Rapid, sensitive detection of bacteria in platelet samples with Fountain Flow Cytometry

Abstract

Background: There is a current need to develop a technique for bacterial screening of platelet donations that is more rapid, sensitive, and economical than alternatives. The objective of this research was to perform a pilot test of the viability of Fountain Flow Cytometry (FFC), for the rapid and sensitive detection of bacteria in platelet donations.

Methods: Platelet samples were inoculated with serial dilutions of five selected bacterial strains. Samples were then centrifuged, reconstituted in buffer, and stained with a live/dead bacterial stain cocktail. The resulting aqueous sample was measured by FFC, in which the sample passed as a stream in front of an LED, which excited the fluorescent labels. Fluorescence was detected with a digital camera as the sample flowed toward it.

Results: Fountain Flow Cytometry enumeration yielded results that were linear with bacterial concentration, having an R² of ≥0.98 with a detection efficiency of 92%±3%. Measurements of uninoculated samples showed a false-positive detection rate at ~400 colony forming units (CFU)/mL. Detection of bacterial concentrations in platelets above this threshold can be made in ~15 minutes, including sample preparation time.

Conclusion: This pilot study supports the efficacy of FFC for the rapid and sensitive screening of platelet donations for bacteria.

Keywords: bacteria; biodetection; cytometry; fountain flow; platelets.

Figure 1

Schematic diagram of an LED‐illuminated Fountain Flow Cytometer. (A) A prototype Fountain Flow Cytometer utilized low‐level detection of bacteria in platelet donations. (B) The FFC optical configuration illustrating 1‐Flow cell with sample inlet (1a) and outlet (1b); 2‐Dichroic mirror; 3‐LED‐focusing lens; and 4‐LED (The CCD camera and 35‐mm lens are seen in A&C.) (C) A schematic representation of Fountain Flow Cytometric detection. A sample of fluorescently tagged cells flows through the flow cell toward the CCD camera and fore optics. The cells are illuminated in the focal plane by an LED. Labeled bacteria pass through the CCD camera focal plane and are imaged by the camera and lens through the flow cell window, as a bandpass filter isolates the wavelength of fluorescence emission. The fluid in which the cells are suspended then passes by the window and out the flow cell drain tube (reproduced with permission9)

Figure 2

Histogram of detections of labeled E. coli from a PBS sample inoculated with bacteria at a concentration of 1040 bacteria/mL. Intensities (digital FFC counts) were computed by summing the individual pixel intensities in a 10 × 10 pixel box

Figure 3

Fountain Flow Cytometer enumeration of bacteria inoculated into fresh apheresis platelet samples diluted by 10× in PBS. Each data point represents a 0.25‐mL sample labeled with SYBR Green I. Diamonds represent FFC counts, equivalent to CFUs in 0.25 mL of the sample. (To obtain the equivalent concentration of bacteria in the original platelet sample, each FFC count must be multiplied by 40.) A square marker represents the FFC counts predicted from hemocytometry calibration. Shown are Fountain Flow Cytometer measurements of platelet samples inoculated with serial dilutions of (A) E. coli, (B) S. epidermidis, (C) S. agalactiae, (D) S. marcescens, and (E) S. aureus