1. Experience of the Irish Blood Transfusion Service William G. Murphy MD nmd@indigo.ie

2. 2004: 100% testing using 1 x 8 ml sample. 2005: 100% testing using 2 x 7.5 ml samples (aerobic & anaerobic). 2005: extended shelf life to day 7 if day 4 retest was clear. All expired platelets tested with 2 x 10 ml samples to 2010

3. Background Lessons from Yersinia enterocolitica in red cells: Blood collection, processing and storage provides unique microbiological niche(s) Intra-species variation from isolate to isolate Effect of plasma reduction on bacterial clearance or survival [(never) been observed in whole blood.......] Would not introduce additive solution for platelets without bacterial testing

4. Rationale for day 4 retesting 1.Bacterial testing before issue of day 5 platelets improves safety. But does not necessarily improve it to the extent that storage time can be increased. 2.Preparation of platelets can reduce initial level of contamination, increasing sampling error. (Holden et al. Transfusion, 2000)

5. Holden et al. Transfusion, 2000 19 different clinical isolates of coagulase negative Staphylococci spiked into whole blood within 4 hours sampled before and after overnight storage at 22 0 C, from the buffy coat, the pooled buffy coat, and the final filtered product

6. Holden et al. Transfusion, 2000 detectable contamination fell from 15/19 after spiking to 3/19 after filtration for inocula of 1 – 10 cfu/ml and from 16/18 after spiking to 3/18 after filtration for inocula of 10 – 100 cfu/ml

7. Rationale for day 4 retesting………cont’d 3.A test with ~ 99.5% sensitivity to detect a bacterium growing from 1 cfu per bag after manufacture……….. to 10,000 cfu per bag by end day 7………… That can detect 10 cfu per sample volume of 10 mls in 300 mls…….. Needs 92 hours of culture in the bag before sampling (assuming log-linear growth) (Murphy & Smyth Transfusion & Apheresis Science 2001)

9. Rationale A 2 x 7.5 ml sample taken on day 4 of shelf life meets these criteria, and allows > 36 hours of BacTAlert culture time before day 5 expiry and extended storage begins, (and can be reserved for platelets that are needed – CMV neg etc)

10. Growth of organisms in platelets may not be log linear…………..

11. Fig. 1 An isolate of Staphylococcus capitis was detected from a pooled platelet concentrate during the initial pilot project. It was not detected on day 1 or day 4 of screening, but the day 7 (postexpiry) sample was positive. The isolate was inoculated at 1–10 CFU/ml (n = 3) and 10–100 CFU/ml (n = 3) into fresh platelet concentrates on day 2 of shelf life. The numbers on the growth curves indicate the concentration of bacteria (CFU/ml) in quantitative cultures from samples taken daily from day 2 onwards.

12. Fig. 1 An isolate of Staphylococcus capitis was detected from a pooled platelet concentrate during the initial pilot project. It was not detected on day 1 or day 4 of screening, but the day 7 (postexpiry) sample was positive. The isolate was inoculated at 1–10 CFU/ml (n = 3) and 10–100 CFU/ml (n = 3) into fresh platelet concentrates on day 2 of shelf life. The numbers on the growth curves indicate the concentration of bacteria (CFU/ml) in quantitative cultures from samples taken daily from day 2 onwards.

13. Sampling: Class D cleanroom; Inoculation: Specialised technologists; Microbiological safety cabinet Immerse caps in bactericidal/sporocidal agent before inoculation

14. Sampling: Apheresis: minimum 14 hour hold after collection (mean of 17 hours) Pooled platelets: >12 hours after manufacture; >30 hours after venepuncture

15. POSITIVES: False: signal in the machine, no bacteria in the bottle. Confirmed: Bacteria in bottle and in the bag, another component of the donation, or the recipient. Unconfirmed: Bacteria in the bottle, but not in the bag; no bag; no split; no reaction; not in another component.

16. Data to August 2007 43,230 platelet units have been screened prior to issue. (15,033 using 8 mls in a single bottle; 28,197 using 2 bottles) 14 confirmed and 21 non-confirmed positives on the initial test. Total positive rate of 0.08%

17. Positive screening tests Confirmed positives Unconfirmed positives Total positives Apheresis platelets N = 12,823 (donations) 4 (0.03%)10 (0.08%)14 (0.11%) Pooled platelets N = 30,407 10 (0.03%)11 (0.04%)21 (0.07%) Total platelets N = 43,230 14 (0.03%)21 (0.05%)35 (0.08%)

18. Positive screening tests Confirmed positives Unconfirmed positives Total positives Day 1 screen N = 43,230 14 (0.03%)21 (0.05%)35 (0.08%) Day 4 retest N = 3310 3 (0.09%)4 (0.12%) Retest at outdate N = 8282 7 (0.09%)11 (0.13%)18 (0.22%) 1 (0.03%)

19. BACTERIA Number of isolates Day 1 screeningDay 4 retestOutdate retest Coagulase- negative Staphylococci 2013 (0.7-5.8) 1 (0.8) 6 (0.1-1.1) P acnes1810 (4.4-5.9) 2 (3.7, 3.5) 6 (4.3 -6.6) Anaerobic Streptococcal spp. 32 (2.1, 2.9) 1 (2.1) Bacillus spp.32 (0.9, 2.3) 1 (0.6) Proteus mirabilis 11 (0.9) Brevibacterium sp 11 (4.8)

20. False negative rate of the initial screening test was taken as the positive rate at outdate: 18/8282 (= 0.002) The sensitivity of the screening test was calculated as the number of observed positives / probable total number of positives %: observed total positive% observed total positive % + false negative %

21. Sensitivity of the screening test = 29.23%; (95% C.I.: 19.35% to 39.1%)

22. Positive screening tests 12 of 35 contaminated units were transfused: i.e. screening effectiveness was ~66% (no reactions reported)

23. Estimating the number of bacteria in the initial contaminating inoculum: Of 24 two-bottle positives - 11 were Propionibacterium or strict anaerobes; 13 should grow in both bottles: none did 8 grew in aerobic culture only 5 grew in anaerobic culture only.

24. Mean number of bacteria in the platelet units at sampling 1.386 (SE: 1.414) cfu per test volume i.e. less than 60 cfu per platelet unit in most instances.

25. Conclusion: Low sensitivity due to low numbers of bacteria and delayed or slow growth means that sampling will never reach an acceptable level of detection no matter how large the sample or sensitive the test. Leading to morbidity, recalls, lost products.

26. Apheresis data Nov 2004 to Aug 2012 Apheresis Platelets Total tested (donations) # Confirmed PosOrganisms Day 153872196 x P. acnes 1 x Propionibacterium sp 2 x Staph capitis 2 x Staph epidermidis 2 x Coag Neg Staph 1 x Staph aureus 1 x Proteus mirabilis 1 x Bacteroides thetaiotamicron 1 x Micrococcus luteus 1 x Kocuria varians 1 x Listeria monocytogenes Day 43287911 x Staph capitis Expiry (no 4 test ) 549321 x Leuconostoc sp 1 x Propionibacterium sp

27. Apheresis (donations) Year apheresis (Day 1) extended life apheresis (Day 4)falsefalse rate non- confirmed Non- confirmed rateconfirmed confirmed rate 20041007010.0991 00.000 2005536529180.14930.0563 200648412328140.19540.08430.042 20075332371880.08830.033 4 (1 x day 4)0.044 200865824554110.099110.09930.027 20097631538190.06950.03820.015 201083645889170.12080.05720.013 201186616076110.07560.04110.007 20126089464200.0000 20.012 5387232879790.091410.047200.023

28. Day 1 v Day 4 results – apheresis & pools Confirmed Results (2004 – Aug 2011 NBC) PoolsApheresis (donations) Quantity tested Quantity Positive Rate % Quantity tested Quantity Positive Rate % Day 1 59509410.06940157160.035 Day 4 617050.0812624210.004 Expiry (Day 6 or 8) 529340.0763479 2*2* 0.057 giveblood.ie You get more than you give

29. Haemovigilance Data for this period (units shipped): Total Apheresis PooledReactions 2012 (est)14,00012,0002,000 0 2005 - 2011162,05595,55166,504 0 2005 (Mandatory Haemovigilance reporting; HV staff in all hospitals) 2004 17,598 8,105 9,493 0 (Testing begins, April) 2003 16,544 6,856 9,6880 2002 15,480 6,363 9,1170 2001 14,131 5,088 9,043 1 Platelet pool, febrile reaction, bag and patient’s blood culture positive for CNS. 2000 13,922 4,871 9,0510

30. IBTS – next steps: Pathogen Inactivation v. Bacterial Testing