1. Plasma free hemoglobin and perfusion – some things to consider Mark Yazer, MD Associate Professor of Pathology, University of Pittsburgh The Institute for Transfusion Medicine

2. Disclosure I have no financial or corporate affiliation with the maker of the Hemobag, Global Blood Resources

3. Encapsulated Encapsulated hemoglobin is good Hb in RBCs is a miracle of nature Perfectly suited to do its main job of gas exchange

4. Normal RBC senescence Normal RBC senescence is good Goodbye world!

5. Freehemoglobin Free hemoglobin is bad

8. NO is not N 2 O

9. Oh NO? Oh yes NO

10. RBCs and Hb are involved in NO transport: Oxygenated RBCs permeable to NO NO 2,3

11. RBCs and Hb are involved in NO transport: Deoxygenated RBCs less permeable to NO NO 2,3

12. Free Hb scavenges NO NO

13. PFHb increases during storage Donadee C et al. Circulation in press 0.13 g/dl 0.21 g/unit

14. PFHb increases during storage Donadee C et al. Circulation in press

15. Free Hb scavenges NO Donadee C et al. Circulation in press Transient increase in rat MAP when NO scavenged Clinical significance? “Whether a transient inhibition in NO signaling is sufficient to increase the risk of multiorgan dysfunction or hemostatic activation in at-risk populations of patients remains to be determined” Minutes

16. How much PFHb is a problem? Vermeulen Windsant IC et al. Kindey Int 2010:913 35 on-pump aortic aneurysm repair patients Post-operative PFHb cutoff value of 10 μmol/ l (0.02 g/dl) had 79% sens, 69% spec for acute kidney injury (AKI) 0.01 g/dl 0.02 g/dl

17. O’Leary MF et al. Transfusion 2011:955 Hemolysis after blood bank cell washing COBE model 2

18. O’Leary MF et al. Transfusion 2011:955 Hemolysis after blood bank cell washing COBE model 1 COBE model 2 Fresenius Free Hb (mg/dl) 50 100 >200 “Our data support a decrease in the expiration time of washed units to 6 hours or less.” NO outcome data presented!! 0.13 g 0.17 g

19. Hemolysis after routine blood bank manipulations Harm S et al. submitted ? COBE Model 1

20. Significant amount of PFHb in unit of washed RBCs Harm S et al. submitted ? COBE Model 1 0.15 g 0.5 g “The significance of these findings, in particular any morbidity from the potentially increased plasma free Hb burden from irradiated and potentially from washed RBCs, needs to be evaluated in clinical studies.”

21. Hemolysis after cell salvage washing Szpisjak DF et al. Anesth Analg 2000:40

23. Hemolysis after post-operative cell salvage Ley JT et al. Transfusion in press Study to compare the extent of RBC membrane damage in 2 post-op cell salvage devices Forty patients undergoing first time, elective TKA were enrolled  Unwashed: 20 had post-op cell salvage with a “flip and drip” device (Suretrans, Davol Inc.)  Washed: 20 had post-op cell salvage with OrthoPAT (Haemonetics) device Samples of the blood to be reinfused were sampled PFHb and mechanical fragility index calculated

24. What is sublethal injury?

25. MFI measures RBC mechanical stress tolerance

26. Mechanical Fragility Index calculation

27. Demographics of patients in study Ley JT et al. Transfusion in press

28. PFHb not lower in washed salvaged blood Ley JT et al. Transfusion in press 30 g

29. Hemolysis after post-operative cell salvage Raval JS et al. Vox Sang 2010:325 The washing device inflicted more sublethal injury on the RBCs than the unwashed device Predicted mean storage age of washed RBCs: 164 days Predicted mean storage age of unwashed RBCs: 6 days

30. Conclusions on washing vs not washing Previously thought that washing would produce a product with less PFHb than unwashed device This was not found in our study Washed device returned significantly more encapsulated Hb Clinical effect of sublethal injury on washed RBCs unknown

31. Blood bank washing also increases MFI Harm S et al. submitted

33. Effect of Hemobag processing on RBCs The Hemobag uses modified ultrafiltration to concentrate the residual whole blood in the CPB-circuit What is the effect of the repeated passes through the hemoconcentrator/ultrafilter on the RBCs? Samolyk KA et al. Perfusion 2005:343

34. Effect of Hemobag processing on RBCs Collected samples before and after processing with Hemobag from 8 patients undergoing on-pump cardiac surgery Sorin DHF0.6 hemoconcentrator/ultrafilter 65 ± 15 years, 6/8 male 5 CABG, 2 valve repair, 1 ascending Aorta repair No patients were transfused while on bypass Average CPB time: 124 ± 34 min Average length of Hemobag processing: 5.56 ± 1.13 min Harm S et al. submitted

35. Hemobag product is highly concentrated Harm S et al. submitted Pre- processing Post- processing mean SD mean SD p Hct (%)22.05 4.07 49.14 6.65<0.0001 Hb (g/dl)7.64 1.31 16.03 2.01<0.0001 Volume of MUF device reservoir (ml) 965.9 223.34 433.1 88.980.0002

36. MFI and PFHb not increased after Hemobag processing Harm S et al. submitted Pre- processing Post- processing mean SD mean SD p MFI 0.20 0.07 0.21 0.06 0.61 % Hemolysis 0.39 0.27 1.14 0.49 0.0008 Total amount of PFHb (g) 0.27 0.24 0.23 0.13 0.64 PFHb concentration (mg/dl) 33.88 22.13 105.0 44.41 0.0006 Total amount of Hb returned (g) NC 34.0 11.61 NC Total amount of PFHb post- processing: Total amount of Hb returned 0.007 0.0032

37. Comparison of PFHb load after washing Kelleher A et al. Anaesthesia online early Cobe BRAT 2 until mid-2006, then Haemonetics CS5 105.0 mg/dl

38. Effect of Hemobag processing on RBCs Comparing RBC parameters after Hemobag processing with data from TKA recovery UnwashedWashedPost-Hemobag Total PFHb (g)0.51 ±0.12 0.55 ±0.35 0.23 ±0.13 Total Hb (g)61 ±12 160 ±103 34 ±11.6 Total PFHb:Total Hb 0.00870.00350.0069 0.20 g/unit Ley JT et al. Transfusion in press

39. Conclusions on PFHb and perfusion Hb belongs in RBCs! NO is best left unscavenged A “safe” level of PFHb is not yet known  Its effects on MAP are transient and of uncertain clinical significance A washed unit is not devoid of PFHb Returning some PFHb with cell salvage seems inevitable Hemoconcentration/ultrafiltration produces a product that is at least on par with other salvage devices  Fragility of intact cells is not increased