1. A summary review provided by the American Red Cross Blood Services Regions serving the North Atlantic Area July, 2003 Bacterial Contamination in Blood Products Risks, Prevention and Detection

2. Bacterial Contamination in Blood Products Agenda What is the Problem? What are the Risks? What Organisms are Associated with Bacterial Contamination? What are the Sources of Contamination? What Corrective Actions are Planned?

3. Recent Advances in Testing Technology 1990-2003 Anti-HCV (1990) Multi-antigen anti-HCV (1992) Anti-HIV1/2, replacing anti-HIV-1 (1992) HIV-1 p24 antigen (1996) HCV/HIV NAT (IND) (1999) Licensed NAT (2003) West Nile Virus (IND) (2003)

4. Comparison of Residual Risks HIV HBV HCV 1996199419921990198819861984 1:100 1:1000 1:10 000 1:100 000 1:1 000 000 1998 2000 Transmission risk, per unit Updated from: Goodnough LT e t al. NEJM 1999;341:126-7 2002 BacterialContamination(platelets) SepticFatalities(platelets) Clinical Sepsis (platelets)

5. Bacterial Contamination of Blood Products First recognized infectious risk of blood transfusion Risk greatly reduced in the 1960s by the use of closed, sterile systems for the collection and storage of blood Recent dramatic improvements in safety from viral screening and testing have reduced the risks from Hepatitis and HIV Bacterial sepsis is now the most common infectious disease event following transfusion

6. Bacterial Contamination of Blood Products Bacterial contamination occurs primarily in room-temperature stored products (platelets) but can occur in red blood cells and plasma also The blood banking community is taking steps to improve prevention and detection of bacterial contamination The American Association of Blood Banks, as well as the College of American Pathologists have established compliance criteria for transfusion services

7. Bacterial Contamination in Blood Products The American Association of Blood Banks has issued two new standards (March, 2003): “5.1.5.1 The blood bank or transfusion service shall have methods to limit and detect bacterial contamination in all platelet components.” “5.1.5.1.1 Standard 5.1.5.1 shall be implemented by March 1, 2004” “5.6.2 The venipuncture site shall be prepared so as to minimize the risk of bacterial contamination. Green soap shall not be used”

8. Bacterial Contamination in Blood Products College of American Pathologist’s Accreditation Checklist (December, 2002): “TRM.44955 Phase 1 Does the laboratory have a system to detect the presence of bacteria in platelet components?”

9. Bacterial Contamination of Blood Products What are the Risks?

10. Risk of clinical sepsis Ness et al, Transfusion 2001;41:857-60. Identified clinical cases of transfusion associated sepsis over a 12 year period, with conversion from 51.7% random donor platelets to 99.4% SDPs The #donors/septic event remained constant at 15,000 throughout the 12 year period, despite the conversion to SDPs Pooled random donor platelets were 5.5-times more likely to cause sepsis than SDPs due to pool size Pooling issues

11. Bacterial Contamination of Blood Products What Bacterial Organisms are associated with Blood Product Contamination?

12. Bacterial species in platelets implicated in clinical sepsis n = 86 Compilation of data from Clin Micro Rev 1994; 7:290-302; Transfusion 2001;41:1493- 99; www.shot.demon.co.uk/toc

13. n = 52 Bacterial species in platelets implicated in septic fatalities reported to the FDA (1976-1998)

14. Differences between the species implicated in septic morbidity and mortality in platelet components S. epidermidis is less commonly observed in septic fatalities and more commonly observed in septic reactions Klebsiella is commonly observed in septic fatalities Gram negative organisms are implicated in more fatalities (60%) than gram positive organisms (40%); gram positives cause a majority of septic reactions (56%)

15. Organisms implicated in sepsis from platelets Approximately 30% are associated with normal skin flora Approximately 56% are gram positive All are aerobic or facultative anaerobes A rare (single case) exception: Clostridium perfringens fatality from a pooled platelet unit Trans Med 1998;8:19-22

16. Bacterial Contamination in Blood Products What are the Sources of Bacterial Contamination?

17. Sources of Bacterial Contamination Skin Surface Contamination Phlebotomy Core Donor Bacteremia Containers and Disposables Environment

18. Avoiding Skin Contamination Diversion of the initial blood flow Improvement in pre-phlebotomy skin cleansing Skin source

19. Diversion of initial blood flow Diversion of initial blood flow into sampling tubes Reduces the load of skin-associated bacteria entering blood container Phlebotomy “core” directed into sampling pouch instead of blood container

20. Clinical data supporting diversion of initial blood flow de Korte et al. Vox Sang 2002;83:13-16 de Korte et al. Vox Sang 2002;83:13-16 Collected blood normally or diverted the first 10mL of whole blood into a satellite bag Collected blood normally or diverted the first 10mL of whole blood into a satellite bag Performed bacterial testing by automated blood culture (BacT/Alert) in a laminar flow hood Performed bacterial testing by automated blood culture (BacT/Alert) in a laminar flow hood Total bacterial prevalence S. epidermidis prevalence StandardCollection 0.35% (0.27- 0.44) 0.14% Collection with diversion 0.21% (0.12- 0.35) 0.03% P-value <0.05<0.02

21. Skin disinfection methods Some agents may reduce the number of surface bacteria more than others Method of application and applicator may have some impact on the extent of reduction of surface bacteria Minimum scrub of 30 seconds required to be effective

22. Impact of Skin Disinfection on surface bacteria Goldman et al, Transfusion 1997;37:309-12 63%

23. Recurrent contamination from the dimpled skin of one plateletpheresis donor Anderson et al., Am J Med 1986;405-11. One donor gave 17 plateletpheresis donations from a scarred dimpled site in the right antecubital fossa Two units were implicated in septic events traced to this donor Four units, including the two units linked to the septic event, were culture positive with coagulase negative Staphylococcus Follow up blood samples obtained from the non- scarred left antecubital fossa were routinely culture negative

24. Recurrent contamination from an asymptomatic bacteremic donor Rhame et al., Ann Intern Med 1973;78:633-41. One plateletpheresis donor was linked to 7 cases of Salmonella cholerae-suis transfusion associated bacterial sepsis; 2 cases were fatal Three of the cases were linked to positive culture of the platelet units The donor had a low-grade bacteremia and unknowingly had Salmonella osteomyelitis of the tibia Donor bacteremia

25. Multiple cases of sepsis from contaminated blood containers Heltberg et al. Transfusion 1993;33:221-7 Högman et al. Transfusion 1993;33:189-91. Serratia marcescens was cultured from three septic patients and their implicated units in Denmark All units were collected using the same lot of blood containers 11 of 1,515 blood products collected using the implicated lot were positive for Serratia marcescens An organism of the same ribotype was isolated from the manufacturing plant The same containers were implicated in Sweden Container source

26. A fatal case of Clostridium perfringens sepsis from a platelet pool McDonald et al., Transfusion Medicine 1998;8:19-22. C. Perfringens is a spore forming facultative anaerobe, found in soil and human intestinal tract Organism recovered from platelet pool; septic recipient was on antibiotics; no organism recovered Patients death was considered a septic event The same serotype of Clostridium was isolated from the arm of 1 of the 4 donors; a subsequent culture of the same arm 6 months later yielded fecal flora The donor was a mother who carried her two toddlers in the crook of her arm Environmental source

27. Bacterial Contamination in Blood Products What Options exist to Prevent and Detect Bacterial Contamination?

28. Bacterial Contamination of Platelets Prevention and Detection Options Donor screening – not feasible except for arm screening. Can’t detect asymptomatic bacteremic donors Arm Preparation-Limited effectiveness of arm scrub Pathogen reduction – not yet available. May not inactivate spore forming organisms Better phlebotomy methods and initial blood diversion Bacterial detection offers best confirmatory option

29. Bacterial Detection Options in Platelet Products Visual examination for discoloration, clumping or abnormal morphology Microscopy Gram stain Acridine orange Measuring Biochemical changes Lowered pH Reduced Glucose Bacterial culture Detection through oxygen consumption Detection through CO 2 production

30. Bacterial Detection Options in Platelet Products Visual Examination Inspect product prior to transfusion for discoloration or abnormal clumping Perform “swirl” procedure to detect morphologic changes in platelets Normal shaped platelets will align with fluid flow and “shimmer” when swirled Contaminated platelets, among others, lose discoid shape and do not “shimmer” when swirled– Not a specific marker for contamination

31. Swirling Low pH Metabolic disturbance No alignment with flow Alignment with flow SENSITIVITY: 75% SPECIFICITY: 95% Leach MF et al. Vox Sang 1998;74(suppl 1):1180.

32. Bacterial Detection Options in Platelet Products Microscopic Methods Gram Stain or Acridine Orange preferred methods Limitations: Must be performed by the Transfusion Service prior to product issue for transfusion Lack sensitivity with low bacterial load

33. Bacterial Detection Options in Platelet Products Measuring Biochemical Changes Measure changes in glucose consumption against a control. Variances of >2 S.D. may indicate bacterial contamination “Dipstick” testing Limitations: Both this method and staining methods are subjective, require high levels of contamination, and must be performed prior to issue by the Transfusion Service

34. Glucose, % Day 0 Storage Time, d after Burstain JM et al. Transfusion 1997;37:255-8. -2 SD Detecting Bacteria in Platelets: Biochemical Changes

35. Chemical Tests - Dipsticks Must be performed immediately before issue because of its relative insensitivity and the need for high bacterial counts

36. Bacterial Detection Options in Platelet Products Blood Culture Methods Two methodologies presently approved by FDA for Quality Control use bioMeriuex BacT/Alert System Pall Biomedical BDS System

37. Bacterial Detection Options in Platelet Products bioMeriuex BacT/Alert System Detects bacterial growth in culture bottles by measuring CO2 production Automated reader continuously monitors samples Sampling interval of >24 hours post phlebotomy Culturing interval of >24 hours post sampling (aerobic and anaerobic cultures) Cultures incubate for 5-7 days; may identify positive cultures post-transfusion FDA-Approved for Q.C. purposes only on Leukoreduced Apheresis Platelets

38. Practical Application of Culturing in a Transfusion Service Laboratory Aubuchon, Dartmouth Experience in first 3 years: 3,927 apheresis units cultured (5 mL into aerobic bottle, BacT/Alert automated system) 23 initial positives (0.5%) in 28 h (10-69) 14 not confirmed on repeat culture 5 not able to be recultured 4 confirmed positives RATE = 1/1,000 units (95% CI: to 1/600)

39. Filter: Stops WBCs+Plts Passes: Bacteria Gas impermeable bag 24 h 24 h at 35C Measure %O 2 in headspace Limit: 19.5% Detecting Bacteria in Platelets: Detection of Growth by O 2 Consumption Pall BDS system

40. Bacterial Detection Options in Platelet Products Pall Biomedical BDS System Detects bacterial contamination by measuring O 2 consumption Automated reader measures O 2 levels in headspace of culture pouch Sampling interval of >24-48 hours Culture performed for >24-30 hours FDA-Approved for Q.C. on leukoreduced platelet concentrates and leukoreduced apheresis platelets

41. Bacterial Detection Options in Platelet Products Limitations of Blood Culture Methods Early sampling/testing may not detect small # bacteria per bag. Approved methods require 24-30 hour wait before sampling Two FDA-Approved methods require bacteria to grow up after sampling to detectable levels, so culture must be done well before planned transfusion (Blood Center) The two time intervals (collection to sampling and sampling to release/transfusion) dominate the logistic considerations

42. Bacterial Detection Options in Platelet Products Limitations of Blood Culture Methods Both options require leukoreduced platelets BacT/Alert requires continued culture after product release Release and recall (BacT/ALERT) or hold to end of culture to release (PALL BDS)

43. Bacterial Detection Options in Platelet Products Limitations of Blood Culture Methods Need to balance the risk of platelet shortages versus the risk of platelet contamination The two available devices are FDA-Approved for Q.C, and not approved as pre-release tests Cost Probable negative impact on outdates Possible extension of platelet storage to seven days or pooling/storing whole blood derived platelets

44. Bacterial Contamination in Transfusable Blood Products AABB Guidance Association Bulletin #03-07 issued May 16, 2003 Provides guidance for methods to limit contamination and to detect contamination

45. AABB Association Bulletin #03-07 May 16, 2003 Methods to Limit Contamination: Careful phlebotomy – No green soap prep Iodine based scrub recommended Consider phlebotomy diversion – “sample first” technologies Consider increased use of apheresis platelets

46. AABB Association Bulletin #03-07 May 16, 2003 Methods to Detect Contamination: Culture methods optimal. Two approved products cited. Other culture methods can be validated. No label claims allowed Due to insensitivity, staining and dipstick methods should be used as close in time to issue as possible Validation of all methods is required “Swirl” procedure useful for inspection but does not by itself meet AABB Standard 5.1.5.1

47. Bacterial Contamination in Blood Products American Red Cross Bacterial Prevention and Detection Strategy Implement prevention and detection strategies to meet the requirements and timelines of the AABB and CAP Solicit customer feedback to develop efficient and cost-effective implementation strategies Keep customers well-informed during the pre- implementation period