1. Transfusion and urinary markers of systemic oxidative injury in trauma patients Sam Rayner, MD; Watkins Laboratory

2. Background -Transfusion Related Acute Lung Injury (TRALI) Criteria for TRALI No ALI immediately before transfusion Onset of signs/symptoms of ALI during or within 6 hours after the end of transfusion of one or more plasma-containing blood products No temporal relationship to alternative risk factor for ALI* Epidemiology Leading cause of transfusion-related mortality with a rate of 1/1333 to 1/5000 per unit transfused 8% of transfused patients in ICU in 2007 study Prognosis Varying severity Varying reports on mortality (5-35%)

3. Background - TRALI Pathogenesis PMN as Key mediator Models of TRALI “Two-Event” Model Threshold Model Potential Precipitants Antibody mediated Non-antibody mediated Recipient-related Factors Non-Immune Transfusion- Related Factors Sachs UJ. Recent insights into the mechanism of transfusion-related acute lung injury. Current opinion in hematology. 2011;18(6):436-42.

4. Hypothesis and Study Aims Hypothesis: TRALI is likely driven by transfusion- related activation of neutrophils acting as a “second hit” in critically ill patients PMN activation may be reflected in elevations in systemic markers of oxidative stress Platelet activation may play a role in TRALI Primary aim - Examine transfusion and urinary markers of oxidative stress and platelet activation Secondary aim – Examine relation of rates of ALI to urinary levels of markers of oxidative stress and platelet activation

5. Methods - Age of Transfused blood and Lung injury After transfusion Study (ATLAS study) Ongoing clinical trial of HMC trauma patients Primary goal: role of blood product storage time and cell-derived microparticles in development of trauma-associated ALI Secondary goal: sampling blood/urine from trauma patients at defined time-points for ongoing and future analysis in the context of transfusion and lung injury

6. Methods - ATLAS Study Mix of transfused/non-transfused Trauma patients in ICU at HMC meeting criteria Sampling (Baseline, Early, Late) Urine < 12 hours from injury (prior to transfusion if transfused), at 24-36 hours, and at 72 hours Biomarkers 8-hydroxy-2-deoxyguanosine (8-OHdG) 11-dehydro-Thromboxane-B2

7. Methods Urine Collection Centrifuge @300g x 15’ Supernatant -> -80°C Thawed x1 to aliquot Creatinine Measurement Allows standardization for concentration Biomarkers reported in unit/mg Cr Measured via Colorimetric Reaction

8. Methods Competitive ELISA: 8-hydroxy-2- deoxyguanosine / Thromboxane Samples diluted 1:100-1:1000 fold Run in duplicate according to manufacturers instructions (see graphic)

9. Results - Cohort Demographics Sample Size39 Age (years)46 (19) Male29 (74%) Caucasian Race 38 (97%) Death2 (5%) ALI9 (23%)

10. Results - General Transfusion Data 54%

11. Results: Cr and 8-OHdG ELISA Cr assay consistent, values wnl (50-300 mg/dl) 8-OHdG results No difference in baseline values between transf/non- transf Trend towards high values at 24-36 hours in transfused group (not statistically significant) 8-OHdG - issues Control values in literature: 5-15 ng/mg Cr with mass- spec and 10-40s ng/mg Cr with EIA Our control values, however, averaged 69.4 ng/mg Cr, Vendor’s averaged 138 ng/mg creatinine

12. Results - Distribution of 8-OHdG Values

14. Results – Changes in 8OH-dG from baseline to early

15. Results – Changes in 8OH-dG from baseline to late

16. Conclusions Our chosen assay did not detect significant change in 8OH-dG levels with transfusion Trend towards higher levels at 24-36 hours in transfused group Assay likely limited by antibody non-specificity, which could mask difference Currently, disease severity not taken into account

17. Future Directions LC/MS ELISA More samples with 8-OHdG Kit vs making Elisa vs LC/MS only Further statistical analysis – adjust for disease severity TxB2 assay ongoing Blood component analysis of transfused blood Urinary markers of neutrophil activation Urine biomarkers in “healthy” patients after transfusion

18. Acknowledgements Puget Sound Blood Center UW Internal Medicine / School of Medicine Tim Watkins Edward Tran, Emily VandenEkart Xiaoyun Fu Alice, Thomas

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