardized degree of soft tissue injury. Laparoscopic ports were placed to include two 12mm ports in the right lower quadrant, one 5mm port in the left lower quadrant and one 12mm port in the left lower quadrant. An intra-abdominal balloon transducer (Cook Quantum TTC Colonic Balloon Dilator 6FR, 5.5cm x 1.6cm) was placed into the left subdiaphragmatic area for intra-abdominal pressure monitoring. The abdomen was then closed with a bespoke incorporated 30mm trocar for XSTAT 30™ deployment at upper midline of the incision and the animal underwent a ten-minute stabilization period. Blood samples were taken following stabilization to confirm no significant physiologic changes occurred.

Following stabilization, the abdomen was insufflated to 15mmHg and laparoscopic liver injury was created in all animals by transecting approximately 70% of the left lateral lobe of the liver. The abdomen was desufflated, all ports were removed with the exception of the XSTAT 30™ trocar, and the skin was reapproximated using staples. Free bleeding was allowed to occur for a period of 10 minutes during which animals were randomized one of three groups: Control–No intervention (CON), XSTAT 30™ with free pellets (FP), XSTAT 30™ with pellets in radiopaque bags (BP). This control was specifically chosen as this model has previously been demonstrated to have lethal effects with no intervention [10]. Randomization schedule was achieved by selecting an unlabeled, sealed envelope that contained one of the three experimental groups and had been prepared by a third-party individual.

All animals underwent infusion of up to 2 boluses of 300mL of Hextend at initial intervention if MAP<50 and SBP<90mmHg. For animals randomized to the FP group, six XSTAT 30™ applicators were deployed through the central 30mm trocar to replicate a penetrating injury while still being able to complete the laparoscopic transection. In the BP group, six XSTAT 30™ devices were deployed within multiple radiopaque bags (33–34 pellets/bag, 3 bags/applicator for a total of 100 pellets/applicator). The total number of pellets administered was the same (600 pellets) regardless of experimental group. The XSTAT 30™ pellets whether in bags or free were deployed per manufacturers guidelines. Animals in all groups were then observed for 180 minutes in a simulated pre-hospital phase.

Following the pre-hospital phase, the animals underwent a standardized damage control surgery (DCS) for primary control of hemorrhage and received up to 3L of whole blood resuscitation to simulate a balanced blood product resuscitation. Upon entering the abdomen, the pellets or bags overlying the liver injury were removed until manual control of the liver could be achieved. Once definitive hemorrhage control was determined by placing atraumatic liver clamps on the transected edge of the liver, the remaining pellets and bags within adjacent areas were collected. Pellets deployed in bags were counted to ensure complete removal and the entire abdomen was inspected for any remaining pellets. Shed blood was collected following removal of pellets. The liver was then packed to control for any additional bleeding. The abdomen was closed with a Bogota bag made from a 1L bag of NS and the animal was observed for an additional 300 minutes of ICU time [11]. During the ICU phase of the experiment, a critical care algorithm was utilized to maintain a normotensive state and to correct any electrolyte abnormalities. If the animal’s SBP<90mmHg, and Hgb≥7g/dL they received fresh frozen plasma (FFP). If the animal’s Hgb<7g/dL they received pRBCs. If the animal’s pressures were unresponsive to two subsequent boluses either FFP or pRBCs and their SVR<80% of baseline, norepinephrine was initiated at 0.02 mcg/kg/min. Insulin and 50mL of 50% Dextrose was administered for potassium≥5.5mmol/L. For glucose<3.0mmol/L, 50mL of 50% Dextrose was administered.

Subsequent DCS, the animals underwent fluoroscopy of the entire abdomen to identify any remaining pellets that were not collected at the time of surgery. If pellets or bags were identified by radiologic imaging, they were noted and retrieved during necropsy.

At the completion of the experiment, animals were euthanized with 1mL/10lb Euthanasia Solution (MWI Animal Health, Boise, ID, USA) and underwent necropsy and gross pathological assessment. Tissue samples were collected from the terminal ileum, kidney, liver, pancreas, lung and heart. These samples were sent for histological analysis by a certified veterinary pathologist.

Data collection and study endpoints

Arterial blood samples were taken pre-surgery (baseline), end of prep and stabilization (T = 0), intervention (T = 10), T = 30, T = 45 and then time intervals of 30 minutes beginning at T = 60 until end of experiment (T = 480) or at the time of euthanasia. Data collected for the H10 primary endpoint included survival at end of pre-hospital and end of study. Secondary endpoints for H10 included indices of cardiovascular and cardiopulmonary function including recorded measurements of cardiac output, intra-abdominal pressure changes, shed blood and remaining pellets within the abdomen. Data collected for the H20 primary endpoint were serum lactate. Secondary endpoints included recorded indices of tissue oxygenation and oxygen consumption, blood gas and chemistry, clotting and coagulation status, and histopathological examination of lung, apex of the heart, kidney, liver, pancreas and bowel for evidence of ischemic injury.

Statistical analysis

Baseline and end pre-hospital data points were compared using ANOVA. End-study data points were compared using Student’s t-test. Categorical data was analyzed with Fischer's exact test. Clinical and laboratory values were measured, and analyzed by ANOVA with post-hoc pairwise comparisons where appropriate (Holm-Sidak). Kaplan-Meier analysis was performed for survival. All statistical analysis was completed using SigmaPlot 12 (SyStat Software Inc.).

Results

Baseline animal characteristics

There were no statistical differences in baseline characteristics including animal weight (53±2kg), spleen weight, and preparation time (Table 1). Baseline lactate was similar amongst groups (p = 0.325). There was no significant difference in pH, hemodynamic parameters, blood counts, chemistries or viscoelastic parameters of coagulation (Table 1).

Table 1

Baseline characteristics for Free Pellets (FP), Bagged Pellets (BP) and no intervention (CON).

Variable	FP (n = 8)	BP (n = 8)	CON (n = 8)	p-value
Weight (kg)	53 ± 2	53 ± 2	53 ± 2	0.935
Total Prep (min)	98 ± 23	93 ± 22	110 ± 29	0.387
Normal Saline (mL)	161 ± 48	148 ± 41	175 ± 56	0.555
Spleen Weight (g)	476.6 ± 120.6	502.4 ± 95.7	498.4 ± 103.5	0.875
Baseline Physiology
Heart Rate (bpm)	102 ± 16	96 ± 12	106 ± 26	0.563
Systolic BP (mmHg)	99 ± 9	94 ± 8	94 ± 9	0.329
MAP (mmHg)	66 ± 3	64 ± 6	67 ± 6	0.376
Diastolic BP (mmHg)	49 ± 4	50 ± 5	53 ± 5	0.373
Central Venous Pressure (mmHg)	6 ± 2	6 ± 2	7 ± 1	0.32
EtCO2	41 ± 1	41 ± 1	41 ± 1	0.943
Systemic Vascular Resistance	830 ± 77	869 ± 146	948 ± 172	0.238
Cardiac Output (L/min)	5.7 ± 0.6	5.5 ± 1.1	5.2 ± 1.1	0.548
Mixed Venous Saturation	62 ± 12	64 ± 13	67 ± 8	0.711
Temperature (°C)	37.8 ± 0.8	37.7 ± 0.5	37.7 ± 0.7	0.859
pO2 (mmHg)	78 ± 20	77 ± 15	79 ± 15	0.976
pCO2 (mmHg)	42.0 ± 3.5	45.3 ± 3.9	42.3 ± 1.8	0.096
Potassium (mmol/L)	4.1 ± 0.3	4.1 ± 0.1	4.1 ± 0.4	0.987
Ionized Calcium (mmol/L)	1.29 ± 0.07	1.35 ± 0.04	1.29 ± 0.06	0.087
pH	7.454 ± 0.036	7.437 ± 0.041	7.435 ± 0.020	0.489
Serum Lactate (mmol/L)	2.0 ± 0.2	2.0 ± 0.7	1.7 ± 0.3	0.325
Hgb (g/dL)	10.3 ± 0.7	10.5 ± 0.9	10.3 ± 0.9	0.845
Leukocytes	20.71 ± 6.32	21.23 ± 5.46	20.97 ± 4.64	0.982
Platelets	319 ± 83	356 ± 113	331 ± 56	0.699
Clotting Time (sec)	718 ± 223	714 ± 238	853 ± 206	0.381
Clot Formation Time (sec)	241 ± 206	252 ± 121	333 ± 190	0.532
α (°)	56 ± 16	53 ± 14	46 ± 17	0.447
Maximum Clot Firmness (mm)	60 ± 5	61 ± 7	59 ± 7	0.789
LI30 (%)	97 ± 2	99 ± 1	99 ± 1	0.078

All values are expressed as mean ± standard deviation.

Injury characteristics

Percent transection of the left lobe of the liver was not significant amongst groups (p = 0.980) and injury duration did not differ (p = 0.270). Intervention duration was significantly different between FP (12±2s) and BP (16±2s), p<0.001 (Table 2).

Table 2

Injury, damage control surgery, and resuscitation characteristics for Free Pellets (FP), Bagged Pellets (BP) and no intervention (CON).

Variable	FP (n = 8)	BP (n = 8)	CON (n = 8)	p-value
Injury/Intervention Characteristics
Injury Duration (sec)	39 ± 8	38 ± 8	45 ± 11	0.270
Transection (%)	69.5 ± 8.4	69.2 ± 9.4	68.7 ± 8.2	0.980
Shed Blood (mL)	1757.2 ± 381.3	1838.0 ± 360.8	2014.9 ± 482.7	0.454
Intervention Duration (sec)	12 ± 2	16 ± 2	-	<0.001
Systolic BP (mmHg)	52 ± 21	36 ± 14	50 ± 17	0.150
MAP (mmHg)	32 ± 12	22 ± 10	32 ± 10	0.109
EtCO2	29 ± 9	21 ± 8	28 ± 7	0.139
Lactate (mmol/L)	3.0 ± 1.1	3.5 ± 0.8	3.0 ± 1.1	0.548
End Pre-Hospital (T = 180)
All animals Hextend (mL)	975 ± 212	1013 ± 223	900 ± 278	0.639
Survivors Hextend (mL)	975 ± 212	1100 ± 155	1050 ± 212	0.498
Damage Control Surgery (DCS)
Manual Control (sec)	215 ± 89	165 ± 43	65 ± 1	0.051
DCS Duration (min)	18 ± 3*	9 ± 2	14 ± 9	0.001
Missed Pellets (animals)	2	0	-
Average Missed Pellets (n)	6.5	-	-
Fresh Whole Blood (mL)	831 ± 175	806 ± 135	823 ± 173	0.958
Intensive Care Phase
Fresh Frozen Plasma (mL)	563 ± 259	417 ± 303	500	0.35
50% Glucose (mL)	88 ± 44	100 ± 32	150	0.156
Insulin/50% Glucose (n)	2 ± 1	2 ± 1	3	0.049
8.4% Sodium Bicarbonate (n)	0*	0*	2	<0.001
Epinephrine (mg)	0.25 ± 0.35*	0.17 ± 0.30*	3.25	<0.001
Norepinephrine (mg)	0	383 ± 938	105.6	0.263
Lactated Ringers (mL)	681 ± 47	670 ± 134	687	0.816

All values are expressed as mean ± standard deviation.

*demonstrates significance in comparison to CON with p <0.05.

Hemodynamic parameters and lactate following injury did not differ among groups. Shed blood was not statistically different among groups (p = 0.454), Table 2.

End pre-hospital (T = 180)

There was no significant difference in all physiologic measures including lactate, mean arterial pressure (MAP), end tidal CO2, potassium, and hemoglobin levels. Intra-abdominal pressures did not differ between groups. Coagulation parameters did not differ among groups. All FP animals, 6/8 BP animals and 2/8 CON animals survived to simulated hospital arrival. In comparison to CON, survival was significantly higher for FP (p = 0.015) at 180 minutes. Over half of the CON animals expired prior to 2 hours, Fig 1.

Fig 1

Kaplan-Meier survival curve at 480 minutes.

Damage control surgery

There was no difference observed in time to manual control of the liver hemorrhage among groups, however, FP manual control (215 ± 89s) was observed to be elevated in comparison to BP (165 ± 43s) and CON (65 ± 1s), however did not achieve statistical significance, p = 0.051 (Table 2). Damage control surgery duration was significantly longer in FP animals (18 ± 3min) in comparison to BP animals (9 ± 2min), p = 0.001(Table 2). Amount of Fresh Whole Blood (FWB) transfused during DCS did not differ, p = 0.958 (Table 2).

Intensive care phase

There was no significant difference detected in the amount of LR or FFP administered among groups (Table 2). Only one CON animal survived to the end of the study while all FP and BP animals that survived simulated pre-hospital, survived to the end of the study. The surviving CON animal required more CON and sodium bicarbonate in comparison to the FP and BP groups during the ICU period (Table 2). There was no significant difference in pressor requirement between FP and BP groups, nor was there a difference in glucose and Insulin/D50. Upon fluoroscopy of the abdomen, there were two animals with documented pellets in the abdomen that were not retrieved at the time of DCS via visual inspection (7 pellets in one animal and 4 in the other animal), Fig 2. All pellets identified by radiologic imaging were successfully identified and removed during necropsy. There were no remaining bags noted during fluoroscopy in the BP group.

Fig 2

X-ray image of retained pellets within the abdomen following damage control surgery.

Arrow denotes one of seven pellets with radiopaque X.

End study (T = 480)

Both FP and BP had significantly improved survival in comparison to CON at 480 minutes (p = 0.003 and p = 0.018, respectively), Fig 1. With exception of survival, statistical comparisons of all three groups was not possible due to CON sample size (n = 1) and therefore comparisons were made only between FP and BP groups. Lactate levels trended towards baseline values in both the FP and BP groups (Fig 3). There was no significant difference in ischemic pathology in the terminal ileum, kidney, pancreas, liver, lung or cardiac apex amongst all groups. However of note, we observed ischemic changes in the terminal ileum as evidence by apoptotic bodies in the presence or absence of mucosal ulcerations (FP 8/8; BP 7/8). For the two animals with pellets in the abdomen for a full eight hours, there was no evidence of adhesions or serosal injury to the bowel.

Fig 3

Serum lactate.

† denotes significance from respective baseline value.

Discussion

This study is the first to demonstrate a novel off-label use for the use of XSTAT 30™ in mitigation of non-compressible abdominal hemorrhage in the setting of simulated prolonged pre-hospital care. Not only did we observe improved survival with deployment of XSTAT 30™ in both the bagged and free pellet formats in comparison to CON, but additionally the physiologic penalty incurred during injury and simulated pre-hospital period was reversible with modern damage control techniques. While a great deal more translational evidence will be required to bring XSTAT 30™ to the clinical armamentarium it does not diminish the fact that the availability of any non-endovascular-based intervention for pre-hospital NCTH management has the potential to revolutionize combat casualty care in Anti-access Area Denial theaters of operation, particularly those where immediate tactical evacuation to Role II or Role III care is not available. The potential for application in austere civilian environments is significant including the stabilization of patients with NCTH arriving to Level IV & V trauma centers that subsequently required transport to higher echelons of care.

One of most interesting features of this study was the indeterminate mechanism of action for XSTAT 30™. We predicted that intracavitary pressures at or near the site of injury would be elevated as previously observed with alternative intracavitary or external adjuncts for NCTH management [12]. Despite an observable space-filling effect of expanded sponges at the wound site, concomitant increases of pressure were not observed. Additionally, confounding was the lack in any decrease in intracavitary shed blood volumes in animals that received XSTAT 30™ in either form as compared to CON alone. We therefore surmised that the mechanism for XSTAT 30™ in the management of NCTH must be highly localized.

It is reasonable to predict that XSTAT 30™ control of hemorrhage at the site of injury is discrete and that it may enhance and localize concentration of blood products due to the rapid absorption of serum during sponge expansion, promoting more effective and robust clot formation. With either mechanistic theory, XSTAT 30™ would likely minimize reperfusion injury incurred with use of current treatments for NCTH, as any ischemia to tissues impacted by XSTAT 30™ use will also be discrete and localized [6, 13]. This imparts a significant benefit over the use of aortic occlusion systems like Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) for the management of NCTH in extended pre-hospital periods as the secondary injury and physiologic derangements that is observed with prolonged occlusion is mitigated [5, 6].

The fundamental limitation of endovascular approaches to pre-hospital NCTH management revolves around the narrow window of therapeutic aortic occlusion time before critical metabolic thresholds for secondary injury are exceeded [14]. We must additionally temper these results in the context that the study was large-animal translational in design and blinding of the investigators to the intervention was not possible. However, this study exemplifies the need for a departure from complete endovascular occlusion for extended pre-hospital transport times in casualties where control of non-compressible bleeding is the principal concern.

Here we used a model of simulated pre-hospital care for 180 minutes. This is a substantial increase in pre-hospital duration from our previous study using REBOA in the same model [15]. Despite the 2 hour increase in pre-hospital care in the absence of balanced blood product resuscitation, the metabolic burden of the animals in the current study at initiation of DCS was half that seen in the animals that underwent REBOA for one third of the total pre-hospital time in our previous work [15]. Modified applications of the REBOA catheter have been developed to extend its usefulness in prolonged field care, specifically intermittent REBOA. Kuckelman et al. have demonstrated usefulness of intermitment REBOA in a similar solid organ injury model with 100% survival though only up to two-hours of simulated pre-hospital observation [16]. While investigators continue to refine endovascular techniques for NCTH the authors want to emphasize that perhaps a non-endovascular approach may be better suited to austere environments.

While endovascular occlusion of the aorta is the prominent technological advance for abdominal NCTH in recent years, alternatives to this approach have also seen significant investment including expanding intracavitary foams [12]. These products address NCTH through space filling and/or acute increases in intracavitary pressure [12]. While this approach has merit in the context of extended field care there are significant complications to include serosal injury to the bowel and adhesive disease with prolonged exposure [12]. Interestingly, in the execution of this study some XSTAT 30™ pellets remained in the abdomen following DCS and initial repair of the injury despite a thorough examination of the abdomen, however at the end of the 8 hour study we did not identify any serosal injury or adhesions. In addition, the risk of retained pellets is low given the radiopaque markers on both free and bagged pellets, which were easily identified by radiographic evidence.

This study generated evidence that free XSTAT 30™ pellets have the potential to increase time for access to the injury site for definitive control. XSTAT 30™ devices with pellet-containing radiopaque bags were created to circumvent the need for individual pellet retrieval and streamline injury site access. While this design change to XSTAT 30™ pellet application addresses potential issues regarding pellet retrieval time, in this study it led to a reduction in overall survival likely attributed to changes in pellet liquid absorption and expansion dynamics though we did not collect or measure indices that would prove or disprove this theory. However, as demonstrated in a postpartum hemorrhage ewe model which used pellets within a bag, there was no mention of saturation or expansion dysfunction [9]. Rodriguez et al also demonstrated 100% survival in all ewes that received this intervention of bagged pellets during postpartum hemorrhage, which differs from our current results [9].

This study is not without limitation. The placement of the bespoke XSTAT 30™ trocar, which was located in the epigastric region medial to where the liver laceration would be created is not necessarily comparable to a penetrating traumatic injury that may have a different trajectory. Future studies will need to be implemented to observe the migration of XSTAT 30™ pellets to the bleeding source if placed in a different quadrant within the abdomen. Secondly, there is no current method of placement for prehospital application. The proposed mechanism of deployment would be similar to the administration of intraabdominal foam or diagnostic peritoneal lavage, in which the provider would perform abdominal cutdown for trocar placement, however, this still needs to be tested. Third, our model employed Hextend as a control, which is no longer the gold standard for pre-hospital resuscitation used in our previous studies of NCTH interventions and may not be appropriate for future studies in the context for prolonged field care applications. Despite the “black box” warning associated with Hextend, the investigators selected Hextend as a pre-hospital positive control to maintain continuity in our current program of research which includes multiple and varying adjuncts for NCTH. In our opinion this selection was also appropriate in order to coincide with the current Tactical Combat Casualty Care guidelines when blood products are not available (worst-case scenario for pre-hospital resuscitation) [1]. While these guidelines incorporate FWB or 1:1:1 resuscitation as the ideal fluid of choice, it may not be available in an austere environment or rural hospital [17].

Conclusion

XSTAT 30™ may be a viable intervention to address NCTH in prolonged pre-hospital care without secondary ischemic complications and metabolic derangements as demonstrated by improved survival in comparison to fluid resuscitation alone. XSTAT 30™ pellets can be identified, either visually or upon radiographic imaging, and removed prior to definitive abdominal closure in the laboratory setting. Furthermore, continued development of XSTAT 30™ within bags is a potential avenue for improvement in pellet retrieval and reduction in total operative time for damage control surgery. Future studies will aim to determine any infectious or harmful effects with prolonged exposure of pellets within the abdomen and will also include further development of XSTAT 30™ within bags along with an intraperitoneal introducer device. Despite these promising results, there is a great deal of continued research and development required to move XSTAT 30™ from the experimental to operational application as a solution set for NCTH and off label use should not be considered until further research is complete.

Depiction of XSTAT 30™ with Free Pellets (FP) and pellets in radiopaque bags (BP).

Subpanel a: Compact and expanded pellets in radiopaque bags. Subpanel b: Pellets in radiopaque bags in-situ. Subpanel c: Compact free pellets. Subpanel d: Expanded free pellets. Subpanel e: Expanded free pellets in-situ.

(TIFF)

Click here for additional data file.

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

12 Sep 2019

PONE-D-19-14707

Novel Use of XSTAT 30™ for Mitigation of Lethal Non-Compressible Torso Hemorrhage in Swine

PLOS ONE

Dear Dr Ross,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Oct 27 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Zsolt J. Balogh, MD, PhD, FRACS

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. At this time, we request that you  please report additional details in your Methods section regarding animal care, as per our editorial guidelines: 1) Please provide the source of the animals, 2) Please explain whether the animals gained consciousness at any point during the experiment or if they were anaesthetised through out the experiment until the time of euthanasia or death. Thank you for your attention to these requests.

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have described an animal experiment where Xstat was used for NCTH.

I have several comments

1. Why was Hextend used as a control group? Its use is not recommended by TCCC, whole blood would have been the correct comparison group.

2. How often does a penetrating wound have a 30 mm opening? This is an important part of the model as access to the peritoneal cavity will likely be a limiting feature in the prolonged field care environment.

3. Are the number of pellets the same between the FP and BP groups?

4. The 30 mm part was placed in the upper abdomen, right over the liver injury, facilitating the pressure action of the pellets. As described by the authos this is a serious limitation of the model. In the prehospital setting the medics will not know if the bleeding site is right below a skin wound, in fact there is little relation of the superficial wound to the actual bleeding organ/vessel.

5. The authors state “Future studies will need to be implemented to observe the migration of XSTAT 30™ pellets to the bleeding source if placed in a different quadrant within the abdomen.” Why do the authors feel the pellets will migrate, unless there is a new capability of these pellets, please remove this statement.

6. Before any suggestion of use of these pellets for NCTH, the authors must perform studies were the pellets are placed remotely from the injury.

7. The authors have studies pellet function in low pressure liver injury model, the easiest to control. Before any recommendation of use, the authors must study the use of pellets in a high pressure iliac artery (lower quadrant) injury model with pellets placed remotely (upper quadrant). In other words create the worst case scenario versus the best.

8. The first sentence of the conclusion and that of the abstract is irresponsible. “XSTAT 30™ may be a viable intervention to address NCTH in prolonged pre-hospital care without secondary ischemic complications and metabolic dyshomeostasis as demonstrated by improved survival in comparison to fluid resuscitation alone.” As stated above the authors have created a model of the best case, pellets placed right over the liver with a “bespoke”30 mm trocar. Before any suggestion that this is ready for battlefield use this must be replicated in studies that more accurately reflect prehospital conditions.

Reviewer #2: Thank you for an excellent piece of work, very relevant and very well described.

I have two questions which I think need further detail in your write up (first two) and two which are non critical, just need clarification although you may have already described these elsewhere (last two).

1. how exactly are the pellets inserted? you describe towards the end that they went in through the port close to the liver injury and that insertion elsewhere in other quadrants is yet to be tested, but a little more detail as to how exactly the operator inserts them (and maybe some pictures) would help the reader understand how this might be feasible in the future with a PHC applicator. Also what do they look like in and out of the bags? Please ignore this if described elsewhere, but maybe reference the description?

2. Regarding the comment “There was no significant difference in ischemic pathology in the terminal ileum, kidney, pancreas, liver, lung or cardiac apex amongst all groups.”.... does this mean there was no damage in all groups or that there was some damage but no difference? If the latter, it would be useful to see a description of any ischaemic changes noted.

3. do retained pellets cause any harm or are they reabsorbed? (you may have already described this in a previous paper).

4. no difference in shed blood: did this include the pigs that died? did they have laparotomy and measurement of shed blood, i would presume so but didn’t see it described.

Thanks

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Dr Samy Sadek

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

8 Jan 2020

Reviewer 1 - Comment 1: Why was Hextend used as a control group? Its use is not recommended by TCCC, whole blood would have been the correct comparison group.

Authors’ Response 1: Hextend was used as a control group for two reasons. The first is that, Hextend represents the “worst case scenario” for resuscitation fluid in the field. If a device is effective in managing NCTH in the presence of a clear fluid resuscitation then it is reasonable to hypothesize that it would be effective in combination with resuscitation fluids like whole blood, plasma, or a multi-function resuscitation fluid. The second reason is that Hextend was used as the control for research conducted in the efficacy of different devices for the management of NCTH including REBOA and the Abdominal Aortic Junctional Tourniquet Torso Plate. The continued used of Hextend in these types of studies allows for comparison with historical studies of device efficacy. Hextend is still a component of the TCCC guidelines for resuscitation in the absence of availability of whole blood. This was addressed in the discussion of the original manuscript discussion. It is also of note that the United States Army Special Operations Command Combat Development Directorate was aware of and concurred with the design of this study.

Comment 2: How often does a penetrating wound have a 30 mm opening? This is an important part of the model as access to the peritoneal cavity will likely be a limiting feature in the prolonged field care environment.

Authors’ Response 2: Entry and exit would sizes are variable dependent on the type of ordinance or fragment that causes the injury. The authors recognize that access to the peritoneal cavity may be variable dependent on injury, however, RevMedx is designing an access device to accompany the XSTAT30 for use in NCTH. XSTAT30 is another tool in the armamentarium for treatment of NCTH pre-hospital and may not necessarily be appropriate for all comers.

Comment 3: Are the number of pellets the same between the FP and BP groups?

Authors’ Response 3: Yes.

Comment 4: The 30 mm part was placed in the upper abdomen, right over the liver injury, facilitating the pressure action of the pellets. As described by the authos this is a serious limitation of the model. In the prehospital setting the medics will not know if the bleeding site is right below a skin wound, in fact there is little relation of the superficial wound to the actual bleeding organ/vessel.

Authors’ Response 4: The authors concur that there is little relation of superficial would to potential intra-peritoneal bleeding or organ injury. The port was not placed directly above the liver injury. The port was placed midline while the injury was left lateral and deep to the port site. In addition, the port could not have facilitated pressure action of the pellets as the pellets in the tested application do not function via application of pressure as determined by placement of pressure transducers near the site of the injury. The authors suspect that the sponges do not require immediate delivery in direct proximity to the injury site as evidenced by their migration toward the site of bleeding from the application site.

Comment 5: The authors state “Future studies will need to be implemented to observe the migration of XSTAT 30™ pellets to the bleeding source if placed in a different quadrant within the abdomen.” Why do the authors feel the pellets will migrate, unless there is a new capability of these pellets, please remove this statement.

Authors’ Response 5: As changes in intra-abdominal pressure were not observed at the injury site in response to sponge expansion, the authors hypothesize that a different mechanism of action is at play. Specifically, the authors observed sponges in contact with the injury site despite the fact that they were not deployed directly above the site of injury. We suspect that as the pellets absorb plasma and expand that they move countercurrent to the source of bleeding. The concentration of red cells and platelets at the site of bleeding through absorption of plasma by the sponges may speed clotting and provide a matrix from which clot could propagate and would be reinforced (analogous to rebar in concrete). The authors intend to study this phenomenon in vivo.

Comment 6: Before any suggestion of use of these pellets for NCTH, the authors must perform studies were the pellets are placed remotely from the injury.

Authors’ Response 6: The authors concur. An assessment of efficacy of the XSTAT sponges when placed further from the injury site would be interesting, particularly in the context of mechanism of action.

Comment 7: The authors have studies pellet function in low pressure liver injury model, the easiest to control. Before any recommendation of use, the authors must study the use of pellets in a high pressure iliac artery (lower quadrant) injury model with pellets placed remotely (upper quadrant). In other words create the worst case scenario versus the best.

Authors’ Response 7: The authors concur that an exploration of XSTAT use in a named arterial model of NCTH would be beneficial, both to understanding its range of efficacy and to better understanding its mechanism of action and the potential for sponge migration to the source of bleeding. We would however comment that the liver injury model employed in this experiment is both venous and arterial in nature. The left lateral lobe transection model described results in the transection of two significant arterioles that have been measured at up to 3mm in diameter and result in significant arterial bleeding. We

Comment 8: The first sentence of the conclusion and that of the abstract is irresponsible. “XSTAT 30™ may be a viable intervention to address NCTH in prolonged pre-hospital care without secondary ischemic complications and metabolic dyshomeostasis as demonstrated by improved survival in comparison to fluid resuscitation alone.” As stated above the authors have created a model of the best case, pellets placed right over the liver with a “bespoke”30 mm trocar. Before any suggestion that this is ready for battlefield use this must be replicated in studies that more accurately reflect prehospital conditions.

Authors’ Response 8: The authors opine that the statement is accurate as written – the operating word being “may”. The principal investigator for the authors’ laboratory has investigated numerous interventions for NCTH and hemorrhage-induced cardiac arrest including REBOA, SAAP, AAJT and XSTAT and in the context of those investigations is confident in the interpretation of the results of this study and the opinions that XSTAT may be a viable intervention for NCTH. However, the authors do not want readers to overestimate the device’s readiness for battlefield application, nor would we condone or suggest its use off-label by deployed medics therefore we have expanded the paragraph appropriately to include caution.

Additionally, the authors would argue that this model reflects the “best case”. The experiment was designed to limit confounding variables in application method and timing. While the trocar was bespoke, it was not placed directly over the site of the injury. There was in fact a measurable distance between the midline and site of injury. In addition, the injury was not in plane with the direction of application. The transection portion of the liver and site of bleeding was transverse to the application of sponges from the XSTAT. In addition, the transection portion of the left lobe remained in anatomical alignment with the rest of the liver, limiting direct access of sponges to the site of bleeding. The authors concur that further study is required and as such, studies are ongoing.

Reviewer 2 - Comment 1: how exactly are the pellets inserted? you describe towards the end that they went in through the port close to the liver injury and that insertion elsewhere in other quadrants is yet to be tested, but a little more detail as to how exactly the operator inserts them (and maybe some pictures) would help the reader understand how this might be feasible in the future with a PHC applicator. Also what do they look like in and out of the bags? Please ignore this if described elsewhere, but maybe reference the description?

Authors’ Response 1: XSTAT sponges were deployed per manufacturer’s instruction through a 30mm trocar placed at the midline and lateral to the site of liver injury. The methods have been revised to better clarify mode of insertion and deployment. We have added a supplemental figure that shows the deployed pellets in free and bagged forms in situ.

Comment 2: Regarding the comment “There was no significant difference in ischemic pathology in the terminal ileum, kidney, pancreas, liver, lung or cardiac apex amongst all groups.”.... does this mean there was no damage in all groups or that there was some damage but no difference? If the latter, it would be useful to see a description of any ischaemic changes noted.

Authors’ Response 2: As only 1 Hextend animal survived to end of study we were unable to make comparisons to the treated groups. In the case of the treated groups, ischemia injury was of note in the terminal ileum and has been better described in the revisions. The authors are unclear as to the source of the ischemia as SHAM controls were not included in the experimental design.

Comment 3: do retained pellets cause any harm or are they reabsorbed? (you may have already described this in a previous paper).

Authors’ Response 3: At this time there is a lack of data to answer the reviewer’s question. The sponges have been produced in a bioabsorbable form but have not been tested. Investigation into the impact of pellet retention is ongoing.

9 Apr 2020

PONE-D-19-14707R1

Novel Use of XSTAT 30™ for Mitigation of Lethal Non-Compressible Torso Hemorrhage in Swine

PLOS ONE

Dear Dr Ross,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by May 24 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Zsolt J. Balogh, MD, PhD, FRACS

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have responded. However they argue that this intervention is revolutionary. see the sentence in the discussion. " The availability of a non-endovascular-based intervention for pre-hospital NCTH management has the potential to revolutionize combat casualty care in Anti-access Area Denial theaters of operation, particularly those where immediate tactical evacuation to Role II or Role III care is not available." This reviewer is extremely familiar with swine anatomy and i have personally cared for thousands of casualties in the prehospital and hospital combat casualty care area. This approach has promise. However the authors must temper their assessment of its potential usefulness, until further work is done.

They need to articulate in the abstract and the discussion the many and significant limitations of this approach. The experiment is sound and I do not have any issues with the data driven conclusions. There is promise here. However I do have significant issue with the tenor of this paper as currently written. The OHSU trauma group, led by Dr Schreiber has significant experience in hemorrhage control studies. I would suggest including in the group a physician that has actually taken care of trauma patients, at the faculty level, and preferably who has deployed to the combat setting. They have recently presented swine work with 3 hour survival with a competing approach, and have presented those data in a very balanced fashion. Until this approach is evaluated in an arterial injury and with the 30 mm bespoke trocar placed as far away as possible from the injury, this can not in any way be recommended. The problem with this is that the pellets are currently cleared for an extremely limited intervention. Medics could read this paper and deploy them tomorrow. The bag, according to these data is not necessary. I consider even the suggestion that this is an intervention to be considered is irresponsible. Additionally, placing a 30 mm trocar into the abdomen of a trauma patient is an advanced surgical procedure. It will require surgical level anesthesia and intubation.

The authors compare the value of their approach to REBOA, using very old references. (ref 15), if they are going to do this type of comparison, they need to include modern references that describe partial or intermittent occlusion. I would suggest they delete most of this discussion as they do not have comparative data.

The authors need to add to the limitations that they were not blinded to the intervention and thus the OR and ICU care could be biased.

Obviously I strongly agree with the last sentence of the paper and feel it should be replicated in the abstract... there is a great deal of continued research and development required to move XSTAT 30™ from the experimental to operational application as a solution set for NCTH and off label use should not be considered until further research is complete.

The senior author has great experience in the lab. this is a well done study with interesting results. Please just review the paper, temper the discussion and in no way encourage any use until more demanding studies are completed.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

16 Jun 2020

We have revised the manuscript along the overarching theme of concerns presented by the reviewer and therefore did not do a point by point response. We would also like to thank the reviewer for their continued thoughtful comments and questions.

22 Jul 2020

PONE-D-19-14707R2

Novel Use of XSTAT 30™ for Mitigation of Lethal Non-Compressible Torso Hemorrhage in Swine

PLOS ONE

Dear Dr. Ross,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 05 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Zsolt J. Balogh, MD, PhD, FRACS

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: the authors have chosen to not highlight their responses, which is unusual.

Since they choose to continue to compare their approach to REBOA and cite animal studies that were published in 2014, it is appropriate to suggest they read and cite more recent data which directly address the issues they raise, and safely extend the survival out to 240 minutes. One of them is from their own Division.

Zilberman-Rudenko J, Behrens B, McCully B, et al. Use of Bilobed Partial Resuscitative Endovascular Balloon Occlusion of the Aorta (pREBOA) is Logistically Superior in Prolonged Management of a Highly Lethal Aortic Injury. J Trauma Acute Care Surg. 2020;10.1097

Kuckelman JP, Barron M, Moe D, et al. Extending the golden hour for Zone 1 resuscitative endovascular balloon occlusion of the aorta: Improved survival and reperfusion injury with intermittent versus continuous resuscitative endovascular balloon occlusion of the aorta of the aorta in a porcine severe truncal hemorrhage model. J Trauma Acute Care Surg. 2018;85(2):318-326.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

27 Aug 2020

Reviewer Comment 1 - the authors have chosen to not highlight their responses, which is unusual.

Authors’ Response 1 – the authors submitted a set of revisions with “track changes” enabled.

Reviewer Comment 2 - Since they choose to continue to compare their approach to REBOA and cite animal studies that were published in 2014, it is appropriate to suggest they read and cite more recent data which directly address the issues they raise, and safely extend the survival out to 240 minutes. One of them is from their own Division.

Zilberman-Rudenko J, Behrens B, McCully B, et al. Use of Bilobed Partial Resuscitative Endovascular Balloon Occlusion of the Aorta (pREBOA) is Logistically Superior in Prolonged Management of a Highly Lethal Aortic Injury. J Trauma Acute Care Surg. 2020;10.1097

Kuckelman JP, Barron M, Moe D, et al. Extending the golden hour for Zone 1 resuscitative endovascular balloon occlusion of the aorta: Improved survival and reperfusion injury with intermittent versus continuous resuscitative endovascular balloon occlusion of the aorta of the aorta in a porcine severe truncal hemorrhage model. J Trauma Acute Care Surg. 2018;85(2):318-326.

Authors’ Response 2 – The reviewer is correct in that the authors compare intra-abdominal XSTAT use to REBOA. However, partial REBOA, EVAC and intermittent REBOA are in the early stages of development and are not practically applicable to the first responder or medic at this time and furthermore do not necessarily add context to the discussion of XSTAT as described in this study.

The authors are aware of the Zilberman-Rudenko publication. The authors reject the reference as the experimental design is seriously flawed. An aortic punch model of hemorrhage is irrelevant regarding REBOA as it is a well-established clinical contraindication for the use of REBOA. The authors have discussed this issue with various other clinical and research thought-leaders in trauma and are confident in this choice.

Authors’ Response 3 – We have added to the discussion to include mention of alternative implementations of REBOA including a more recent Kuckelman reference that uses a similar solid organ injury model.

16 Oct 2020

PONE-D-19-14707R3

Novel Use of XSTAT 30™ for Mitigation of Lethal Non-Compressible Torso Hemorrhage in Swine

PLOS ONE

Dear Dr. Ross,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The paper clearly has the potential to contribute to the field, and the reviewers have laid out a clear path to strengthen the manuscript and bring a perspective to the study and the broader field that could be quite important. I really encourage you to look more holistically at the work and current approaches and carefully consider the reviewers' suggestions. They are supportive, and you need to be responsive to the suggestions.

==============================

Please submit your revised manuscript by Nov 30 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Erin Lavik

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have persisted in comparing their approach to reboa, yet have provided inadequate balance in their presentation. This is unfortunate, as comparing their experimental data to reboa is not needed in this precclinical paper. Lastly, when pacing a reboa, either in the ED or prehospital, it is unknown if an aortic injury is present.

Reviewer #3: Thank you for the opportunity to review this work by Bonanno and colleagues on a novel application of hemostatic pellets in an uncontrolled torso hemorrhage swine model. The authors have conducted a rigorous evaluation of this novel approach and have some very interesting results. I have a few comments and questions for the authors that I hope they will address.

1) Although I think I understand the meaning of the term "dyshomeostasis" I am not certain this is a very common term and would suggest that it is not specific enough to be considered an endpoint for your 2nd hypothesis. I would suggest using a different term to convey your meaning.

2) Similarly, I would suggest that your 2nd hypothesis needs to be more clear. What specific markers of physiologic derangement do you consider clinically meaningful here? Please revise the terminology in your hypothesis.

3) I would argue that for each hypothesis you should have a primary endpoint; so on p7, lines 148-152, I would identify one or a couple parameters as your primary endpoint for your 2nd hypothesis. Then, for secondary outcomes, you should be more specific about elements like "indices of cardiovascular and cardiopulmonary function." For example, did you measure cardiac output in some manner? Or vasopressor use? Or ventilator settings?

4) In your conclusions, I disagree with your statement that "XSTAT 30 pellets can be easily identified and removed prior to definitive abdominal closure...." In my experience with these pellets, once they get saturated with blood, they are actually challenging to find, consistent with your finding that pellets were left behind in 2 animals. You also did not study definitive closure (I would not consider necropsy the same as definitive closure); so please revise this comment accordingly.

Minor issues:

1) I would suggest mentioning the concept of intra-abdominal foam in the introduction

2) The background of your abstract is a bit awkward. For example, "co-morbidities" should probably be better described as "adverse outcomes" related to prolonged ischemia and subsequent reperfusion.

and then detail your specific endpoints in your methods section.

3) Can you provide more detail on your randomization approach?

4) Methods, p.4. The protocol number is unnecessary. Lines 83-91 can be eliminated by referencing your prior work.

5) Please specify the total (or approximate total) of pellets injected in the FP animals and then list a total number of pellets per animal in FP and BP in either the narrative or one of your tables.

6) Results, p.9. I would not consider a 4 second difference clinically meaningful. Consider measuring this in minutes which would show no difference.

7) Results, p.11. Can you indicate the number of animals and the group assignments for the TI ischemic changes? Was this due to hypoperfusion?

8) Figure 3. Legend. Do you mean "respective"? Did you also compare FP and BP at each timepoint?

Thank you again for the opportunity to review this interesting study.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: Yes: Jeremy W. Cannon, MD, SM

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

20 Oct 2020

Author Responses to Reviewers’ Comment and Questions

Reviewer #1: The authors have persisted in comparing their approach to reboa, yet have provided inadequate balance in their presentation. This is unfortunate, as comparing their experimental data to reboa is not needed in this precclinical paper. Lastly, when pacing a reboa, either in the ED or prehospital, it is unknown if an aortic injury is present.

Authors’ Response: The authors continue to discuss the points raised by the reviewer at great length and have enjoyed the collegial academic debate. It is possible, at this stage, that an academic impasse has been reached. There is an endovascular approach to the management of NCTH and there are approaches that are not endovascular; XSTAT & Foam. While foam technology is a better analogue for comparison, ResQ foam is no longer considered a viable option by the US DoD and USASOC and therefore not an appropriate technological context for XSTAT. The authors would suggest that REBOA is an important “contrast” as opposed to a comparison with XSTAT. Known or suspected aortic injury remains a critical contraindication to placement of a REBOA catheter and in the authors’ opinions exemplifies why REBOA is an important technology that provides contrast to the XSTAT for management of NCTH. We would again like to thank Reviewer 1 for his/her critical input and response to our work.

Reviewer #3: Thank you for the opportunity to review this work by Bonanno and colleagues on a novel application of hemostatic pellets in an uncontrolled torso hemorrhage swine model. The authors have conducted a rigorous evaluation of this novel approach and have some very interesting results. I have a few comments and questions for the authors that I hope they will address.

Authors’ Response: The authors would like to thank Dr. Cannon for his thorough review of their manuscript and have addressed his comments in the following:

RQ1: Although I think I understand the meaning of the term "dyshomeostasis" I am not certain this is a very common term and would suggest that it is not specific enough to be considered an endpoint for your 2nd hypothesis. I would suggest using a different term to convey your meaning.

AR1: The authors have changed the wording of the abstract and the manuscript to appeal to a broader readership including the replacement of the word “dyshomeostasis” with “derangement(s)”. Additionally, the authors recognize that the second hypothesis was incompletely expressed and have revised it significantly to reflect specific metabolic parameters as evidence of the null hypothesis.

RQ2: Similarly, I would suggest that your 2nd hypothesis needs to be more clear. What specific markers of physiologic derangement do you consider clinically meaningful here? Please revise the terminology in your hypothesis.

AR2: The authors concur and have revised accordingly (see above).

RQ3: I would argue that for each hypothesis you should have a primary endpoint; so on p7, lines 148-152, I would identify one or a couple parameters as your primary endpoint for your 2nd hypothesis. Then, for secondary outcomes, you should be more specific about elements like "indices of cardiovascular and cardiopulmonary function." For example, did you measure cardiac output in some manner? Or vasopressor use? Or ventilator settings?

AR3: The authors concur with the recommendation for organization and clarity of endpoints for each hypothesis and have revised accordingly. All endpoints (primary and secondary for each hypothesis) were recorded and analyzed and have been indicated as such in the specified paragraph.

RQ4: In your conclusions, I disagree with your statement that "XSTAT 30 pellets can be easily identified and removed prior to definitive abdominal closure...." In my experience with these pellets, once they get saturated with blood, they are actually challenging to find, consistent with your finding that pellets were left behind in 2 animals. You also did not study definitive closure (I would not consider necropsy the same as definitive closure); so please revise this comment accordingly.

AR4: The authors recognize that there is a difference between clinical application and controlled research regarding the performance of medical devices. Reasonably, the authors can only comment on direct observations from the model and this is what the original text indicated. However, the authors concur that the conclusions do not necessarily reflect translational research outcomes and encroach upon the clinical experience. In light of that recognition, the authors have significantly revised the conclusions to reflect opinions based on the research setting.

Minor issues:

RC1: I would suggest mentioning the concept of intra-abdominal foam in the introduction

At the time of the initial draft of this manuscript the authors considered including discussion of the prominent intra-abdominal foam device in the background section of the manuscript. As our laboratory was directly involved in USASOC validation testing of ResQ foam devices prior to the experimentation with XSTAT we felt that the discussion of intra-abdominal foam was not relevant to this manuscript. At that time, SOCOM had expressed strong disinterest in the foam device and an intent to discontinue support for its development. As it would not be pursued by the US DoD for further use in military casualty care we thought it irrelevant as a contextual background for XSTAT. Since the submission of this manuscript, the US DoD has invested heavily in foam technology with a new industry partner. Unfortunately, at this time we are unable to discuss that work as it is proprietary to the industry partner and publication of foam translational data has not occurred in the public space. Future publications regarding XSTAT for NCTH will certainly discuss alternative NCTH technologies as circumstances allow.

RC2: The background of your abstract is a bit awkward. For example, "co-morbidities" should probably be better described as "adverse outcomes" related to prolonged ischemia and subsequent reperfusion.

and then detail your specific endpoints in your methods section.

AR2: The authors concur and have revised the abstract accordingly. The second partial sentence in this comment is unclear and therefore has not been addressed.

RC3: Can you provide more detail on your randomization approach?

AR3: The manuscript has been revised to include randomization method.

RC4: Methods, p.4. The protocol number is unnecessary. Lines 83-91 can be eliminated by referencing your prior work.

AR4: The authors have removed the protocol number as suggested. After some deliberation, the authors chose to leave the methods section referenced by the reviewer in the manuscript for completeness as they are critical to the reader understanding how physiologic parameters presented in the results were recorded.

RC5: Please specify the total (or approximate total) of pellets injected in the FP animals and then list a total number of pellets per animal in FP and BP in either the narrative or one of your tables.

AR5: This is an excellent catch by the reviewer. The authors have clarified pellet number total administration in the text of the methods.

RC6: Results, p.9. I would not consider a 4 second difference clinically meaningful. Consider measuring this in minutes which would show no difference.

AR6: The authors recognize and agree that a four second difference is not meaningful at the level of definitive care. However, four seconds is highly relevant to a medic providing “care under fire”. This parameter was discussed directly with USASOC CDD during the design of the study and therefore has been retained as written.

RC7: Results, p.11. Can you indicate the number of animals and the group assignments for the TI ischemic changes? Was this due to hypoperfusion?

AR7: These data were indicated in the manuscript and are found on P12 line 233 of the revised manuscript. The authors did not have the appropriate data to speculate the source of the ischemic changes.

RC8: Figure 3. Legend. Do you mean "respective"? Did you also compare FP and BP at each timepoint?

AR8: The authors have updated the Figure 3. Legend appropriately. Yes, the authors compared FP and BP at each time point though no significant differences were detected between groups.

23 Oct 2020

Novel Use of XSTAT 30™ for Mitigation of Lethal Non-Compressible Torso Hemorrhage in Swine

PONE-D-19-14707R4

Dear Dr. Ross,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Erin Lavik

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

6 Nov 2020

PONE-D-19-14707R4

Novel Use of XSTAT 30 for Mitigation of Lethal Non-Compressible Torso Hemorrhage in Swine

Dear Dr. Ross:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Erin Lavik

Academic Editor

PLOS ONE