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...

The reliability of the assay of endogenous erythroid colony (EEC) formation in serum-free, cytokine-free collagen-based media was investigated in a multicentric study including 140 patients with polyglobuly (80 polycythemia vera (PV), 54 secondary erythrocytosis (SE), six idiopathic erythrocytosis (IE)) and 10 healthy donors. In each center, EEC assays were performed in parallel with progenitor cells from bone marrow (BM) and peripheral blood (PB); two commercialized media and 'low' and 'high' cell plating densities were tested. Negativity of EEC assays was considered certain only when sufficient BFU-E growth was obtained in control cultures with cytokines. In the two media, EEC formation was specific - never observed in cultures of healthy donors or SE patients - and comparable. BM EEC assays were positive (presence of eythroid colonies) for 75% ('low' plating) to 100% ('high' plating) of PV patients; PB EEC assays were positive for 83.3% ('low' plating) to 93.7% ('high' plating) of PV patients (differences not significant). Depending on the medium, 86.2-93.7% of patients with a positive BM EEC assay had a positive PB EEC assay. Hence, a standardized collagen-based EEC assay can be performed with either BM or PB progenitors; the EEC assay described here is positive for at least 75% of PV patients when a single EEC assay is performed, and for at least 94% of PV patients when both BM and PB EEC assays are performed.

Macrophage colony stimulating factor (CSF-1 or M-CSF) is involved in haemopoiesis and probably in mouse gestation. Sexual steroids induce its production by uterine glandular epithelial cells and its receptor (product of the protooncogene C-FMS) is expressed on placental trophoblastic cells. We measured M-CSF serum levels in 119 pregnant women and in eight women undergoing ovarian hyperstimulation for in vitro fertilization. M-CSF increased early (4-8 weeks) and progressively during gestation. Its rapid elevation during the course of ovarian hyperstimulation suggests that its synthesis is probably induced by sexual steroids. This locally produced M-CSF could play a role in human pregnancy and in the pathogenesis of thrombocytopenias observed during pregnancy.

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Fluorescent labeled monoclonal antibodies (mAbs) against CD36 are routinely used as monocyte, erythroid, or platelet markers in clinical cytometry. CD36 has recently been proposed by various authors as a valuable marker helping to enumerate leukocyte's subpopulations by flow cytometry. However, it is known that binding of CD36 may induce platelet activation and formation of platelet's rosettes on leukocytes, resulting in false expression of platelet markers on white blood cells. To study this phenomenon, we have combined classical flow cytometry and a new quantitative flow imaging technique with the ImageStream 1 analyzer. We show that CD36 ligation induces activation of platelets with CD62 expression and their adhesion on leukocytes due to CD62 and CD162 interactions. Preincubation of whole blood samples with either anti-CD62 or anti-CD162 antibodies could prevent formation of these rosettes. Our approach also emphasizes the fact that immunomorphological analysis of cell events with ImageStream technology is a useful tool to validate the specificity of marker's labeling or to elucidate incoherent results obtained with classical flow cytometry. We thus propose to prevent false platelet labeling on leukocytes by preincubation with either anti-CD62 or anti-CD162 antibodies when using CD36 mAbs. ; platelet-leukocyte rosettes EXPRESSION of platelet markers on the surface of leukocytes, such as blast cells of acute leukemia is very often difficult to demonstrate by flow cytometry. Indeed, formation of platelet's rosettes on the surface of white blood cells (WBCs) can never be excluded, mainly when labeling is performed on whole blood samples and when using a combination of antibodies for multicolor flow cytometry. These labeling artifacts are very likely to be due to in vitro platelet activation during the labeling procedure. Indeed, among antibodies able to activate platelets are those against CD36, such as NL07, OKM5, OKM8, or FA6-152 (1-4).CD36, also called GPIV, is one of the numerous receptors expressed at the surface of platelets. CD36 belongs to the class B scavenger receptor family, which includes the receptor for selective cholesteryl ester uptake, scavenger receptor class B type I (SR-BI), and lysosomal integral membrane protein II (LIMP-II) (5). CD36 has a molecular weight of 53 kDa (6) and is expressed on an extensive range of cells and tissues, including microvascular endothelial cells, monocytes and macrophages, dendritic cells, adipocytes, keratinocytes, cardiac and skeletal muscle, retinal pigment epithelium, microglia, cells of the erythroid lineage (7), breast, gut, renal epithelium, and platelets (8). The protein is heavily N-linked glycosylated, a modification that