Comparison of single and dual platform methodologies for the estimation of CD34+ hematopoietic progenitor cells: Correlation with colony assay

Abstract

In this study three assays for the enumeration of CD34+ progenitors were compared: 1) a modified version of the Milan protocol, used in the standard dual-platform format; 2) a dual-platform version of the ISHAGE protocol; 3) the ProCOUNT software version 2.0/ProCOUNT kit. The assays were compared to validate the accuracy of CD34+ cell counts in mobilized peripheral blood (PB), apheresis products (AP), and cord blood (CB). The ProCOUNT protocol uses reference beads for absolute CD34+ cell counting, whereas CD34 counts by other techniques are derived from a separate leukocyte count performed by a hematology analyzer. A good correlation between the ISHAGE and ProCOUNT methods was obtained for estimation of CD34+ counts in PB (n=42 samples analyzed) and AP (n=35)--except for samples having a leukocyte count >25 x 10(9)/L or a CD34 count <0.0025 x 10(9)/L)--while a suboptimal correlation between the methods was observed for CB (n=30). The ProCOUNT system proved to be effective in reducing the variability in CD34+ cell counting and appeared to be useful for intralaboratory methodology standardization. The main disadvantage of the ProCOUNT assay was its inability to calculate CD34 counts in leukopenic samples and in CB samples showing a high erythroblast count. As far as the correlation with hematopoietic colonies is concerned, data collected from apheresis samples showed a good correlation between the three flow cytometry methods and colony-forming unit granulocyte-macrophage (CFU-GM) counts, confirming the value of the flow cytometric test as a real-time, truly predictive test to measure the hematopoietic potential of the graft. In summary, all methods are suitable for enumeration of most PB samples, while the single-platform methodology should be preferred for the analysis of AP and CB. We also found the dual-platform format of the ISHAGE method precise and accurate for the estimation of CD34+ cells from CB samples. Based on these data it can be concluded that the single-platform flow cytometry assay format should be the preferred approach for CD34+ stem cell enumeration in different types of samples.

Instrument‐ and protocol‐dependent variation in the enumeration of CD34+ cells by flow cytometry

“…The interprotocol variability issue has been addressed in the past by several groups. Moretti and coworkers 13 reported a good correlation between the ISHAGE protocol and a commercial single‐platform system (ProCOUNT, BD Biosciences, San Jose, CA) when analyzing peripheral blood and leukapheresis samples, as long as the white blood cell (WBC) count did not exceed 25 × 10 9 per L. Correlation was reported as suboptimal when analyzing cord blood samples. Piedras‐Ross and coworkers 10 also found a good correlation between the use of the dual‐platform Milan protocol 5 and the single‐platform ProCOUNT method in peripheral blood and apheresis products, but an underestimation of CD34+ cells was observed by the use of the single‐platform method in marrow samples 14 .…”

Long-term outcome of cord blood transplantation from unrelated donors as an initial transplantation procedure for children with AML in Japan

“…Use of HLA-A and -B serology and low-resolution allelic DRB1 typing showed that most of the children had either one (65%) or two (13%) disparities with their graft ( Table 2). The CD34 þ cell counts were analyzed by the two most commonly used CD34 þ cell quantification methods (ISHAGE protocol and ProCount; Becton Dickinson, Franklin Lakes, NJ, USA) 23 at 11 regional CB banks.…”

Good and poor CD34+ cells mobilization in acute leukemia: analysis of factors affecting the yield of progenitor cells

“…CD34 enumeration was performed on viable cells with a dual-platform version of the ISHAGE protocol; 22 briefly, the WBC were measured with an automated hematology analyzer and, if necessary, samples were diluted; 100 ml aliquots of the sample, containing between 5 Â 10 6 and 10 Â 10 6 cells, were incubated for 30 min at 41C with 10 ml of phycoerythrin (PE)-conjugated anti-CD34 (HPCA-2), 10 ml of fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (2D1) (BD Biosciences) and 20 ml of 7-aminoactinomycin D (final concentration 1 mg/ml). After incubation, the red cells were lysed and cells were analyzed by fluorescenceactivated cell sorting (FACScan, Becton Dickinson) using the CellQuest Software.…”