1. Bacterial Testing of Platelet Components: 2008 Update William B. Lockwood, PhD, MD Clinical Professor Department of Pathology & Laboratory Medicine University of Louisville Director, Transfusion Services & Tissue/Bone Bank University of Louisville Hospital Director, Transfusion Services, Tissue/Bone Bank & Coagulation Lab Norton Hospital & Kosair Children’s Hospital Louisville, Kentucky wblock01@gwise.louisville.edu 502-852-5857

2. 2SCACM Conference Jan. 20, 2009 Objectives Describe the current FDA regulations and accreditation agency standards for testing platelet collections for bacterial contamination Describe the current FDA regulations and accreditation agency standards for testing platelet collections for bacterial contamination Compare & contrast 3 methods of bacterial detection in platelet collections Compare & contrast 3 methods of bacterial detection in platelet collections Describe a validation plan for use of non-FDA approved bacterial detection methods Describe a validation plan for use of non-FDA approved bacterial detection methods PACE Program Number: 362-001-09 SCACM is approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E. Program.

3. 3SCACM Conference Jan. 20, 2009 Whole Blood Collection Set ca. 1900 ca. 1900 Whole Blood Glass Bottle ca. 1940 ca. 1940 TODAY!

4. 4SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components Known to occur since 1900 when using vented glass bottles for whole blood collection Known to occur since 1900 when using vented glass bottles for whole blood collection Continued with advent of plastic blood containers in 1950s Continued with advent of plastic blood containers in 1950s Platelets stored refrigerated had less of a contamination problem, but were not efficacious on transfusion*! Platelets stored refrigerated had less of a contamination problem, but were not efficacious on transfusion*! *Murphy S, Gardner FH. NEJM 1969;380:1094-98

5. 5SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components Additional studies on platelet storage medium, storage temperature, pH, type of agitation, volume of platelets in container and size of container, plastic used for platelet bag from 1960s-1980s Additional studies on platelet storage medium, storage temperature, pH, type of agitation, volume of platelets in container and size of container, plastic used for platelet bag from 1960s-1980s Changes included: Changes included: Gentle horizontal agitation Gentle horizontal agitation Room temperature (RT) storage- 3 days to 5 days (1981); 5 days to 7 days (1984) Room temperature (RT) storage- 3 days to 5 days (1981); 5 days to 7 days (1984) Reduction from 7 day to 5 day RT storage (1986)* Reduction from 7 day to 5 day RT storage (1986)* *Shiffer CA et al. Blood 1986;67:1591-94

6. 6SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components US Food & Drug Administration (FDA) via blood regulatory department Center for Biologics Evaluation & Research (CBER) held industry workshops in 1995 & 1999 to discuss platelet bacterial contamination US Food & Drug Administration (FDA) via blood regulatory department Center for Biologics Evaluation & Research (CBER) held industry workshops in 1995 & 1999 to discuss platelet bacterial contamination Literature continues to report increasing platelet bacterial contamination (BaCon study of American Red Cross Blood Services) Literature continues to report increasing platelet bacterial contamination (BaCon study of American Red Cross Blood Services)

7. 7SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components Bacterial contaminated blood components cause of >10% (77/694) of recipient fatalities reported to FDA from 1985-1999* Bacterial contaminated blood components cause of >10% (77/694) of recipient fatalities reported to FDA from 1985-1999* 1 st multicenter study of bacterial contamination of blood components published by American Red Cross** 1 st multicenter study of bacterial contamination of blood components published by American Red Cross** *Workshop on bacterial contamination of platelets.Bethesda: FDA, Center for Biologics Evaluation and Research,September 24, 1999. Available from http://www.fda.gov/cber/minutes/workshop- min.htm. **Kuehnert MJ, et al. Transfusion-transmitted bacterial infection in the United States, 1998 through 2000. Transfusion 2001;41:1493-99

8. 8SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components BaCon study 23,711,169 RBCs 1,804,725 single donor platelets (SDP) 1,033,671 Pooled platelets (WBDP)

9. 9SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components Transfusion-transmitted bacteremia rates: SDP= 1:100,000 WBDP= 1:100,000 RBC= 1:8,000,000

10. 10SCACM Conference Jan. 20, 2009 1:100,000 1:1,000,000Incidence rate Bacterial vs. Virus: Infectious risk in the blood supply HIV Hepatitis C HTLV 1/2 Bacteria - Platelets Bacteria - Red Cells West Nile Virus Hepatitis B HIV Ab 1985 P 24 1996 HIV PCR 2000 HCV Ab 1990 HCV PCR 2000 HTLV 1/2 1989 WNV NAT 2003 HBsAg 1971 Anti HBsAg 1987 1:2,000 1:20,000 No Practical Screening Tests Currently Available No Practical Screening Tests Currently Available Source: Ilert WE, et al. Transfusion Medicine 1995, 5:57-61 Brecher et al. Clinical Microbiology Reviews, 2005, 195-204

11. 11SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components Sources of Bacterial Contamination Skin Surface Contamination Phlebotomy Core Donor Bacteremia Containers and Disposables Environment

12. 12SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components ORGANISMS INVOLVED Exogenous Staphylococcus epidermidis Staphylococcus aureus Diphteroids spp Micrococcus spp Pseudomonas spp Bacillus cereus Propionibacterium acnes Flavobacterium spp Normal skin

13. 13SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components ORGANISMS INVOLVED Endogenous OsteomyelitisStaphylococcus S. cholera suis Staphylococcus spp TeethStreptococcus viridans TeethStreptococcus viridans Serratialiquefaciens Yersiniaenterocolitica IntestinesSalmonella spp IntestinesSalmonella spp Campylobacter spp

14. 14SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components Prevention and Detection Options Donor screening – not feasible except for arm screening. Can’t detect asymptomatic bacteremic donors Donor screening – not feasible except for arm screening. Can’t detect asymptomatic bacteremic donors Arm Preparation-Limited effectiveness of arm scrub Arm Preparation-Limited effectiveness of arm scrub Better phlebotomy methods and initial blood diversion Better phlebotomy methods and initial blood diversion Bacterial contamination testing offers best confirmatory option Bacterial contamination testing offers best confirmatory option Pathogen Inactivation--INTERCEPT Pathogen Inactivation--INTERCEPT

15. 15SCACM Conference Jan. 20, 2009 FDA Regulations FDA is silent about Whole-blood Derived Platelets (WBDP) bacterial testing guidelines [FDA guidelines are recommendations but are not legally binding until published as a Code of Federal Regulations (CFR)] FDA is silent about Whole-blood Derived Platelets (WBDP) bacterial testing guidelines [FDA guidelines are recommendations but are not legally binding until published as a Code of Federal Regulations (CFR)] HOWEVER- establishments requesting a Biological License Application (BLA) or Supplement (BLS) must follow the Title 21 CFR Parts 200 and 600 HOWEVER- establishments requesting a Biological License Application (BLA) or Supplement (BLS) must follow the Title 21 CFR Parts 200 and 600

16. 16SCACM Conference Jan. 20, 2009 FDA Regulations Part 200 relates to Good Manufacturing Practice, current (cGMP) of biologics Part 200 relates to Good Manufacturing Practice, current (cGMP) of biologics Part 600 relates to blood components - safety, quality, purity, potency, identification Part 600 relates to blood components - safety, quality, purity, potency, identification Part 640- platelets Part 640- platelets Subsections itemize various manufacturing regulations, - source, donor suitability, collections, QC Subsections itemize various manufacturing regulations, - source, donor suitability, collections, QC QC elements- platelet count of donor, donor weight, pH at outdate (≥6.2), sterility testing, etc QC elements- platelet count of donor, donor weight, pH at outdate (≥6.2), sterility testing, etc Manufacturer’s requirements for automated plateletpheresis collection Manufacturer’s requirements for automated plateletpheresis collection

17. 17SCACM Conference Jan. 20, 2009 FDA Regulations Revised regulation effective in 2008 (1 st was in 1988, draft in 2005!!) Revised regulation effective in 2008 (1 st was in 1988, draft in 2005!!) “Revisions to the Requirements Applicable to Blood, Blood Components and Source Plasma”* “Revisions to the Requirements Applicable to Blood, Blood Components and Source Plasma”* Pertains to those establishments that want to license their blood component Pertains to those establishments that want to license their blood component THEREFORE, FDA regulates platelet “manufacturing” through the BLA/BLS!! THEREFORE, FDA regulates platelet “manufacturing” through the BLA/BLS!! Also, approval of manufacturer’s devices through Pre-Market Approval 510(k) application regulates equipment used in all phases of blood component production Also, approval of manufacturer’s devices through Pre-Market Approval 510(k) application regulates equipment used in all phases of blood component production *Federal Register: August 16, 2007 (Volume 72, Number 158)

18. 18SCACM Conference Jan. 20, 2009 CAP Accreditation Checklist 1 st accreditation group to require bacterial testing of platelets! 1 st accreditation group to require bacterial testing of platelets! CAP Checklist - 2002 CAP Checklist - 2002 Phase I deficiency Phase I deficiency Revised 12/29/2004 to Phase II deficiency Revised 12/29/2004 to Phase II deficiency

19. 19SCACM Conference Jan. 20, 2009 CAP Accreditation Checklist 2008 Phase II TRM.44955 Phase II N/A YES NO Does the laboratory have a validated system to detect the presence of bacteria in platelet components? NOTE: For random donor platelets, any of the following testing methods satisfy this checklist question: detection of decreased pH or glucose by analytic instrument or dipstick; gram stain; acridine orange stain. Though of low sensitivity, these methods may detect units that are heavily contaminated by bacteria. Culture or FDA-approved commercial detection systems have greater sensitivity. The swirling technique is not recommended because of its very low sensitivity.

20. 20SCACM Conference Jan. 20, 2009 CAP Accreditation Checklist 2008 Two commercial systems have been cleared by the FDA for in- process quality control culturing of platelet units; one detects the growth of bacteria by their generation of CO2, and the other detects growth by their consumption of O2. Another system has been cleared for bacterial detection by fluorescent staining. If this testing is performed by the supplier of platelet components, the transfusion service can satisfy this checklist requirement by having an agreement with the supplier to notify the transfusion service if any units suspected of containing bacteria have been transferred to the transfusion service.

21. 21SCACM Conference Jan. 20, 2009 AABB Accreditation AABB Standards for Blood Banks & Transfusion Services, 23 rd ed, Effective May 1, 2004 AABB Standards for Blood Banks & Transfusion Services, 23 rd ed, Effective May 1, 2004 Implement standard 5.1.5.1: Implement standard 5.1.5.1: The blood bank or transfusion service shall have methods to limit and detect bacterial contamination in all platelet components. Standard 5.6.2 applies Standard 5.6.2- Protection Against Contamination: Standard 5.6.2- Protection Against Contamination: The venipuncture site shall be prepared so as to minimize risk of bacterial contamination. Green soap shall not be used.

22. 22SCACM Conference Jan. 20, 2009 AABB Accreditation con’t Standard 5.1.5.1.1 (Standard 5.1.5.2, 25 th ed, 2008) Standard 5.1.5.1.1 (Standard 5.1.5.2, 25 th ed, 2008) When a true-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 5.2.2 and 7.1 to 7.1.4 apply. When a true-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 5.2.2 and 7.1 to 7.1.4 apply. Standard 5.2.2: Donor Notification of Abnormal Findings and Test Results (Standard 5.2.3, 25 th ed, 2008) Standard 5.2.2: Donor Notification of Abnormal Findings and Test Results (Standard 5.2.3, 25 th ed, 2008) Standards 7.1-7.1.4: Nonconformances Standards 7.1-7.1.4: Nonconformances

23. 23SCACM Conference Jan. 20, 2009 AABB Accreditation con’t Standard 5.6.6.1 (25 th ed, 2008) Standard 5.6.6.1 (25 th ed, 2008) 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.

24. 24SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components Bacterial detection tests 3 devices are cleared for quality control monitoring of platelet collection process of leukoreduced platelets  BioMeriuex BacT/ALERT®  Pall eBDS  hemoSystems Scansystem™ Other non approved and non validated methods are also being used to meet the AABB standard for bacterial detection

25. 25SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components FDA concerns with bacterial detection as currently applied () FDA concerns with bacterial detection as currently applied (Vostal JG, Jan. 28, 2005 CBER workshop) Test performance characteristics unknown Use of non-validated tests (glucose and pH by dipstick, swirling) Non-standardized methodology even with culture-based devices Potential for excessive false positives or negatives Less reliable methods are used on whole blood derived platelets creating a two tiered safety system for apheresis and whole blood derived platelets

26. 26SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components Manual tests- validation required for pH & glucose Manual tests- validation required for pH & glucose pH pH Glucose Glucose Swirling Swirling

27. 27SCACM Conference Jan. 20, 2009

28. 28SCACM Conference Jan. 20, 2009 Devices approved for QC detection of platelet bacterial contamination- Pall BDS

29. 29SCACM Conference Jan. 20, 2009 Pall eBDS Sample set/Oxygen Analyzer Sterile weld platelet component to set Fill pouch with ~3 mL of product Disconnect sample pouch from set and incubate at 35°C for 24-30 hrs Measure the O 2 content in the air above the plasma sample with insertion of analyzer probe into pouch LED display will read PASS or FAIL

30. 30SCACM Conference Jan. 20, 2009 Pall eBDS Pall Medical eBDS Brochure, 2004

31. 31SCACM Conference Jan. 20, 2009 BioMeriuex BacT/ALERT® Colorimetric technology/Sensor Culture bottles CO 2 release causes sensor bottle to turn yellow Instrument measures & detects color change, analyzes data to determine positivity, alerts when positive culture

32. 32SCACM Conference Jan. 20, 2009 hemoSystems Scansystem™ Scansystem Platelet Kit/Scansystem Analyzer After sample processing in platelet kit, bacteria are stained with a green fluorescence and retained on the surface of a dedicated membrane (platelets are aggregated and lysed) Membrane is inserted in analyzer and scanned by an Argon laser Each fluorescent spot on the membrane will be detected and analyzed Analyze 3 platelet components (SDP, WBDP)

33. 33SCACM Conference Jan. 20, 2009 hemoSystems Scansystem™ Scansystem™ Brochure accessed at www.hemosystem.com

34. 34SCACM Conference Jan. 20, 2009 Preparing Platelets for Culture Sterile Connection Device

35. 35SCACM Conference Jan. 20, 2009 Connecting Platelets to Syringe Set

36. 36SCACM Conference Jan. 20, 2009 Tubing from syringe set Tubing from platelet bag

37. 37SCACM Conference Jan. 20, 2009 Aliquoting Platelet Sample from Transfusion Bag (Suspected Transfusion Reaction ) Syringe set SDP Transfusion set

38. 38SCACM Conference Jan. 20, 2009 Aliquoting Platelet Sample for Bacterial Detection Testing

39. 39SCACM Conference Jan. 20, 2009 Aliquoting Platelet Sample for Bacterial Detection Testing

40. 40SCACM Conference Jan. 20, 2009 Inoculating Culture Bottle (pediatric size)

41. 41SCACM Conference Jan. 20, 2009 Sterile Connection Port

42. 42SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components-2008 ARC Study of bacterial contamination before & after implementation of sample diversion pouch (Pall Acrodose with eBDS) Prestorage pooling of WBDP with culture testing Jan 2003-December 2006 Benjamin RJ, et al. Transfusion 2008;48:2348-55

43. 43SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components-2008 Benjamin RJ, et al. Transfusion 2008;48:2348-55

44. 44SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components-2008 Benjamin RJ, et al. Transfusion 2008;48:2348-55

45. 45SCACM Conference Jan. 20, 2009 Bacterial Contamination of Blood Components-2008 Benjamin RJ, et al. Transfusion 2008;48:2348-55 PSP=prestorage pooled WBDP

46. 46SCACM Conference Jan. 20, 2009

47. 47SCACM Conference Jan. 20, 2009 Blood Transfusion Safety- FDA Blood Transfusion Fatality Report http://www.fda.gov/Cber/

48. 48SCACM Conference Jan. 20, 2009 Why test at point of issue ? Wide variation in duration of Lag Phase Wide variation in rate of log Phase Growth Source: Goodnough LT et al. NEJM 1999, Klein et al.JAMA, vol 274, issue 1368 –1373, November 1,2005, Goodman et al. Bacterial Contamination of blood Components: Risk, Strategy and regulation, Hematology 2003

49. 49SCACM Conference Jan. 20, 2009 New FDA Approved Device for Transfusion Services The Abbott Verax Platelet PGD® Test A rapid, qualitative immunoassay for the detection of Aerobic and Anaerobic; Gram-positive and Gram-negative bacteria in leukocyte reduced apheresis platelets (SDP) ~30 minute TAT SDP must have undergone QC culture by blood supplier!!

50. 50SCACM Conference Jan. 20, 2009 New Device for Transfusion Services

51. 51SCACM Conference Jan. 20, 2009 New Device for Transfusion Services 1. Rapid ~ 25 minutes (3 min hands on) 3. Sensitivity ~ 10 3 CFU/ mL Single-use disposable test Verax rapid Platelet PGD ® Test 2. Positives typically < 10 minutes 4. Specificity > 99.7%

52. 52SCACM Conference Jan. 20, 2009 Pan Genera Rapid Test Format Test Features 2. Shared 300 uL sample addition well 3. Separate GP and GN read windows 4. Procedural controls for GP & GN Procedural Control Procedural Control 1. ABS housing holding GP/GN test strips Sample Well Gram-Negative Read Window Gram-Positive Read Window

53. 53SCACM Conference Jan. 20, 2009 Methods Comparison Verax PGD BacT/ALERT Pall eBDS Technology Technology Conserved Bacterial Ag Immunoassay Culture CO2 Measure Aerobic Culture O2 measure Sample Volume 500 uL 4-20 mLs 3-5 mLs. Time to Result 10 – 30 min (positives typically within 10 minutes) 24 – 96 hours 24 – 30 hours Detect Aerobic and Anaerobic bacteria? Yes Yes, but time varies Misses Anaerobes Clinical Specificity 99.7%99.2-99.8% ~ 99% Source: Abbott, Biomerieux and Pall Medical web site.

54. 54SCACM Conference Jan. 20, 2009 Validation of Bacterial Detection Methods pH & glucose tests are analytically insensitive (Yomatovian R, Brecher ME. Transfusion 2005;45:647-8) pH & glucose tests are analytically insensitive (Yomatovian R, Brecher ME. Transfusion 2005;45:647-8) Validation required Validation required Variable plastic bags Variable plastic bags Variable anticoagulants Variable anticoagulants Variable handling Variable handling Gram stain insensitive unless 10 6 CFU/mL Gram stain insensitive unless 10 6 CFU/mL Facilities may not have FDA approved equipment Facilities may not have FDA approved equipment Validate for QC use Validate for QC use Sensitivity Sensitivity Types of organisms Types of organisms Growth time Growth time

55. 55SCACM Conference Jan. 20, 2009 Snyder JW, et al. BACTEC detection of bacteria in platelet pools. ASM 05-GM-A-4146; 2005

56. 56SCACM Conference Jan. 20, 2009 SUMMARY Bacterial contamination of blood components continues to pose a threat to transfusion recipients Progress to prevent adverse reaction to blood transfusions due to bacterial contamination continues to be seen Microbiologists now play a major role in this progress!!

57. 57SCACM Conference Jan. 20, 2009 UNIVERSITY OF LOUISVILLE MEDICAL CENTER, LOUISVILLE, KENTUCKY THANK YOU!!!!