Page 71 - JSOM Fall 2025

TABLE 2 Variables to be Captured with linear and polynomial terms to determine the relation-
Demographics • Admission diagnosis ship between the volume of blood transfused and the amount
• Age of antibiotic in the plasma.
• Height/body mass/BMI
• Military status Sample Size Estimates
• Medical and surgical history
• Sex We will enroll participants in a 1:2 ratio, pairing each partici-
• Social History pant receiving three or more units of blood products with two
Timing • Time of blood product transfusions “control” subjects. Control subjects are defined as participants
• Time of hospital arrival who receive two or fewer blood products with antibiotics.
• Time of injury Partial enrollments (e.g., missed draws, deaths during the en-
• Time of fluid infusions rollment period) will be included in the analysis for available
• Time of prehospital and in-hospital drug
administration concentrations. We are estimating that we will need 208 par-
Drug concentration • Immediately post-infusion ticipants with all samples measured.
• 30min post-infusion
• 60min post-infusion Extraction Methods
• 2hr post-infusion Participant blood will be collected, centrifuged, and the plasma
• 4hr post-infusion (100μL) pipetted into 1.5mL tubes and stored at –80°C. The
• 12–18hr post-infusion antibiotics in plasma will be extracted using either of two
Scheduled laboratory • Blood gas values methods: 1) 1mL of 90% methanol, 100 mM ammonium car-
studies • Complete blood counts (hemoglobin,
hematocrit, white blood cell counts) bonate (ampicillin, ceftriaxone, ertapenem, piperacillin, clin-
• Coagulation studies (PT, INR, PTT, TEG) damycin, cefazolin) or 2) 90% ethanol, 100mM ammonium
• Haptoglobin carbonate (sulbactam, tazobactam, ampicillin, ceftriaxone,
• Lactate ertapenem). Extraction fluid will be added to the frozen sam-
• Metabolic studies (electrolytes, blood urea
nitrogen, creatinine, liver function studies) ples. The samples will then be vortexed and centrifuged (5000g
Prehospital • Anticoagulants for 5min). The supernatant will be removed to a new tube and
medications • Blood products dried by a centripetal dryer. Samples will be stored at –20°C
• Electrolytes for liquid chromatography with tandem mass spectrometry
• IV fluids analysis and run in a batch with standard curves for each an-
• Tranexamic acid alyte (antibiotic). The standards and solvents will come from
• Antibiotics MilliporeSigma, U.S. Pharmacopeia (USP), European Director-
Hospital medications • Anticoagulants ate for the Quality of Medicines & HealthCare, ThermoFisher,
within ±12hr of • Blood products
antibiotic infusion • Electrolytes and any other qualified suppliers. Standard curves will be gen-
• IV fluids erated (linear regression), and slope, intercept, r2, lowest limit
• Tranexamic acid of detection (LLOD) and carryover are developed as part of
• Antibiotics the validation. Linear curves typically fall within the 10nM
Major procedures • Central line placement to 100µM range. Internal standards will be added to the ex-
within 24hr of • Chest needle decompression traction fluid to monitor the entire procedure from extraction
antibiotic infusion • Chest tube
• Compressive hemorrhage procedures to measurement. Internal standards will be chosen as chemicals
(e.g., liver packing) of similar structure and chemical and physical properties, such
• Estimated blood loss during each that they extract and chromatograph in a manner similar to the
operative procedure analyte in question.
• Exploratory laparotomy
• Fracture stabilization
• Hemorrhage control interventions Liquid Chromatography/Tandem Mass
• Interventional radiology procedures Spectrometry Methods (LC-MS/MS)
• Intubation Dried samples are brought up in 200µL of 10% acetonitrile,
• Irrigation and debridement 0.1% formic acid and run (10µL) by liquid chromatography
• Thoracotomy
• REBOA (Ultimate 3000, ThermoFisher Scientific, Waltham, MA) on
Blood products • Cryoprecipitate Kinetex C18(2) Polar 2.1×150mm column (Phenomenex, Tor-
within 12hr of • Packed red blood cells rance, CA) at 250µL/min with a increasing acetonitrile/0.1%
antibiotic infusion • Plasma formic acid gradient for separation of individual antibiotics.
• Platelets Gradient is 12% Mobile Phase B (MPB) for 1.5min, then
• Whole blood ramped to 32% for 1min, held for 1min, then ramped to 95%
Outcome data • Discharge status in 4min. The total time for the run is 10min. Tandem Mass
• Hospital days
• Intensive care unit days Spectroscopy (Quantiva, ThermoFisher, Waltham, MA) is used
• Time to death in the Multiple Ion Monitoring mode for specific parent and
• Ventilator days daughter analytes. Plasma sample unknowns are compared
INR = international normalized ratio; PT = prothrombin time; PTT = to linear curves generated by standards for final quantitation
partial thromboplastin time, REBOA = resuscitative endovascular bal- µg/L. Standard curves are generated for each antibiotic.
loon occlusion of the aorta; TEG = thromboelastography.
Discussion
differences between the transfusion volume groups at each
time point with the appropriate correction for multiple com- The findings from this study will have a significant impact on
parisons, and compare the change in concentration between trauma medicine in both civilian and military settings. In a
the groups. Additionally, we will conduct a regression analysis military setting, combat injuries often involve large volumes of

Antibiotic Concentrations after Massive Transfusion | 69