Comparison of flow cytometry and heterotrophic plate count methods for dialysis water microbial monitoring

Abstract

Water is an essential component of renal replacement therapy by dialysis. Haemodialysis patients undergoing 4-hour dialysis sessions, thrice weekly, may be exposed to more than 360 L of dialysis fluid per week. Dialysis water and ultrapure dialysis fluids have been established as a prerequisite for online convective therapies (namely haemodiafiltration), to improve biocompatibility of the dialysis system and to reduce inflammation profile of the dialysis patients. The microbial quality of dialysis water should be monitored regularly to demonstrate the effectiveness of the disinfection protocol. Current water and dialysis fluids microbial quality assessment is typically done via heterotrophic plate counts (HPC) and endotoxins analysis. Plate counts methodology provides information on the microbial content of the water only after a considerable incubation time (typically 7 days). Flow cytometry (FCM), as a rapid alternative method, provides the possibility of real-time corrective actions and greater patient safety. In this study, we compared the outcomes of dialysis water microbial quality in an existing dialysis clinic, using two different measurement methods, HPC and FCM, to test the possible benefits of applying FCM as a valid alternative method for dialysis water microbial monitoring. We conclude that FCM offers higher sensitivity than HPC for microbial monitoring of dialysis water, potentially enabling earlier corrective actions. More extensive and larger studies are needed, namely, to evaluate the possible added value of FCM method in dialysis fluids monitoring. If the FCM method is confirmed, it will be necessary to establish maximum allowable levels and typical action levels for dialysis water and dialysis fluids quality when using FCM.

Keywords: Dialysis water; Flow cytometry; Haemodialysis; Heterotrophic plate count; Microbiological monitoring; Water microbiology.

Fig. 1

Dialysis water treatment system and reference sampling points (drawing generated in Adobe Illustrator 2025, version 29.0.1; https://www.adobe.com/uk/products/illustrator.html).

Fig. 2

Display of a single measurement results with the FCM device used in this study (courtesy of BWT Aqua AG, Switzerland). 1. Measured parameters are displayed. 2. The FL2 vs. FL1 dot plot shows all detected events according to the amplitude of their fluorescence signals FL1 (535 nm, X-axis) and FL2 (715 nm, Y-axis). The red polygon defines the gates. 3. The SSC vs. FL1 dot plot shows only cells inside the gates, according to their fluorescence signal FL1 (535 nm) and scattered light signal SSC (488 nm). 4. The FL1 histogram shows all cells inside the gates, binned according to their fluorescence in FL1. 5. Export saves this result to a USB stick. 6. Re-Gate result allows to move the gates and recalculate cell counts. 7. Rename: ability to rename the result. 8. Delete: the result is deleted permanently (requires confirmation). FL1 - Fluorescence signal 1 = Green fluorescence emission of the SYBR Green I (525 nm). FL2 - Fluorescence signal 2 = Red fluorescence emission of Propidium Iodide (715 nm). SSC - Side scatter signal = Scattered light, increases with the size of the cell.

Fig. 3

Scatter plot with the mean value and the corresponding confidence interval for sampling points SP1 (a), SP2 (b), SP3 (c), and SP4 (d).

Fig. 4

Scatter plot with the mean value and the corresponding confidence interval for the combined SP2, 3 and 4 (dialysis water sampling points).

Fig. 5

Cost estimate per sample (FCM vs. HPC). Assumptions: 4 sampling points per clinic for dialysis water monitoring plus 4 additional dialysis fluids samples monitoring, each measured in duplicates, monthly sampling; initial invest for FCM device incl. spare cartridge, installation and qualification of 81,000 EUR, plus full supplier validation package (18,000 EUR); 10-year device lifetime period with respective “per-sample allocation”; annual maintenance and service cost (5,000 EUR); 1,000 samples per cartridge refill, cartridge lifetime 9 months (if less than 1,000 samples are analyzed within 9 months, the rest is “wasted”); cost of cartridge refill of 2,900 EUR, as per manufacturer information; the authors assumed an average cost for external HPC assay of 20 EUR per sample. Sample transport and logistics costs were not considered for either method.