Minimization of CD34+ Cell Enumeration Variability Using the ProCOUNT Standardized Methodology

Abstract

The dose of cells expressing the surface antigen CD34 (CD34+) has been shown to be a reliable predictor of the time to engraftment following transplantation of PBPC to support high-dose chemotherapy. However, evaluation of rare cells is complicated by a number of factors, including the variability in operator and technical procedures. Recently, Becton Dickinson Immunocytometry Systems introduced a new CD34+ cell analysis system, the ProCOUNT cell enumeration kit, which automates the analysis of CD34+ cells and minimizes the variabilities of this procedure. We have evaluated the ProCOUNT system in comparison to a standard CD34 cell analysis (based on the Milan approach) using leukapheresis products from patients and normal donors mobilized with chemotherapy plus recombinant human G-CSF (rhG-CSF) or with rhG-CSF alone. In addition, we compared these analyses using CD34+ cell-selected mobilized leukapheresis products with purities of 75% or greater. The standard CD34 cell analysis methodology quantitated the frequency of cells identified as CD45+, low side scatter, and CD34+. A high correlation coefficient was obtained between the ProCOUNT methodology and the standard CD34 cell analysis methodology for cells obtained from leukapheresis products mobilized with chemotherapy plus rhG-CSF (r = 0.98), rhG-CSF alone (r = 0.96), and CD34+-selected mobilized leukapheresis products (r = 0.83). A comparison was also made between technicians using both analysis methods. Whereas the correlation coefficient between two technicians using the standard methodology was r = 0.77, the correlation coefficient was much higher when using ProCOUNT (r = 0.99). These data demonstrate that the use of ProCOUNT is associated with less variability between data analyzed by different operators. Also, ProCOUNT is consistent with existing CD34+ cellular analysis methodologies. An additional advantage is the ability to determine the absolute concentration of CD34+ cells, thereby allowing calculation of total CD34+ cell numbers without using WBC counts, which also have inherent errors. The ProCOUNT system provides an automated analysis procedure that minimizes the variables in CD34+ cell analysis and may be useful for standardization of methodology between laboratories.

Absolute CD4+ T‐lymphocyte and CD34+ stem cell counts by single‐platform flow cytometry: the way forward

“…Firstly, the individual single‐platform approaches gave similar CVs, with all values being lower than the dual‐platform approach. Secondly, the data for ProCount users supports the findings of McNiece et al (1998 ), who observed minimal variation between operators at a single site and suggested that the ProCount kit may be useful for standardization between laboratories. However, it should be noted that ProCount, although producing the lowest inter‐laboratory CVs, still has software limitations with suboptimal samples that require operator intervention ( Peel et al , 1998 ).…”

Absolute Values of Dendritic Cell Subsets in Bone Marrow, Cord Blood, and Peripheral Blood Enumerated by a Novel Method

“…The use of TruCount tubes loaded with beads (BDIS) decreased both the inter-and intralaboratory variability when 60 specimens from HIV type 1-infected donors were shipped to five laboratories. The acquisition of cellular events along with TruCount beads has been demonstrated to be superior to standard methods in enumerating rare cells, as well as for CD34 + progenitor cells in cord blood [48] and in mobilized leukopheresis products [49] with less variability between data analyzed by different operators. The absolute DC numbers obtained by our lyse/no wash method appear to be in range with some other flow cytometric DC enumeration studies [44,45].…”

Standardized, unrelated donor cord blood transplantation in adults with hematologic malignancies

“…Thawing of CB units was performed by Rubinstein et al's method, 21 with minor modifications as described elsewhere. 9 Samples for cell counts, quantitation of CD34 ϩ cells, 22 cell viability, clonogenic assays, 23 and microbiology were drawn directly from the bag before infusion.…”