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-PE1CRTH2-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. ' International Society for Analytical CytologyKey terms white blood cell differential; flow cytometry; screening for hematological malignancies SCREENING for hematological disorders is routinely performed by counting circulating cells with hematology analyzers. But identification of circulating white blood cells (WBCs) by electronic counters is limited to only five cell-types: lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Moreover, although most cell hematology analyzers are very good in detection of quantitative abnormalities, qualitative recognition of abnormal WBCs is poor, and microscopic examination of blood smears is needed for most cases to ascertain the presence of abnormal circulating cells. Therefore, both electronic WBC count and microscopic inspection of blood smears are needed to establish a reliable WBC differential. This traditional scheme, referred to as traditional or standard cytology, was set up in the 70s.Standard cytology is based on the expertise of cytologists and technicians, which is noticeably variable. Blood smear reviewing is time consuming and difficult to standardize. A recent study shows that, in a median institution among 263 hospitals and independent laboratories, manual review of peripheral blood smears were performed on 26.7% of specimens. The authors raised clearly the question of how to reduce the rate of manual peripheral blood smear review and to improve the efficiency of generating blood cell count results (1). In our institutions (JF, FL), <5% of reviewed blood smears will lead to further investigations. Last but not leas...

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 1 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 plasmacells 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. V C 2010 International Clinical Cytometry Society

Telomerase catalytic subunit (hTERT) exerts important cellular functions including telomere homeostasis, genetic stability, cell survival and perhaps differentiation. However, the nature of external or internal signals, which regulate hTERT expression in tissues, remains poorly understood. Thus, whereas it has been described that hTERT gene is regulated along the differentiation of primitive myeloid progenitors, the effect of specific cytokines on telomerase expression in each myeloid lineage is currently unknown. Based on these considerations, we have investigated hTERT expression in erythroid cells treated with erythropoietin (EPO) and transforming growth factor b (TGFb), as putative positive and negative regulators, respectively. We describe here that EPO activates hTERT gene transcription in in vitro-expanded primary erythroid precursors as well as in UT7 erythroleukemia cells. In UT7 cells, this study shows also that EPO acts through a JAK2/STAT5/c-myc axis. In contrast, TGFb blocks EPO signaling downstream of c-myc induction through a Smad3-dependent mechanism. Finally, hTERT appears to be efficiently regulated by EPO and TGFb in an opposite way in erythropoietic cells, arguing for a role of telomerase in red blood cell production.

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