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Working Party Transfusion-Transmitted Infectious Diseases (WP-TTID) Chair: Silvano Wendel, Brazil

Working Party Transfusion-Transmitted Infectious Diseases (WP-TTID) Chair: Silvano Wendel, Brazil . Subgroup Bacteria Chair: Thomas Montag, Germany Co-Chair: Erica Wood, Australia. International Validation Study on Blood Bacteria Standards.

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Working Party Transfusion-Transmitted Infectious Diseases (WP-TTID) Chair: Silvano Wendel, Brazil

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  1. Working PartyTransfusion-TransmittedInfectious Diseases(WP-TTID)Chair: Silvano Wendel, Brazil Subgroup BacteriaChair: Thomas Montag, Germany Co-Chair: Erica Wood, Australia International Validation Study on Blood Bacteria Standards

  2. Background of International Validation Study on Blood Bacteria Standards:Why do we need a panel of transfusion-relevant bacteria?

  3. Bacterial contamination of PCs 1:2.000 Septic fatalities PCs 1:140.000 Infection Risk in Platelets: bacterial vs viral risk per unit 2 1:10 HCV 3 1:10 HBV 4 1:10 5 HIV 1:10 1:230.000 6 1:10 1:5.000.000 1:5.000.000 2000 1984 1988 1992 1996 2004 2006 according to Mike Busch, modified

  4. Crux in Bacterial Contamination of Blood Components Usually, bacteria are contaminating blood donations in a very low count(10 to 100 CFU per bag corresponding to 0.03 to 0.3 CFU / ml).Thereafter, they can grow up in platelets to 108 to 109 CFU / ml(in dependency on species and strain). Artificial contamination of platelets imitating “real life” conditions:-> contamination with 0.03 CFU / ml-> storage at 22.5 °C under agitation

  5. Kinetic of microbial growth in blood components Objective validation and assessment of methods for improvement of microbial safety of cellular blood components (Screening, Pathogen Reduction) requires: -> defined (growing) strains -> “real life” conditions, i.e. bacteria multiplying in the matrix (acute spiking may produce uncertain results) CFU/ml 1,000,000,000 100,000,000 10,000,000 1,000,000 100,000 10,000 1,000 100 10 1 0.1 bacterial growth curve lag phase 0.03 CFU/ml T0 Blood donation

  6. Growth of different bacteria strains in platelet concentrates

  7. Consequence: The established bacterial reference strains (e.g. from ATCC) are not (automatically) applicable for validation studies regarding methods for improvement of microbial safety of cellular blood components. Decision:Development of PEI Blood Bacteria Standards during the past 10 years

  8. Blood Bacteria Standards (References) are able to growin PCs up to high counts (what is not automatically given in case of reference strains like ATCC strains). • Strains grow up in PCs independent on donor properties (tested for relevant multiplication in PCs from at least 100 different donors). • The standards are deep frozen, ready to use, stable, and shippable (manufactured by a special procedure). • They are defined in count and consist mainly of living cells (as a rule > 95% living cells). • The standards allow “real life” spiking of blood components, i.e. artificial contamination with ~ 10 CFU per bag corresponding to 0.03 CFU per millilitre. • Thus, Blood Bacteria Standards are a feasible tool for objective validation and assessment of methods for screening and pathogen reduction in blood components. What are Blood Bacteria Standards ?

  9. Prototype Certificate of one of the Blood Bacteria Standards

  10. Prototype Certificate of one of the Blood Bacteria Standards

  11. Prototype Certificate of one of the Blood Bacteria Standards

  12. Partners of InternationalValidation Study on Blood Bacteria Standards

  13. International Validation Study on Blood Bacteria Standards 14 partner laboratories from 10 countries

  14. Study Design

  15. B B B B C C C C D D D D A A A A A B C D 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL A D2 A D1 A D3 A D1 A D2 A D4 A D5 A D6 A D3 A D1 A D4 A D5 A D6 A D3 A D1 A D2 A D4 A D5 A D6 A D3 A D2 A D6 A D4 A D7 A D5 A D3 A D5 A D3 A D2 A D3 A D? A D1 A D2 A D3 A D4 A D1 A D5 A D7 A D8 A D1 A D2 A D3 A D4 A D4 A D6 A D8 A D2 A D5 A D5 A D6 A D7 A D8 A D1 A D2 A D6 A D5 A D7 A D2 A D4 A D5 A D6 A D3 A D1 A D2 A D4 A D5 A D4 A D6 A D3 A D1 A D5 A D4 A D4 A D2 A D6 A D1 A D2 A D5 A D3 A D3 A D1 A D2 A D4 A D6 A D6 A D1 A D2 A D4 A D5 A D6 A D3 A D2 A D3 A D? A D1 A D3 A D4 A D6 A D3 A D5 A D6 A D3 A D1 A D2 A D4 A D1 A D5 A D3 A D1 A D2 A D4 A D5 A D6 A D1 A D2 A D4 A D5 A D6 A D6 A D7 A D4 A D8 A D2 A D8 A D4 A D5 A D6 A D3 A D2 A D2 A D3 A D2 A D? A D1 A D2 A D3 A D8 A D4 A D5 A D7 A D1 A D6 A D3 A D3 A D8 A D1 A D4 A D1 A D1 A D5 A D6 A D7 A D6 A D1 A D2 A D4 A D5 A D6 A D3 A D1 A D2 A D4 A D5 A D6 A D3 A D1 A D2 A D4 A D5 A D6 A D2 A D5 A D3 A D6 A D2 A D4 A D5 A D6 A D3 A D2 A D3 A D1 A D2 A D3 A D? A D5 A D5 A D4 A D4 A D2 A D3 A D8 A D7 A D6 A D1 A D3 D1 D1 D1 D1 D5 D5 D5 D5 D2 D2 D2 D2 D6 D6 D6 D6 D3 D3 D3 D3 D7 D7 D7 D7 D4 D4 D4 D4 D8 D8 D8 D8 Complete protocol (4 standards) Sum: 4 x 190 = 760 agar plates according to Melanie Störmer, PEI, and Bernd Lambrecht, GRC

  16. Results ofInternational Validation Study on Blood Bacteria Standards

  17. Part 1Identification of blinded Bacteria Standards * In one case, determination as Staphylococcus delphinii instead of Staphylococcus epidermidis (most likely to the commercial identification kit used) but identified at least as coagulase-negative Staphylococcus.

  18. Part 2Enumeration of Blood Bacteria Standards Data from South Africa to be added.

  19. Part 2Enumeration of Blood Bacteria Standards Data from South Africa to be added.

  20. Part 2Enumeration of Blood Bacteria Standards Data from South Africa to be added.

  21. Part 2Enumeration of Blood Bacteria Standards Data from South Africa to be added.

  22. Part 2Enumeration of Blood Bacteria Standards

  23. Part 3Growth of Blood Bacteria Standards in PC(summarised results) Table does not consider all details, e.g. growth in case of contamination of PCs applying 100 CFU per bag but no growth applying 10 CFU per bag, repetitions, pooled or apheresis PCs (will be described in detail in study report).

  24. WHO Collaborative Centres Meeting(Reference Materials Blood & IVD) 2009 February 17th to 19thLangen (PEI), Germany:Decision on start of formalised procedure for submission of Blood Bacteria Standards (Transfusion-relevant Bacterial Panel) to WHO Expert Committee for Biological Standardisation (ECBS).

  25. Relevant remark: The Blood Bacteria Standards are applicable for the novel Advanced Therapy Medicinal Products (ATMPs: Cell Therapeutics, Gene Therapeutics and combinations).

  26. The Blood Bacteria Standards are applicable for the novel Advanced Therapy Medicinal Products (ATMPs), e.g. Cell Based Medicinal Products.Current topic:Use of Blood Bacteria Standards in BEST Study on Sterility Testing of Stem Cells(decision in Krakow, Poland, April 24th, 2009)

  27. PEIMelanie StoermerUtta SchurigSven-Boris NicolJulia BrachertUte Sicker Rekia BeshirChristian Schneider The study partners The study coordinating group WHOAna PadillaGabriele UngerMichael ChudyHeiner Scheiblauer I have to say “Thank you very much!” to: and to you for attention

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