Does blood transfusion alter the balance between hemostasis and thrombosis?

1. Does blood transfusion alter the balance between hemostasis and thrombosis? Neil Blumberg MD University of Rochester, Rochester, NY SABM Annual Meeting, Los Angeles CA September 21, 2013

2. Hemostasis is a balance of platelet, endothelial cell and coagulation factor activation Bleeding Hemostasis Thrombosis Thanks to Katie Lannan

3. Why would transfusions affect hemostasis and thrombosis? • Other than the obvious effects of hemostatic components like plasma, platelets and cryoprecipitate • When we transfuse RBC, we increase viscosity, alter shear stress, decrease nitric oxide, etc. • We are infusing “stuff” other than RBC—microparticles, supernatant, storage altered red cells • We create iatrogenic immune complexes by transfusing ABO mismatched platelets and plasma

4. Outline of the talk • Epidemiologic, in vitro and clinical trial evidence demonstrate RBC transfusion’s association with thrombosis (BAD) • RBC transfusion can prevent stroke in homozygous SS disease (GOOD) • ABO mismatched transfusions may impair hemostasis (BAD) • Infusion of DNA and histones predisposes to thrombosis (BAD) and leukoreduction reduces the amount of infused DNA and histones (GOOD) • For those keeping score that’s about even 

5. RBC and Platelet Transfusions are associated with thrombosis in cancer patients

6. Logistic regression of the probability of thrombosis (THROMBO) in pediatric cardiac surgery patients vs. storage duration of the oldest RBC transfused (washed, LR transfusions) Washed transfusion study arm p = 0.03 Unpublished Observations--Cholette, Blumberg, et al.

7. Logistic regression of the probability of thrombosis (THROMBO) in pediatric cardiac surgery patients vs. storage duration of the oldest RBC transfused (unwashed, LR transfusions) Unwashed transfusion study arm p = 0.33 Unpublished Observations-- Cholette, Blumberg, et al.

8. Stored red cell supernatant and sediment microparticles shorten thromboelastograph R time– likely due to increased thrombin generation R time Transfusion 49:1569-1579 (2009)

9. Prevention of stroke with regular red cell transfusion in sickle cell anemia NEJM 339: 5-11 (1998)

10. Prevention of post-operative complications in sickle cell anemia through prophylactic red cell transfusion Lancet 381: 930-938 (2013)

11. Complications are more frequent with a liberal transfusion strategy in GI Bleeding NEJM 368: 11-21 (2013)

12. Effect of washing when the oldest red cell transfused is ≤ 15 days of storage • Washed is superiorto unwashed when ≤15 day storage blood is transfused Transfusion 52[Supplement]: 51A (2012)

13. Effect of washing when the oldest red cell transfused is ≥ 28days of storage • Washed is inferiorto unwashed when ≥ 28day storage blood is transfused Transfusion 52[Supplement]: 51A (2012)

14. Conclusions: Transfusions and Thrombosis • Transfusions are associated with a higher rate of thrombosis in surgical and cancer patients † Whether this is due to sicker patients receiving transfusions, or partial cause and effect is unknown † There are pro-thrombotic in vitro effects of stored red cells, stored supernatant and platelets † There are settings where transfusion of normal red cells prevents thrombosis (sickle cell anemia)

15. Rationale for considering whether transfusion can lead to impaired hemostasis and bleeding •ABO antigens are present on all cells, in particular platelets, red cells, endothelial cells, as well as on plasma proteins including factor VIII. •All group O, A, B and donor blood contains some amount of anti-A and/or anti-B †Platelets, cryoprecipitate and plasma are often given with no regard to ABO matching with the recipient •Diseases with circulating immune complexes often are associated with increased bleeding and thrombosis

16. ABO Blood Group • A glycosyltransferase creates the A antigen by adding a terminal N-acetylgalactosamine (Gal-Nac) to H antigen • B glycosyltransferase creates the B antigen by adding a terminal Galactose (Gal) to H antigen

18. ABO Blood Group • ABO blood group antigens are present on/in: • RBCs • Platelets • Endothelial cells • Plasma • Body fluids

19. ABO Blood Group • The total amount of plasma transfused with each type of blood product: • 30 mL for each RBC unit • 150 mL for a ten unit pool of cryoprecipitate • 250 mL for FFP • 250 mL for a five unit pool or apheresis single donor platelet

20. ABO non-identical transfusions are routine practice • Immunohematology 2007; 23: 20 (L Cooling) • 40% of platelet transfusions are ABO non-identical • Most commonly due to limited inventory and the desire to minimize product wastage

21. Days with bleeding-46 adult patients with acute leukemia receiving ABO non-identical platelet transfusions and 67 receiving ABO identical platelet transfusions during 1985-95 ABO non-identical platelet transfusions 2.3 ± 5.5 (1 SD) ABO identical group 0.75 ± 1.7 (p=0.034 by unpaired t test). Bone Marrow Transplant. 2005 Nov;36(9):747-55 (JM Heal)

22. ABO Mismatched Platelet Transfusions are Associated with Inferior Outcomes in Patients Undergoing Cardiac Surgery Transfusion 41: 790 (2001)

23. Exposure of Platelets to ABO incompatible Plasma Increases PFA-100 Closure Time Transfusion 53: 382-93 (2013) M Refaai

24. Group O (1:1024 titer anti-A) Group O (1:128 titer anti-A) Exposure to anti-A in O plasma decreases % platelet aggregation of A platelets Normal Saline (control) Group A plasma (control) Transfusion 53: 382-93 (2013)

25. Exposure of A platelets to anti-A also causes additional functional changes: • Impaired platelet spreading • Reduced thrombin generation Transfusion 53: 382-93 (2013)

26. Conclusion •In vitro modeling demonstrates that platelet dysfunction is caused by anti-A/platelet antigen interactions •Clot formation is slower and weaker in whole blood when A whole blood is mixed at clinically relevant ratios with O plasma. •These findings provide a potential mechanism for increased bleeding, multi-organ failure and mortality after ABO mismatched platelet and plasma transfusions

27. ABO—no such thing as a universal donor if non-red cell issues considered? • ABO mismatched transfusions are associated with increased platelet refractoriness, increased bleeding, increased multi-organ failure, increased lung injury and increased mortality • ABO mismatched transfusions overall may be associated with increased febrile and allergic transfusion rates and increased rates of alloimmunization to red cell antigens

28. Some revolutionary ideas about thrombosis Relevant to transfusion as well, some think! Denisa Wagner PhD Harvard University

29. Courtesy Dr. Denisa Wagner

30. Courtesy Dr. Denisa Wagner

31. Courtesy Dr. Denisa Wagner

32. Courtesy Dr. Denisa Wagner

33. Courtesy Dr. Denisa Wagner

34. Venous thrombi contain both DNA and vWF Courtesy Dr. Denisa Wagner

35. Courtesy Dr. Denisa Wagner

36. Courtesy Dr. Denisa Wagner

37. Do neutrophils release NETs during storage of red blood cell units?? T. Fuchs, R. Kaufman, D. Wagner et al Published in Transfusion on-line April 2013 doi: 10.1111/trf.12203

38. Leukocytes release DNA during storage of RBC units (Freshly outdated) Courtesy Dr. Denisa Wagner

39. RBC units contain high-molecular weight, extracellular histone-DNA complexes DNA H3 fragments MPO is also part of the complex---NETs Courtesy Dr. Denisa Wagner

40. Transfusion? Courtesy Dr. Denisa Wagner

41. Transfusion and Thrombosis—A NET benefit to Leukoreduction? • Non-leukoreduced blood is rich in DNA and histones, implicated in acute lung injury, sepsis and thrombosis in animal models • Leukoreduction may have a benefit in reducing the thrombogenic potential of transfused red cells and other components

42. Acknowledgments Hematology: Dr. Joanna Heal Dr. Jacob Rowe Dr. Jane Liesveld Dr. Gordon Phillips Dr. Charles Francis SMH Coagulation Lab: Dr. Jim Corsetti Bob Miller Ed Masel Zachary Boldt Anesthesiology/POCT: Dr. Mike Eaton Dr. Bob Mooney Bonnie McGorty SMH Blood Bank: Dr. Majed Refaai Dr. Scott Kirkley Debbie Masel Kelly Henrichs Dr. Dan Ryan Donna Davidson Dr. Denisa Wagner Phipps Lab: Dr. Rick Phipps Dr. Sherry Spinelli Ann Casey Stephen Pollock