Evaluation of an easy and affordable flow cytometer for volumetric haematopoietic stem cell counting

Abstract

Background: Accurate estimation of haematopoietic stem cell (HSC) counts by flow cytometry may be difficult in laboratories in which sophisticated equipment and staff with specific expertise are not available. Affordable flow cytometers that can perform basic functions may help to overcome these difficulties. In this study we compared HSC and leucocyte counts determined by volumetric and bead-based protocols performed with the small, low-cost Accuri(®) C6, with those obtained with two gold-standard instruments, the four-colour FACSCalibur(®) and the eight-colour FACSCantoII(®), our reference flow cytometers.

Materials and methods: With the three cytometers we tested, in parallel, 111 consecutive samples from cord blood, peripheral blood from patients with myelofibrosis and myeloproliferative syndromes, fresh and thawed HSC collected by apheresis and bone marrow products. The findings were compared with one-way ANOVA, Bland-Altman analysis and linear regression.

Results: The results of HSC and leucocyte enumeration by the three devices were strongly correlated (r(2)>0.99; p<0.0001). ANOVA performed on different subgroups of samples did not reveal significant differences between HSC count determined by the C6 bead-based and reference flow cytometers in any of the subgroups. Regarding the C6 volumetric protocol, a statistically significant difference was observed only in the cord blood subgroup. Time for instrument set-up, calibration and analysis was slightly longer with Accuri(®) C6 (40 min) than with FACSCantoII(®) (30 min).

Discussion: Accuri(®) C6 is a reliable instrument for HSC enumeration in fresh samples, using both volumetric and bead-based approaches, although the volumetric protocol on cord blood samples needs to be improved. The Accuri(®) C6 is easy to use, does not require profound knowledge of flow cytometry and could be employed in an urgent setting. Its performance may be improved by more efficient calibration and shorter analysis time.

Figure 1

Bland-Altman analysis performed comparing both C6 volumetric (A) and C6 bead-based (B) protocols vs reference flow cytometers in all samples without cord blood (upper panels, n=69) and in the cord blood subgroup only (lower panels, n=42).