Enlargement of WHO Repository Transfusion Relevant Bacteria Reference Strains - Report on experimental preparatory work and study design
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1 WP-TTID Chair: Silvano Wendel, Brazil Subgroup on Bacteria Chair: Thomas Montag, Germany Co-Chair: Erica Wood, Australia Enlargement of WHO Repository Transfusion Relevant Bacteria Reference Strains - Report on experimental preparatory work and study design Dr. Melanie Störmer
2 Overview Enlargement of Repository TRBR Candidates - Enlargement of Repository Transfusion- Relevant Bacteria Reference Strains Experimental Preparatory Work Production of Defined Frozen Suspensions (DFS) Study Design - Enlargement of Repository
3 Candidates Enlargement of Repository Transfusion-Relevant Bacteria Reference Strains Extraordinary Meeting on occasion of AABB Annual Meeting, Baltimore MD, USA, October 9 th, 2010
4 ISBT WP-TTID, Subgroup Bacteria, Extraordinary Meeting On occasion of AABB Annual Meeting, Baltimore MD, USA, October 9 th, 2010 Participants No. Name Facility Country 1 Morris Blajchman McMaster University Canada 2 Dirk de Korte Sanquin Blood Supply Foundation, Amsterdam The Netherlands 3 Robert Duncan FDA, Rockville USA 4 Ray Goodrich Caridian BCT, Denver USA 5 Luisa Gregori FDA, Rockville USA 6 Salim Haddad FDA, Rockville USA 7 Michael Jacobs Case Western Reserve University, Cleveland USA 8 Lisa Jarvis SNBTS Scotland 9 Susanne Marschner Caridian BCT, Denver USA 10 Thomas Montag Paul-Ehrlich-Institute, Langen Germany 11 Thomas Mueller German Red Cross, NSTOB, Springe Germany 12 Sandra Ramirez Canadian Blood Service, Ottawa Canada 13 Paolo Rebulla Centro di Medicina Transfusionale, Terapia Cellulare e Criobiologia, Milano 14 Melanie Stoermer Paul-Ehrlich-Institute, Langen Germany 15 Jaroslav Vostal FDA, Rockville USA 16 Silvano Wendel Hospital Sirio Libanês, São Paulo Brazil 17 Erica Wood Australian Red Cross Blood Service, Melbourne Australia Italy 18 Roslyn Yomtovian Louis Stokes Veterans Administration Medical Center, Cleveland USA
5 Enlargement of Repository Recommendations of ISBT Bacteria Group
6 1 St. aureus and 1 Coagulase-negative Staphyloccus (CNS) 1 aerobic spore forming bacterium (isolated spores) no anaerobic bacteria 1 Klebsiella species 1 Pseudomonas species 1 Streptococcus species no fungi Enlargement of Repository Recommendations of ISBT Bacteria Group no red cells (RBC) (Yersinia enterocolitica)
7 Enlargement of WHO Repository - Candidates Asking partners for providing candidates for enlargement! Species Validation Study 2008/2009 Staphylococcus epidermidis Streptococcus pyogenes Escherichia coli Klebsiella pneumoniae Enlargement candidates Bacillus thuringiensis spores Enterobacter cloacae Morganella morganii Proteus mirabilis Pseudomonas fluorescens Salmonella cholerae-suis Serratia marcescens Staphylococcus aureus Streptococcus dysgalactiae
8 Enlargement of WHO Repository Provided Isolates Provided Transfusion Reaction Investigation Isolates Partner Strain Sourc e Sandra Ramirez- Arcos Canadian Blood Service, Ottawa, Canada Code PEI- Code Bacillus cereus PC CBS PEI-A-57 Bacillus cereus PC CBS PEI-A-58 Bacillus cereus PC CBS PEI-A-59 Proteus mirabilis PC CBS A PEI-A-55 Serratia marcescens PC CBS PEI-A-60 Carl McDonald NHS Blood and Transplant, London, United Kingdom Enterobacter cloacae RBC PT/BAC/38/07/M PEI-A-43 Group G Streptococcus (dysgalactiae) Group G Streptococcus (dysgalactiae) PPC PT/BAC/69/08/M PEI-A-37 APC PT/BAC/44/08/H PEI-A-42 Streptococcus bovis APC PT/BAC/29/06/T PEI-A-41
9 Enlargement of WHO Repository - Candidates Validation Study 2008/ Staphylococcus epidermidis 2. Streptococcus pyogenes 3. Escherichia coli 4. Klebsiella pneumoniae Enlargement Candidates 5. Bacillus thuringiensis spores 6. Enterobacter cloacae 7. Morganella morganii 8. Proteus mirabilis 9. Pseudomonas fluorescens 10. Salmonella cholerae-suis 11. Serratia marcescens 12. Staphylococcus aureus 13. Streptococcus dysgalactiae (G) Buffer 14. Bacillus cereus 15. Bacillus cereus 16. Bacillus cereus 17. Streptococcus bovis 18. Streptococcus dysgalactiae (G) Exclusion after pre-characterisation (no ability to grow in PCs)
10 Experimental Preparatory Work Production of Defined Frozen Suspensions (DFS)
11 Production of Defined Frozen Suspensions (DFS) New Isolate Cultivation Identification of Species (GRAM, API, 16S DNA Sequencing) Inclusion into Master Bacteria Bank, MBB Production of Mastersuspension MBB+1 Frozen Suspension used for production of new lots of DFS Setting of Dilution of MBB+1 Growth characteristics (computer based impedance curves) of dilution steps Selection of dilution growing within a workable time
12 Production of Defined Frozen Suspensions Setting Point of Harvest Impedance curves of selected dilution In parallel enumeration by plate assay: Growth curve Production of new Lot Harvest at the defined time point and enumeration 24 hours after freezing Proof of Identity & Batch to Batch consistency Gram-stain, API, Sequencing of bacterial 16S rrna, DNA-Fingerprint Stability Testing during storage (enumeration) Growth ability in PCs Growth in PCs (>100 different donors) Growth independence on innate immunity Bacteria Reference
13 Enlargement of WHO Repository - Candidates Validation Study 2008/ Staphylococcus epidermidis 2. Streptococcus pyogenes 3. Escherichia coli 4. Klebsiella pneumoniae Enlargement Candidates 5. Bacillus thuringiensis spores 6. Enterobacter cloacae 7. Morganella morganii 8. Proteus mirabilis 9. Pseudomonas fluorescens 10. Salmonella cholerae-suis 11. Serratia marcescens 12. Staphylococcus aureus 13. Streptococcus dysgalactiae (G) Buffer 14. Bacillus cereus 15. Bacillus cereus 16. Bacillus cereus 17. Streptococcus bovis 18. Streptococcus dysgalactiae (G) Growth ability
14 Study Design Enlargement of Repository
15 Enlargement: Study Design PART 1 Pre-Characterisation Candidates will be sent to the participating laboratories Identification Enumeration Growth in PC Selection of 6 Candidates for Enlargement of Repository PART 2 Enlargement Study 15
16 PART 1 Advisement: Saving PCs Enlargement of WHO Repository Pre-charactersation of growth ability in fresh frozen plasma instead of PCs Artificial contamination of plasma (100%, 35%) in cell culture bottles (100 cfu/bottle) and incubation at 22.5 C for 4 days Daily sampling for automated culture Enumeration after 4 days incubation
17 PART 1 Advisement: Saving PCs Enlargement of WHO Repository Pre-charactersation of growth ability in fresh frozen plasma instead of PCs Artificial contamination of plasma (100%, 35%) in cell culture bottles (100 cfu/bottle) and incubation at 22.5 C for 4 days Daily sampling for automated culture Enumeration after 4 days incubation Conclusion: PCs are necessary Some strains did not grow in FFP
18 Proposal: Study Design PART 1 Pre- characterisation 9 Candidates will be sent to the participating laboratories Starting as soon as possible Results up to May 2012 Selection of 6 candidates Circulation of Study Design Enlargement Growth in PC (n=5) Selection of 6 Candidates for Enlargement of Repository Submission of Study Design to ECBS ( ) PART 2 Enlargement Study Winter 2012 Submission of results on Growth in PCs (n=?) 18
19 Thank You! Discussion: Study Design Enlargement
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