Very low levels of surface CD45 reflect CLL cell fragility, are inversely correlated with trisomy 12 and are associated with increased treatment-free survival.

It has recently been suggested that the percentage of smudge cells on blood smears from patients with chronic lymphocytic leukemia (CLL) could predict overall survival. However, smudge cells are a cytological artifact influenced by multiple physical factors not related to CLL. To identify simple parameters reflecting CLL cell fragility, we studied CD45 expression in a series of 66 patients with Binet stage A CLL. Decreased CD45 expression was specific for CLL cells when compared to 44 patients with a leukemic phase of B-cell non Hodgkin lymphoma and 42 control B-cells. CD45 expression was markedly decreased for all patients with CLL with high percentages of smudge cells. CLL cells with the lowest CD45 expression were the most sensitive to osmotic shock. Very low levels of CD45 expression were significantly associated with lack of CD38 expression, absence of trisomy 12, and with increased treatment free survival time. Altogether, these results demonstrate that low levels of CD45 expression are specific to CLL cells and reflect cell fragility, suggesting that this is an important intrinsic biological feature that determines disease course.

A simple method for detection of major phenotypic abnormalities in myelodysplastic syndromes: expression of CD56 in CMML.

Using a very simple flow cytometry protocol, we found that CD36 and CD117 on granulocytes and CD56 on monocytes were the major bone marrow phenotypic aberrations in patients with myelodysplasia, including CMML. CD56 on monocytes was associated with CMML. Importantly, phenotypic aberrations were lost on blood cells, except for CD56.

PGE(2) receptor subtype functionality on immature forms of human leukemic blasts.

The ability of prostaglandin E2 (PGE2) to regulate the immune system is well documented. PGE2 effects are mediated through interactions with four distinct membrane EP receptors (EP(1-4)). We investigated, for the first time, the functionality of EP receptors on immature forms of blast cells of acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients. RT-PCR experiments documented the presence of the four EP receptor subtype transcripts in leukemic blasts of AML M0, AML M1, AML M2 and ALL patients. Western blot analysis only documented the presence of the EP2 receptor. Functional assays (cAMP production, calcium flux) confirmed Western blot results, i.e., the presence of functional EP2 receptors. Results of the present study suggest that the mechanism used by PGE2 to influence blast physiology is mediated through the EP2 receptor subtype, and subsequently through a cAMP-elevating effect. Results obtained with M0-2 subtypes have to be necessarily extended to more differentiated phenotype.

Hypoxia maintains and interleukin-3 reduces the pre-colony-forming cell potential of dividing CD34(+) murine bone marrow cells.

OBJECTIVE: The aim of this study was to determine whether the combination of a sizable generation of colony-forming cells (CFC) with the maintenance of their progenitors (pre-CFC) ensured by incubation in hypoxia is associated with a certain degree of cell cycling, ultimately responsible for "self-renewal" of pre-CFC. The effects of interleukin-3 (IL-3) on the cycling and CFC-generation potential of pre-CFC also was investigated. MATERIALS AND METHODS: In severely hypoxic (0.9% O(2)) murine bone marrow cell cultures containing stem cell factor, interleukin-6, and granulocyte colony-stimulating factor, pre-CFC maintenance was characterized by the culture-repopulating ability assay, an in vitro analogue of the marrow-repopulating ability assay. The proliferative history of CD34(+) cells in primary cultures was determined by PKH2 staining and related to their CFC-generation potential. In some experiments, subsets of CD34(+) cells sorted on the basis of the number of cell divisions (0, 1, >1) were independently characterized. RESULTS: In hypoxia, the numbers of CFC and CD34(+) cells were markedly reduced, whereas pre-CFC were maintained better than in air. Addition of 5-fluorouracil to hypoxic cultures for 2 days suppressed their CFC-generation potential. The CFC-generation potential of divided CD34(+) cells was maintained or increased with respect to that of undivided cells in hypoxia but not in air. IL-3 decreased CFC-generation potential at both oxygen concentrations. IL-3 also increased the number of CD34(+) cells that divided more than once in hypoxia, decreasing their CFC-generation potential. CONCLUSIONS: Maintenance of CFC-generation potential in hypoxia occurs mainly in a subset of cycling progenitors, despite their proliferation (self-renewal). IL-3 decreased CFC-generation potential by increasing the rate of pre-CFC proliferation beyond the first cycle, which probably results in their differentiation and loss of CFC-generation potential.

A GEIL flow cytometry consensus proposal for quantification of plasma cells: application to differential diagnosis between MGUS and myeloma.

BACKGROUND: Flow cytometry is the sole available technique for quantification of tumor plasma-cells in plasma-cell disorders, but so far, no consensus technique has been proposed. Here, we report on a standardized, simple, robust five color flow cytometry protocol developed to characterize and quantify bone marrow tumor plasma-cells, validated in a multicenter manner. METHODS: CD36 was used to exclude red blood cell debris and erythroblasts, CD38 and CD138 to detect plasma-cells, immunoglobulin light chains, CD45, CD56, CD19, and CD117 + CD34 to simultaneously characterize abnormal plasma-cells and quantify bone marrow precursors. This approach was applied in nine centers to 229 cases, including 25 controls. RESULTS: Tumor plasma-cells were detected in 96.8% of cases, all exhibiting an immunoglobulin peak over 1g/L. Calculation of a plasma-cells/precursors (PC/P) ratio allowed quantification of the plasma-cell burden independently from bone marrow hemodilution. The PC/P ratio yielded the best results in terms of sensitivity (81%) and specificity (84%) for differential diagnosis between MGUS and myeloma, when compared with other criteria. Combination of both the PC/P ratio and percentage of abnormal plasma-cells allowed the best differential diagnosis, but these criteria were discordant in 25% cases. Indirect calculation of CD19 negative PC/R ratio gave the best results in terms of sensitivity (87%). CONCLUSION: This standardized multiparameter flow cytometric approach allows for the detection and quantification of bone marrow tumor plasma-cell infiltration in nearly all cases of MGUS and myeloma, independently of debris and hemodilution. This approach may also prove useful for the detection of minimal residual disease.

"6 markers/5 colors" extended white blood cell differential by flow cytometry.

Electronic white blood cell (WBC) differential by standard cytology (hematology analyzer and visual inspection of blood smears) is limited to five types and identification of abnormal cells is only qualitative, often problematic, poorly reproducible, and labour costing. We present our results on WBC differential by flow cytometry (FCM) with a 6 markers, 5 colors CD36-FITC/CD2-PE+CRTH2-PE/CD19-ECD/CD16-Cy5/CD45-Cy7 combination, on 379 subjects, with detection of 12 different circulating cell types, among them 11 were quantified. Detection of quantitative abnormalities of whole leucocytes, neutrophils, eosinophils, basophils, monocytes, or lymphocytes was comparable by FCM and by standard cytology in terms of sensitivity and specificity. FCM was better than standard cytology in detection and quantification of circulating blast cells or immature granulocytes, with a first lineage orientation in the former case. All cases of lymphocytosis, with lineage assignment, were detected by FCM. FCM identified a group of patients with excess of CD16pos monocytes as those having an inflammatory syndrome. WBC differential by FCM is at least as reliable as by standard cytology. FCM superiority consists in identification and systematic quantification of parameters that cannot be assessed by standard cytology such as lineage orientation of blast cells or lymphocytes, and expression of markers of interest such as CD16 on inflammatory monocytes.

Hypoxia modifies proliferation and differentiation of CD34(+) CML cells.

We previously showed that hypoxia (1% O(2)) favors the self-renewal of murine and human normal hematopoietic stem cells. This study represents the first attempt to characterize the effects of hypoxia on the maintenance of chronic myeloid leukemia (CML) progenitors. CD34(+) cells isolated from apheresis products of CML patients were incubated in hypoxia (1% O(2)) and normoxia (20% O(2)). After 8 days of culture, their proliferation, capacity for colony-forming-cell (CFC) generation in secondary cultures (pre-CFC), and phenotype (CD34 and platelet-activating factor receptor [PAF-R]) were compared with those of normal cells, and tyrosine phosphorylation in CML cells was measured. Hypoxia inhibits the proliferation of CD34(+) cells and preserves the pre-CFC capacity and cell-surface CD34 expression of CML cells better than normoxia. The PAF-R expression, which was absent on freshly isolated cells, was detected at the cell surface in both populations after 8 days of culture, but with a lower percentage of positive cells in CML cell cultures. Incubation in hypoxia suppressed the PAF-R expression of normal cells and increased it in CML cells, resulting in a similar expression in the two populations. These effects could be linked to inhibition by hypoxia of the tyrosine hyperphosphorylation of cellular proteins, a major hallmark of CML cells.