Evaluation of the platelet counting by Abbott CELL-DYN SAPPHIRE haematology analyser compared with flow cytometry.

The Abbot Cell-Dyn Sapphire is a new generation haematology analyser. The system uses optical/fluorescence flow cytometry in combination with electronic impedance to produce a full blood count. Optical and impedance are the default methods for platelet counting while automated CD61-immunoplatelet analysis can be run as selectable test. The aim of this study was to determine the platelet count performance of the three counting methods available on the instrument and to compare the results with those provided by Becton Dickinson FACSCalibur flow cytometer used as reference method. A lipid interference experiment was also performed. Linearity, carryover and precision were good, and satisfactory agreement with reference method was found for the impedance, optical and CD61-immunoplatelet analysis, although this latter provided the closest results in comparison with flow cytometry. In the lipid interference experiment, a moderate inaccuracy of optical and immunoplatelet counts was observed starting from a very high lipid value.

Characterization of two novel cell lines, DERL-2 (CD56+/CD3+/Tcry5+) and DERL-7 (CD56+/CD3-/TCRgammadelta-), derived from a single patient with CD56+ non-Hodgkin's lymphoma.

Two novel IL2-dependent cell lines, DERL-2 and DERL-7, were established from a patient with hepatosplenic gammadelta T cell lymphoma. This patient presented, at diagnosis, two discrete populations of CD56+ cells, one TCRgammadelta+, the second lacking T cell-restricted antigens. The cell lines derived displayed features corresponding to the two cellular components of the disease: DERL-2 was CD56+/CD3+/TcRgammadelta+ while DERL-7 was CD56+/CD3-/TcRgammadelta-. Along with CD56, the two cell lines shared the expression of CD7, CD2, CD158b and CD117. Karyotype analysis showed that both cell lines were near-diploid, with iso-7q and loss of one chromosome 10. In addition, DERL-2 showed 5q+ in all metaphases analyzed, while DERL-7 revealed loss of one chromosome 4. Genotypically, both cell lines shared the same STR pattern at nine loci and demonstrated an identical rearranged pattern of the T cell receptor genes beta, gamma and delta, with respect to the original tumor cells. These data indicated that both cell lines and the original neoplastic populations were T cell-derived and arose from a common ancestor. Among a large panel of cytokines tested, only SCF was able to substitute IL2 in supporting cell proliferation. Moreover, SCF and IL2 acted synergistically, dramatically enhancing cell growth. These cell lines may represent a model to further analyze the overlap area between T and NK cell malignancies, and may provide new information about the synergistic action of IL2 and SCF on normal and neoplastic T/NK cells.

Complete remission in acute myeloid leukaemia with t(8;21) following treatment with G-CSF: flow cytometric analysis of in vivo and in vitro effects on cell maturation.

A 75-year-old patient diagnosed as having acute myeloid leukaemia with t(8;21) received G-CSF alone as induction therapy. Complete remission was achieved following 2 weeks of treatment. Flow cytometric analysis, performed by CD45 technique modified by the introduction of preliminary gating with LDS-751, confirmed the disappearance of blast cells along with myeloid maturation. Finally, in vitro studies demonstrated that G-CSF, as compared to other differentiation inducers, was able to induce a striking effect toward neutrophilic differentiation.

In vitro exposure of acute promyelocytic leukemia cells to arsenic trioxide (As2O3) induces the solitary expression of CD66c (NCA-50/90), a member of the CEA family.

Arsenic trioxide (As2O3) is a useful drug for the treatment of acute promyelocytic leukemia (APL), acting through a complex mechanism involving the induction of apoptosis. We investigated by flow cytometry whether in vitro treatment of APL leukemic cells with As2O3 determined specific surface membrane changes. Twelve APL bone marrow aspirates were analyzed following 7 days of in vitro treatment with As2O3 (0.25, 0.5 and 2.5 microM) with regard to the expression of a series of differentiation antigens. Twelve acute myeloid leukemia (AML) samples of non-APL morphotype were analyzed as controls. Exposure of APL as well as non-APL samples to any concentration of As2O3 did not affect the expression of beta2 integrins (CD11a and CD11b), CD45 isoforms (RA, RB and R0), CD44/H-CAM, CD33 and the CEA-related antigen family members CD66ade and CD66b, thus failing to disclose any maturating effect. Of interest, in all APL samples (but not in AML) every tested dose of As2O3 determined a dramatic upregulation of CD66c display; intermediate concentration (0.5 microM) of As2O3 increased the median percentage of CD66c+ cells from 5% in control cultures (25th-75th percentile 2-12%) to 80% in drug-exposed cultures (25th-75th percentile 58-90%) (P<0.001). The induction of solitary expression of CD66c is a new finding which demonstrates As2O3 capability of generating phenotypic changes absolutely restricted to APL cells Moreover, these results provide experimental basis for considering the involvement of the newly described CD66 signalling pathway in As2O3-driven programmed cell death.

CD66c antigen expression is myeloid restricted in normal bone marrow but is a common feature of CD10+ early-B-cell malignancies.

CD66c is a surface (and intracellular) molecule bound to the membrane by a glycosyl-phosphatidylinositol anchor. While its expression on peripheral granulocytes is well recognized, less is known about its distribution in early steps of normal and neoplastic hematopoiesis. We analyzed by flow cytometry cell surface expression of CD66c on bone marrow cells from 4 healthy subjects and on bone marrow or peripheral blood cells from 127 patients with newly diagnosed hematologic malignancies: 70 de novo acute myeloid leukemias (AML), 6 refractory anemias with excess of blasts in transformation, 3 myeloid and 3 lymphoid blastic phases of chronic myelogenous leukemia, 33 B-lineage and 6 T-lineage acute lymphoblastic leukemias (B- and T-ALL), and 3 B-cell and 3 T-cell non-Hodgkin's lymphomas in the leukemic phase. We found that in normal bone marrow CD66c expression was myeloid restricted, reaching its highest level on promyelocytes. As for de novo AML, slight expression of CD66c was found on 6/25 (24%) AML-M4 and only occasionally in other subgroups. In 9 out of 10 cases of acute promyelocytic leukemia, CD66c was totally absent, but antigen expression was easily detectable following in vitro exposure to all-trans retinoic acid. Among lymphoid malignancies, CD10+ early-B-ALL consistently expressed the molecule (20/23 cases, or 87%) whereas both CD10- early-B ALL and SmIg+ B-ALL completely lacked it. Finally, dual staining with CD66c and CD10 proved to be a suitable tool for distinguishing even low percentages of residual leukemic cells (CD10+/CD66c+) from normal regenerating early-B cells (CD10+/CD66c ) in CD10+ early-B-ALL induced into remission.

Immunophenotypic analysis enables the correct prediction of t(8;21) in acute myeloid leukaemia.

Immunophenotypic findings from 14 patients affected by acute myeloid leukaemia (AML) with t(8;21) were compared to those obtained from 79 AML patients with normal or other aberrant karyotypes. Classic lineage markers, adhesion molecules, surface enzymes, stem-cell-related antigens and HLA-DR were investigated. Following evaluation by the Mann-Whitney test, we found that t(8;21) AMLs showed a significantly higher expression of CD19, CD34, CD56, CD45RA and CD54. Conversely, blasts from patients in the control group significantly expressed higher levels of CD45RO, CD33, CD36, CD11b and CD14. In order to split the data at the best cut-off point to achieve the most homogeneous subset with regard to cytogenetic pattern, i.e. t(8;21) or not, the CART (Classification and Regression Trees) method was applied. In the univariate analysis by CART, statistically significant differences were found when CD19 was dichotomized at 10%, CD34 at 37%, CD45RA at 84%, CD54 at 21%, CD56 at 12%, CD36 at 14%, CD45RO at 25%, CD11b at 18% and CD14 at 12%. Once cut-off points were established by CART, we applied the logistic regression model to establish which combination of two or more antigens was most predictive for t(8;21). The combination CD19-CD34 at the cut-off points indicated above correctly classified 92/93 cases (98.9%). The addition of any other antigen combination to the CD19/CD34 model failed to improve the level of prediction. We conclude that AML with t(8;21) displays an exclusive immunophenotype that is highly predictive of the cytogenetic pattern.

JURL-MK1 (c-kit(high)/CD30-/CD40-) and JURL-MK2 (c-kit(low)/CD30+/CD40+) cell lines: 'two-sided' model for investigating leukemic megakaryocytopoiesis.

Two novel cell lines (JURL-MK1 and JURL-MK2) have been established from the peripheral blood of a patient in the blastic phase of chronic myelogenous leukemia. The cells grow in a single cell suspension with doubling times of 48 h (JURL-MK1) and 72 h (JURL-MK2). Cytogenetic analysis has shown that JURL-MK1 is hypodiploid whereas JURL-MK2 is near triploid and that both cell lines retain t(9;22). Moreover, JURL-MK1 and JURL-MK2 have a bcr/abl-fused gene with the same junction found in the patient's fresh cells, and both cell lines express the b3/a2 type of hybrid bcr/abl mRNA. The morphology and immunophenotype of these cell lines are reminiscent of megakaryoblasts. In both lines, a limited but consistent percentage of cells expresses gpIIbIIIa (CD41a), gpIIIa (CD61) and CD36, with no expression of gplb (CD42b), glycophorin A, hemoglobin and CD34. Both cell lines are clearly positive for CD33, CD43, CD45RO and CD63, while CD13, CD44, CD54, CD30 and CD40 are specific features of JURL-MK2. Among cytokine receptors, CD117/SCF-R is strongly displayed by a large fraction of JURL-MK1 cells but is hardly detectable on about 20% JURL-MK2 cells. Both cell lines are clearly positive for CD25/IL2R alpha, while a marked expression of CD116/GM-CSF-R and CDw123/IL3R alpha is restricted to JURL-MK2. Induction of cell differentiation in vitro has demonstrated that TPA is able to modulate the JURL-MK1 phenotype, causing an increased expression of platelet-associated antigens. The JURL-MK2 phenotype is easily modulated by both TPA and DMSO, which cause an increased expression of CD41a and CD117 accompanied by a decreased expression of CD30. Proliferation studies demonstrated that JURL-MK1 cell growth is enhanced by stem cell factor, while JURL-MK2 proliferation is unaffected by this cytokine. JURL-MK1 and JURL-MK2 are two novel cell lines with divergent biological features, representing a 'two-sided' model for investigating new aspects of megakaryocytopoiesis.

Differential regulation of GPI-linked molecules on leukaemic promyelocytes treated in vitro with all-trans retinoic acid.

It has been demonstrated that certain cell-surface proteins are anchored to the cell membrane by a unique structure known as the glycosylphosphatidylinositol (GPI) anchor whose absence has been reported on blood cells from patients with paroxysmal nocturnal haemoglobinuria. We have investigated the expression of CD16/Fc(tau)R-III and CD66b GPI linked molecules at the surface of blast cells from five acute promyelocytic leukaemia (APL) patients before and after in vitro stimulation with all-trans retinoic acid (ATRA). We observed that whereas CD66b antigen exhibited a strong ATRA-driven up-regulation in all cases studied, CD16 expression was unaffected by the treatment with the drug.

All-trans retinoic acid (ATRA) and the regulation of adhesion molecules in acute myeloid leukemia.

A review of recent information on the expression and the ATRA-driven modulation of cell surface adhesion molecules of acute myelogenous leukemia blast cells is presented. Cytofluorometric studies on fresh blast cells have demonstrated that CD11a, CD11b CD11c, CD15, CD45RO and CD54 expression is significantly lower in acute promyelocytic leukemia (APL) than is acute myeloid leukemia of other subtypes (AML). In vitro treatment with ATRA dramatically modifies the adhesion phenotype of APL blast cells, promoting a consistently striking up-regulation of CD11b, CD11c, CD15, CD65, CD54, and CD38. Which is in general, poorly demonstrable in AML. The behaviour of CD15s is variable and fully independent from CD15 and CD65 in induction experiments, suggesting a differential enzyme regulation within the selectin ligand system. ATRA is capable, in both APL and AML, of producing a switch from the high- (RA) to the low- (RO) molecular weight isoform of CD54, Moreover, treatment with this retinoid exerts a negative regulation of the membrane expression of CD49e, CD58 and CD11a in APL as well as in AML. Of particular interest is the fact that the negative effect on CD1 1a expression generates an asynchronous phenotype in APL (CD11a-, CD11b+, CD15+), undetectable on normal maturing myeloid cells. In the last part of this review the possible implications of adhesion molecule modulation in the pathogenesis of ATRA syndrome are discussed.

Stem cell factor receptor (c-kit, CD117) is expressed on blast cells from most immature types of acute myeloid mallignancies but is also a characteristic of a subset of acute promyelocytic leukaemia.

Investigating 208 patients with acute haematological malignancies, we found that stem cell factor receptor (SCFR) was expressed on high numbers of blast cells from the vast majority of patients (93%) with refractory anaemia with excess of blasts in transformation. SCFR was also detected in 62% of AMLs, in which it was directly associated to the expression of CD7, interleukin 6 receptor and CD34, and inversely to that of CD11b and CD14. SCFR-positive cases were preferentially represented in AML-M1 (70%) and in AML-M2 (83%) subsets, whereas only 45% of the remaining samples (M3-M4-M5) exhibited SCFR positively. Interestingly, 50% of cases with acute promyelocytic leukaemia expressed SCFR and this molecule was heterogenously regulated by in vitro treatment with all-trans retinoic acid.

Expression of the leucocyte common antigen (LCA, CD45) isoforms RA and RO in acute haematological malignancies: possible relevance in the definition of new overlap points between normal and leukaemic haemopoiesis.

The membrane expression of CD45RA and CD45RO on fresh leukaemic cells taken from 529 cases of acute haemopoietic malignancies, including 117 B-origin acute lymphoblastic leukaemia (B-origin ALL), 37 T-origin acute lymphoblastic leukaemia (T-origin ALL0, 297 de novo acute myeloid leukaemia (AML), 42 refractory anaemia with excess of blasts in transformation (RAEB-T) and 36 myeloid blastic phase of chronic myelogenous leukaemia (CML-BP-my), was analysed. B-origin ALLs were characterized by the lack of the RO isoform along with the consistent presence of RA. Conversely, a differential expression of the two isoforms was detected in different subsets of T-origin ALL, in that T-stem cell leukaemias (T-SCL: CD7+, CD4-, CD8-, CD1-) preferentially expressed CD45RA whereas conventional T-acute lymphoblastic leukaemias (T-ALL: CD7+, CD4+ and/or CD8+ and/or CD1+) were consistently marked by CD45RO. Within myeloid malignancies, most of AMLs displayed CD45RA, while a substantial group of CML-BP-my preferentially exhibited CD45RO. As a general rule, a reciprocal exclusion of the two isoforms was observed in AML as well as in ALL. Nevertheless, a frequent coexpression of CD45RA and CD45RO was observed in CD14+ AML. In vitro treatment with all-trans retinoic acid (ATRA) was able to promote a switch from CD45RA to CD45RO expression in 27 de novo AML, independently from morphological subtyping. To our knowledge, this is the first report on CD45 isoform expression in a large series of patients with acute leukaemia. The knowledge of the differential expression of CD45RA and CD45RO can ameliorate our classificative approach to haematological malignancies, as well as disclose new multiple overlap points between normal and leukaemic cell differentiation.

Expression and ATRA-driven modulation of adhesion molecules in acute promyelocytic leukemia.

On fresh leukemic cells taken from 30 patients with acute promyelocytic leukemia (APL) the membrane expression of a series of adhesion molecules including beta 2 integrins (CD11a/LFA-1, CD11b/Mac-1), selectin ligands (CD15/Le(x), CD15s/Le(x)) and tyrosine-phosphatase isoforms (CD45RA, CD45R0) was analyzed. The expression of these molecules was also studied in nine of these patients following the APL cells' culture with and without all-trans retinoic acid (ATRA). The fresh APL promyelocytes expressed CD45RA and CD15s on their surface, while CD11a, CD11b, CD15, and CD45R0 were constantly absent. In vitro treatment with ATRA consistently increased the expression of CD15, CD11b, and CD45R0 on leukemic promyelocytes; these changes were paralleled by a decrease of CD45RA display. The expression of sialylated antigen CD15s was fully independent from CD15 suggesting a differential enzymatic regulation within this selectin ligand system. ATRA was, however, incapable of promoting the up-regulation of CD11a in APL. As a result, asynchronous phenotype (CD11a-, CD11b+, CD15+, CD15s+/-, CD45RA-, CD45R0+) was generated that is normally undetectable on maturing myeloid cells. In order to provide a further control a case of acute agranulocytosis was also investigated, in which > 75% bone marrow cells were arrested at the promyelocyte stage; these bone marrow cells showed a surface phenotype identical to non-leukemic promyelocytes (CD11a+, CD11b+, CD15+, CD45R0+, CD45RA-) with a spontaneous ability to differentiate in vivo towards the more mature stages of myeloid differentiation. We therefore suggest that in fresh and ATRA-induced APL cells distinct, regular phenotypic changes are identifiable that are probably associated with t(15;17) and not seen in normal and activated bone marrow.

All-trans retinoic acid promotes a differential regulation of adhesion molecules on acute myeloid leukaemia blast cells.

In the present study we investigated the membrane expression of selectin ligands (CD15/Le(x), CDw65/VIM2, CD15s/sLe(x), beta 2 integrins (CD11a/LFA-1, CD11b/Mac-1) and CD45 phosphatase isoforms (CD45RA, CD45O) on leukaemic cells from 28 patients with acute myeloid malignancies cultured with and without all-trans retinoic acid (ATRA). Within each adhesion system. ATRA was able to differentially regulate distinct molecules. Furthermore, it was able to exert effects specific for acute promyelocytic leukaemia (APL) blast cells, as well as to induce a series of non-cytotype-restricted phenotypic changes. An impressive feature of ATRA induction was a simultaneous increase in the expression of CD15, CDw65 and CD11b on leukaemic promyelocytes. The sialylated antigen CD15s, however, showed results independent from the other two carbohydrates (CD15 and CDw65), suggesting a differential enzymatic regulation within the selectin ligands system. In spite of the well-recognized expression of CD11a throughout all stages of normal myeloid differentiation, APL blast cells were found to virtually lack LFA-1 expression. Moreover, ATRA was unable to promote an up-regulation of this antigen in APL, while inducing a frequent down-modulation in non-APL cases constitutively expressing this antigen. In APL cases ATRA generated an asynchronous phenotype (CD15+, CDw65+, CD11b+, CD11a-), undetectable on normally maturing myeloid cells, but consistent with the concept that incomplete differentiation, in terms of surface molecule expression, can be sufficient to obtain therapeutic results.

Expression and ATRA-driven modulation of adhesion molecules in acute promyelocytic leukemia.

On fresh leukemic cells taken from 30 patients with acute promyelocytic leukemia (APL) the membrane expression of a series of adhesion molecules including beta 2 integrins (CD11a/LFA-1, CD11b/Mac-1), selectin ligands (CD15/Le(x), CD15s/sLex) and tyrosine-phosphatase isoforms (CD45RA, CD45R0) was analyzed. The expression of these molecules was also studied in nine of these patients following the APL cells' culture with and without all-trans retinoic acid (ATRA). The fresh APL promyelocytes expressed CD45RA and CD15s on their surface, while CD11a, CD11b, CD15, and CD45R0 were constantly absent. In vitro treatment with ATRA consistently increased the expression of CD15, CD11b, and CD45R0 on leukemic promyelocytes; these changes were paralleled by a decrease of CD45RA display. The expression of sialylated antigen CD15s was fully independent from CD15 suggesting a differential enzymatic regulation within this selectin ligand system. ATRA was, however, incapable of promoting the up-regulation of CD11a in APL. As a result, asynchronous phenotype (CD11a-, CD11b+, CD15+, CD15s+/-, CD45RA-, CD45R0+) was generated that is normally undetectable on maturing myeloid cells. In order to provide a further control a case of acute agranulocytosis was also investigated, in which > 75% bone marrow cells were arrested at the promyelocyte stage; these bone marrow cells showed a surface phenotype identical to non-leukemic promyelocytes (CD11a+, CD11b+, CD15+, CD45R0+, CD45RA-) with a spontaneous ability to differentiate in vivo towards the more mature stages of myeloid differentiation. We therefore suggest that in fresh and ATRA-induced APL cells distinct, regular phenotypic changes are identifiable that are probably associated with t(15;17) and not seen in normal and activated bone marrow.

Immunodiagnosis of acute leukemia displaying ectopic antigens: proposal for a classification of promiscuous phenotypes.

The nature of the blast cells in 163 cases of acute leukemia was investigated by immunophenotyping, with particular emphasis on the expression of "ectopic" surface membrane structures. Although no antigen included in our panel except CD3 revealed absolute lineage restriction, immunological typing allowed a definite characterization of blast cells in more than 90% of cases. Four groups of patients were identified (A, B, C, D) with different degrees of antigen ectopic expression. We classified as group A leukemias (74%) those expressing conventional antigenic patterns, in absence of cross-lineage markers. Samples classified as group B (18%) showed a single ectopic membrane specificity, apparently discordant with the overall composite phenotype; such a "low-grade deviation" did not prevent a definite immunodiagnosis. Pattern C specimens (5%) revealed a promiscuous coexpression of markers related to different lineages (biphenotypic leukemias), whereas group D included unclassifiable phenotypes, characterized by no antigen or DR-only expression. Our findings suggest the possibility of interpreting complex phenotypic constellations of membrane markers in a consistent and logical manner.