Measurement of imatinib uptake by flow cytometry.

One of the essential parameters of targeted therapy efficiency in cancer treatment is the amount of drug reaching the therapeutic target area. Imatinib (IM) was the first specifically targeted drug to be developed and has revolutionized the treatment of patients with chronic myeloid leukemia (CML). To evaluate cellular uptake of IM, we developed a method based on the chemical structure of the molecule and using the natural UV fluorescence that we quantified by flow cytometry. In two CML cell lines, we obtained a satisfactory relationship between intracellular IM (ICIM) levels and media concentrations, and we found a strong correlation between ICIM at 1 h and IM efficacy at 24 h, demonstrating that ICIM at 1 h might be a relevant predictive parameter of cell sensitivity. Our method was more sensitive than the standard physicochemical method. We applied our method to primary cells and found cell morphology-dependent IM accumulation. Moreover, in CML cells from patients at diagnosis, IM accumulation was heterogeneous. In all cases, ICIM at the single-cell level was much higher than in culture media arguing in favor of a predominantly active uptake process. We developed a simple method directly applicable to primary cells that has shown two major advantages: only a small number of cells are required, and cell subsets can be identified according to morphological criteria and/or the presence of particular antigenic sites. This method provides a new tool to assess CML cell sensitivity to IM, and ICIM levels in native CML cells could be used to monitor therapeutic response.

Glucocerebrosidase deficiency dramatically impairs human bone marrow haematopoiesis in an in vitro model of Gaucher disease.

One of the cardinal symptoms of type 1 Gaucher Disease (GD) is cytopenia, usually explained by bone marrow (BM) infiltration by Gaucher cells and hypersplenism. However, some cases of cytopenia in splenectomized or treated patients suggest possible other mechanisms. To evaluate intra-cellular glucocerebrosidase (GlcC) activity in immature progenitors and to prove the conduritol B epoxide (CBE)-induced inhibition of the enzyme, we used an adapted flow cytometric technique before assessing the direct effect of GlcC deficiency in functional assays. Among haematopoietic cells from healthy donors, monocytes showed the highest GlcC activity but immature CD34(+) and mesenchymal cells also had significant GlcC activity. CBE greatly inhibited the enzyme activity of all cell categories. GlcC-deficient CD34(+) cells showed impaired ability to proliferate and differentiate in the expansion assay and had lower frequency of erythroid burst-forming units, granulocyte colony-forming units (CFU) and macrophage CFU progenitors, but the effect of GlcC deficiency on megakaryocyte CFU lineage was not significant. GlcC deficiency strongly impaired primitive haematopoiesis in long-term culture. Furthermore, GlcC deficiency progressively impaired proliferation of mesenchymal progenitors. These data suggest an intrinsic effect of GlcC deficiency on BM immature cells that supplements the pathophysiology of GD and opens new perspectives of therapeutic approach.

[XE-2100: platelet clumps or polymorphonuclear neutrophil aggregates, that is the question]. / XE-2100: amas de plaquettes ou de leucocytes, telle est la question...

Leukoagglutination of polymorphonuclear neutrophils is described as a rare phenomenon but its incidence is certainly underestimated. We report here 4 cases showing why this phenomenon is sometimes difficult to detect. In each case, the only flag reported from the hematology analyser XE-2100 (Sysmex corporation) was 'Platelet clumps?'. The graphic patterns were not suggestive of leukoagglutination but rather evoked platelet clumps. The 4 white blood cell differential scattergrams were absolutely normal and the 4 cases were not associated with leukoneutropenia. Two patients had clumps located only at the edges of the smears, and automated imaging processes, scanning only the center of the smears, may also contribute to the failure of recognising this phenomenon.

Characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow.

OBJECTIVE: Adult bone marrow (BM) mesenchymal stem/progenitor cells (MS/PC) are a potentially useful tool for cell therapy and tissue repair. However, the identification of cell subsets rich in MS/PC from fresh BM has not been described. We have developed a means of identifying such subsets from untouched bone marrow. MATERIAL AND METHODS: First, MS/PC were enriched by short-time adherence (D(1-3)) before any cell division to evaluate the efficiency of CD73, CD105, CDw90, and CD49a antigens to select highly purified CD45(-)CD14(-) fluorescence-activated sorted subsets enriched in clonogenic mesenchymal cells. Then, we adapted this method to unmanipulated BM mononuclear cells (MNC). RESULTS: Short-time (D(1-3)) adherent CD45(-)CD14(-) cells expressing CD73 or CD49a antigens contained all the CFU-F, even though the CD105(+) and CDw90(+) subsets comprised less than half the total. In fresh unmanipulated BM MNC, CD73 and CD49a were also highly discriminative and allowed up to a 3 log enrichment of CFU-F when compared to BM MNC. Normal culture conditions upregulated most of the tested antigens. CONCLUSION: The CD45(-)CD14(-)/CD73(+) and CD45(-)CD14(-)/CD49a(+) phenotypes identified subsets containing all the CFU-F and sufficiently enriched to detect them in fresh BM, enabling evaluation of mesenchymal content of BM collections for cell therapy.

Normal human bone marrow CD34(+)CD133(+) cells contain primitive cells able to produce different categories of colony-forming unit megakaryocytes in vitro.

OBJECTIVE: To evaluate the megakaryocyte potential of normal bone marrow (NBM) CD34(+)CD133(+) cells, a subset offering a possible alternative for clinical CD34 immunoselection, we evaluated their colony-forming unit megakaryocyte (CFU-Mk) content and their ability to produce clonogenic Mk progenitors in comparison with the CD133(-) subset. MATERIALS AND METHODS: Sorted NBM CD34(+)CD133(+) and CD34(+)CD133(-) subsets were evaluated for Mk clonogenic capacity before and after in vitro proliferation in serum-free liquid culture containing kit ligand, Flt3 ligand, thrombopoietin, interleukin-3, and interleukin-6. The segregation of CFU-Mk according to the expression of CD34, CD133, and CD41 was compared between fresh BM cells and expanded cells. RESULTS: Although the fresh NBM CD133(-)CD34(+) subset included two thirds CFU-Mk, only the CD133(+) subset contained primitive cells able to produce all categories of CFU-Mk in vitro. Immunophenotyping confirmed that CD41 antigen is nonspecific for Mk lineage and showed that the usual CD34(+)CD41(+) subset does not specifically define a CFU-Mk population. The segregation of CFU-Mk before and after expansion according to CD34, CD41, or CD133 was modified in relation with down-regulation of CD34 and CD133 antigens and up-regulation of CD41 antigen. CONCLUSIONS: The NBM CD133(+) subset contains primitive cells able to generate CFU-Mk, a subset probably relevant to platelet recovery after infusion. The alteration of antigen expression during in vitro proliferation calls for caution in the identification of the different categories of Mk subsets produced and in the assessment of their predictivity for in vivo platelet production.

CD34+CDw90(Thy-1)+ subset colocated with mesenchymal progenitors in human normal bone marrow hematon units is enriched in colony-forming unit megakaryocytes and long-term culture-initiating cells.

OBJECTIVE: The progress made in the supportive care of allografts and the identification of mesenchymal stem cells in adult human bone marrow (BM) has prompted renewed interest in the use of BM as a form of cell therapy. With the aim of optimizing the collection of BM cells, we evaluated the hematopoietic and mesenchymal immature cell contents of BM hematon units (HUs), which usually are eliminated during graft processing. MATERIALS AND METHODS: Hematopoietic CD34+ progenitors from HU and buffy coat (BC) compartments were characterized in short-term culture. The sorted CD34+CDw90(Thy-1)+ primitive subset was assessed in colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, then further characterized by the expression of additional antigens. In parallel, we evaluated the colony-forming unit fibroblast (CFU-F) number and phenotyped the fresh adherent (D1-3) cells. RESULTS: The plating efficiencies of CD34+ cells derived from HU and BC were identical. However, the HU CD34+CDw90(Thy-1)+ subset was enriched in colony-forming unit megakaryocyte (2.3x), LTC-IC (4.6x), and cells coexpressing CD105 (5x). We found a higher frequency of CFU-F (4.7x), considered to be the mesenchymal stem cell-containing population, correlated with an enrichment in fresh adherent (CD45/GPA)-CD14- cells. CONCLUSIONS: We show for the first time that functional properties of the CD34+CDw90+ subset are related to its in vivo location in HU, which may represent the BM mesenchymal reserve compartment. The location in HU of 35.6%, 59.1%, and 58.7% of CD34+ cells, CD34+CDw90+ LTC-IC, and CFU-F, respectively, justifies the development of a procedure to collect them in order to reduce the therapeutic BM volume.

Phosphorylation of spleen tyrosine kinase at tyrosine 348 (pSyk³48) may be a marker of advanced phase of Chronic Myeloid Leukemia (CML).

We investigated Syk as a potential marker of CML progression. We observed a significant over-expression of Syk mRNA and constitutive phosphorylation of Syk Y348 in blast cells from six AP or BP-CML, but not in 15 CML in chronic phase. We could follow in vivo the recurrence of pSyk(348) throughout blast cell escape, despite observing storage of dasatinib in blast cells. A combination of dasatinib and R406 did not improve therapeutic efficacy in vitro. Our results strongly suggest that Syk activation could be a relevant biomarker of disease progression and dasatinib resistance but is probably not a molecular target.

Proposal of a score combining red blood cell indices for early differentiation of beta-thalassemia minor from iron deficiency anemia.

This study evaluates the diagnostic reliability of 11 red blood cells indices, together with our new index, referred to as '11T', in differentiation of beta-thalassemia minor (BTm) from iron deficiency anemia (IDA). A total of 129 patients with microcytic anemia were involved in a retrospective study, 80 with IDA and 49 with BTm. Using an automatic program, we calculated 11 discrimination indices and a new score, 11T, based on the results of these 11 tests. To confirm the interest of 11T, we performed a prospective study with 53 patients. 11T was most effective in differentiating BTm from IDA: 11T had the highest Youden's index (83%) and the best percentage of correctly identified patients (93%) and gave optimal performance in our prospective study. We proposed a score, 11T, which is able to discriminate between IDA and BTm with high specificity and high sensitivity in order to conduct the appropriate confirmatory examination.

Effects of imiglucerase treatment on traumatic fracture and bone and blood abnormalities in a patient with previously untreated type 1 gaucher disease.

OBJECTIVE: This letter reports on the effect of enzyme replacement therapy with imiglucerase on bone healing and bone and blood abnormalities in a woman with previously untreated type 1 Gaucher disease (GD). METHODS: The 49-year-old patient had been diagnosed with GD at the age of 28 years and had previously undergone splenectomy. She presented with pseudarthrosis 14 months after sustaining a traumatic fracture of the tibia and fibula. Therapy was begun with imiglucerase 60 U/kg q2wk. The effects of treatment on bone healing were monitored radiographically, and effects on blood and bone marrow biology were monitored by hemograms, myelograms, and hematopoietic and mesenchymal clonogenic assays. RESULTS: Objective bone healing was observed starting in the third month of imiglucerase treatment. Blood abnormalities normalized and bone marrow parameters improved over the first 9 months, including a decrease in Gaucher cells, an increase in bone marrow CD34+ cell cloning efficiency, and the appearance of mesenchymal progenitors. CONCLUSION: This research letter reports the results of hematologic and bone evaluations during enzyme replacement therapy with imiglucerase in a woman with previously untreated type 1 GD who presented with a traumatic fracture.

Photopheresis in pediatric graft-versus-host disease after allogeneic marrow transplantation: clinical practice guidelines based on field experience and review of the literature.

BACKGROUND: Extracorporeal photochemotherapy (ECP) gives positive results in the management of graft-versus-host disease (GVHD), but in children, specific difficulties can outweigh this benefit. These difficulties must be taken into consideration when establishing a standardized reproducible procedure for implementation under a quality management plan. STUDY DESIGN AND METHODS: Twenty-seven children underwent ECP for severe acute GVHD (aGVHD) or chronic GVHD (cGVHD) after allogeneic marrow transplantation. Data were collected prospectively, with particular emphasis placed on technical, biologic, immunologic, clinical, and long-term follow-up issues. RESULTS: The 27 children underwent a total of 750 sessions. Mononuclear cells were collected on a commercially available apheresis system (COBE Spectra, Gambro BCT). Overall survival was 73 percent, and ECP led to significant improvement in 21 of the 27 patients (11 with complete response and 10 with partial response, i.e., >50% of organ involvement). Tolerance was good overall, the main limiting factors being vascular access and the psychological impact of repeated apheresis procedures. Children weighing less than 25 kg were not more susceptible to side effects. CONCLUSION: A specifically pediatric-dedicated and -experienced team faces only limited difficulties when treating children with GVHD by ECP. Overall, ECP is efficient and well tolerated. Our experience was therefore pooled together with available pediatric data to establish clinical practice guidelines. These guidelines consider ECP as a first-line therapy in Grade IV aGVHD (in association with conventional pharmacologic approaches) and limited cGVHD and as a second-line therapy in steroid-resistant Grades II to III aGVHD and extensive cGVHD.

Mesenchymal content of fresh bone marrow: a proposed quality control method for cell therapy.

The scarcity of mesenchymal stem cells (MSC) in bone marrow (BM) has justified their ex vivo expansion before therapeutic use, but a method to evaluate the quality of initial mesenchymal content and track the modifications induced by graft processing has not yet been proposed. The aim of this study was to establish such a procedure. Flow cytometric and functional assay methods were modified to count CD45(-) CD14(-)/CD73(+) subsets containing all MSC and used them to study BM from spongy bone (SB) and iliac crest aspirate (ICA). These methods detected the target subsets in all BM suspensions derived from SB (n = 154) and ICA, (n = 44) with a satisfactory correlation between immuno-phenotyping and functional tests by low-density plating. We noted a higher overall MSC frequency in SB cell suspensions but a lower plating efficiency of CD45(-) CD14(-)/CD73(+) SB cells under standard culture conditions. We propose a cell quality control on un-manipulated BM cell suspensions to quantify the mesenchymal compartment with regard to varying donor factors, such as age and sampling site, that influence expansion and define a therapeutic threshold value. Furthermore, we were able to confirm differences in plating efficiency and proliferative capacity between two BM origins.

Cell culture medium composition and translational adult bone marrow-derived stem cell research.

For most therapeutic strategies using MSC, the preliminary amplification is carried out in media containing fetal calf serum (FCS). The theoretical health risk of using a xenogenic serum, a recent practice for which we have limited data, cannot be underestimated, while amplification using human serum (HS) remains controversial. At present, the available information on multipotentiality, self-renewal, and transplantability does not permit the selection of FCS rather than HS. Cellular modifications observed during cell passage seem to indicate a gradual impairment of cells in relation to native MSC, suggesting the making of short cell cultures without necessarily trying to reinfuse a high number of MSC in patients. With this approach, the volume of HS required would remain limited. While clinical studies have already started, many problems remain, such as evaluating the quality of the initial mesenchymal compartment and the biological properties of the cell suspension with FCS compared to those with HS, and depending on culture time.

Successful extracorporeal photochemotherapy for chronic graft-versus-host disease in pediatric patients.

A total of 254 extracorporeal photochemotherapy (ECP) procedures were performed in 8 children (median age 10 years; range 5-15) with extensive resistant chronic graft-versus-host disease (GVHD). ECP was carried out in the pediatric environment using a Cobe Spectra separator and UV-MATIC irradiator. A peripheral venous with a single-lumen permanent central catheter access (69% of ECP-apheresis) or a dual-lumen permanent central catheter access (26% of ECP-apheresis) were used preferentially. A median platelet decrease of 17% (0-71) (p = 0.0001) and median hemoglobin level decrease of 15 g/L (0-31)(p = 0.0001) were noted following each ECP-apheresis. However, none of the patients had profound thrombocytopenia or anemia. Two minor episodes of catheter related-bacteriemia (Staphylococcus aureus) were noted (2310 catheter-days). A negative correlation was found between lymphocyte collection efficacy (median = 38%) and pre ECP-apheresis lymphocyte count (r = 0.4, p = 0.00001). The median of 5 x 10(7) lymphocytes/kg (0.1-50.10(7)/kg) was irradiated in each procedure. All patients are alive and well, and 7/8 experienced a dramatic improvement in their cutaneous status. Liver and gut disease resolved completely in 4/6 and 5/5 patients, respectively. In all patients, a concomitant immunosuppressive therapy was stopped (5/8) or considerably reduced (3/8). Five patients with more than 2 years follow-up after discontinuation of ECP are in remission with no immunosuppression treatment. They have normal growth rates and normal school and sport activity. Our study shows that ECP is beneficial, well tolerated, and can be safely used for chronic GVHD treatment even in young children with low body weight and a poor performance status. We believe that having a dedicated pediatric environment together with an experienced, motivated, and specifically pediatric team is of crucial importance for improving patient's acceptance of this long-term therapeutic program.