Mona Hansson

CD34+ cell subpopulations detected by 8-color flow cytometry in bone marrow and in peripheral blood stem cell collections: application for MRD detection in leukemia patients

Fast development in polychromatic flow cytometry (PFC) makes it possible to study CD34+ cells with two scatter and eight fluorescence parameters. Minimal residual disease (MRD) is determined as persistence of leukemic cells at submicroscopic levels in bone marrow (BM) of patients in complete remission. MRD can be present in collections of hematopoietic stem cell from blood (HSC-B). Using PFC, we have defined patterns of antigen expression in CD34+ cell subpopulations in BM and applied them as templates in MRD analysis. Twelve BM samples from hospital control (HC) patients with no signs of hematological malignancy were studied using five 8-color monoclonal antibody combinations detecting subsets of CD34+ cells. These patterns have been used as templates to determine levels of MRD in HSC-B collections from six AML patients. Several subsets of CD34+ precursor cells were found to be present at very low frequencies (<10−4) in BM and/or HSC-B collections. All six HSC-B collections from AML patients showed MRD by 8-color technique and only three by previously applied 3-color method. The 8-color technique showed promising results in efficient detection of different CD34+ subpopulations of HSC-B and in MRD quantification. Monitoring of MRD should become a part of quality control of HSC-B collections.

Increase of monocytes predicts mobilization of peripheral stem and progenitor cells after chemotherapy followed by G‐CSF administration

Abstract: Mobilization of primitive haematopoietic cells to the peripheral blood was studied in 25 patients with haematological malignancies. The optimal level of peripheral stem cells (PSC), defined by their surface expression of CD34, was significantly higher after mobilization with G‐CSF, either following chemotherapy or alone (median: 123 × 106/l and 143 × 106/l of CD34+ cells respectively) than without administration of G‐CSF subsequent to chemotherapy (median: 27 × 106/l of CD34+ cells). An individual variation in when optimal mobilization of CD34+ cells and myeloid progenitors occurs after chemotherapy and G‐CSF administration was noted (median: day 12, range 7–24 days), which makes it difficult to predict when PSC collections in a given patient should be performed. In this study, chemotherapy followed by G‐CSF administration resulted in a short lasting (2–3 days) peak appearance of CD34+ cells that could predicted by a 2‐fold increase in absolute numbers of monocytes, as compared to the previous day. After the peak level of CD34+ cells in the blood was reached, no further increase in monocytes was seen. The identification of an increase in monocytes, to be used as a predictive variable for when optimal mobilization of PSC will occur in a given patient, may be particularly useful in the individual timing of PSC collections from non‐hospitalized patients.

Generation of dendritic cells from peripheral blood of patients at different stages of chronic myeloid leukemia

We report a method to generate dendritic cells (DC) from frozen leukapheresis products of patients with chronic myeloid leukemia (CML), using sterile culture bags and serum-free culture medium, ie conditions feasible for re-infusion into the patient as part of immunother-apeutic protocols. Leukapheresis products were stored from harvests performed either at diagnosis (13 patients) or after chemotherapy with subsequent granulocyte colony stimulating factor (G-CSF) administration (9 patients), for Peripheral Blood Stem Cell (PBSC) collections. In the presence of optimal concentrations of GM-CSF (50 ng/ml) and IL-4 (40 ng/ml) CML progenitors differentiated on day 7 and 14 of culture to DC, expressing CD1a, HLA-DRand CD86 surface antigens. Mature DCs exhibited on average 12-fold higher allo-stimulatory capacity for CD4+ and CD8+ cells compared to non-cultured PBMC in mixed lymphocyte reaction (MLR). Only DCs obtained from CML patients at diagnosis exhibited bcr/abl fusion gene when tested by fluorescentin situ hybridization (FISH). CD34-selection on leukapheresis products from diagnosis (7 patients) resulted in later maturation of DCs (after 14–15 d), compared to the non-selected PBMC. CD34-selection significantly increased the DC growth, and improved the allo-stimulatory capacity in MLR (on average on day 14, 3.5- and 2.3-fold, respectively). Large differences were observed between individual patients and different leukapheresis products from the same patient. Our report demonstrates the possibility to generateex vivo autologous functionally active DC in CML in a way that allows their clinical application as immunotherapeutic agents.

Effects of intercept pathogen inactivation on platelet function as analysed by free oscillation rheometry

INTRODUCTION The Intercept Blood System, using InterSol as additive solution, is used for inactivation of contaminating pathogens in PCs, thus reducing the risk for transfusion transmitted infection and making it possible to prolong the storage period. This study aimed at investigating the ability of Intercept treated platelets to induce clot formation, as measured by coagulation time using free oscillation rheometry (FOR), and to compare with that of platelets in concentrates with the additive solution T-Sol or plasma. METHODS Seventy-four single-donor platelet units were diluted in InterSol (n=27) or T-Sol (n=47) to a mean plasma concentration of 38%. The Intercept treatment was performed by addition of amotosalen HCl to the InterSol PCs followed by UVA irradiation and treatment with a compound adsorption device (CAD). Forty-six units were collected and stored in 100% plasma for comparison. Clotting time was measured by FOR in fresh PCs (within 26h after collection) after stimulation by a platelet activator. Soluble P-selectin was analysed as a marker of platelet activation in the Intercept and T-Sol PCs. RESULTS The clotting time was shorter for Intercept treated platelets compared to platelets in T-Sol and plasma (p<0.05). There was no difference in clotting time between T-Sol and plasma PCs. Soluble P-selectin was higher for Intercept platelets than platelets in T-Sol (p<0.05). CONCLUSIONS The platelets treated with the Intercept procedure had good clot promoting capacity.

Autologous peripheral blood stem cells: collection and processing

The rapid development in the area of collecting and processing autologous peripheral blood stem cells (PBSC) is reflected by the escalating number of patients treated with PBSC, and by the increasing amount of literature on the subject. Clinical experience suggests that among the variables with a negative influence on mobilization of PBSC, the most important may be the amount of previous stem cell toxic chemotherapy. In selecting patients suitable for autologous PBSC support, the requirement of an adequate anti-tumor therapy has to be weighed against the risk of chemotherapy related stem cell toxicity which will result in inability to collect a sufficient amount of PBSC. The general consensus is that a sufficient PBSC-autograft should contain 2-5 × 106 CD34+ / kg body weight, but attempts to provide a recommended optimal or threshold level are hampered by the lack of standardized methods for CD34+ cell enumeration. In addition, the time to haematological recovery depends both on the dose of infused CD34+ cells and also on the amount of previous chemotherapy, which affects both the quality of the graft and the supportive microenvironment of the host. The quality of the autograft may also be contaminated by malignant cells, even if the biological significance of tumor cell detection in the PBSC graft has not yet been established. Recent development of methods forin vitro purging and selection of CD34+ cells for clinical use have provided the means to avoid or reduce reinfusion of malignant cells. Future directions of clinical research include the ability to define and enumerate the proportion of stem cells versus committed progenitor cells among the CD34+ cells in a PBSC collection, which will be important to ensure rapid engraftment as well as long term haematopoiesis.