Magali Donnard

“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.

Platelet-activating Factor and Normal or Leukaemic Haematopoiesis

Platelet-activating factor (PAF), a phospholipid mediator with a wide range of actions on mature leukocytes, acts directly during early human haematopoiesis by affecting the growth of haematopoietic progenitors and indirectly, by modulating cytokine synthesis by bone marrow stromal cells. At this time, its role during leukaemic diseases remains speculative. The lack of membrane PAF receptor (PAF-R) on leukaemic blasts suggest that this receptor represents a marker of mature cells and its membrane induction a consequence of cell maturation. While the couple PAF/PAF-R has been largely studied using B cell lines, few results are available using B cells of patients with haematopoietic malignancies casting some doubts concerning the potential role (if any) of this molecule during leukaemic diseases.

5-LOX, 12-LOX and 15-LOX in immature forms of human leukemic blasts

Several reports have demonstrated an important role of leukotriene B(4) (LTB(4)) in the immune system. We investigated whether leukemic blasts from acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients produced LTB(4), 12- and 15-hydroxyeicosatetraenoic acids (12-HETE and 15-HETE) and whether these compounds affected blast proliferation and apoptosis. Leukemic blasts from AML M(0-2) and ALL patients expressed 5-LOX, 12-LOX and 15-LOX transcripts. Quantitative polymerase chain reaction indicated that 5-LOX transcripts were far more abundant than 12-LOX and 15-LOX ones. Leukemic blasts expressed 5-LOX activating protein (FLAP) transcripts and produced LTB(4) in response to calcium ionophore. In contrast no 15-HETE production was found. Calcium ionophore-stimulated leukemic blasts produced 12-HETE but also released thromboxane A(2) suggesting that contaminating platelets accounted for the release of these compounds. No significant effect of LTB(4), 12-HETE or 15-HETE could be documented on leukemic blast growth and on their apoptose rate. Results of the present study indicate that immature form of leukemic blasts produce LTB(4). However, the three major lipoxygenase metabolites of arachidonic acid; i.e., LTB(4), 12-HETE or 15-HETE, had no evident effect on their growth and apoptosis. We may speculate that LTB(4)-derived blast cells might initiate, augment or prolong tissue inflammation and damages by affecting the marrow and blood cytokine network.

Cyclooxygenase-1, but not -2, in blast cells of patients with acute leukemia.

V eronique Truffinet, Magali Donnard, Christelle Vincent, Jean Luc Faucher, Dominique Bordessoule, Pascal Turlure, Franck Trimoreau and Yves Denizot* Universit e de Limoges, Centre National de la Recherche Scientifique, CNRS UMR 6101, Limoges, France Laboratoire d’H ematologie, CHU Dupuytren, Limoges, France Service d’H ematologie Clinique et de Th erapie Cellulaire, CHU Dupuytren, Limoges, France

Both CD62 and CD162 antibodies prevent formation of CD36‐dependent platelets, rosettes, and artefactual pseudoexpression of platelet markers on white blood cells: A study with ImageStream®

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 leukocytes subpopulations by flow cytometry. However, it is known that binding of CD36 may induce platelet activation and formation of platelets 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® 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 markers 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.

Functional EP2 receptors on blast cells of patients with acute leukemia

on their cAMP synthesis.Over a period of 1 year, blood samples recovered on EDTAwere obtained from 12 consecutive patients (6 men, 6 women;mean age, 66 years) at diagnosis according to the Helsinki recom-mendations. Blood samples from patients with more than 85%(range, 85–97%) blast cells as circulating leukocytes were used.Leucocytosis ranged from 11 to 210 G/l. The population (gradedaccording to the French-American-British classification) consistedof 2 AML0, 3 AML1, 2 AML2, 1 AML3, 1 AML4, 1 AML5 and2 ALL2. Cytogenetic data were available for 11 patients. Six hada normal karyotype. The 5 remaining patients were heterogeneousand comprised one complex karyotype, one with a Philadelphiachromosome, one with a t(15;17), one with an unidentified markerand one with a monosomy 7. Blood mononuclear cells were iso-lated by separation on a Ficoll gradient and washed 2 times withHank’s balanced salts solution (HBSS). The blast purity (mean,98%; range, 94–99%) was controlled by flow cytometry analysis(XL II; Coulter, Margency, France). Blast viability (> 95%) wasjudged by Trypan blue exclusion. Blasts (3 610 cells) werestimulated in HBSS with PGE

Membrane and Intracellular Platelet-Activating Factor Receptor Expression in Leukemic Blasts of Patients with Acute Myeloid and Lymphoid Leukemia

Platelet‐activating factor (PAF), a phospholipid mediator with a wide range of actions on mature leukocytes, acts through PAF‐receptors (PAF‐Rs) on the membranes of responsive cells. No results are available concerning the putative presence of PAF‐Rs on leukemic blasts. Using multiparameter flow cytometry, we assessed intracellular and membrane PAF‐Rs on blast cells of acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients. Membrane PAF‐Rs were documented in 7/15 cases of ALL and 0/28 cases of AML. Putative intracellular PAF‐Rs were found in blasts of 8/8 ALL and 13/13 AML patients. Vitamin D3 and dimethyl sulfoxide that induced the expression of PAF‐Rs on the membrane of the human promyelocytic leukemia cell line, HL60, failed to induce their expression on the membranes of CD34+ AML blasts. The lack of membrane PAF‐Rs on the membranes of AML blasts confirms that these receptors represent a marker of mature cells and that their membrane induction is a consequence of cell maturation and differentiation.

Detection of functional platelet-activating factor receptors on leukemic B cells of chronic lymphocytic leukemic patients

Although platelet-activating factor receptors (PAF-R) are reported on normal B cells, few results are available concerning leukemic ones. We demonstrated functional PAF-R on cell and nuclear surfaces of leukemic B cells of chronic lymphocytic leukemic (CLL) patients. Analysis of 102 patients revealed dramatic differences for their membrane PAF-R expression, a result that might be related to their plasma IL-4 levels. In the light of the potent immunoregulatory role of PAF on B cell physiology, it is suggested that the presence or absence of PAF-R on leukemic B cells may profoundly affect their in vivo behavior.

Platelet-activating factor does not stimulate cAMP formation from immature forms of freshly isolated leukaemic blasts

To the Editor,PAF is a phospholipid mediator that sparks off awide range of immunoregulatory actions on maturehaematopoietic cells [1]. Intracellular and nuclearPAF-receptors (PAF-R) are reported [2–5]. Theybelong to the G-protein-coupled family. Studieshighlight the presence of PAF-R transcripts innormal haematopoietic CD34

Presence of membrane platelet-activating factor receptors on B cells of chronic B cell leukaemia patients.

Platelet-activating factor (PAF) is an inflammatory phospholipid molecule that acts in vitro on B cell activation, proliferation and immunoglobulin synthesis [1]. PAF acts through membrane PAF receptors (PAF-R) [2]. Recently, we have highlighted their presence on B cells of chronic lymphocytic leukaemia patients [3] and their virtual absence on leukaemic blasts of patients with acute B lymphoid leukaemia [4] suggesting that PAF-R might represent a marker of B cell differentiation and maturation. Taken together, these results lead us to examine for the presence of PAF-R in other mature B cell leukaemias. Blood was obtained from 42 untreated patients according to the Helsinki recommendations. Thirteen patients had mantle B cell lymphoma (6 men, 7 women, mean age 72 years), 15 had a villous or non-villous marginal zone B cell lymphoma (11 men, 4 women, mean age 74 years), 5 had follicular B cell lymphoma (2 men, 3 women, mean age 62 years), 5 had plasma cell leukaemia (2 men, 3 women, mean age 72 years) and 4 had prolymphocytic or prolymphocytoid B cell leukaemia (2 men, 2 women, mean age 77 years). PAF-R were investigated using flow cytometry as previously reported [3,4]. As shown in Fig. 1, B cells of patients with mantle B cell lymphoma, marginal zone B cell lymphoma, follicular lymphoma, prolymphocytic/prolymphocytoid leukaemia and plasma cell leukaemia expressed PAF-R. Using a cut off of 20% PAF-Rþ cells, PAF-R were found in 20% (1/5) of patients with follicular lymphoma, 50% (2/4) of patients with a prolymphocytic/prolymphocytoid leukaemia, 60% (3/5) of patients with a plasma cell leukaemia, 69% (9/13) of patients with a mantle cell lymphoma and 80% (12/15) of patients with a marginal zone B cell lymphoma. Fifty seven (15/26) and 68% (11/16) of patients with a k and a l chain expressed PAF-R, respectively, indicating that the nature of the light chain did not interfere with the presence or absence of membrane PAF-R. This short clinical study highlights PAF-R in several types of chronic (mature) B cell malignancies. It clearly strengthens the hypothesis that the expression of membrane PAF-R is a marker of B cell differentiation and maturation. While the physiological meaning of PAF-R on leukaemic B cells remains an open question, it