Evaluation of trypan blue stain in the TC20 automated cell counter as a point-of-care for the enumeration of viable cryptococcal cells in cerebrospinal fluid

Abstract

Cerebrospinal fluid (CSF) culture can determine a quantitative viability of Cryptococcus yeasts; however, culture has a long turnaround-time. The TC20 automated cell counter (Bio-Rad) is a benchtop instrument used to count cells in 30 seconds. In vitro studies suggest trypan blue staining can distinguish between viable and dead cryptococcal yeasts. We hypothesized that trypan blue staining with automated cell counting may provide rapid quantification of viable CSF Cryptococcus yeasts. In sum, 96 HIV-infected participants with cryptococcal meningitis were enrolled and provided 194 CSF specimens in Kampala, Uganda. Cryptococcosis was diagnosed by CSF cryptococcal antigen (CRAG). CSF was stained with trypan blue and quantified yeasts with the TC20 cell counter. We compared the log10 transformed cell counter readings with gating of 4-10 μm versus log10 quantitative Cryptococcus cultures/ml. TC20 showed more positive results (95.4%) overall than culture (78.4%) with reference to CSF CRAG. TC20 had higher readings compared to culture in most cases with only a 25% level of agreement between the two methods. TC20 had a poor correlation to culture throughout the 14 days of antifungal therapy. The median of log10 transformed counts were 5.22 (IQR = 4.79-5.44) for the TC20 and 3.99 (IQR = 2.59-5.14) for culture. Overall, a linear regression showed no significant relationship between the TC20 and culture (r = -0.0025; P = .92). TC20 automated cell counting with trypan blue staining was poorly predictive of the quantitative CSF culture and could not be used as a substitute for quantitative culture.

Utility investigation of automated techniques in hematopoietic progenitor cell count and viability assessment in the Good Manufacturing Practice (GMP) setting

“…In our study, when the two different automated cell counters were compared by means of cell count results, a significant correlation value of 0.72 was observed, in concordance with that of Camacho-Fernández et al [11]. On the other hand, Kwizera et al [5] counted Cryptococcus yeast cells in cerebrospinal fluid culture by trypan blue staining and rapidly quantified viable cells with an automated cell counter. The study of Kwizera et al [5] is the first article practicing the comparison of different automated cell counting methods on human stem cells and mainly focusing on the validation of the repeatability of results.…”

“…The use of such techniques force the researchers to analyze the reliability of the automated systems. A number of previous studies have compared the various cell count and viability assessment methods [5,6,[9][10][11]17]. Several studies have focused on cell count and viability of human cells.…”

“…hemocytometer) or by the use of automatic devices which are operated by certain principles such as the automated trypan blue exclusion [1,2] and radio frequency [3,4]. Viability assessment is also possible via the trypan blue exclusion technique [5]. Also some manual methods-i.e.…”

“…In this manner, automated techniques present themselves to be utile in performing both cell count and viability estimation, and when necessary being able to give both results realtime. Previously, researchers made various comparisons of manual and automated cell count and viability techniques in different cell types [2,5,6,[9][10][11]. However, none of these studies show comparison between automatic and/or manual cell count and viability techniques by means of hematopoietic progenitor cells.…”

Emerging concepts in HIV-associated cryptococcal meningitis

“…Trypan blue staining and automated cell counting using the TC20 automated cell counter has been evaluated in Uganda, but found to be poorly predictive of quantitative CFU counts (16). Flow cytometry using a BD LSRFortessa flow cytometer did provide a rapid and accurate measurement of fungal burden in patients with CM in a small pilot study in South Africa (17), providing proof or principle for the technique, and if combined with a cryptococcal viability stain (18), could potentially be used to assess the response to treatment.…”