Vox Sanguinis (2014) 107, 192–195 © 2014 International Society of Blood Transfusion DOI: 10.1111/vox.12148
SHORT REPORT
Evaluation of a universal point-of-issue assay for bacterial detection in buffy coat platelet components S. Ramirez-Arcos, Y. Kou & H. Perkins Canadian Blood Services, Ottawa, ON, Canada
Received: 22 November 2013, revised 6 January 2014, accepted 24 February 2014, published online 27 March 2014
Bacterial contamination of platelet concentrates poses a major post-transfusion infectious risk. This study was aimed at evaluating the efficacy of the BacTx assay (Immunetics Inc.) for bacterial detection in leucocyte-reduced buffy coat platelet pools and for its sensitivity in detecting clinical isolates, including bacteria that form surface-attached aggregates (biofilm positives). Platelet pools were inoculated at bacterial concentrations of 08–13 CFU/ml. The BacTx assay detected all species at concentrations ≥103 CFU/ml within 20–69 h of platelet incubation. Detection of slow-growing and biofilm-forming strains was delayed in comparison with the other strains. This assay could be used as a point-of-issue method to increase the safety of the platelet supply. Key words: BacTx, biofilm-positive bacteria, platelet contaminants.
Introduction Testing of platelet concentrates (PCs) for bacterial contamination has considerably increased the safety of the platelet supply worldwide [1, 2]. However, platelet contaminants still escape detection as exemplified by reports of false-negative transfusion cases [2]. To further reduce the occurrence of adverse transfusion reactions due to bacterially contaminated PCs is a challenge. Development of more sensitive screening methods for use by the blood suppliers, implementation of pathogen reduction technologies, and using a rapid screening test prior to transfusion are options which have different advantages and disadvantages. Additional challenges are posed by bacteria that form aggregates able to attach to the inner surface of platelet containers or to platelets (biofilm positives). We have shown that biofilm formation increases the risk of missed detection during routine platelet screening [3]. Furthermore, biofilm-negative bacteria can convert to a biofilmpositive phenotype when incubated in PCs, displaying increased pathogenicity [4].
Correspondence: Sandra M. Ramirez-Arcos, Development Scientist, Canadian Blood Services, 1800 Alta Vista Drive, Ottawa, ON, K1G 4J5, Canada E-mail: [email protected]
192
AABB Bulletin #12-04 recommends several strategies, including additional testing later in the platelet shelf life, to optimize platelet transfusion practice. During the 82nd Meeting of the Food and Drugs Administration (FDA) Blood Products Advisory Committee, it was agreed that point-of-care tests should have a sensitivity of 103–104 colony-forming units per millilitre (CFU/ml). The BacTx assay has received FDA clearance for bacterial testing in leucocyte-reduced apheresis and whole-blood-derived platelet concentrates prepared by the platelet-rich-plasma method that are pooled within 4 h of transfusion. This system provides universal bacterial detection in a single colorimetric assay targeting the bacterial cell wall component peptidoglycan (http://www.immunetics.com/bactx. html). The BacTx assay has a sensitivity >103 CFU/ml and has been shown to be effective for bacterial detection in apheresis and pooled PCs prepared in plasma or additive solutions [5–7]. Herein, the BacTx assay was evaluated for its efficacy in detecting bacterial clinical isolates in buffy coat platelet pools.
Materials and methods Detailed Materials and Methods are described in supplementary material. Briefly, outdated (7–11 days old) buffy coat platelet pools that had tested negative during initial screening were assayed with the BacT/ALERT and BacTx assay systems to confirm sterility. The pools were
Biofilm formation
Pos
Pos
Neg
Neg
Not tested
Not tested
Pos
Pos
Neg
Neg
Bacterial Strain
S. marcescens CBS07/2005 [3]
S. marcescens 12/2004 [3]
S. marcescens CBS11/2010 [3]
S. marcescens CBS12/2010 [3]
E. coli CBS11001
E. coli CBS07007
S. epidermidis CBS6043 [10]
S. epidermidis CBS0726 [9]
S. epidermidis CBS6010 [9]
S. epidermidis CBS6038 [9]
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
Rep 7 11 7 11 8 7 8 7 11 11 11 11 10 10 10 10 10 10 10 10
Platelet age at spiking 08 16 22 22 2 22 1 19 1 14 1 14 13 55 63 54 48 58 3 45
Initial concentration (CFU/ml)
Table 1 Bacterial testing of buffy coat platelet pools with the BacTx assay
Pos Pos Neg Pos Pos Pos Pos Pos Neg Pos Pos Pos Neg Neg Neg Neg Neg Pos Neg Neg
BacTx Result
DAY 1
20 23 24 28 23 23 28 23 24 20 20 20 285 27 285 27 28 28 28 28
Time (h) 13E+04 55E+05 88E+02 56E+03 15E+05 58E+05 16E+04 25E+03 < 10 18E+03 11E+03 75E+02 48E+02 67E+00 23E+02 38E+02 59E+02 12E+03 < 10 31E+02
Concentration (CFU/ml)
© 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107, 192–195 Pos Pos
Pos Neg Pos Pos Pos
Pos
Pos
BacTx Result
DAY 2
52 46
46 51 46 45 47
44
44
Time (h)
77E+03 21E+04
11E+04 63E+02 65E+03 59E+03 12E+04
76E+05
≥ 50E+06
Concentration (CFU/ml)
Pos
BacTx Result
DAY 3
69
Time (h)
17E+04
Concentration (CFU/ml)
Bacterial screening of buffy coat platelets with the BacTx assay 193
194 S. Ramirez-Arcos et al.
inoculated with 10 clinical bacterial isolates listed on Table 1, at a target concentration of 2–5 CFU/ml. Platelet pools were tested with the BacTx assay following the algorithm shown in Fig. 1. Ethical approval for this study was granted by the Canadian Blood Services Research Ethical Board.
Results and discussion In this study, we demonstrate that the BacTx assay is able to detect clinical bacterial isolates in buffy coat platelet pools within 20–69 h of platelet incubation at concentrations ≥103 CFU/ml (Table 1). Other research conducted with the BacTx assay has shown a similar efficiency for bacterial detection in other types of platelet components including apheresis and whole-blood-derived platelet pools prepared by the platelet-rich-plasma method [5–7]. Although targeted to 2–5 CFU/ml, the initial bacterial concentration in the PCs ranged from 08 to 13 CFU/ml (Table 1). Of the 63 BacTx assays performed both on negative control pools and on sterile pools prior to inoculation, no false-positives were observed. Also, there were no contaminated pools that were missed by the BacTx assay, and therefore, no false-negatives were documented during the development of this study. Additional information on the sensitivity and specificity of the BacTx assay should be obtained in a larger study.
• •
Rep 1
CONTROL
Notably, the BacTx assay was able to detect S. marcescens CBS07/2005 and 12/2004, which were involved in false-negative fatal transfusion reactions (Table 1) [3]. Detection of S. epidermidis, known for its slow-growing patterns in PCs, was achieved on days 2–3 postspiking, 1 day later than detection of its Gram-negative counterparts (Table 1). It has been reported that detection of Gram-positive skin flora is usually delayed in comparison to fast-growing organisms [2, 8]. The time of detection for biofilm-positive S. marcescens 12/2004 and S. epidermidis CBS6043 was increased by 1 day in comparison with other strains (Table 1). This result might be due to attachment of bacteria to the inner surfaces of platelet containers which delays bacterial cell release into the supernatant of the PCs as previously reported by our laboratory [9].
Conclusion This study shows that the BacTx assay is efficient in uncovering contaminant bacteria in buffy coat platelet pools. There could be variation in the time of detection between strains of the same species which is intrinsic to the genetic and physiological characteristics of each bacterium. Importantly, detection of a variety of Gram-positive and Gram-negative organisms, including strains involved in fatal transfusion reactions and potentially virulent biofilm-forming isolates, can be attained by the
Three outdated (7-11 days old) buffy coat platelet pools BacT/ALERT® and BacTx® sterility testing
Rep 2
Target concentration: 2-5 CFU/ml
Platelet spiking
Sample for BacTx® testing and bacterial concentration twice (at ~9 AM and ~5 PM) on daily basis for a maximum of five days
If negative bacterial detection with the BacTx® Assay, continue testing
If positive bacterial detection with the BacTx® Assay, repeat test two more times
Control continued to be tested until test units yielded positive results
≥ 2 tests should be positive for a definite positive BacTx® result
Fig. 1 Experimental design. The algorithm followed to test platelet components in this study is shown. PCs were tested for sterility before spiking with one of 10 clinical bacterial isolates listed in Table 1. BacTx was performed twice on daily basis, for a maximum of 5 days, until positive bacterial detection was attained. For detailed description of the methods related to this figure, please see supporting information (Ramirez-Arcos et al., Fig. S1).
© 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107, 192–195
Bacterial screening of buffy coat platelets with the BacTx assay 195
BacTx system. Therefore, this assay is a universal, rapid and sensitive point-of-issue method that could be used to further increase the safety of transfusable PCs.
Acknowledgements The authors are grateful to volunteer blood donors and Canadian Blood Services Sites for providing platelet pools for this research. We thank Ms. Adriana Zapata (Research Assistant, Canadian Blood Services) for technical support during the development of this study. S. marcescens 12/2004 was kindly provided by Drs. Michael Jacobs and Roslyn Yomtovian (Case Western Reserve University). All
authors contributed significantly to the design of the experiments, the interpretation of the results and writing of the manuscript. Funding for materials to conduct this research was provided by Immunetics Inc. Canadian Blood Services and Health Canada also provided financial support for this study; however, the views expressed herein do not necessarily represent those of the Canadian federal government.
Conflict of Interest The authors declare that this study was partly funded by Immunetics Inc.
References 1 Corash L: Bacterial contamination of platelet components: potential solutions to prevent transfusion-related sepsis. Expert Rev Hematol 2011; 4:509–525 2 Ramirez-Arcos S, Goldman M: Bacterial Contamination; in Popovsky M (ed.): AABB Transfusion Reactions, 4th edn. Bethesda, MD, AABB Press, 2012:153–181 3 Greco-Stewart VS, Brown EE, Parr C, et al.: Serratia marcescens strains implicated in adverse transfusion reactions form biofilms in platelet concentrates and demonstrate reduced detection by automated culture. Vox Sang 2012; 102:212–220 4 Hodgson SD, Greco-Stewart V, Jimenez CS, et al.: Enhanced pathogenicity of biofilm-negative Staphylococcus
epidermidis isolated from platelet preparations. Transfusion 2014; 54: 461–470. 5 Krueger NX, Han A, Mitchell L: Slow growing bacteria strains in platelet units evade detection with culture systems but are detected by the BacTxTM Assay, a rapid test for bacterial contamination in platelet units. Transfusion 2009; SP470:228A 6 Jacobs R, Good E, Heaton A, et al.: Time of detection of bacterial contamination with the immunetics BacTx test in leukocyte reduced apheresis platelet units inoculated with representative bacterial species. Vox Sang 2013; 105(Suppl. 1):34 7 Morel P, Deschaseaux M, Sauget M, et al.: Bacterial detection in platelet
concentrates: evaluation of the BacTx test. Transfusion 2013; 53:200A 8 Palavecino EL, Yomtovian RA, Jacobs MR: Bacterial contamination of platelets. Transfus Apher Sci 2010; 42:71– 82 9 Greco CA, Zhang JG, Kalab M, et al.: Effect of platelet additive solution on bacterial dynamics and their influence on platelet quality in stored platelet concentrates. Transfusion 2010; 50:2344–2352 10 Greco C, Mastronardi C, Pagotto F, et al.: Assessment of biofilm-forming ability of coagulase-negative staphylococci isolated from contaminated platelet preparations in Canada. Transfusion 2008; 48:969–977
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1 Bacteria used in this study were isolated from contaminated platelet components.
© 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107, 192–195
SHORT REPORT
Evaluation of a universal point-of-issue assay for bacterial detection in buffy coat platelet components S. Ramirez-Arcos, Y. Kou & H. Perkins Canadian Blood Services, Ottawa, ON, Canada
Received: 22 November 2013, revised 6 January 2014, accepted 24 February 2014, published online 27 March 2014
Bacterial contamination of platelet concentrates poses a major post-transfusion infectious risk. This study was aimed at evaluating the efficacy of the BacTx assay (Immunetics Inc.) for bacterial detection in leucocyte-reduced buffy coat platelet pools and for its sensitivity in detecting clinical isolates, including bacteria that form surface-attached aggregates (biofilm positives). Platelet pools were inoculated at bacterial concentrations of 08–13 CFU/ml. The BacTx assay detected all species at concentrations ≥103 CFU/ml within 20–69 h of platelet incubation. Detection of slow-growing and biofilm-forming strains was delayed in comparison with the other strains. This assay could be used as a point-of-issue method to increase the safety of the platelet supply. Key words: BacTx, biofilm-positive bacteria, platelet contaminants.
Introduction Testing of platelet concentrates (PCs) for bacterial contamination has considerably increased the safety of the platelet supply worldwide [1, 2]. However, platelet contaminants still escape detection as exemplified by reports of false-negative transfusion cases [2]. To further reduce the occurrence of adverse transfusion reactions due to bacterially contaminated PCs is a challenge. Development of more sensitive screening methods for use by the blood suppliers, implementation of pathogen reduction technologies, and using a rapid screening test prior to transfusion are options which have different advantages and disadvantages. Additional challenges are posed by bacteria that form aggregates able to attach to the inner surface of platelet containers or to platelets (biofilm positives). We have shown that biofilm formation increases the risk of missed detection during routine platelet screening [3]. Furthermore, biofilm-negative bacteria can convert to a biofilmpositive phenotype when incubated in PCs, displaying increased pathogenicity [4].
Correspondence: Sandra M. Ramirez-Arcos, Development Scientist, Canadian Blood Services, 1800 Alta Vista Drive, Ottawa, ON, K1G 4J5, Canada E-mail: [email protected]
192
AABB Bulletin #12-04 recommends several strategies, including additional testing later in the platelet shelf life, to optimize platelet transfusion practice. During the 82nd Meeting of the Food and Drugs Administration (FDA) Blood Products Advisory Committee, it was agreed that point-of-care tests should have a sensitivity of 103–104 colony-forming units per millilitre (CFU/ml). The BacTx assay has received FDA clearance for bacterial testing in leucocyte-reduced apheresis and whole-blood-derived platelet concentrates prepared by the platelet-rich-plasma method that are pooled within 4 h of transfusion. This system provides universal bacterial detection in a single colorimetric assay targeting the bacterial cell wall component peptidoglycan (http://www.immunetics.com/bactx. html). The BacTx assay has a sensitivity >103 CFU/ml and has been shown to be effective for bacterial detection in apheresis and pooled PCs prepared in plasma or additive solutions [5–7]. Herein, the BacTx assay was evaluated for its efficacy in detecting bacterial clinical isolates in buffy coat platelet pools.
Materials and methods Detailed Materials and Methods are described in supplementary material. Briefly, outdated (7–11 days old) buffy coat platelet pools that had tested negative during initial screening were assayed with the BacT/ALERT and BacTx assay systems to confirm sterility. The pools were
Biofilm formation
Pos
Pos
Neg
Neg
Not tested
Not tested
Pos
Pos
Neg
Neg
Bacterial Strain
S. marcescens CBS07/2005 [3]
S. marcescens 12/2004 [3]
S. marcescens CBS11/2010 [3]
S. marcescens CBS12/2010 [3]
E. coli CBS11001
E. coli CBS07007
S. epidermidis CBS6043 [10]
S. epidermidis CBS0726 [9]
S. epidermidis CBS6010 [9]
S. epidermidis CBS6038 [9]
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
Rep 7 11 7 11 8 7 8 7 11 11 11 11 10 10 10 10 10 10 10 10
Platelet age at spiking 08 16 22 22 2 22 1 19 1 14 1 14 13 55 63 54 48 58 3 45
Initial concentration (CFU/ml)
Table 1 Bacterial testing of buffy coat platelet pools with the BacTx assay
Pos Pos Neg Pos Pos Pos Pos Pos Neg Pos Pos Pos Neg Neg Neg Neg Neg Pos Neg Neg
BacTx Result
DAY 1
20 23 24 28 23 23 28 23 24 20 20 20 285 27 285 27 28 28 28 28
Time (h) 13E+04 55E+05 88E+02 56E+03 15E+05 58E+05 16E+04 25E+03 < 10 18E+03 11E+03 75E+02 48E+02 67E+00 23E+02 38E+02 59E+02 12E+03 < 10 31E+02
Concentration (CFU/ml)
© 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107, 192–195 Pos Pos
Pos Neg Pos Pos Pos
Pos
Pos
BacTx Result
DAY 2
52 46
46 51 46 45 47
44
44
Time (h)
77E+03 21E+04
11E+04 63E+02 65E+03 59E+03 12E+04
76E+05
≥ 50E+06
Concentration (CFU/ml)
Pos
BacTx Result
DAY 3
69
Time (h)
17E+04
Concentration (CFU/ml)
Bacterial screening of buffy coat platelets with the BacTx assay 193
194 S. Ramirez-Arcos et al.
inoculated with 10 clinical bacterial isolates listed on Table 1, at a target concentration of 2–5 CFU/ml. Platelet pools were tested with the BacTx assay following the algorithm shown in Fig. 1. Ethical approval for this study was granted by the Canadian Blood Services Research Ethical Board.
Results and discussion In this study, we demonstrate that the BacTx assay is able to detect clinical bacterial isolates in buffy coat platelet pools within 20–69 h of platelet incubation at concentrations ≥103 CFU/ml (Table 1). Other research conducted with the BacTx assay has shown a similar efficiency for bacterial detection in other types of platelet components including apheresis and whole-blood-derived platelet pools prepared by the platelet-rich-plasma method [5–7]. Although targeted to 2–5 CFU/ml, the initial bacterial concentration in the PCs ranged from 08 to 13 CFU/ml (Table 1). Of the 63 BacTx assays performed both on negative control pools and on sterile pools prior to inoculation, no false-positives were observed. Also, there were no contaminated pools that were missed by the BacTx assay, and therefore, no false-negatives were documented during the development of this study. Additional information on the sensitivity and specificity of the BacTx assay should be obtained in a larger study.
• •
Rep 1
CONTROL
Notably, the BacTx assay was able to detect S. marcescens CBS07/2005 and 12/2004, which were involved in false-negative fatal transfusion reactions (Table 1) [3]. Detection of S. epidermidis, known for its slow-growing patterns in PCs, was achieved on days 2–3 postspiking, 1 day later than detection of its Gram-negative counterparts (Table 1). It has been reported that detection of Gram-positive skin flora is usually delayed in comparison to fast-growing organisms [2, 8]. The time of detection for biofilm-positive S. marcescens 12/2004 and S. epidermidis CBS6043 was increased by 1 day in comparison with other strains (Table 1). This result might be due to attachment of bacteria to the inner surfaces of platelet containers which delays bacterial cell release into the supernatant of the PCs as previously reported by our laboratory [9].
Conclusion This study shows that the BacTx assay is efficient in uncovering contaminant bacteria in buffy coat platelet pools. There could be variation in the time of detection between strains of the same species which is intrinsic to the genetic and physiological characteristics of each bacterium. Importantly, detection of a variety of Gram-positive and Gram-negative organisms, including strains involved in fatal transfusion reactions and potentially virulent biofilm-forming isolates, can be attained by the
Three outdated (7-11 days old) buffy coat platelet pools BacT/ALERT® and BacTx® sterility testing
Rep 2
Target concentration: 2-5 CFU/ml
Platelet spiking
Sample for BacTx® testing and bacterial concentration twice (at ~9 AM and ~5 PM) on daily basis for a maximum of five days
If negative bacterial detection with the BacTx® Assay, continue testing
If positive bacterial detection with the BacTx® Assay, repeat test two more times
Control continued to be tested until test units yielded positive results
≥ 2 tests should be positive for a definite positive BacTx® result
Fig. 1 Experimental design. The algorithm followed to test platelet components in this study is shown. PCs were tested for sterility before spiking with one of 10 clinical bacterial isolates listed in Table 1. BacTx was performed twice on daily basis, for a maximum of 5 days, until positive bacterial detection was attained. For detailed description of the methods related to this figure, please see supporting information (Ramirez-Arcos et al., Fig. S1).
© 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107, 192–195
Bacterial screening of buffy coat platelets with the BacTx assay 195
BacTx system. Therefore, this assay is a universal, rapid and sensitive point-of-issue method that could be used to further increase the safety of transfusable PCs.
Acknowledgements The authors are grateful to volunteer blood donors and Canadian Blood Services Sites for providing platelet pools for this research. We thank Ms. Adriana Zapata (Research Assistant, Canadian Blood Services) for technical support during the development of this study. S. marcescens 12/2004 was kindly provided by Drs. Michael Jacobs and Roslyn Yomtovian (Case Western Reserve University). All
authors contributed significantly to the design of the experiments, the interpretation of the results and writing of the manuscript. Funding for materials to conduct this research was provided by Immunetics Inc. Canadian Blood Services and Health Canada also provided financial support for this study; however, the views expressed herein do not necessarily represent those of the Canadian federal government.
Conflict of Interest The authors declare that this study was partly funded by Immunetics Inc.
References 1 Corash L: Bacterial contamination of platelet components: potential solutions to prevent transfusion-related sepsis. Expert Rev Hematol 2011; 4:509–525 2 Ramirez-Arcos S, Goldman M: Bacterial Contamination; in Popovsky M (ed.): AABB Transfusion Reactions, 4th edn. Bethesda, MD, AABB Press, 2012:153–181 3 Greco-Stewart VS, Brown EE, Parr C, et al.: Serratia marcescens strains implicated in adverse transfusion reactions form biofilms in platelet concentrates and demonstrate reduced detection by automated culture. Vox Sang 2012; 102:212–220 4 Hodgson SD, Greco-Stewart V, Jimenez CS, et al.: Enhanced pathogenicity of biofilm-negative Staphylococcus
epidermidis isolated from platelet preparations. Transfusion 2014; 54: 461–470. 5 Krueger NX, Han A, Mitchell L: Slow growing bacteria strains in platelet units evade detection with culture systems but are detected by the BacTxTM Assay, a rapid test for bacterial contamination in platelet units. Transfusion 2009; SP470:228A 6 Jacobs R, Good E, Heaton A, et al.: Time of detection of bacterial contamination with the immunetics BacTx test in leukocyte reduced apheresis platelet units inoculated with representative bacterial species. Vox Sang 2013; 105(Suppl. 1):34 7 Morel P, Deschaseaux M, Sauget M, et al.: Bacterial detection in platelet
concentrates: evaluation of the BacTx test. Transfusion 2013; 53:200A 8 Palavecino EL, Yomtovian RA, Jacobs MR: Bacterial contamination of platelets. Transfus Apher Sci 2010; 42:71– 82 9 Greco CA, Zhang JG, Kalab M, et al.: Effect of platelet additive solution on bacterial dynamics and their influence on platelet quality in stored platelet concentrates. Transfusion 2010; 50:2344–2352 10 Greco C, Mastronardi C, Pagotto F, et al.: Assessment of biofilm-forming ability of coagulase-negative staphylococci isolated from contaminated platelet preparations in Canada. Transfusion 2008; 48:969–977
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1 Bacteria used in this study were isolated from contaminated platelet components.
© 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107, 192–195
