1. Transfusion Related Acute Lung Injury (TRALI): Clinical and Laboratory Aspects David Stroncek, MD Chief, Laboratory Services Section Department of Transfusion Medicine Clinical Center, NIH, Bethesda, Maryland

2. Disclaimer-1 The views expressed are those of the presenter and do not necessarily represent the position of the National Institutes of Health or the Department of Health and Human Services.

3. Disclaimer-2 The views expressed are those of the presenter and do not necessarily represent the position of anyone else. TRALI is controversial!

4. Overview Definition and clinical features Pathophysiology Female donors Leukocyte antibodies Leukocyte activating agents Patient factors Testing and TRALI

5. Transfusion Related Acute Lung Injury (TRALI): What is it? Severe shortness of breath within 4 to 6 hours of a transfusion No signs of fluid overload Pulmonary infiltrates on chest x-ray

6. TRALI: NHLBI Working Group Definition TRALI New ALI Onset of symptoms or signs is within 6 hours after the transfusion of plasma containing blood components ALI (Acute Lung Injury) Acute onset Chest x-ray: Bilateral infiltrates Pulmonary artery occlusion pressure ≤18 mm Hg or lack of clinical evidence of left atrial hypertension Hypoxemia: Ratio of PaO 2 /FiO2 ≤300mm Hg or O 2 saturation of less ≤90% on room air Clinical Diagnosis Crit Care Med 2005;33:721-726

7. TRALI: Incidence and Products Incidence From 1 in 1,000 to 1 in 10,000 units transfused Products implicated Fresh Frozen plasma, platelets, and red cell concentrates More likely to be associated with FFP and platelets SD plasma is not thought to cause TRALI Sachs UJ et al. Transfusion 2005;45:1628-31

8. TRALI: Clinical Features Dyspnea and hypoxemia Fever Hypotension or hypertension Chest x-ray: bilateral infiltrates, “white out”

9. TRALI: Treatment Hypoxemia Supplemental oxygen Intubation and mechanical ventilation Hypotension Intravenous fluids Agents to increase blood pressure Corticosteroids Moore SB. Critical Care Medicine 2006; 34: S114-117

10. TRALI: Clinical Course Symptoms generally resolve in 24 to 48 hours Symptoms may resolve before diagnosis is made Mortality 10% to 50% Moore SB. Critical Care Medicine 2006; 34: S114-117 Rana R et al. Transfusion 2006;46:1478-1483

11. Why the concern about female donors?

12. A Prospective Study: Cardiopulmonary Reactions to Plasma for Multiparous Donors 100 ICU Patients 1 unit Donor Control Plasma 1 unit Multiparous Donor Plasma PaO 2 :FiO 2 ratio BP, Heart rate Temp 1 unit Multiparous Donor Plasma 1 unit Control Donor Plasma PaO 2 :FiO 2 ratio BP, Heart rate Temp End Palfi M, Berg S, Ernerudh J, Berlin G. Transfusion 2001;41:317-322 Multiparous = 3 or more births

13. Multiparous vs Control Plasma (n=100) BeforeAfterp PaO 2 :FiO 2 Ratio Control32.7 ± 13.832.8 ± 12.8NS Multip33.8 ± 12.831.1 ± 13.1<0.001 Mean Arterial Pressure (mmHg) Control78.6 ± 13.981.8 ± 14.9<0.01 Multip81.2 ± 14.280.4 ± 14.5NS Palfi et al. Transfusion 2001;41:317-322

14. Multiparous vs Control Plasma: Reactions TRALI One case Multiparous donor unit Granulocyte antibodies; No HLA antibodies Mild Reactions (4) 3 Pulmonary (multiparous units) 1 Fever only (control unit) 1 of 3 multiparous donors units had granulocyte, but no HLA antibodies

15. TRALI in ICU Patients Single Institution Retrospective case-control study All new cases of respiratory failure within 6 hr of a transfusion over 1 year Number of patients studied: TRALI (n = 24), fluid overload (n = 25), and controls (n = 124) TRALI patients were more likely to receive Plasma-rich products Larger volumes of plasma Plasma from female donors Rana R et al. Transfusion 2006;46:1478-1483

16. What Causes TRALI? Donor factors Leukocyte antibodies Product storage factors Bioactive lipids CD40L Patients factors

17. 1957: Evidence that Leukoagglutinins Cause Transfusion Reactions One severe reaction Infusion of 50 mL of blood with leukoagglutinins caused vomiting, diarrhea, chills, fever, hypotension, tachypnea, dyspnea, cyanosis and leukopenia within 45 minutes. Symptoms resolved the next day, but a chest x-ray showed bilateral pulmonary infiltrates and a small pleural effusion. Two days the chest x-ray was normal. Two mild reactions Infusion of 250 mL of plasma containing weak leukoagglutinins to two subjects cause mild reactions. Brittingham TE. Vox Sang 1957; 2:242-248

18. Leukocyte Antigens Implicated in TRALI Human Neutrophil Antigens HNA-1 HNA-2 HNA-3 Human Leukocyte Antigens HLA Class I HLA Class II Leukoagglutinins HLA antibodies Neutrophil-specific antibodies

19. On the basis of case reports in the 1960’s and 1970’s the concept that leukoagglutinins causes pulmonary transfusion reactions takes hold …both our cases suggest that the acute pulmonary edema was related to a leukoagglutinin, but such a relationship was not established. (JS Thompson NEJM 1971:284:1120-1125) …both the responsible donors were multiparous, raising concern about the use of whole blood from multiparous donors… (JS Thompson NEJM 1971:284:1120-1125)

20. 1980’s: the term TRALI was first used and the idea that leukocyte antibodies cause TRALI becomes widely accepted 1983 (Popovsky, Able, and Moore) A series of 5 cases of pulmonary transfusions reactions 19 implicated donors One donor for each case had an HLA antibody TRALI defined 1985 (Popovsky and Moore) 36 cases Leukocyte antibodies in 89% HLA antibodies in 65%

21. Interpreting Antibody Test Results 2 RBCs 4 FFP 1 Platelet TRALI Incidence of leukocyte antibodies in blood donors HLA Antibodies 4% to 7% of all donors Up to 21% of females with 3 for more pregnancies Neutrophil Antibodies Less than 0.1% of donors It is difficult to interpret the results of testing TRALI implicated donors for HLA antibodies without including a control group

22. Leukocyte Antibodies, TRALI, and Leukopenia Leukocyte antibody transfusion associated with leukopenia and TRALI Anti-HNA-1b Yomtovian et al. Lancet 1984;1:244-6 Anti-HLA class I and II, 3 cases Nakagawa and Toy. Transfusion 2004;44:1689-94 Anti-HLA class I and II, 2 cases Marques et al. Am J Hematol 2005;80:90-1 Leukocyte antibodies transfusions associated with leukopenia and transfusion reactions, but not TRALI Anti-HNA-2a Fadeyi et al. Transfusion. 2007;47:545-50

23. Look-Back Studies Antibody# Donors # Transf All Reactions Serious Reactions Author HNA-3a136158Kopko et al HNA-3a12500Davoren et al HNA-2a139120Fadeyi et al HLA Class I and II 21810Nicolle et al HLA Class I and II 1600Fontaine et al HLA Class I and II 110311Toy et al

24. Leukocyte Antibodies in Blood Donors Study Design 1043 donors 633 previously pregnant females 410 males Tested for both HLA and neutrophil antibodies Results No neutrophils antibodies No HLA antibodies in males HLA antibodies in 62 (9.8%) of females Look-back 211 components: 187 RBCs, 61 platelets, 48 FFP 1 case of TRALI (RBCs, multispecific HLA class I and class II) Maslanka et al. Vox Sanguinis 2007:92:247-249

25. Animal Models of TRALI AntibodyAnimalPMNsComplementAuthors Neutrophil Antibodies HNA-3aRabbit++Seeger et al HNA-2aRat+-Sachs et al HLA Antibodies MHC Class IMouse+-Looney et al

26. Bioactive Lipids Accumulate during the storage of cellular blood products (lysophosphatylcholine, L- PC) Prime neutrophils: primed neutrophils have a greater response to activating agents Enhance neutrophil-mediated lung injury in animal models Prospective and retrospective studies have found greater levels of bioactive lipids in TRALI implicated units or post-transfusion sera from TRALI patients than controls Silliman et al. Transfusion. 1997;37:719-26 Silliman et al. Blood. 2003;101:454-62

27. Soluble CD40L Released by platelets Levels increase in stored platelets Primes neutrophils Inhibition of CD40-CD40L system reduces acute lung injury in animal models A case-control study found higher sCD40L levels in TRALI-implicated units than in control units Khan et al. Blood. 2006;108:2455-62

28. Patient Factors TRALI is more common in Surgery patients Patients with hematological malignancies and cardiac disease Moore. Crit Care Med 2006; 34: S114-S117 Silliman et al. Blood 2003; 101:454-462

29. Two-Hit TRALI Model 1.Patient conditions  activation of pulmonary endothelium  sequestration of neutrophils  priming of neutrophils (adhesion) 2.Infusion of leukocyte antibody or biological response modifier  activates primed neutrophils  neutrophils damage pulmonary endothelium CC Silliman. Crit Care Med 2006;34:S124-S131

30. Testing for TRALI-Associated Factors

31. What and When to Test? What?When? HLA antibodiesDonor SerumAt Donation HNA AntibodiesDonor SerumAt Donation Bioactive lipids ProductAt Transfusion CD40L ProductAt Transfusion

32. Type of Assays and Commercial Availability TypeCommercial Kit Available? HLA antibodiesSolid PhaseYes HNA AntibodiesCellularNo Bioactive lipidsCellularNo CD40LELISAYes

33. HLA Class I and II Antibody Testing Antigen Immune affinity chromatography Recombinant technology Solid phase assays ELISA Microbeads-flow cytometry Microbeads-modified flow cytometry Other High throughput testing is possible Most HLA antibodies containing products do not cause TRALI

34. HNA Antibody Testing Antigen Intact neutrophils (short life span) Assays Agglutination Immunoflourescence-flow cytometry Monoclonal antibody capture Mixed passive hemagglutination Problems with solid phase HNA-3a has not been characterized at a molecular level No monoclonal antibody to HNA-3a

35. Bioactive Lipids Antigen Intact neutrophils Assay Respiratory burst by stimulated neutrophils Other Threshold for causing TRALI is not known

36. CD40L Assays ELISA Other Available as a research assay Threshold for causing TRALI is not known

37. Summary HLA antibodies Testing donor samples is straight-forward A positive result has a low predictive value of TRALI HNA antibodies Testing donors requires working with fresh neutrophils A positive result has a higher predictive value for a transfusion reaction Bioactive lipids Testing donors requires working with fresh neutrophils Testing products at the time of transfusion is challenging CD40L Testing is straight-forward Testing products at the time of transfusion is challenging

38. Conclusions Donor, product, and patient factors have been implicated in TRALI No single factor is highly predictive of TRALI Testing for HLA antibodies and CD40L is feasible Testing for neutrophil antibodies and bioactive lipids is possible but more difficult

39. Current Practices and Protocols: Department of Transfusion Medicine, Clinical Center, NIH Practices Transfusion male plasma Transfuse female AB apheresis plasma if negative for neutrophil antibodies Defer TRALI implicated donors if an antibody to a characterized neutrophil antigen is identified Protocol Comparison of the incidence of transfusion reactions in recipients of platelet components with and without HLA antibodies