Enumeration of CD34(+) hematopoietic progenitor cells for clinical transplantation: comparison of three different methods

Three different methods for determination of CD34(+) cells in G-CSF-mobiliz ed peripheral blood were compared. The methods mere: the Milan/Mulhouse pro tocol, the ISHAGE guidelines for CD34(+) cells enumeration and our own prot ocol. The procedure we have adopted is essentially a Milan/Mulhouse protoco l-derived methodology combined with a multiparametric approach using the PA INT-A-GATE software analysis program. The samples were collected from 70 pa tients affected by acute leukemia, non-Hodgkin's lymphoma, Hodgkin's lympho ma, myeloma and breast cancer who were scheduled to receive autologous PBSC transplantation. PBSC collection was performed following mobilization with subcutaneous G-CSF at 5-10 mu g/kg/day. A minimum target of 2 x 10(6)/kg C D34(+) cells was considered an acceptable harvest to ensure a safe transpla nt. On average, three aphereses per patient were performed and a total of 2 04 apheresis samples mere analyzed. Regression analysis of the percentage a nd absolute number of CD34(+) cells, as calculated with each method, achiev ed an excellent correlation in spite of methodological differences. In fact , both CD34(+dim) and CD31(+)CD15(-) events were included in our gating str ategy. In the setting of a triple staining associating CD34, CD38 and CD45, we identified a variable fraction of CD34(+)CD38(+)CD45(-) cells which wou ld be otherwise undetected due to its CD45 negativity. To this end, we used a new technology referred to as laser-scanning cytometry (LSC) which allow ed the isolation and morphological identification of CD34(+)CD45(-) cells. By comparing CD34(+)CD45(+) and CD34(+)CD45(-) cells, we found that they sh are a common morphology, thus confirming the hypothesis that the latter are to be considered for CD34(+) cell calculation. The median number of CD34() cells/kg, as calculated by the three methods, was: 4.79 x 10(6)/kg (range 1-570) for the Milan/Mulhouse protocol, 3.9 x 10(6)/kg (range 0.8-498) for the ISHAGE one, and 5.17 x 10(6)/kg (range 2-599) for our protocol. The me dian time to ANC and PLT engraftment was 11 (range 9-24) and 20 (range 10-7 0) days, respectively, Our protocol achieved the best correlation between C D34(+) cells/kg and time to ANC/PLT recovery according to the Spearman's ra nk test (r = -40 and P < 0.015 for ANC, r = -46 and P = 0.005 for PLT). We conclude that (1) CD45 does not appear the ideal partner of HPCA-2 for dete rmination of hematopoietic progenitors in mobilized peripheral blood; and ( 2) for clinical application, a single staining with 8G12 appears simple, re liable and feasible when rigorous procedures for sample preparation and acq uisition are followed and an adequate software for multiparametric analysis is available.