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Thread: Micromegakaryocytes, megathrombocytes, myeloid leukemia picture - blood histology atlas

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    Default Micromegakaryocytes, megathrombocytes, myeloid leukemia picture - blood histology atlas

    Introduction
    Myelodysplastic syndromes (MDS) are abnormal proliferations of hematopoietic stem cells that retain their capacity to differentiate but do so in an inefficient manner, so that bone marrow (BM) and peripheral blood cells show various morphological abnormalities and the numbers of mature blood cells are variably reduced. These disorders are characterized clinically by BM failure, followed by a progressive impairment in the ability of myelodysplastic stem cells to differentiate and an increasing risk of evolution into acute myeloid leukemia (AML).

    Since dysplasia is the pathologic hallmark and various chromosomal abnormalities may be found in these patients, the current diagnostic approach includes peripheral blood and BM morphology, BM histology, and cytogenetics. In vitro cultures of hematopoietic progenitors can provide additional useful information for the diagnosis of MDS, since colony formation is poor or absent in most of these patients. Thus, overt marrow dysplasia, clonal cytogenetic abnormality, and reduced in vitro progenitor cell growth allow a conclusive diagnosis of MDS. This combination is, however, found in only some MDS patients, who tend to be those with more advanced disease. In many instances, cytogenetics is not informative and BM cultures are not performed, so that the diagnosis of MDS is based entirely and exclusively on morphological criteria.

    Flow cytometry immunophenotyping, a reliable method for quantitative and qualitative evaluation of hematopoietic cells, is playing an increasingly crucial role in the diagnosis and management of acute leukemias.6 MDS patients have been found to have abnormal expression of several surface antigens, as indicated by either the intensity of fluorescence or the percentage of positive cells. Findings of recent studies suggest that flow cytometry immunophenotyping might provide useful information in management of MDS patients

    The purpose of this study was to develop a flow-cytometric approach to the evaluation of erythroid and myeloid dysplasia in patients with MDS.

    Patients and controls
    This study enrolled patients followed at the Division of Hematology, IRCCS Policlinico San Matteo and University of Pavia Medical School, Pavia, Italy. The procedures followed were in accordance with the Ethical standards of the institutional committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 1983. The cytological diagnosis of MDS was initially made according to the FAB criteria, all cases were then reclassified according to the WHO classification.3 Patients with RAEB-T were excluded from this study.
    Morphological analysis of marrow specimens was performed by two or three independent cytologists, who were blinded to the cytometric data. A total of 500 BM nucleated cells per sample were assessed. The evaluation of the erythroid lineage was based on the detection of megaloblastic changes, nuclear lobulation, multinuclearity, and cytoplasmic granules/inclusions. In the myeloid lineage, the following abnormalities were considered: bizarre nuclear shape, hypo- or agranularity, nuclear/cytoplasmic asynchrony, and pseudo-Pelger anomaly. Micromegakaryocytes, small binucleated megakaryocytes, megakaryocytes with small round separated nuclei, and megathrombocytes were considered signs of megakaryocytic dysplasia.
    Micromegakaryocytes, megathrombocytes, myeloid leukemia picture attachment.php?s=cd4a2851cbe0bb17bc50fe84706af096&attachmentid=1430&d=1439592947

    The degree of erythroid or myeloid dysplasia was defined according to the following morphological scoring system: 0 (absence of dysplasia, 0–2% of abnormal cells), (mild dysplasia, 3–9% of abnormal cells), (moderate dysplasia, 10–29% of abnormal cells), and (severe dysplasia, 30% or more abnormal cells).

    Cytogenetic analysis was performed at diagnosis, using standard G-banding with trypsin-Giemsa staining, and karyotypes were classified using the International System for Cytogenetic Nomenclature Criteria. FISH studies were performed as described previously. The International Prognostic Scoring System (IPSS) was employed to define prognosis according to Greenberg et al.

    This study involved two cohorts of patients and controls: (a) a 'learning cohort', whose investigations were aimed at defining discriminant immunophenotypic markers; (b) a 'testing cohort' on which we examined whether our discriminant markers were able to reproduce what we had found in the learning cohort.

    Flow cytometry studies
    K3-EDTA BM aspirate specimens were collected and stained within 3 h using a whole-blood lysis technique (FACS lysing solution, Becton Dickinson, San José, CA, USA) and a panel of direct conjugated monoclonal antibodies (MoAb) according to standard procedures described previously.
    Six-parameter, four-color flow cytometry was performed with a FACSCalibur flow cytometer (Becton Dickinson) equipped with a 15-nW argon laser (excitation at 488 nm). Daily instrument quality control, including fluorescence standardization, linearity assessment, and spectral compensation, were performed to ensure identical operation from day to day.

    At least 50 000 events were acquired for each sample in linear mode for side scatter (SSC) and in log mode for fluorescent signals; isotype-matched negative controls were used in all assays. Data were collected and analyzed with CellQuest software (Becton Dickinson).

    The MoAb panel employed was as follows: fluorescein isothiocyanate (FITC)-conjugated anti CD3, CD5, CD7, CD15, CD16, CD34, and CD71; phycoerythrin (PE)-conjugated anti CD4, CD8, CD10, CD13, CD19, CD33, CD34, CD56, and anti-glycophorin A (GlyA); allophycocyanine (APC)-conjugated CD14, CD19 and CD45; Tri-Color (TC, PE-Cyanine5)-conjugated anti CD45. All MoAbs were purchased from Becton Dickinson except CD45-TC, which was obtained from Caltag Laboratories (Burlingame, CA, USA). In addition, Laser Dye Styryl (LDS751, Molecular Probes, Leiden, The Netherlands) was used for nuclear cell identification.

    The CD45 marker was tested in each combination to allow a primary gating of BM cell subsets based on CD45 antigen expression and SSC laser light diffraction as described.22 Orthogonal SSC was compared with that obtained for mature granulocytes in healthy control peripheral blood specimen processed in the same day. Cell subsets recognized by this method were blast cells (CD45lowSSClow), lymphocytes (CD45highSSClow), monocytes (CD45highSSCintermediate), and granulocytes (CD45highSSChigh). CD34, CD3, CD19, CD14 and CD33 populations were back-gated to determine whether the analysis gates were appropriate.

    Nucleated red cells were gated using a combination of CD71/GlyA/LDS751/CD45. All MoAbs were analyzed on all cell populations defined in the CD45/SSC dot-plot. Gating strategy to identify erythroid precursors was based on nuclear staining (LDS751) to exclude cellular debris and nonviable cells: erythroblasts were defined in LDS751+ gate as GlyA+/CD45low-/SSClow cells.

    References:
    Leukemia - Flow cytometry evaluation of erythroid and myeloid dysplasia in patients with myelodysplastic syndrome











    Last edited by Medical Photos; 08-14-2015 at 10:55 PM.

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