Bacterial Safety of Platelets: Has the Time for Pathogen Inactivation/Reduction Finally Arrived?

1. Bacterial Safety of Platelets: Has the Time for Pathogen Inactivation/Reduction Finally Arrived? Roslyn Yomtovian, M.D. September 21, 2013

2. Disclosures Dr. Yomtovian has received funding as an investigator/consultant at Case Western Reserve University and University Hospitals of Cleveland to investigate bacterial contamination of platelets from Gambro, Hemosystem, Immunetics, Pall Corporation, GenPrime, Verax and Fenwal. She also serves as a consultant to Verax, and Immuntics.

3. Objectives • Provide an historical overview of the • problem of platelet bacterial contamination • in transfusion practice • Present the current status of platelet • bacterial contamination risk in the context • of transfusion practice • Discuss strategies, past and present, to • reduce and/or eliminate the problem of • platelet bacterial contamination and • emphasize the superiority of pathogen • inactivation/reduction.

4. Historical Perspectives Bacterial contamination of blood components is the oldest transfusion-associated infectious risk and is proving to be the most difficult to eradicate. Yomtovian, Palavecino. Bacterial Contamination of Blood Components - History and Epidemiology. In: BrecherME,ed. Bacterial and Parasitic Contamination of Blood Components. AABB Press, 2003

5. Transfusion Associated Exposures Bacterial contamination Brecher ME, Hay SN. Bacterial contamination of blood components. ClinMicrobiol Rev 2005;18:195-204

6. A Saga of > 70 Years – WHY ? • Lack of widespread recognition • Incomplete understanding of clinical significance • Distraction of attention by viral agents • Clinical symptoms often obscured and highly variable especially in patients at highest risk (platelet recipients) • Bacteria not detectable at time of donation  requires a new testing paradigm/approach • Viral assays not applicable to bacterial detection • Heterogeneity of bacteria makes a single, uniform solution unlikely • Uncertainties, fear and expense

7. Preservation of Stored Blood with Sulfanilamide: First Effort to Prevent Bacterial Contamination • Advised that careful attention be given to the potential for bacterial contamination of stored blood as it becomes more useful as a therapeutic agent • Pointed out the susceptibility of blood to harbor bacteria noting a 5% contamination rate of refrigerated blood stored for 10 days • Suggested the most likely source of bacteria is from the phlebotomy process, noting (prophetically)… Novak. JAMA 1939;113:2227-2229

8. Preservation of Stored Blood with Sulfanilamide: First Effort to Prevent Bacterial Contamination - Con’t “Complete sterilization of the skin previous to phlebotomy is impossible, since there are always a few bacteria which escape the bactericidal action of any cutaneous antiseptic. The needle in its course through the skin may encounter these viable organisms, and the chance for introducing them into the bottle with the blood is high...The detection of contamination in stored blood is an uncertain procedure, since the number of organisms is usually small…The possibility of using sulfanilamide…is worthy of consideration.”

9. Fatal Transfusion Reactions from Massive Bacterial Contamination of Blood Bacterial growth in stored blood is best retarded by immediate and continuous refrigeration… Detection of bacterial contamination in stored blood or its products is a problem for which no satisfactory solution has been found. Borden and Hall. NEJM 1951; 245:760-765.

10. Effects and Clinical Significance of Bacterial Contaminants in Transfused Blood Cultured 1697 consecutive bottles of blood after storage at 40C for 24 hrs finding 2.2% to be contaminated. Concluded that the entrance of bacteria into blood appears to be inevitable. Braude, Sanford, Bartlett, et al. J Lab Clin Med 1952; 39:902-916.

11. A Study of Bacteria Implicated in Transfusion Reactions and of Bacteria Isolated from Blood Products Summarized 18 instances of severe or fatal reactions involving blood or blood components coming to the agency’s attention noting that.. “This number could be considered as a fraction of the total number that are occurring, since they were heard about incidentally.” An additional 29 isolates of gram neg rods and 44 isolates of gram pos cocci were identified on submitted surveillance cultures. Biologics Control Lab, US Public Health Service Pittman. J Lab Clin Med 1953;42:273-288.

12. FDA Contributions to Reduction of Bacterial Contamination in Platelet Products Within the US • March 2001 BPAC – Optimal design for diversion pouch system which reduced PBC by 50% • Feb 2002 – Cleared BacT/ALERT device for quality control testing for PBC of LR-Apheresis Platelets • March 2003 – Approved PASSPORT Study in hope of extending shelf-life of platelets to 7-days and expressed disapproval of non-validated testing • 2005 – Cleared pre-storage pooling system for testing platelet pools • Has cleared 2 culture based and 2 point of care tests Office of Blood Research and Review, Center for Biologics Evaluation and Review, FDA; Epstein JS, Vostal JG. Transfusion 2013;53:232-233.

13. Platelet Transfusions Utilizing Plastic Equipment Described a procedure for the use of plastic equipment for platelet transfusion and in particular noted that, the plastic bag and attachments may be prepared and kept sterile indefinitely. Gardner, Howell, Hirsch. J Lab & Clin Med 1954; 43:196-207

14. Platelet Preservation - Effect of Storage Temperature on Maintenance of Platelet Viability Deleterious Effect of Refrigerated Storage A “shelf” life of 96 hours would introduce into the use of platelet transfusions a degree of flexibility that has not been possible with refrigerated storage. Several questions remain to be answered before one can be wholly enthusiastic about this approach. The first is that one should know if the risk from inadvertent bacterial contamination will be greatly increased at this higher temperature; we encountered no such difficulties in our studies. Murphy and Gardner. NEJM 1969; 280:1094-1098.

15. Detection and Quantitation of Bacteria in Platelet Products Stored at Ambient Temperature • Up to 1.6% of platelet units contaminated • The risk of bacterial proliferation may warrant a review of current methods of platelet collection and of ambient temperature platelet storage. Certainly caution should be exercised in the use of platelet products stored in this manner, especially in recipients with impaired host defense mechanisms. Buchholz, Young, Friedman, et al. Transfusion 1973; 13:268-275.

16. Over 3 million platelet units are transfused in the form of 1.8 million apheresis units and 0.26 million pools (approx 0.1 million pooled at production, and 0.16 million at issue) Bacterial contamination rates are similar for apheresis and whole-blood derived units pooled at issue, with contamination rate per transfusion 4–6-fold higher for pool transfusions Estimated that over 500 bacterially contaminated apheresis units and a similar number of RDP units are transfused each year The fatality rate associated with bacterial contamination of platelets is estimated to be 2 deaths per million units transfused ( 6 deaths per year) The rate of septic transfusion reactions is estimated to be 10–13 cases per million units transfused (30–40 cases per year) U.S. Platelet Transfusions Niu MT, et al. Transfus Med Rev 2006; 20:149–57 Whitaker BI, Sullivan M. http://www.aabb.org/apps/docs/05nbcusrpt.pdf Morrow JF, et al JAMA 1991; 266:555–8. Kuehnert MJ, et al. Transfusion 2001; 41:1493–9. Eder AF, et al. Transfusion 2007; 47:1134–42. Jacobs MR, et al. Transfusion 2011; 51:2573-82

17. Bacterial Contamination of Platelet Pools -- Ohio, 1991 January 24, 1992 / 41(03);36-37 From June 27 through July 30, 1991, four episodes of bacterial contamination of platelet pools occurred in an Ohio hospital and were reported by the hospital through the Food and Drug Administration (FDA) to CDC. Morbidity & Mortality From Platelet Bacterial Contamination • Case Outcomes: • Death – Pseudomonas aeruginosa • Bilobar pneumonia – Bacillus sp • Severe febrile reaction – Bacillus sp • Positive Blood Culture – Coagulase negative staph

18. Platelets Contaminated with Gram Positive Organisms

19. Platelets Contaminated with Gram Negative Organisms

20. Platelets Contaminated with Gram Negative Organisms

21. No active surveillance performed Bacterial contamination of platelets UHCMC Cleveland 1991-2012 N=75 Yomtovian R, et al. Transfusion 2006;46(5):719-30. Jacobs MR, et al. 2008. Clin Infect Dis 46(8):1214-20. Yomtovian R, et al. AABB 2011 abstract SP410 Jacobs MR, et al. Transfusion. 2011, 51:2573-82 Updated to 2012

22. Relationship between bacterial load, species virulence, and transfusion reaction N=46 12 10 11 10 10 10 Staphylococcus epidermidis 9 10 Staphylococcus aureus 8 10 Staphylococcus lugdunensis 7 10 Staphylococcus warneri Bacterial count (cfu/ml) 6 10 Viridans group streptococcus 5 10 Streptococcus bovis 4 10 Bacillus cereus 3 10 Pseudomonas aeruginosa 2 10 Serratiamarcescens 10 0 Life- 0 1 2 3 4 5 6 None Severe Fatal Mild Moderate 7 threatening Reaction grade Jacobs, M. R., C. E. Good, H. M. Lazarus, and R. A. Yomtovian. 2008. Relationship between Bacterial Load, Species, Virulence, and Transfusion Reaction with Transfusion of Bacterially Contaminated Platelets. Clin Infect Dis46:1214-20.

23. What are the Strategies to Interdict Platelet Bacterial Contamination? • Preventative strategies to • reduce or eliminate bacteria • Detection Strategies to identify • and interdict bacteria

24. Current AABB Standards on Platelet Bacterial Contamination - 1 5.1.5 Sterility Aseptic methods shall be employed to minimize the risk of microbial contamination of blood and blood components. Equipment and solutions that come into direct contact with blood or blood components shall be sterile and pyrogen-free. Single-use equipment shall be used whenever possible. 5.1.5.1* The blood bank or transfusion service shall have methods to to limit and detect or {inactivate (26th Ed, Nov, 2009)} bacteria in all platelet components.Standard 5.6.2 applies. 5.1.5.1.1 Detection methods shall either be approved by the FDA or be validated to provide sensitivity equivalent to FDA- approved methods. 5.1.5.2 When a true-culture positive result is obtained and an appropriate specimen is available, additional testing to identify the organism shall be performed. Additional testing and follow-up shall be defined. Standards 5.2.4 and 7.1 to 7.1.4 apply. * 5.1.5.1 first appeared in the 22nd Ed of the Standards but instead of being effective Nov 1, 2003, it became effective March 1, 2004

25. Current AABB Standards on Platelet Bacterial Contamination - 2 5.6.2 Blood Collection – Protection Against Contamination The venipuncture site shall be prepared so as to minimize risk of bacterial contamination. Green soap (USP) shall not be used. 5.6.2.1 Blood collection containers with draw line (inlet) diversion pouches shall be used for any collection of platelets, including whole blood from which platelets are made. (Became requirement in 25th Edition of Standards, May 1, 2008). 5.2.4 Donor Notification of Abnormal Findings and Test Results 7.1 Nonconformances

26. Phlebotomy Preparation Diversion of early blood draw – significant impact on Coagulase Negative Staphylococci Exposure to leukocytes during early storage – appears to be reduced bacterial contamination in buffy coat stored units Pathogen Inactivation/Reduction Strategies Methods to Reduce Bacteria

27. MetabolicSubstrates/Products Glucose ( 107) pH ( 107)  Cell Growth  Culture Flasks ( 102) Culture Plates ( 102 ) % Oxygen in Air ( 102 - 103) Cell Markers Observations Clumping/color Swirling ( 107) Gram Stain ( 105-6) Wright Stain ( 105-6) Acridine Orange Stain ( 104) Methods to Detect Bacteria (CFU/mL)

28. The Impact of the 1994 Early Platelet Culture Mandate • The impact of this mandate has been limited and disappointing resulting in many ‘breakthrough’ cases: • Early culture of apheresis platelets (together with sample diversion) has reduced PBC by only ≈ 25%. • Thus, patients continue to receive bacterially contaminated platelets with resulting morbidity & mortality Methods to Reduce PBC • Palavecino, E. L., R. A. Yomtovian, and M. R. Jacobs. 2010. Bacterial contamination of platelets. TransfusApherSci 42:71-82

29. Culture Detection Limit using 8 ml Culture Volume at 24 h Growth of bacteria in platelets based on one viable organism in a 400 ml unit (0.0025 organisms/ml) Generation time of most bacteria in platelets at 220C is 1-4 h Palavecino, E. L., R. A. Yomtovian, and M. R. Jacobs. 2010. Bacterial contamination of platelets. TransfusApherSci 42:71-82.

30. Limitations of Culture Testing TargetCaptured Sampling Incubation Sampling Incubation POSITIVE Too few targets to reliably capture an organism in sample OR FALSE NEGATIVE TargetMissed Courtesy of Dr. Michael Jacobs

31. Detection of Platelet Bacterial ContaminationTwo Paradigms of Test Application • Near Donor Collection, Culture Methodologies • generally require a large sample inoculum • low bacterial load  false negs ›› “breakthrough cases” • pos result may occur following transfusion (BacT/ALERT) • risk of false pos during sampling process • holding time required  may delay platelet availability • generally performed by the Blood Center • At/Near Transfusion Issue, Point of Care Methodologies • generally use smaller sample inoculum • low bacterial load may result in false neg results • reflects bacterial status at time of issue • testing at time of issue  may delay platelet release • generally performed by the Transfusion Service

32. Prepared platelet suspension Gram positive result Gram negative result Validity controls Technical Approaches for Bacterial Detection (CFU/mL) – Verax Biomedical Platelet PGD® Test • Lateral flow immunoassay format • Detects lipotechoic acid (LTA) in GP bacteria • Detects lipopolysaccharide (LPS) in GN bacteria • Differentiates GN and GP organisms in one assay • Results in approximately 30 minutes • Limits of bacterial detectability ≈ 104-106 CFU/mL • Multisite study found 1:2314 units + for PBC at or near time of issue (Jacobs et al. Detection of platelet bacterial contamination in prestorage culture-negative apheresis platelets on day of issue with the Pan Genera Detection test. Transfusion 2011; 51:2573-2582)

33. Technical Approaches for Bacterial Detection (CFU/mL) – ImmuneticsBacTx® Test • Enzyme-based qualitative colorimetric assay using an automated reader • Ligand that detects bacterial peptidoglycans, a ubiquitous component of bacterial cell walls • Detects but does not differentiate GN and GP organisms • Results in less than 60 minutes • Limits of bacterial detectability ≈ 104-105CFU/mL • The BacTx® Assay was more sensitive overall, by more than an order of magnitude in several instances compared to the PGD® Assay. (Heaton et al. Evaluation of the ImmuneticsBacTx® Assay for Detection of Bacterial Contamination in Apheresis and Pooled Random Donor Platelet Units. Transfusion 2013. Submitted for publication.

34. Shortcomings of Detection Paradigm • A new model for hospitals who would • now be responsible for at issue testing – much resistance • Remaining uncertainty of test results • Expense

35. Pathogen Inactivation/Reduction The ‘Penultimate Paradigm Shift’ Obstacles to Implementation • Perception of Toxicity • A Totally New Technology • Fear and Uncertainty • Loss of Platelet Function Associated • with Treatment Process • Possible Need for more Platelet • Transfusions • Expense

36. Pathogen Inactivation/Reduction Strategies Photochemical Treatment Processes • Intercept System (Cerus Corp, • Concord, CA) • Mirasol Pathogen Reduction Technology • (Caridian BCT, Concord, CO) • THERAFLEX UV-Platelets (MacoPharma, • Mouvaux, France)- Does not require a • photoactive agent

37. Employs the synthetic psoralen compound amotosalen HCL (S-59) • The S-59, a heterocyclic compound intercalates into nucleic acids • Upon UVA irradiation it forms a covalent bond, a monoadduct • Upon further illumination, the monoadduct combines with a second nucleic acid strand forming a crosslink • Residual S-59 is removed with a compound absorption device (CAD) filter • The above renders the target incapable of replication and eventually leads to the death of the target • In Europe received CE Mark in 2006 Intercept System From: Wollowitz. Fundamentals of the psoralen-based Helinx ™ techology for inactivation of infectious pathogens and leukocytes in platelets and plasma. 2001; SeminHematol 38)suppl 11):4-11.

40. Riboflavin, vitamin B-2 is added to platelets after transfer of platelets to an illumination bag • The bag is then illuminated with UVA and UVB irradiation resulting in the formation of free radicals which are toxic to nucleic acids • Residual riboflavin is not removed with a CAD as in the case of the intercept system • In Europe, received CE Mark in 2008 Mirasol System

42. Uses an ethylene vinyl acetate treatment bag, under agitation to allow penetration of short-length UVC radiation • Uses 254nm wavelength (in max range of DNA/RNA absorption and therefore an additional photosensitizer is not needed) • Following treatment, platelets are transferred to a polyvinyl chloride bag • In Europe, received CE Mark in 2009 and has been accepted for clinical trials in Germany Theraplex System From: Seghatchian et al: Characteristics of the THERAPLEX UV-Platelets Inactivation system – An Update. Transf and ApherSci 2012; 46:221-229.

43. Mechanism of action of THERAFLEX UV-Platelets: short-wave UVC light is directly absorbed by DNA/RNA nucleobases, thus inactivating pathogens predominantly by formation of intra- and inter-strand cyclobutane pyrimidine and pyrimidine–pyrimidone dimers. From: Seghatchian et al: Characteristics of the THERAPLEX UV-Platelets Inactivation system – An Update. Transf and ApherSci 2012; 46:221-229.

44. Efficacy of Platelet Pathogen Inactivation/Reduction Technologies From: AuBuchon and Prowse. Pathogen Inactivation. The Penultimate Paradigm Shift. AABB Press, Bethesda, MD 2010; In: Sweeney and Lozano. Platelet Transfusion Therapy – Pathogen Inactivation Of Platelets. pp. 119-176; AABB Press, Bethesda, MD 2013

45. Diffusion of PI/PR Technology

46. Diffusion of New Technology - An Illustrative Study: Controlling Scurvy in the British Navy Many believe that advantageous innovations will sell themselves, that the obvious benefits will be widely realized and the innovation will diffuse rapidly. Seldom is this the case. Most innovations diffuse at a disappointingly slow rate, at least in the eyes of the inventors who create them and promote them. Scurvy control illustrates how slowly an obviously beneficial innovation spreads. In the early days of long sea voyages, scurvy killed more sailors than did warfare accidents and other causes. In 1601 a sea captain James Lancester commanded 4 ships that that sailed from England to India. He served 3 tps of lemon juice to sailors on one of the 4 ships and none to the sailors on the other “control” ships. On the control ships 110/278 sailors died and the remainder were transferred to the treatment ship in order to finish the voyage. Despite the clarity of the results, it took this innovation nearly 200 years – 1795 - to become routine practice in the British Navy. from: Everett M. Rogers, Diffusion of Innovations, 5th Edition, Free Press, NY, 2003

47. Has Pathogen Reduction/ Inactivation Reached a Tipping Point? Gladwell defines a tipping point as "the moment of critical mass, the threshold, the boiling point".

48. Quickly emerging as a viable strategy for dealing definitively • with the problem of platelet bacterial contamination • Emerging as a strategy for dealing with unknown and emerging • pathogens (at least initially) • Emerging as a strategy for dealing with known pathogenic agents • to reduce the initial level of infectivity and eliminate some • redundant testing • Useful as a new paradigm to eliminate the problem of TA-GVHD • But, need to solve the application of PR/PI to RBCs In the US Where do we Stand with Platelet Pathogen Reduction/Inactivation?

49. Transfusion-Transmitted Bacterial Infection of Platelets - The End of an Era??? • bacterial contamination is an ongoing, recurrent complication of primarily platelet transfusion therapy • until recently, no systematic approach, in the US, to reduce or eliminate this problem has been defined, suggested or implemented • a single ideal preventative strategy has not been developed • there are, numerous strategies, alone or together, which will reduce and may eliminate the occurrence and/or magnitude • prevention strategy(ies) may prove cost-enhancing if linked with a seven day platelet storage product • a pathogen-inactivation strategy while theoretically the most attractive is not yet on the horizon (2003) opening the gate for novel approaches at the front and back-end of platelet storage Above points presented at a talk at the AABB in 2003 The good news is that today, a pathogen-inactivation/reduction strategy finally is emerging as a viable solution to the problem of platelet bacterial contamination as well as the overall infectious risks associated with transfusion therapy in general