scholarly journals Oncogenesis of CRLF2 Overexpression and Effect of JAK2 Inhibitor in CRLF2 Overexpressed B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2757-2757
Author(s):  
Yan Gu ◽  
Chunhua Song ◽  
Sinisa Dovat ◽  
Qinglong Guo ◽  
Qinyu Ge ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cox Model ◽  
Lymphoblastic Leukemia ◽  
Normal Bone Marrow ◽  
Jak2 Inhibitor ◽  
Normal Bone

Backgroud: Cytokine receptor-like factor 2 (CRLF2) plays an important role in the development of normal B lymphocytes, which can mediate the proliferation of early B cells. However, the diect oncogenic effect of CRLF2 overexpression in acute lymhpoblastic leukemia (ALL) is far yet to be clarified. Here, we explored the effect of CRLF2 overexpression on cell proliferation and the effect of the novel JAK2 inhibitor on B-ALL cells with CRLF2 overexpression. Methods: The 83 patients with newly-diagnosed ALL (56 B-cell and 27 T-cell ALL; range from 14 to 77 years old) between June 2008 and June 2016 were studied at Zhongda Hospital Southeast University. The 21 normal bone marrow subjects were enrolled as controls. The qPCR method is developed for detection CRLF2 expression and the CRLF2 overexpression was determined with a cutoff value more than the highest sample of normal bone marrow control. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Event-free survival (EFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. CRLF2 F232C gain-of-function mutant which we previously reported or CRLF2 were expressed in Nalm6 and 697 B-ALL cells with lentiviral transduction. WST-1 cell proliferation assay and in vitro clonogenic assay were performed upon JAK2 inhibitor (BBT594) treatment. Nalm6-CRLF2-luc, Nalm6-F232C-luc, and Nalm6-vector-luc cells were injected via tail vein into the NSG mice. The leukemia engraftment was monitored once a week by living imaging. Results: The expression of CRLF2 in patietns with ALL was significantly higher than the normal control (P<0.0001). Patients with CRLF2 overexpression had a significantly higher WBC count (53*10^9/L vs. 29.5*10^9/L, P=0.041). Survival analysis showed that the patients with CRLF2 overexpression had a worse EFS and OS, the differences were statistically significant (11 months vs. 26 months, P=0.043 and 15 months vs. 32 months, P=0.015). Also, the CRLF2 expression is determined with flow cytometry after staining with FITC-CRLF2 antibody in 28 samples. The correlation analysis was performed on the CRLF2 expression detected by qPCR and flow cytometry, respectively. A significant positive correlation of the two methods was observed(r=0.957, P<0.0001). These data not only indicate that CRLF2 overexpression is a marker of poor outcome, but also reveal the qPCR might be a simple and quick method for screening CRLF2 overexpression in the clinic compared to flow cytometry which is commonly used. We further found that expression of CRLF2 or CRLF2 F232C mutant into Nalm6 and 697 B-ALL cells dramatically increase the CRLF2 mRNA level, which is 69 times than vector-only control. Moreover, CRLF2 or CRLF2 F232C significantly promotes the cell proliferation of Nalm6 and 697 cells compared to vector only (P<0.001). In addition, JAK2 inhibitor (BBT594) treatment showed the significant dose-dependent cell proliferation arrest and clonogenic inhibition in CRLF2 or CRLF2 F232C overexpressed Nalm6 and 697 cells compared to vector-only control. Furthermore, in vivo we observed the 5-fold higher signal intensity of leukemia engraftment in the mice injected with Nalm6-CRLF2-luc or Nalm6-F232C-luc compared to that of Nalm6-vector-luc control 1-3 weeks after the injection(P<0.001). The Nalm6-CRLF2-luc and Nalm6-F232C-luc infiltrations were observed in bone marrow, central nervous system, liver and spleen of the mice. Conclusion: We showed that CRLF2 overexpression could enhance the proliferation and infiltration of human B-ALL cells, and for the first time indicated that JAK2 inhibitor could suppress the cell proliferation and clonogenesis of the CRLF2 overexpressed B-ALL cells. Our data provide direct evidence of the oncogenic role of CRLF2 overexpression and the new therapeutic potential for targeting CRLF2 overexpressed B-ALL with JAK2 inhibitor. Disclosures No relevant conflicts of interest to declare.

Immunoreactivity of MIC2 (CD99) and Terminal Deoxynucleotidyl Transferase in Bone Marrow Clot and Core Specimens of Acute Myeloid Leukemias and Myelodysplastic Syndromes

2006 ◽  
Vol 130 (2) ◽  
pp. 153-157 ◽  
Author(s):  
Loveleen C. Kang ◽  
Cherie H. Dunphy
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Myelodysplastic Syndromes ◽  
Lymphoblastic Leukemia ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Normal Bone Marrow ◽  
Cell Precursor ◽  
Normal Bone ◽  
Myeloid Leukemias

Abstract Context.—MIC2 (“thymus leukemia”) antigen has been shown to be expressed by T cells and monocytes, as well as B cells and granulocyte-lineage cells. It is most intensely expressed by the most immature thymus T-lineage cells and is more intensely expressed by CD34-positive/CD33-positive myeloid cells (compared to more mature myeloid cells) and the earliest CD34-positive/CD10-positive B-cell precursor cells (compared to cells of later B-cell precursor stages). CD99 (MIC2) is characteristically expressed in precursor B- and T-cell lymphoblastic lymphomas/leukemias, as well as in Ewing sarcoma/primitive neuroectodermal tumors (ES/PNET). It has also been shown to be expressed in a few terminal deoxynucleotidyl transferase (TdT)–positive myeloid processes, but has been uniformly negative in TdT-negative myeloid processes. A more recent study showed that 43% of acute myeloid leukemias (AMLs) and 55% of chloromas express CD99, concluding that CD99 is commonly expressed in AML and rarely seen in myeloproliferative disorders, myelodysplastic syndromes, or normal bone marrow. Although this study speculated that MIC2 expression was probably not limited to TdT-positive AML, there was no comparison with TdT reactivity in this study. Objective.—Since AML and high-grade myelodysplastic syndrome may occasionally be difficult to distinguish morphologically from acute lymphoblastic leukemia and ES/ PNET, we undertook a study to analyze MIC2 expression in conjunction with TdT reactivity in distinguishing AML or high-grade myelodysplastic syndrome from acute lymphoblastic leukemia and ES/PNET. Design.—We studied bone marrow core and clot paraffin specimens from AML (classified according to criteria of the World Health Organization; n = 49), myelodysplastic syndromes (n = 4), precursor B-cell acute lymphoblastic leukemia (n = 4), ES/PNET (n = 1), and normal bone marrow (n = 3) with MIC2 (CD99) and TdT immunohistochemistry. Results.—Overall, CD99 was expressed in 24 (49%) of 49 AML cases, including all (11/11) TdT-positive cases. CD99 was expressed in all subtypes of AML except M5. Myelodysplastic syndromes and normal bone marrow specimens were uniformly CD99 negative. Expression of TdT was limited to a subset of AML-M0, -M1, -M2, and -M4, and AML with multilineage dysplasia. Conclusions.—In contrast to a previous study, CD99 expression was not restricted to TdT-positive hematologic proliferations. In particular, the CD99-positive M3 and M7 AMLs were TdT negative. An M5 AML may likely be excluded based on a uniform TdT-negative/CD99-negative immunophenotype. In addition, in our experience, CD99 should be routinely evaluated on bone marrow clots, owing to decreased reactivity or loss of reactivity in rapid decalcifying (RDO) solution–decalcified specimens.

scholarly journals Distinct Expression Patterns of CD123 and CD34 on Normal Bone Marrow B-Cell Precursors (“Hematogones”) and B Lymphoblastic Leukemia Blasts

2009 ◽  
Vol 132 (4) ◽  
pp. 573-580 ◽  
Author(s):  
Nagwa M. Hassanein ◽  
Felisa Alcancia ◽  
Kathryn R. Perkinson ◽  
Patrick J. Buckley ◽  
Anand S. Lagoo
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Expression Patterns ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
B Cell Precursors

scholarly journals Expression of the three myeloid cell-associated immunoglobulin G Fc receptors defined by murine monoclonal antibodies on normal bone marrow and acute leukemia cells

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1951-1956
Author(s):  
ED Ball ◽  
J McDermott ◽  
JD Griffin ◽  
FR Davey ◽  
R Davis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Acute Leukemia ◽  
Cell Sorting ◽  
Lymphoblastic Leukemia ◽  
Mononuclear Phagocytes ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Normal Bone Marrow ◽  
Normal Bone

Monoclonal antibodies (MoAbs) have been prepared recently that recognize the three cell-surface receptors for the Fc portion of immunoglobulin (Ig), termed Fc gamma RI (MoAb 32.2), Fc gamma R II (MoAb IV-3), and Fc gamma R III (MoAb 3G8) that are expressed on selected subsets of non-T lymphocyte peripheral blood leukocytes. In the blood, Fc gamma R I is expressed exclusively on monocytes and macrophages, Fc gamma R II on granulocytes, mononuclear phagocytes, platelets, and B cells, and Fc gamma R III on granulocytes and natural killer (NK) cells. We have examined the expression of these molecules on normal bone marrow (BM) cells and on leukemia cells from the blood and/or BM in order to determine their normal ontogeny as well as their distribution on leukemic cells. BM was obtained from six normal volunteers and from 170 patients with newly diagnosed acute leukemia. Normal BM cells were found to express Fc gamma R I, II, and III with the following percentages: 40%, 58%, and 56%, respectively. Cell sorting revealed that both Fc gamma R I and Fc gamma R II were detectable on all subclasses of myeloid precursors as early as myeloblasts. Cell sorting experiments revealed that 66% of the granulocyte-monocyte colony-forming cells (CFU-GM) and 50% of erythroid burst-forming units (BFU-E) were Fc gamma R II positive with only 20% and 28%, respectively, of CFU-GM and BFU-E were Fc gamma R I positive. Acute myeloid leukemia (AML) cells expressed the three receptors with the following frequency (n = 146): Fc gamma R I, 58%; Fc gamma R II, 67%; and Fc gamma R III, 26% of patients. Despite the fact that Fc gamma R I is only expressed on monocytes among blood cells, AML cells without monocytoid differentiation (French-American-British [FAB]M1, M2, M3, M6) were sometimes positive for this receptor. However, Fc gamma R I was highly correlated with FAB M4 and M5 morphology (P less than .001). Fc gamma R II was also correlated with FAB M4 and M5 morphology (P = .003). Cells from 11 patients with acute lymphoblastic leukemia were negative for Fc gamma R I, but six cases were positive for Fc gamma R II and III (not the same patients). These studies demonstrate that Ig Fc gamma R are acquired during normal differentiation in the BM at or before the level of colony-forming units. In addition, we show that acute leukemia cells commonly express Fc gamma R.

scholarly journals The influence of a dosage regimen of dexamethasone on detection of normal B-cell precursors in the bone marrow of children with BCP-ALL at the end of induction therapy

2020 ◽  
Vol 19 (1) ◽  
pp. 53-57
Author(s):  
E. V. Mikhailova ◽  
T. Yu. Verzhbitskaya ◽  
J. V. Roumiantseva ◽  
O. I. Illarionova ◽  
A. A. Semchenkova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Induction Therapy ◽  
Therapy Group ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Intermittent Therapy ◽  
Continuous Therapy ◽  
B Cell Precursors

Minimal residual disease (MRD) monitoring by flow cytometry at the end of induction therapy is one of the key ways of a prognosis assessment in patients with acute lymphoblastic leukemia (ALL). In B-cell precursor ALL (BCP–ALL), this method of MRD detection is complicated due to the immunophenotypic similarity between leukemic cells and normal B-cell precursors (BCPs). A decrease in intensity of induction therapy can lead to a more frequent appearance of normal BCPs in the bone marrow, which significantly complicates the MRD monitoring. Aim: to assess the incidence of normal BCPs in bone marrow on the 36th day of induction therapy with two different regimens of glucocorticoid (GC) administration according to ALL-MB 2015 protocol. This study was approved by the Independent Ethical Committee and the Academic Council of Dmitriy Rogachev National Medical Research Center of Pediatric Hematology, Oncology, Immunology Ministry of Healthcare of Russian Federation. The study included 220 patients with BCP-ALL who were randomized to two types of GC-based induction therapy: a continuous administration of dexamethasone (n = 139) and an intermittent regimen with a 1-week dexamethasone therapy stop (n = 81). On the 36th day of induction therapy, MRD and normal BCPs were quantified in bone marrow samples by flow cytometry. On the 36th day of treatment, 43.2% of BCP(+) samples were established in the intermittent-therapy group, and 27.3% in the continuous-therapy group (p = 0.016). Comparison of the BCP level in BCP(+) samples revealed the more equitable distribution of BCPs at different developmental stages in the intermittent-therapy group, meanwhile mainly the immature BCPs in a quantity of less than 0.01% were found in the continuous-therapy group. Reduced-intensity induction therapy for patients with BCP-ALL leads to a noticeable increase of normal BCPs in bone marrow at the end of this treatment stage. A higher rate of BCP(+) bone marrow samples hinder the MRD detection due to the immunophenotypic similarity of BCPs and leukemic cells.

scholarly journals Expression of the three myeloid cell-associated immunoglobulin G Fc receptors defined by murine monoclonal antibodies on normal bone marrow and acute leukemia cells

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1951-1956 ◽  
Author(s):  
ED Ball ◽  
J McDermott ◽  
JD Griffin ◽  
FR Davey ◽  
R Davis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Acute Leukemia ◽  
Cell Sorting ◽  
Lymphoblastic Leukemia ◽  
Mononuclear Phagocytes ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Normal Bone Marrow ◽  
Normal Bone

Abstract Monoclonal antibodies (MoAbs) have been prepared recently that recognize the three cell-surface receptors for the Fc portion of immunoglobulin (Ig), termed Fc gamma RI (MoAb 32.2), Fc gamma R II (MoAb IV-3), and Fc gamma R III (MoAb 3G8) that are expressed on selected subsets of non-T lymphocyte peripheral blood leukocytes. In the blood, Fc gamma R I is expressed exclusively on monocytes and macrophages, Fc gamma R II on granulocytes, mononuclear phagocytes, platelets, and B cells, and Fc gamma R III on granulocytes and natural killer (NK) cells. We have examined the expression of these molecules on normal bone marrow (BM) cells and on leukemia cells from the blood and/or BM in order to determine their normal ontogeny as well as their distribution on leukemic cells. BM was obtained from six normal volunteers and from 170 patients with newly diagnosed acute leukemia. Normal BM cells were found to express Fc gamma R I, II, and III with the following percentages: 40%, 58%, and 56%, respectively. Cell sorting revealed that both Fc gamma R I and Fc gamma R II were detectable on all subclasses of myeloid precursors as early as myeloblasts. Cell sorting experiments revealed that 66% of the granulocyte-monocyte colony-forming cells (CFU-GM) and 50% of erythroid burst-forming units (BFU-E) were Fc gamma R II positive with only 20% and 28%, respectively, of CFU-GM and BFU-E were Fc gamma R I positive. Acute myeloid leukemia (AML) cells expressed the three receptors with the following frequency (n = 146): Fc gamma R I, 58%; Fc gamma R II, 67%; and Fc gamma R III, 26% of patients. Despite the fact that Fc gamma R I is only expressed on monocytes among blood cells, AML cells without monocytoid differentiation (French-American-British [FAB]M1, M2, M3, M6) were sometimes positive for this receptor. However, Fc gamma R I was highly correlated with FAB M4 and M5 morphology (P less than .001). Fc gamma R II was also correlated with FAB M4 and M5 morphology (P = .003). Cells from 11 patients with acute lymphoblastic leukemia were negative for Fc gamma R I, but six cases were positive for Fc gamma R II and III (not the same patients). These studies demonstrate that Ig Fc gamma R are acquired during normal differentiation in the BM at or before the level of colony-forming units. In addition, we show that acute leukemia cells commonly express Fc gamma R.

Impaired Precursor B-Cell Maturation/Production In The Bone Marrow: Association With Molecular and Immunophenotypic Markers Of Myeloid Dysplasia In Suspected Low-Grade MDS

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4943-4943
Author(s):  
Charles Repetti ◽  
Hsueh-Hua Chen ◽  
Yongbao Wang ◽  
Vanessa A Jones ◽  
Albert K Ho ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Somatic Mutation ◽  
Conflicts Of Interest ◽  
Statistical Significance ◽  
Sequence Variant ◽  
Low Grade ◽  
Precursor B Cells

Abstract Rationale Myelodysplastic syndromes (MDS) are clonal stem cell disorders that disrupt orderly maturation of multiple hematopoietic lineages. Several studies have suggested that maturation of precursor B cells (hematogones) is also abnormal in MDS. As a result, the presence of normal numbers or increased precursor B cells in bone marrow (BM) is frequently used as a diagnostic feature arguing against a diagnosis of MDS. We compared the presence of myeloid-associated gene mutations and myeloid maturation abnormalities with qualitative and quantitative precursor B cell findings in BM samples submitted for workup of cytopenias or MDS. Methods Seventeen BM aspirate samples with <5% blasts submitted for cytopenia or MDS evaluation were compared with 10 samples having 5% or more blasts and changes diagnostic of MDS or AML. Mutation analysis was performed on genomic DNA using a targeted exome sequencing assay. This assay employs a TruSeq custom amplicon design on the MiSeq platform (Illumina, San Diego, CA). The assay covers the commonly mutated areas of 19 myeloid-associated genes. Somatic mutation status was assigned based on mutation levels, previous association with myeloid neoplasia, and no prior identification in public or internal databases as a normal sequence variant. Flow cytometry using 6-color (CD19/CD34) and 8-color (CD19/10) formats was used to assess lymphoblasts; CD34/13 was used to assess myeloblasts; and CD11b, CD13, CD16, and CD38 were used to assess abnormalities in myelopoiesis. Results  Among the 17 BM samples submitted for cytopenia or MDS evaluation that had <5% blasts, 7 (41%) had immunophenotypic myeloid maturation abnormalities. Ten (59%) of the 17 cases had at least one myeloid-associated somatic mutation, with TET2 and ASXL1being the most commonly mutated genes. The ratio of myeloblasts to B-lymphoblasts, calculated using either CD10 or CD19, was >10:1 in 10/17 (59%) cases. Nine of the 17 (53%) cases had virtually no precursor B cells detected. Discrete abnormalities in more mature myeloid forms were seen in 7/10 (70%) cases with low numbers of B-lymphoblasts but in none of the 7 cases with significant numbers of B-lymphoblasts. MDS-associated mutations were more common in cases with rare B-lymphoblasts (7/9) than in those with higher percentages of precursor B cells (3/8), but the difference did not reach statistical significance (P = 0.15).  Genes mutated in the group with B-lymphoblasts present included ASXL1 (3 cases), DNMT3A (2), TET2 (1) and TP53 (2). Two of these mutated cases presented with isolated thrombocytopenia. By comparison, myeloblast/lymphoblast ratios were >50:1 in all 10 unequivocal MDS/AML samples (>5% blasts); 8 (80%) of these cases had MDS-associated mutations, and 4 (50%) had mutations in multiple genes. Conclusions Decreases in BM precursor B cells in cases of possible low-grade MDS were usually, but not always, associated with the presence of MDS-associated mutations. However, cases with normal or increased precursor B cell numbers also showed MDS-associated mutations although immunophenotypic evidence of myeloid maturation abnormalities was not seen in this group. The identification of a subgroup of cytopenic patients with likely pathogenic mutations in bone marrow precursors but minimal phenotypic evidence of myeloid dysplasia may indicate clonal abnormalities primarily located outside the granulocyte or common stem precursor populations, e.g. restricted to the megakaryocytic lineage. Therefore, the presence of intact precursor lymphoblast and myeloid maturation by higher-dimensional flow cytometry as a primary criterion to argue against a diagnosis of low-grade MDS needs further evaluation, especially when granulocytopenia is absent. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A rare morphological and immunophenotypic presentation of adult B-cell acute lymphoblastic leukemia with blasts in the monocytic zone on CD45 side scatter

2019 ◽  
Vol 5 (3) ◽  
pp. 82
Author(s):  
Shuaeb Bhat ◽  
Saleem Hussain
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Bone Marrow Aspirate ◽  
Lymphoblastic Leukemia ◽  
Prognostic Significance ◽  
Flow Cytometric Analysis ◽  
Blood Film ◽  
High Side

<p class="abstract">We present a case of B-acute lymphoblastic leukemia in an elderly patient who presented with severe weakness and pancytopenia. The patient was a 75 year old Female whose blasts had an unusual morphology in form of coarse azurophilic granules and cytoplasmic blebs and on flow cytometry the blasts were present in the bright CD45 zone with a high side scatter. Bone marrow aspirate sample was subjected to multicolour flow cytometry using Beckman Coulter Navios® which is an 8 colour flow cytometer.  Flow cytometric analysis of the bone marrow aspirate showed blasts in the monocytic zone with a precursor B cell immunophenotype. Complete blood counts showed pancytopenia with peripheral blood film not showing any blasts. Bone marrow aspirate smears showed 20% blasts with coarse azurophilic granules and cytoplasmic blebs. The position of the blasts in this case which were in monocytic zone giving them a bright expression of CD45 and a high side scatter on the CD45 side scatter. This is not the usual position for blasts in B- acute lymphoblastic leukemia as these blasts are less complex. A bright expression of CD45 by blasts in B- acute lymphoblastic leukemia is known to be associated with a poor prognosis but the clinical significance of blasts being bright CD45 with a high side scatter is a very rare occurrence and more number of cases with a similar presentation are required to determine a prognostic significance.</p>

Novel Multiparametric Quantitative Immunocytomorphological Characterisation of Human Bone Marrow Precursor Differentiation and of Haematopoietic Neoplasms Using ImageStream® Cytometry

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4871-4871
Author(s):  
Catherine Claude Martin ◽  
Chantal Jayat-Vignoles ◽  
Jean-Luc Faucher ◽  
Thaddeus George ◽  
Vidya Venkatachalam ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Blood Cells ◽  
High Speed ◽  
Tumour Cells ◽  
Leukemic Cells ◽  
Normal Bone Marrow ◽  
Normal Cells ◽  
Normal Bone ◽  
Abnormal Cells

Abstract The ImageStream technology performs high speed acquisition of brightfield, laser scatter and up to four fluorescent images per cell for several thousands of cells in suspension, thereby enabling simultaneous immunophenotyping and morphology-based measurements. This is the only technology combining cytology and flow cytometry in one single platform. Our aim was to study normal and tumour cells of the haematopoietic lineage with this new technology in order to improve diagnosis of haematological disorders. We have defined cytomorphological criteria of normal bone marrow (n=4) and circulating blood cells (n=40). Cells were multi-colour labelled with both DRAQ5 nuclear stain and CD45 ECD-mAb, and additionally labeled with a combination of mAbs against either CD3/CD19, CD11b/CD16, CD14/CRTH2, or CD71/CD235. Results for normal cells were compared to those obtained by classical cytometry and cytology. We then applied these criteria to samples with patients with circulating leukemic cells, including 1 myelodysplatic syndrome (MDS), 1 myeloproliferative syndrome (MPS), 3 acute lymphoblastic leukaemia (ALL), 2 follicular lymphomas (FL) and 20 chronic lymphocytic lymphomas (CLL). We have created completely new quantitative cytomorphological criteria for classifying blood cells using parameters that measure cellular size and shape, nuclear to cytoplasmic area ratio, nuclear lobe count, SSC texture, the ratio between the size and the major axis of CD45, the ratio between the intensity and the compactness of SSC signal, and the intensity of DRAQ5 labelling, to name a few. Using these criteria, we have characterised normal bone marrow differentiation and normal circulating blood cells. We have obtained a perfect correlation with classical cytology and flow cytometry. Analysis of pathological samples showed that abnormal cells were recognized in all cases. We found an abnormal blast cell compartment and an abnormal monocytic differentiation branch in the case of MDS. We have also defined specific cytomorphological properties that distinguish ALL, FL and CLL tumour cells from normal cells. We also provide data that enumerates the proportion of large cells, of atypical CLL cells and of cells in the G2/M phase. Altogether, these results show that a technology combining cytology and flow cytometry in a single platform leads to the discovery of completely new and quantitative cytomorphological parameters defining each stage of normal cell and each category of abnormal cells of the haematopoietic lineage, opening completely new perspectives for the diagnosis of haematopoietic neoplasms.

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