Circulating autologous stem cells collected in very early remission from acute non‐lymphoblastic leukaemia produce prompt but incomplete haemopoietic reconstitution after high dose melphalan or supralethal chemoradiotherapy

Summary:For the better understanding of engraftment properties after autologous peripheral blood stem cell transplantation (PBSCT), hematopoietic recovery, immune reconstitution and functional capacity of cytokine production in different lymphocyte populations were examined. In a prospective study, we examined 24 patients suffering from different malignancies after autologous PBSCT. The examination intervals were 1, 3, 6 and 12 months after PBSCT. T cells, B cells and NK cells were analyzed using flow cytometry. The expression and kinetics of cytokines in T lymphocytes were evaluated in 10 patients by intracellular staining of cytokines after PMA/ionomycin stimulation. We observed rapid hematopoietic engraftment proceeding to stable long-term reconstitution. For CD3 + lymphocytes, a consistent reconstitution associated with an increase in CD3 + CD8 + cytotoxic T cells was observed, whereas the CD3 + CD4 + helper/inducer T cells remained low (Ͻ200/ l). Impaired B lymphopoiesis with severe depression (Ͻ1%) was detected 1 month after PBSCT but recovered thereafter (12.8% after 3 months). The percentages of cytokine-producing T cells and the mean fluorescence intensity (MFI) shifts suggested an insufficient capacity for producing IFN␥, in particular for CD3 + CD4 + T cells, compared to healthy volunteers early after PBSCT. Rapid hematopoietic recovery and partly impaired immune reconstitution, especially regarding the regeneration of B lymphocytes and T helper cells, was observed. The CD4 + subpopulation remained low throughout the period of examination, whereas the B cells showed a delayed recovery after 3 months. Cytokine production proved to be sufficient after in vitro stimulation in T cell populations with the exception of IFN␥ synthesis. Bone Marrow Transplantation (2001) 28, 251-257.

Section: Discussionmentioning

confidence: 99%

“…[1][2][3][4][5] G-CSFmobilized peripheral blood stem cells (PBSCs) are becoming an attractive alternative to bone marrow as a stem cell source. The advantages include shortening of severe neutropenia, and lower rate of life-threatening complications.…”

mentioning

confidence: 99%

Immune reconstitution and production of intracellular cytokines in T lymphocyte populations following autologous peripheral blood stem cell transplantation

Schlenke

Sheikhzadeh

Weber

et al. 2001

“…1,2 Autologous peripheral blood stem cell transplantation (PBSCT) became available after the introduction of recombinant growth factors such as G-CSF. 3,4 The collection of hematopoietic progenitor cells by leukapheresis and the rapid engraftment with a shortened period of severe neutropenia are the most pronounced advantages of the PBSCT. While treatment-related mortality is lower for autologous BMT or PBSCT, it lacks the beneficial GVL effect and carries the risk of retransfusing malignant cells.…”

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confidence: 99%

The cytotoxicity of mafosfamide on G-CSF mobilized hematopoietic progenitors is reduced by SH groups of albumin – implications for further purging strategies

Schlenke¹,

Kisro

Deeken

et al. 1999

Summary:The efficacy of mafosfamide purging depends on factors like incubation time, drug and erythrocyte concentration. To determine the influence of protein-bound SH groups in the incubation medium, the cytotoxicity of mafosfamide on G-CSF mobilized CD34 + cells was evaluated by short-term culture assays and drug concentration measurements. 100 mol/ml mafosfamide was incubated for 30 min in five buffers (PBS, PBS with 1%, 5% and 10% BSA and plasma). The mean calculated areas under the concentration-time curves (AUC) were 2489 ؎ 198, 1561 ؎ 286, 976 ؎ 201, 585 ؎ 62 and 605 ؎ 196 mol/l/min. The mean reductions of CFU-GM growth were 79.4%, 73.0%, 62.5%, 30.3%, 6.2% respectively. Similar results were obtained for BFU-E. Regression analysis showed a good correlation between cytotoxicity and AUCs (CFU-GM: r = 0.8195; BFU-E: r = 0.8207). This effect is well explained by the different concentrations of SH moieties in the incubation medium resulting in a higher drug binding capacity. The profound difference between AUCs and CFU-GMs in plasma and 10% BSA cannot be explained by the quantity of SH-groups. It is probably due to an additional enzymatic drug degeneration by the 3Ј-5Јexonuclease subsite of plasma DNA polymerase. In conclusion, the concentration of albumin-associated SH groups strongly influences the cytotoxicity of mafosfamide. It has to be considered as a new and important aspect in ex vivo bone marrow purging.

“…The ever more frequent use of peripheral blood stem cells (PBSC) to reconstitute hematopoiesis after myeloablative therapy [1][2][3][4][5][6][7][8][9][10] has prompted the need for a standardized method for estimation of CD34 + cells in stem cell harvests. Flow cytometry represents a valuable tool to quantitate the progenitors in mobilized peripheral blood and to evaluate in real time the engraftment potential of the cytapheresis product.…”

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confidence: 99%

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

Venditti

Battaglia

Poeta

et al. 1999

Summary: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 protocolderived 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 g/kg/day. A minimum target of 2 × 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 × 10 6 /kg (range 1-570) for the Milan/Mulhouse protocol, 3.9 ؋ 10 6 /kg (range 0.8-498) for the ISHAGE one, and 5.17 ؋ 10 6 /kg (range 2-599) for our protocol. The median time to ANC and PLT engraftment was 11Correspondence: Dr A Venditti, Cattedra di Ematologia, Università 'Tor Vergata', Divisione di Ematologia, Ospedale S Eugenio, Piazzale Umanesimo 10, 00144 Rome, Italy Received 1 February 1999; accepted 29 June 1999 (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 Ͻ 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.