[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.

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.