Characterization of cd34+ cells mobilized to the peripheral blood during the recovery from cyclophosphamide chemotherapy

Abstract

Twenty two patients were treated with cyclophosphamide therapy to mobilize progenitors into the blood. Progenitor cells were quantitated in peripheral blood or leukapheresis products using colony assays and flow cytometric measurement of CD34 + cells. Prompt engraftment of > 500 granulocytes/ul at a median of 13 days was observed in all patients reconstituted with mobilized cells. In four patients where complete sets of serial samples were obtained, the appearance of CD34+ cells preceded the increase in CFU‐GM by 24 to 48 hours. Peak levels of CD34+ cells ranged from 0.6–5% and coincided with the peak increase in CFU‐GM. Mobilized CD34+ cells were predominantly CD33+, CD13+, CD45R+, CD38+, HLA‐DR+ and CD41+. In contrast to bone marrow CD34+ cells, few mobilized CD34 + cells expressed CD71, CD19 or CD10. Long term culture initiating cells (LTC‐IC), capable of reconstituting hematopoiesis in vitro on irradiated stromal layers, were also measured in serial samples from a single patient with high peak levels of CD34 (∼︁5%). LTC‐IC were present in all samples and the ratio of LTC‐IC to CD34 + cells remained similar throughout the post cyclophosphamide recovery phase. In this patient, preferential mobilization of LTC‐IC earlier in the recovery phase was not observed suggesting that during recovery both primitive and committed progenitors are mobilized together. These data indicate that mobilized CD34+ cells represent subpopulations of CD34+ cells with a myeloid phenotype and that the presence of primitive progenitors among these cells confirms clinical reports that they are capable of long term reconstitution.

The low cycling status of mobilized peripheral blood CD34+ cells is not restricted to the more primitive subfraction

“…+ progenitors in terms of a predominance of expression of myeloid antigens in CD34-positive PB progenitor cells over those in BM after administration of granulocyte colony-stimulating factor 23,24 or in steady-state BM. 21 No definite explanation is available at present why few mobilized PBPC are in S phase.…”

A European reference protocol for quality assessment and clinical validation of autologous haematopoietic blood progenitor and stem cell grafts

“…The resulting Milan/Mulhouse manual is still a valuable procedure reference in laboratory practice. 9,10 The subsequent years have resulted in several publications about the standardisation of flow cytometry analysis [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] as well as reports on the clinical implication of the enumeration of CD34 ϩ cells. 24,[26][27][28][29][30][31][32][33][34][35][36][37] The CD34 antigen is stage-specific and identifies cells in the early stages of haemopoietic differentiation.…”

“…Ideally, phases II-III document the usefulness, convincing one or more centres to participate in a phase IV validation trial, which however in this A European Reference Protocol on CD34 ϩ cell enumeration (Table 3) Recently, several reports have presented protocols on flow cytometry enumeration and several companies have announced standard kits for absolute enumeration of CD34 ϩ cells. [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] These protocols seem different at several levels depending on the designer and none has been proved superior to the others, if it fulfills 'the state-of-the-art' recommendation. The major conclusion from the work done over the last decade is that training diminishes the interlaboratory variations with only minor differences between first-and second-line strategies.…”

“…62 One necessary step in such a strategy exemplified by this European protocol (Table 3) is to add the pan-CD45 antibody in order to be able to quantify leukocytes and discriminate these from erythroblasts and non-lysed erythrocytes in the live gate, ie in obtaining an accurate denominator for the CD34% calculation. 13,15,17,63 The number of CD34 ϩ events present in the low SSC region is still calculated on the basis of a minimum population in the range of 50 to 100 events positively stained by a CD34 class III antibody. 5,6,10,15,64 The percentage of CD34 ϩ cells can then be calculated by the number of CD34 ϩ events divided by the denominator (ϭCD45 ϩ leukocytes).…”

“…13,15,17,63 The number of CD34 ϩ events present in the low SSC region is still calculated on the basis of a minimum population in the range of 50 to 100 events positively stained by a CD34 class III antibody. 5,6,10,15,64 The percentage of CD34 ϩ cells can then be calculated by the number of CD34 ϩ events divided by the denominator (ϭCD45 ϩ leukocytes). The total number of CD34 ϩ cells per volume can subsequently be calculated by multiplication of the CD34% and the leukocyte count obtained from a haematology analyzer (the dual platform technique).…”