Antibiotic Concentrations After Massive Transfusion (ACME) Study

Abstract

Background:

Trauma in combat or civilian settings often involves severe hemorrhage and open wounds, which carry a high risk of infection. Current clinical guidelines recommend prophylactic antibiotics for high-risk wounds. Adequate plasma antibiotic concentrations are necessary for tissue penetration, particularly into injured tissue. Blood loss from traumatic hemorrhage may impact plasma antibiotic concentrations. However, the association between blood loss, subsequent blood product transfusion, and antibiotic concentrations remains unclear. We hypothesize that antibiotic concentrations decrease in proportion to the volume of blood transfused, potentially leading to insufficient antibiotic concentrations, placing the injured patient at increased infection risk.

Methods:

We are conducting a prospective, multicenter study that will enroll trauma patients from two large trauma centers: Brooke Army Medical Center and the University of Colorado Hospital. We will enroll participants receiving antibiotics for wound prophylaxis and three or more units of blood products. We will also enroll a control arm comprised of participants receiving the same antibiotics who receive two or fewer units of blood. Blood samples will be collected from participants at predetermined time intervals after antibiotic infusion to assess antibiotic concentrations. Our statistical analysis will focus on the relationship between the volume of blood products administered and antibiotic concentrations. Results will inform the development of antibiotic dosing models for clinicians that adjust for the effects of blood transfusion.

Conclusion:

The goal of this study is to fill a significant gap in trauma care that could potentially lead to optimized antibiotic dosing and improved outcomes for trauma patients.

Introduction

Traumatic injury, whether in military or civilian settings, often leads to hemorrhage, which is a leading cause of potentially preventable death on the battlefield.^1,2 Studies indicate that early blood transfusions can significantly improve survival rates in both settings.^3,4 Additionally, casualties often require extensive volumes of blood products, with 23% of combat casualties receiving any volume of blood products, and nearly 3% reaching supermassive transfusion.^5–7 Casualties receiving transfusions frequently have open wounds, which confer a significant risk of infection. Although these open wounds may not result in immediate death, infections can cause delayed morbidity and mortality. To mitigate this risk and prevent such infections, administration of antibiotics during the early phases of care is recommended by several guidelines, including the Committee on Tactical Combat Casualty Care (CoTCCC) guidelines and Joint Trauma System guidelines.^8,9 However, there is insufficient data guiding clinicians on initial dosing and potential redosing of antibiotics in cases of blood loss and subsequent blood transfusions.

Current antibiotic dosing practices for wound prophylaxis and infections are based on studies in non-hemorrhaging patients in civilian settings. These are frequently based on dosing in healthy volunteers as part of pharmacokinetic analyses required for Food & Drug Administration approval. The limited available data for medication dosing in the cases of volume loss and volume restoration primarily come from studies focused on hemodialysis, plasmapheresis, and exchange transfusions, which have limited applicability to traumatic hemorrhage and are more controlled, preplanned events.¹⁰ Data suggests that antibiotic dosing should be adjusted based on renal and liver function, particularly for elderly patients and those with chronic kidney disease.^11–14 This information is rarely available in real-time during the initial resuscitation period. Unfortunately, data on massive hemorrhage are very limited, despite robust data on dose-reducing practices in the setting of reduced kidney function.

A substantial amount of literature evaluates antimicrobial dosing in cases of kidney replacement therapy (KRT) or hemodialysis.^15,16 Antibiotic underdosing is common in KRT and hemodialysis due to increased volume of distribution and rapid removal, especially for low-protein-binding antibiotics.^17–26 Based on these data, we can speculate that significant blood loss could affect antibiotic dosing in a similar way. However, the physiological changes that occur during KRT or hemodialysis differ from hemorrhage, as these treatments do not rapidly remove fluid from the body in a whole blood volume–loss manner. Moreover, their removal and potential replacement volumes are predictable and known in advance. In the setting of massive hemorrhage, blood replacement is complex, as fluid rapidly redistributes across compartments from the interstitial space into the vasculature to replenish the volume loss.²⁷ The blood loss to blood volume replacement is often subjected to mismatches due to the desire to achieve permissive hypotension and few methods to assess ongoing transfusion requirements. This further complicates physiology when compared to KRT or hemodialysis. Importantly, hemodialysis is often used specifically for drug removal in cases of toxic ingestions.²⁸ Though there is less literature on plasmapheresis, it is important to understand dosing in the setting of plasmapheresis due to its physiological resemblance to blood volume loss.²⁹ The process of plasmapheresis mirrors the effects of massive transfusion, where plasma and other blood components are lost and replaced, though this occurs in a predictable and planned fashion. However, during massive transfusions, all components are turned over, including the proteins that often bind to antibiotics. There are limited studies focused on the effects of plasmapheresis and antibiotics, which highlights the need for hemorrhage-specific data.^30–36 Moreover, alterations in liver function occur as a result of ischemia.

In intraoperative settings for non-trauma patients, changes in antibiotic concentrations may occur due to blood loss and transfusion. Lasko et al. measured the loss of four commonly used antibiotics—vancomycin, piperacillin, ampicillin, and cefazolin—in an ex vivo intraoperative cell salvage (IOCS) and in two patients undergoing liver transplantation. In IOCS, the plasma and waste in blood lost during a surgical procedure are washed, and the red blood cells are reinfused to the patient.³⁷ The use of Cell Saver (Haemonetics, Boston, MA) technology has limited applicability to the trauma setting as it requires specific machines that wash the red blood cells prior to reinfusion and does not mirror a true unprocessed autologous transfusion, which may be used in resource-limited settings. The ex vivo IOCS assessment observed that 2% (SD 1%) of antibiotic inoculated in whole blood was recovered in the IOCS reinfusion bag, whereas 97% (SD 17%) was found in the waste.³⁷ These observations were corroborated by results from two patients undergoing liver transplantation, in which only 2% of antibiotics were recovered.³⁷ Overall, their study concluded that there was a significant loss (95%) of antibiotics in blood when processed through an autologous blood transfusion system and in vivo. This study emphasizes the need for studies on the effect of transfusions on antibiotic and other drug concentrations.

Similarly, Markantonis et al. conducted an investigation in which the gentamicin concentration in serum and tissues was measured with blood loss during colorectal surgery.³⁸ Blood and tissue samples were collected at specific times throughout each procedure. They found that many factors, such as creatinine clearance, fluid administration, and blood loss, contributed to a loss of antibiotic concentration in serum and tissue samples that did not meet the minimum inhibitory concentration required for adequate treatment.³⁸ Swoboda et al. measured the effect of intraoperative blood loss on prophylactic cefazolin and gentamicin serum and tissue concentrations, and similar results were observed.³⁹ A correlation between blood loss and decreased tissue antibiotic concentrations for cefazolin (r=.73, P=.04) and the clearance of gentamicin from the tissues correlated with blood loss (r=.82, P=.01) was observed.³⁹ The results of these studies show how fluid loss and transfusion in the setting of hemorrhage will affect antibiotic concentrations. Additionally, it was concluded that the initial prophylaxis dose of 2mg/kg was insufficient for treatment and recommended a higher dosage of 6mg/kg to account for the antibiotic concentration decrement.

A recently published study discussed the importance of antibiotic prophylaxis during cesarean deliveries.⁴⁰ Fay and Yee point out that the effectiveness of preoperative antibiotic doses can potentially be compromised by significant blood loss during delivery.⁴⁰ They emphasize the importance of redosing antibiotics to ensure proper levels in the bloodstream and tissues.⁴⁰ This is especially critical considering the rising rates of postpartum hemorrhage and the unique pharmacokinetics during pregnancy. Low antibiotic concentrations in plasma can result in insufficient antibiotic distribution in tissues. This is particularly detrimental in cases of devitalized tissues, where significant antibiotic concentrations are needed to achieve effective therapeutic levels.⁴¹ Although there is some literature on the pharmacokinetics of antibiotics in perioperative settings and non-hemorrhaging patients, the pharmacokinetics of antibiotic concentrations and transfusions in trauma patients is understudied. To our knowledge, there is no literature specifically researching the effects of massive transfusions and how they alter the pharmacokinetics in hemorrhagic patients, potentially risking inadequate concentrations for tissue penetration.⁴¹

We hypothesize that the drug plasma concentrations decrease with a direct relationship to the amount of blood transfused during resuscitation after trauma. We seek to understand the relationship between antibiotic concentrations and blood product administration in the setting of hemorrhage. Our study has three proposed aims:

1. Determine the plasma drug concentration at regular intervals during the first 12–18 hours after antibiotic administration.

2. Determine the total volume of blood products (and fluids as covariates) transfused during the first 24 hours after antibiotic administration.

3. Conduct data modeling to explore the correlation between blood transfusions and plasma drug concentrations to inform data-driven dosing models.

Methods

We are conducting a prospective observational study at two major trauma centers. We will conduct regularly scheduled blood draws to assess the pharmacokinetics of antibiotic concentration after transfusions and compare those to controls. Additionally, we will collect routine clinical care data, including interventions, concomitant medications, and outcomes.

Setting

We will enroll patients at two level 1 trauma centers: Brooke Army Medical Center (BAMC) and the University of Colorado Hospital (UCH). BAMC is the Department of Defense’s (DoD) only level 1 trauma center and the largest Military Treatment Facility in the DoD, with nearly 90,000 emergency department (ED) visits annually.⁴² The UCH cares for more than 1,800 trauma cases and is the seventh busiest emergency department in the country, treating over 110,000 patients annually. Both trauma centers frequently use blood products for resuscitation, including the use of whole blood.⁴³ Together, these centers provide a unique opportunity to analyze antibiotic administration and blood product use in diverse patient populations.

Ethics and Data Safety Monitoring

Our study has been approved by the Colorado Multiple Institutional Review Board (COMIRB #23–2559), which serves as the single IRB for this study. BAMC will conduct the study under an institutional agreement for IRB reliance. Our study meets the federal definition and ethical standards for a waiver of informed consent, as it involves no more than minimal risk to subjects, particularly those for whom their condition prohibits the ability to obtain consent safely. We will obtain consent for all potential subjects only when the primary clinical team determines that obtaining consent will not hinder their care. We anticipate some participants may not be able to consent for themselves due to the nature of their injuries. Participants enrolled under a waiver of informed consent will receive a patient information sheet regarding their participation in this study after the fact. All participants will have the right to refuse to participate in this study. Our study has received second-level approval from the Defense Health Agency Office of Human Research Oversight (memo E05020.1a).

Study Procedures

Participants for enrollment in this study will be identified using site-specific trauma alert and activation protocols, along with consultations with the trauma care teams. We will enroll participants who are hospitalized or anticipate hospital admission for acute trauma and receive an antibiotic from our predetermined list (Table 1). Site research staff will identify the participants who meet the inclusion criteria (Table 2). Study team members will collect samples at six designated time intervals after antibiotic administration, striving to complete draws within their designated goal times. Study personnel will extract additional relevant data from the medical records (Table 2).

TABLE 1.

Inclusion and Exclusion Criteria

Inclusion criteria	Exclusion criteria
• Trauma patient • Hospitalized or anticipated hospital admission • Received any dosage of the following antibiotic(s):  – Ampicillin/sulbactam  – Cefazolin  – Cefepime  – Ceftriaxone  – Clindamycin  – Ertapenem  – Levofloxacin  – Metronidazole  – Piperacillin/tazobactam	• Received listed antibiotic(s) within the past 5 half-lives of the drug • <18 years of age • Pregnant • Incarcerated

TABLE 2.

Variables to be Captured

Demographics	• Admission diagnosis • Age • Height/body mass/BMI • Military status • Medical and surgical history • Sex • Social History
Timing	• Time of blood product transfusions • Time of hospital arrival • Time of injury • Time of fluid infusions • Time of prehospital and in-hospital drug administration
Drug concentration	• Immediately post-infusion • 30min post-infusion • 60min post-infusion • 2hr post-infusion • 4hr post-infusion • 12–18hr post-infusion
Scheduled laboratory studies	• Blood gas values • Complete blood counts (hemoglobin, hematocrit, white blood cell counts) • Coagulation studies (PT, INR, PTT, TEG) • Haptoglobin • Lactate • Metabolic studies (electrolytes, blood urea nitrogen, creatinine, liver function studies)
Prehospital medications	• Anticoagulants • Blood products • Electrolytes • IV fluids • Tranexamic acid • Antibiotics
Hospital medications within ±12hr of antibiotic infusion	• Anticoagulants • Blood products • Electrolytes • IV fluids • Tranexamic acid • Antibiotics
Major procedures within 24hr of antibiotic infusion	• Central line placement • Chest needle decompression • Chest tube • Compressive hemorrhage procedures (e.g., liver packing) • Estimated blood loss during each operative procedure • Exploratory laparotomy • Fracture stabilization • Hemorrhage control interventions • Interventional radiology procedures • Intubation • Irrigation and debridement • Thoracotomy • REBOA
Blood products within 12hr of antibiotic infusion	• Cryoprecipitate • Packed red blood cells • Plasma • Platelets • Whole blood
Outcome data	• Discharge status • Hospital days • Intensive care unit days • Time to death • Ventilator days

INR = international normalized ratio; PT = prothrombin time; PTT = partial thromboplastin time, REBOA = resuscitative endovascular balloon occlusion of the aorta; TEG = thromboelastography.

Blood Sampling and Storage

After the antibiotic infusion, blood samples (approximately 1mL) will be collected at six time intervals using standard clinical sample tubes with anticoagulants. The first draw will be collected immediately post-infusion, followed by additional draws at 30 minutes, 60 minutes, 2 hours, and 4 hours. The final draw will be collected between 12 and 18 hours post-infusion. Given the unpredictable nature of trauma care, the first three draws will be allotted a 15-minute draw window, and the final three draws a one-hour draw window. The samples will be stored on ice or in a 4°C refrigerator until centrifugation. The samples will be centrifuged as soon as feasible, after which the plasma will be removed and frozen at −80°C until further analysis. Specimens from both sites will be processed at the United States Army Institute of Surgical Research (USAISR) using a standardized method for storage.

Data Acquisition

Sites will manually collect data from their electronic health record system and local trauma registry. Captured data will then be de-identified and managed using Research Electronic Data Capture (REDCap), a secure, encrypted, HIPAA-compliant server designed for research data management. Additionally, quality assurance will be assessed by trained project managers at each site prior to analysis.

Statistical Analysis

This study will primarily use both descriptive and inferential statistical models to analyze the plasma antibiotic concentrations in relation to blood transfusion status. Our primary analysis will assess the differences in plasma antibiotic concentrations between participants who do and do not receive blood transfusions using a general linear model (repeated measures analysis of variance (ANOVA)). Blood transfusion status will be treated as a between-subjects effect, and time post-infusion will serve as a within-subjects effect. We will compute and report pairwise differences between the transfusion and non-transfusion groups at each time point, making necessary corrections for multiple comparisons.

Our secondary analysis will construct a similar model to compare the plasma antibiotic concentrations among participants who received blood transfusions based on the volumes of blood administered (e.g., massive transfusion, low volume, etc.). We will adjust our model to account for fluctuations in acute changes in kidney function during treatment and include relevant covariates such as the timing of blood transfusion. We will obtain orthogonal polynomial contrasts to examine concentration trends over time, calculate and report pairwise differences between the transfusion volume groups at each time point with the appropriate correction for multiple comparisons, and compare the change in concentration between the groups. Additionally, we will conduct a regression analysis with linear and polynomial terms to determine the relationship between the volume of blood transfused and the amount of antibiotic in the plasma.

Sample Size Estimates

We will enroll participants in a 1:2 ratio, pairing each participant receiving three or more units of blood products with two “control” subjects. Control subjects are defined as participants who receive two or fewer blood products with antibiotics. Partial enrollments (e.g., missed draws, deaths during the enrollment period) will be included in the analysis for available concentrations. We are estimating that we will need 208 participants with all samples measured.

Extraction Methods

Participant blood will be collected, centrifuged, and the plasma (100μL) pipetted into 1.5mL tubes and stored at −80°C. The antibiotics in plasma will be extracted using either of two methods: 1) 1mL of 90% methanol, 100 mM ammonium carbonate (ampicillin, ceftriaxone, ertapenem, piperacillin, clindamycin, cefazolin) or 2) 90% ethanol, 100mM ammonium carbonate (sulbactam, tazobactam, ampicillin, ceftriaxone, ertapenem). Extraction fluid will be added to the frozen samples. The samples will then be vortexed and centrifuged (5000g for 5min). The supernatant will be removed to a new tube and dried by a centripetal dryer. Samples will be stored at −20°C for liquid chromatography with tandem mass spectrometry analysis and run in a batch with standard curves for each analyte (antibiotic). The standards and solvents will come from MilliporeSigma, U.S. Pharmacopeia (USP), European Directorate for the Quality of Medicines & HealthCare, ThermoFisher, and any other qualified suppliers. Standard curves will be generated (linear regression), and slope, intercept, r2, lowest limit of detection (LLOD) and carryover are developed as part of the validation. Linear curves typically fall within the 10nM to 100μM range. Internal standards will be added to the extraction fluid to monitor the entire procedure from extraction to measurement. Internal standards will be chosen as chemicals of similar structure and chemical and physical properties, such that they extract and chromatograph in a manner similar to the analyte in question.

Liquid Chromatography/Tandem Mass Spectrometry Methods (LC-MS/MS)

Dried samples are brought up in 200μL of 10% acetonitrile, 0.1% formic acid and run (10μL) by liquid chromatography (Ultimate 3000, ThermoFisher Scientific, Waltham, MA) on Kinetex C18(2) Polar 2.1×150mm column (Phenomenex, Torrance, CA) at 250μL/min with a increasing acetonitrile/0.1% formic acid gradient for separation of individual antibiotics. Gradient is 12% Mobile Phase B (MPB) for 1.5min, then ramped to 32% for 1min, held for 1min, then ramped to 95% in 4min. The total time for the run is 10min. Tandem Mass Spectroscopy (Quantiva, ThermoFisher, Waltham, MA) is used in the Multiple Ion Monitoring mode for specific parent and daughter analytes. Plasma sample unknowns are compared to linear curves generated by standards for final quantitation μg/L. Standard curves are generated for each antibiotic.

Discussion

The findings from this study will have a significant impact on trauma medicine in both civilian and military settings. In a military setting, combat injuries often involve large volumes of blood loss and subsequent transfusions in field environments. The findings of this study could directly impact combat casualty care by providing the data needed for clinical practice guidelines on dosing and redosing of antibiotics for wound prophylaxis. This will ensure optimal antibiotic dosing for tissue penetration without reaching supratherapeutic levels that can lead to toxic effects. By developing an evidence-based dosing model that accounts for the pharmacokinetic changes caused by massive blood transfusions, civilian providers will be able to dose their patients more accurately and safely. Overall, the results of the ACME study will close a critical and significant knowledge gap by providing the data necessary to revise current antibiotic dosing models in massive transfusions. This will lead to overall better patient outcomes, reduced infections, and set new care standards in civilian and military trauma settings.

Conclusion

Our study aims to address a critical gap in trauma care by assessing the relationship between the pharmacokinetics of antibiotic concentration and transfusions through a multicenter, prospective, observational model. Our findings have the potential to significantly improve patient outcomes through evidence-based dosing guidelines that will ultimately enhance the effectiveness of prophylactic antibiotic therapy in trauma settings.