Feasibility study of implementing flow cytometry in a fecal microbiota transplant preparation unit

Introduction
The development of analytical methods for the control of fecal transplants (FT) is a major objective for preparation units. Cytometric characterization, in addition to microbiological qualification of donations would provide complementary assurance of their quality. Flow cytometry is a method that would allow enumeration of microorganisms and approximation of their viable content in FTs. The aim of this project is to investigate the implementation of this method in a FT preparation unit.

Materials and Methods
One-month test period was carried out, two cytometers: the CyFlow® Cube 6 (Sysmex^®) and the CytoFLEX^® S (Beckman Coulter^®) were tested. Ergonomics, handling and analysis times, ease of use of software, purchase price and consumables costs were compared. Pairs of DNA-targeting markers with different membrane permeability characteristics were used: SYTO™9/Propidium Iodide (PI) and CyStain™ Green/Red. Analyses were performed in triplicate on transplant samples from different routinely used preparation and storage methods, to assess repeatability.

Results
CytoFLEX^® offers reduced analysis time and manipulations (fully automated homogenisation and rinsing). It can be upgraded with additional lasers without instrument modification. Both instruments are compact. The computer is incorporated into the CyFlow^® but the sheath fluid and waste bottles are not. Some CyFlow^® consumables are captive. The price is equivalent and includes full training for both instruments. Sample preparation consists of dilution to 106 with 0.9% NaCl and staining. Analyzed FTs contain between 109 and 1012 microorganisms per gram of stool. Coefficients of variation for viability and enumeration were <13% and <2% respectively. A higher proportion of cells appeared to be PI-stained in frozen FT suspensions without glycerol. Differences in the amount of microorganisms or the proportion of PI-stained cells seem to occur, depending on the donor, especially for frozen native stools.

Conclusion
The equipment tested allowed us to consider that flow cytometry would be suitable for monitoring faecal microbiota transplants. This method provides repeatable and rapid results. To complete this work, we have started a collaboration with a research team to increase our expertise, develop, and validate our method. Flow cytometry will then enable us to implement research protocols and promote innovation, particularly in the development of new galenic forms.

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