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-mobilized peripheral blood were compared. The methods were: the Milan/Mulhouse protocol, the ISHAGE guidelines for CD34+ cells enumeration and our own protocol. The procedure we have adopted is essentially a Milan/Mulhouse protocol-derived methodology combined with a multiparametric approach using the PAINT-A-GATE software analysis program. The samples were collected from 70 patients affected by acute leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, 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 microg/kg/day. A minimum target of 2 x 10(6)/kg CD34+ cells was considered an acceptable harvest to ensure a safe transplant. On average, three aphereses per patient were performed and a total of 204 apheresis samples were analyzed. Regression analysis of the percentage and absolute number of CD34+ cells, as calculated with each method, achieved an excellent correlation in spite of methodological differences. In fact, both CD34+dim and CD34+CD45- events were included in our gating strategy. In the setting of a triple staining associating CD34, CD38 and CD45, we identified a variable fraction of CD34+CD38+CD45- cells which would be otherwise undetected due to its CD45 negativity. To this end, we used a new technology referred to as laser-scanning cytometry (LSC) which allowed the isolation and morphological identification of CD34+CD45- cells. By comparing CD34+CD45+ and CD34+CD45- cells, we found that they share 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 median time to ANC and PLT engraftment was 11 (range 9-24) and 20 (range 10-70) days, respectively. Our protocol achieved the best correlation between CD34+ cells/kg and time to ANC/PLT recovery according to the Spearman's rank 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 determination of hematopoietic progenitors in mobilized peripheral blood; and (2) for clinical application, a single staining with 8G12 appears simple, reliable and feasible when rigorous procedures for sample preparation and acquisition are followed and an adequate software for multiparametric analysis is available.
Venditti
A
A
Cattedra di Ematologia, Università 'Tor Vergata', Divisione di Ematologia, Ospedale S Eugenio, Rome, Italy.
Battaglia
A
A
Del Poeta
G
G
Buccisano
F
F
Maurillo
L
L
Tamburini
A
A
Del Moro
B
B
Epiceno
A M
AM
Martiradonna
M
M
Caravita
T
T
Santinelli
S
S
Adorno
G
G
Picardi
A
A
Zinno
F
F
Lanti
A
A
Bruno
A
A
Suppo
G
G
Franchi
A
A
Franconi
G
G
Amadori
S
S
D018952:Antigens, CD34 / Q000097:blood*; D001772:Blood Cell Count / Q000379:methods*; D001781:Blood Component Removal; D002985:Clinical Protocols; D003114:Colony-Forming Units Assay; D005069:Evaluation Studies as Topic; D005260:Female; D005434:Flow Cytometry; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000097:blood; D008297:Male; D012984:Software; D013194:Staining and Labeling; D014182:Transplantation, Autologous
D018952:Antigens, CD34; D017493:Leukocyte Common Antigens