RAPID BACTERIAL DETECTION METHODS FOR PLATELETS. M. Schmidt
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1 RAPID BACTERIAL DETECTION METHODS FOR PLATELETS M. Schmidt Deutsches Rotes Kreuz Blutspendedienst Baden-Württemberg-Hessen Goethe Universität Frankfurt am Main Institut für Transfusionsmedizin und Immunhämatologie
2 Schrezenmeier et al Transfusion, 2007;47: Hourfar et al Transfusion, 2008;48: Rapid bacterial detection methods for platelets 2 German Red Cross bacteria and virus study Bacterial risks Viral risks Contaminated platelets 1:1.428 Septic reactions 1: HBV HCV HIV 1 : 360,000 (CI: million) 1 : million ( million) 1 : 4.3 million ( million)
3 Fatalities due to transmission of bacteria 4x PRC, 4xAPC, 4xPPC 3 Anzahl der Fälle (n) Fraglicher Fall Bestätigter Fall Tod des Empfänger durch Transfusion von EKs Tod des Empfängers durch Transfusion von TKs Funk et al Transfus Med Hemoth, 2011 Jahre
4 Funk et al Transfus Med Hemoth, 2011 Rapid bacterial detection methods for platelets Bacteria strains in blood components 4 PRC storage temperature 2-10 C PC storage temperature 21 C 6/8 contaminated PCs were transfused at the end of shelf life Strains in PRCs: Serratia marcenses Staphylococcus aureus Yersinia enterocolitica (2 cases) Strains in PCs: Klebsiella pneumoniae (2 cases) Staphylococcus aureus Staphylococcus epidermidis Streptococcus pyogenes (3 cases) Enterococcus cloacae
5 Schmidt et al Manuscript in preparation Rapid bacterial detection methods for platelets Growth kinetics of K pneumoniae 5 Growth kinetics in PRCs Growth kinetics in PCs Bacterial concentration in CFU/ml time (h) Bacterial concentration in CFU/ml time (h)
6 Votum 38 working group blood Reduction of platelet shelf life to 4 days
7 Dreier et al Clinical Chemistry, 2009 Rapid bacterial detection methods for platelets 7 First description of bactiflow
8 8 Investigation of pool platelets and apheresis platelets on day 3, day 4 and day 5 after spiking with BacT/ALERT, Bactiflow, inhouse FACS and 16s DNA NAT Spiking day 1 day 2 day 3 day 4 day 5 Time (days) W. Sireis Vox Sanguinins 2011
9 9 Results Bactiflow with apheresis platelets Counts (n) Decision line A B C A B C A B C A B C A B C A B C A B C K. pneumoniae (PEI) K. pneumoniae (GRC) S. epidermidis S. aureus S. pyogenes B. cereus S. marcescens W. Sireis Vox Sanguinins 2011
10 10 Results BacT/ALERT with apheresis platelets Maximum incubation time in BacT/ALERT Time (h) A B C A B C A B C A B C A B C A B C A B C K. pneumoniae (PEI) K. pneumoniae (GRC) S. epidermidis S. aureus S. pyogenes B. cereus S. marcescens W. Sireis Vox Sanguinins 2011
11 11 Comparison of all results with rapid bacterial detection systems for pool platelets BacT/ALERT Bactiflow FACS method 16s DNA method Bacterial strains Day 3 Day 4 Day 5 Day 3 Day 4 Day 5 Day 3 Day 4 Day 5 Day 3 Day 4 Day 5 K. pneumoniae PEI K. pneumoniae BRC S. epidermidis 7 / 10 8 / 10 S. aureus 0 / 10 9 / 10 9 / 10 S. pyogenes 9 / 10 7 / 10 3 / 10 2 / 10 9 / 10 B. cereus 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 S. marcescens W. Sireis Vox Sanguinins 2011
12 12 Comparison of all results with rapid bacterial detection systems for apheresis platelets W. Sireis Vox Sanguinins 2011
13 13 Zelos x100 extraction system
14 Comparison of automated NAT systems 14
15 Description of screening process: Pooling (up to 10 samples per pool) 15 Extraction Zelos x100 Amplification ABI 7500
16 German Red Cross BAK PCR Kit POS controls: 16
17 German Red Cross BAK PCR Kit Internal controls: 17
18 18 Investigation of pool platelets and apheresis platelets on day 2, day 3 and day 4 after spiking with 16s DNA NAT Spiking day 1 day 2 day 3 day 4 Time (days)
19 19 Data for buffy coat derived minipool platelets ID-NAT MP-NAT with pools of 5 samples MP-NAT with pools of 10 samples Bacterial strain day 2 day 3 day 4 day 2 day 3 day 4 day 2 day 3 day 4 K. pneumoniae 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 K. pneumoniae Bayern 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 S. epidermidis 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 S. aureus 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 S. pyogenes 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 B. cereus 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 S. marcesens 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10
20 20 Data for apheresis platelets ID-NAT MP-NAT with pools of 5 samples MP-NAT with pools of 10 samples Bacterial strain day 2 day 3 day 4 day 2 day 3 day 4 day 2 day 3 day 4 K. pneumoniae 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 K. pneumoniae Bayern 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 S. epidermidis 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 S. aureus 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 S. pyogenes 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 B. cereus 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 S. marcesens 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5
21 Specificity of the German Red Cross bacteria NAT system 21 Specificity was investigated for buffy coat derived minipool platelets as well as for apheresis platelets on individual samples (volume 6ml) Samples were collected at the end of platelet shelf life Buffy coat derived minipool - platelets Apheresis platelets Total number of products Initial reactive products 0 0 confirmed reactive products 0 0 confirmed positive products 0 0 Specificity >99,5% >99,5%
22 PCR/Bactiflow PCR 4 5 BacT/ALERT without screening Donation day 1 after donation day 2 after donation day 3 day 4 after after donation donation Time (days) day 5 after donation day 6 after donation
23 Issue of PCs in BDS Baden-Württemberg - Hessen 23 Schrezenmeier et al. Transfusion 2007;47:644
24 Data bacterial screening since October Total number of investigation (N) (34,4%) Platelets shelf life extended Platelets released on day 5 Platelets discarded on day 6
25 Data bacterial screening Bactiflow (IR app. 2%) 25 30" 25" Total number tested 2,050 platelets No confirmed reactive samples 20" 15" 10" ULM" OST" FFM" MA" Gesamt" 5" 0" IR" IR"(%)" WR" WR"(%)"
26 Data bacterial screening NAT (IR app. 0.2%) 26 2,5" 2" FFM#PCR# Total number tested 1,045 platelets No confirmed reactive samples 1,5" 1" FFM"PCR" 0,5" 0" IR" IR"(%)" WR" WR"(%)"
27 27 Diversion of the first 30 to 40 ml TIME (days)
28 28 Diversion of the first 30 to 40 ml Strategy 1: Culture tests in combinafon with the negafve- to- date concept TIME (days)
29 29 Diversion of the first 30 to 40 ml Strategy 2: Pathogen reducfon methods Strategy 1: Culture tests in combinafon with the negafve- to- date concept TIME (days)
30 30 Diversion of the first 30 to 40 ml Strategy 3: Release on day 1 and day 2 aler donafon without tesfng Strategy 2: Pathogen reducfon methods Rapid bacterial detecfon systems on day 3 Strategy 1: Culture tests in combinafon with the negafve- to- date concept TIME (days)
31 31 Diversion of the first 30 to 40 ml Strategy 4: ReducFon of platelet shelf life Strategy 3: Release on day 1 and day 2 aler donafon without tesfng Strategy 2: Pathogen reducfon methods Rapid bacterial detecfon systems on day 3 Strategy 1: Culture tests in combinafon with the negafve- to- date concept TIME (days)
32 Acknowledgement 32 E. Seifried W. Sireis B. Rüster M.K. Hourfar K. Gubbe G. Capalbo U. Mayr-Wohlfart U.M. Liebscher H. Schrezenmeier H. Klüter M. Heiden E.-M. Schneider T. Montag M. Störmer M. Nübling
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