Basics of flow cytometry–based sterility testing of platelet concentrates

Mohr, H.; Lambrecht, Bernd; Bayer, Anette; Spengler, Hans-Peter; Nicol, Sven-Boris; Montag, Thomas; Müller, Thomas H.

doi:10.1111/j.1537-2995.2005.00668.x

Cited by 50 publications

(44 citation statements)

References 33 publications

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“…Not only are NAT assays complex, but their time to result (approximately 4 h) is still relatively long for a rapid method. In contrast, flow cytometry for bacterial testing of PLTs has been demonstrated to be a rapid and feasible approach (17)(18)(19)(20). This method can be used either as a rapid test or, alternatively, in combination with a preincubation procedure (17,19 ).…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, our newly developed protocol for PLT screening on the BactiFlow platform is easy to perform and offers an acceptable time to result (maximum 1 h) combined with high sensitivity (Table 2). In comparison, the first flow cytometer-based assays for bacterial screening used the 1-step sample preparation procedure, i.e., simultaneous platelet lysis and bacteria labeling with the membrane-permeable dye thiazole orange (17,18 ). This procedure is the most rapid and comfortable approach, but separation of bacteria population and platelet debris is complicated for several bacteria species, e.g., Klebsiella sp., due to an overlap of bacterial fluorescence signals and a high background from platelet debris.…”

Section: Discussionmentioning

confidence: 99%

“…Rapid methods for bacterial detection in PLTs should be easy to perform and not time-consuming to ensure the feasibility of performing them just before transfusion (13 ). These requirements can be met for bacterial detection by nucleic acid amplification techniques (NATs) (3,14,15 ), qualitative immunoassay [Pan Genera Detection (PGD)] (13,16 ), and fluorescence-activated cell sorting (FACS) (17)(18)(19)(20).…”

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confidence: 99%

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Novel Flow Cytometry–Based Screening for Bacterial Contamination of Donor Platelet Preparations Compared with Other Rapid Screening Methods

2009

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Background: Bacterial contamination is the major infectious hazard associated with transfusion of platelet preparations (PLTs). Routine testing for bacterial contamination in PLTs has become common, but transfusion-transmitted bacterial sepsis has not been eliminated. Here, we describe a novel flow cytometry–based method for point-of-issue screening of PLTs for bacterial contamination. Methods: We used the BactiFlow flow cytometer to detect and count bacteria based on esterase activity in viable cells. We compared the assay to incubation (BacT/Alert culture system) and rapid nucleic acid–based or immunoassay (reverse transcription PCR, Pan Genera Detection) methods. Results: We established a protocol for bacterial screening of PLTs consisting of enzymatic digestion and centrifugal filtration for the elimination of viable platelets and selective labeling of bacteria with fluorescent esterase substrate (ChemChrome V23). Results from the BactiFlow showed an excellent correlation (r = 0.9923 E. coli, r = 0.9736 S. epidermidis) to traditional plate count results. The lower detection limit of the assay was determined to be 150 CFU/mL, and the time to result was <1 h. Conclusions: Our study demonstrates that BactiFlow flow cytometry is suitable for rapid screening of PLTs for bacterial contamination and fulfils the requirements for a point-of-issue testing of PLTs with acceptable time to result, specificity, sensitivity, and cost.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Novel Flow Cytometry–Based Screening for Bacterial Contamination of Donor Platelet Preparations Compared with Other Rapid Screening Methods

2009

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“…A method based on reagents from BD Biosciences (Becton Dickinson GmbH, Heidelberg, Germany) has been evaluated for the investigation of platelet concentrates [29]. First, a 50-l volume of platelet concentrates is added to a BD True Count tube with a defined number of fluorescent beads.…”

Section: Bacterial Screening In Platelets By Facs Methodsmentioning

confidence: 99%

Implementation of Bacterial Detection Methods into Blood Donor Screening – Overview of Different Technologies

Schmidt

Sireis

Seifried³

2011

Transfus Med Hemother

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Background: Through the implementation of modern technology, such as nucleic acid testing, over the last two decades, blood safety has improved considerably in that the risk of viral infection is less than 1 in a million blood transfusions. By contrast, the residual risk of transfusion-associated bacterial infection is stable at approximately 1 in 2,000 to 1 in 3,000 in platelets. To improve blood safety with regard to bacterial infections, many countries have implemented bacterial screening methods as part of their blood donor screening programmes. Methods: Bacterial detection methods are clustered into three groups: i) culture methods in combination with the ‘negative-to-date’ concept, ii) rapid detection systems with a late sample collection, and iii) bedside screening tests. Results: The culture methods are convincing because of their very high analytical sensitivity. Nevertheless, false-negative culture results and subsequent fatalities were reported in several countries. Rapid bacterial systems are characterised as having short testing time but reduced sensitivity. Sample errors are prevented by late sample collection. Finally, bedside tests reduce the risk for sample errors to a minimum, but testing outside of blood donation services may have risks for general testing failures. Conclusion: Bacterial screening of blood products, especially platelets, can be performed using a broad range of technologies. Each system exhibits advantages and disadvantages and offers only a temporary solution until a general pathogen inactivation technology is available for all blood components.

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“…However, transfusion-transmitted bacterial sepsis has still not been completely eliminated, with septic complications observed particularly with older PCs (6,8,11,12,20,26,32,34). At present, the detection of microbiological contamination in PCs can be divided into two major methodological concepts (19): (i) incubation or cultivation methods and (ii) rapid detection methods, such as nucleic acid amplification techniques (NAT) (9,16,28), fluorescence-activated cell sorting (FACS) (10,13,17,30,31), or immunological detection methods (Pan Genera Detection [PGD] system) (24, 25).…”

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The Pan Genera Detection Immunoassay: a Novel Point-of-Issue Method for Detection of Bacterial Contamination in Platelet Concentrates

et al. 2010

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Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk in transfusiontransmitted sepsis. Recently the Pan Genera Detection (PGD) system was developed and FDA licensed for screening of bacterial contamination of PCs directly prior to transfusion. The test principle is based on the immunological detection of lipopolysaccharide (for Gram-negative bacteria) or lipoteichoic acid (for Grampositive bacteria). In the present study we analyzed the applicability of this method with regard to detection limit, practicability, implementation, and performance. PCs were spiked with Staphylococcus aureus, Bacillus subtilis, and five different Klebsiella pneumoniae strains, as well as eight different Escherichia coli strains. The presence of bacteria was assessed by the PGD immunoassay, and bacteria were enumerated by plating cultures. Application of the PGD immunoassay showed that it is a rapid test with a short hands-on time for sample processing and no demand for special technical equipment and instrument operation. The lower detection limits of the assay for Gram-positive bacteria showed a good agreement with the manufacturer's specifications (8.2 ؋ 10 3 to 5.5 ؋ 10 4 CFU/ml). For some strains of K. pneumoniae and E. coli, the PGD test showed analytical sensitivities (>10 6 CFU/ml) that were divergent from the designated values (K. pneumoniae, 2.0 ؋ 10 4 CFU/ml; E. coli, 2.8 ؋ 10 4 CFU/ml). Result interpretation is sometimes difficult due to very faint bands. In conclusion, our study demonstrates that the PGD immunoassay is an easy-to-perform bedside test for the detection of bacterial contamination in PCs. However, to date there are some shortcomings in the interpretation of results and in the detection limits for some strains of Gram-negative bacteria.

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Basics of flow cytometry–based sterility testing of platelet concentrates

Abstract: Sterility testing of PCs by FACS is a feasible approach. The present data suggest incubating PC samples for 20 to 24 hours at 37 degrees C before testing. For slow-growing bacteria, the incubation period must be prolonged by 1 to 2 days.

Cited by 50 publications

References 33 publications

Novel Flow Cytometry–Based Screening for Bacterial Contamination of Donor Platelet Preparations Compared with Other Rapid Screening Methods

Novel Flow Cytometry–Based Screening for Bacterial Contamination of Donor Platelet Preparations Compared with Other Rapid Screening Methods

Implementation of Bacterial Detection Methods into Blood Donor Screening – Overview of Different Technologies

The Pan Genera Detection Immunoassay: a Novel Point-of-Issue Method for Detection of Bacterial Contamination in Platelet Concentrates

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