Participants Identification No. % Evaluation. Mitotic figure Educational Erythrocyte precursor, abnormal 1 0.
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1 Cell Identification Mitotic figure Educational Erythrocyte precursor, abnormal BMD-02 The arrowed cell is a mitotic figure. It was correctly identified by 99.5% of the participants. A cell containing a mitotic figure is variable in size and the cytoplasm contains characteristics of the resting cell. However, the nucleus differs from the resting cell, appearing as a dark irregular mass, often with a clear central zone and variable shape with irregular projections. In certain phases of mitosis, individual chromosomes may become visible, as seen in the current example. Differentiation from a degenerating cell relies on evaluation of the nuclear features. A degenerating cell contains pyknotic spherical nuclear fragments, lacking individual chromosomes versus the compact nucleus of a mitotic figure. 3
2 Neutrophil with Pelger-Huët nucleus Educational (acquired or congenital) Neutrophil, segmented or band Educational Neutrophil with dysplastic nucleus Educational and/or hypogranular cytoplasm Neutrophil, giant band or giant metamyelocyte Educational BMD-03 The arrowed cell is a neutrophil with a Pelger-Huët nucleus. It was correctly identified by 67.7% of the participants as either neutrophil with Pelger-Huët nucleus (acquired or congenital) or neutrophil with dysplastic nucleus and/or hypogranular cytoplasm. The arrowed cell contains a bilobed nucleus with two round lobes connected by a thin filament, characteristic of a Pelger-Huët nucleus or Pelger-Huët cell. These cells may occur in the autosomal dominant genetic disorder known as Pelger-Huët anomaly or may be seen in acquired disorders and are thus known as pseudo- Pelger-Huët neutrophils. Pseudo- Pelger- Huët neutrophils may be associated with neoplastic processes such as myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms and acute myeloid leukemias such as seen in our current case. Dysplasia within the myeloid lineage is characterized by hypolobation or hypersegmentation of the nucleus, hypogranular cytoplasm and small cell size. Pseudo- Pelger-Huët neutrophils may also be associated with certain drug therapies (sulfonamide, colchicines, mycophenolate mofetil) and infections (HIV, Mycoplasma pneumonia). These features differ from the normal neutrophilic band in that the nucleus of a band may become twisted or folded on itself but does not constrict to become a filament, which is seen in the arrowed cell. Unlike normal segmented neutrophils, the two lobes of the cell seen here are equal in size and round shape with no further segmentation. Additionally the chromatin in the current cell shows more condensed chromatin than normal neutrophilic bands and segmented forms. Evaluation of the current aspirate smear shows that there are frequent forms with similar nuclear features. 4
3 Megakaryocyte or precursor, Educational abnormal Megakaryocyte or precursor, normal Educational Osteoclast Educational Plasma cell, abnormal (malignant, Educational myeloma cell) Blast cell Erythrocyte precursor, abnormal/ dysplastic nuclear features Macrophage (histiocyte) BMD-04 The arrowed cell is abnormal megakaryocyte. It was correctly identified by 81.2% of the participants. Dysplasia in the megakaryocytic lineage is characterized by abnormalities in cell morphology and topography. Morphological abnormalities include micromegakaryocytes (dwarf megakaryocytes), megakaryocytes with non-lobated nuclei and/or separated nuclei. Micromegakaryocytes are abnormally small megakaryocytes typically 20 μm or less, having a N:C ratio of 1:1 or 1:2. Micromegakaryocytes may be hypolobated or have multiple small lobes. These morphological features can also be appreciated on bone marrow biopsy (H&E stained) slides. Abnormalities in megakaryocytic topography are best appreciated on bone marrow biopsy slides and include abnormal clustering and trabecular location. A normal mature megakaryocyte is 25 to 50 μm and has numerous nuclear lobes which are connected by bands or chromatin threads. The arrowed cell differs from a normal megakaryocyte as it lacks numerous nuclear lobes and contains separated nuclei. When an abnormal megakaryocyte contains separated nuclear lobes it may resemble an osteoclast. Unlike the arrowed megakaryocyte, osteoclasts are very large, approximately 100 um, typically have multiple uniformly-sized separated nuclei and the cytoplasm typically has frayed margins with fine reddish-purple granules. 5
4 Blast cell Educational Myeloblast with Auer rod Educational Erythrocyte precursor, abnormal/ dysplastic nuclear features Erythrocyte precursor, normal Lymphocyte, normal Neutrophil, promyelocyte, abnormal with/without Auer rod(s) BMD-05 The arrowed cells are blast cells. They were correctly identified by 95.2% of the participants. A blast cell is large and measures 10 to 20 μm and has a high N:C ratio of 7:1 to 1:1. The nuclei are round to oval, occasionally indented or folded with fine chromatin and one or more prominent nucleoli. The cytoplasm is scant to moderate in amount, pale to moderately basophilic and generally agranular although cytoplasmic granules may be seen. The identification of Auer rods within the cytoplasm indicates a myeloid lineage. Otherwise, lineage specificity cannot be determined morphologically and is thus evaluated by flow cytometric analysis, immunohistochemical analysis and, less commonly, cytochemical staining. The arrowed blast cells show myeloid antigen expression by flow cytometry indicating a myeloid lineage but do not contain Auer rods. 6
5 Lymphocyte, normal Educational Erythrocyte precursor, normal Educational Erythrocyte precursor, abnormal/ dysplastic nuclear features Erythrocyte precursor with megalo- blastic changes/maturation Megakaryocyte or precursor, abnormal BMD-06 The arrowed cell is a normal lymphocyte. It was correctly identified by 92.9% of the participants. Lymphocytes are small cells measuring 7 to 15 μm with a high N:C ratio of 5:1 to 2:1. Lymphocyte nuclei are round to oval and unlike a blast cell, contains dense or coarsely clumped chromatin and lacks a prominent nucleoli. The scant cytoplasm is typically pale to moderately basophilic and agranular. Lymphocytes are normal constituents of the bone marrow numbering 10-15% of the nucleated cells in adults. Higher numbers of lymphocytes as well as hematogones are seen in bone marrow aspirates of infants and young children. Lymphocytes may resemble erythroid precursors such as a late basophilic normoblast or early polychromatophilic normoblast. Unlike mature lymphocytes, the chromatin pattern in a basophilic normoblast typically does not have significant clumping, exhibiting more thickened, coarse chromatin. The cytoplasm appears somewhat denser with a royal blue color with increasing grey tones in the later forms. The early polychromatophilic normoblasts exhibits increased graying of the cytoplasm. Kathryn Rizzo, DO, PhD Hematology and Clinical Microscopy Resource Committee 7
6 Bone Marrow Interpretation AML with myelodysplasia-related changes 1. Dysplastic granulopoiesis can include: Hypogranular cytoplasm - - Small cell size - - Hypo-segmented nuclei All of the above Hypogranular cytoplasm and Hypo-segmented nuclei only The correct answer is all of the above. At least 10% of cells in the myeloid or erythroid lineage must demonstrate dysplastic features by WHO 2008 criteria to qualify as significant. Dysplastic granulopoiesis affects the nucleus as well as the cytoplasm of myeloid precursors. Nuclear abnormalities can include hypolobation, such as with the pseudo Pelger-Huët anomaly, or hypersegmentation of the nuclear lobes. The cytoplasm of dysplastic myeloid cells can be hypogranular, agranular or contain pseudo Chediak-Higashi granules or Auer rods. Ineffective granulopoiesis can also be manifested by small size of the cell. Determining the presence of dysplasia in any cell line can be affected by the quality of the smear preparation as well as the quality of the stain.myeloid cells especially can appear to be hypogranular with pale staining of a smear which highlights the need for well stained and well prepared smears. 2. Dyspoiesis of erythroid precursors can include: Ring sideroblasts Karyorrhexis - - Internuclear bridging Cytoplasmic blebbing - - All of the above Ring sideroblasts, Karyorrhexis and Internuclear bridging only The correct answer is ring sideroblasts, karyorrhexis and internuclear bridging. Dysplastic erythropoiesis can affect the nucleus in the form of nuclear budding, internuclear bridging, karyorrhexis, multinuclearity and megaloblastoid changes. Cytoplasmic features can include ring sideroblasts, as defined with a Prussian blue stain for iron. Cytoplasmic vacuoles can also be seen, although vacuoles can be associated with the toxic effects of alcohol, as well as with Parvovirus infection. PAS (Periodic-acid-Schiff) staining in the cytoplasm can be present in the form of fine or coarse globules. 8
7 3. Criteria for a diagnosis of AML with myelodysplasia-related changes can include: Myelodysplastic syndrome-related cytogenetic abnormalities are present e.g. -7/del(7q). All of the above % blasts are counted in the bone marrow aspirate and dysplasia is present in one cell lineage Dysplasia is present in at least 20% of cells of at least two cell lineages The history includes prior cytotoxic therapy for an unrelated disease - - The correct response is Myelodysplastic syndrome-related cytogenetic abnormalities are present e.g. -7/del(7q). WHO 2008 morphologic criteria for a diagnosis of AML with myelodysplasia related changes include the presence of dysplasia in at least 50% of the cells in at least two, not one, bone marrow cell lines, which would eliminate the third and fourth choice listed in the responses. Cytogenetic abnormalities can include -7/del(7q), as in the first and correct response listed, -5/del(5q-) or a complex karyotype. This diagnosis also requires the presence of >20% blasts in the peripheral blood or bone marrow by differential count. To determine the blast percentage in the bone marrow, a 500 cell differential count of the aspirate smear or touch imprint of the biopsy, is recommended, while a 200 white blood cell differential count of the peripheral blood is recommended. 4. Cytogenetic abnormalities associated with AML with myelodysplasia-related changes can include any of the following EXCEPT: -5/del(5q) del(11q) t(5;17)(q33;p13) t(3;5)(q25;q34) t(16;16)(p13.1;q22) The correct response is t(16;16)(p13.1;q22) and is not associated with this subtype of AML. This particular question highlights in part the need for obtaining a sample for cytogenetic studies during the bone marrow biopsy procedure. Correlation of cytogenetic results with those of the morphologic findings in the bone marrow or peripheral blood are needed to achieve the correct classification of an acute myeloid leukemia and can also provide prognostic information. The pathologist responsible for the case would receive the results and correlate them with the cytologic findings. Subsequent bone marrow evaluations after chemotherapy should also include a cytogenetic evaluation to determine the presence or absence of the particular cytogenetic abnormality associated with the leukemia. 9
Participants Identification No. % Evaluation. Mitotic figure Educational Erythrocyte precursor, abnormal/
Cell Identification BMD-09 Participants Identification No. % Evaluation Mitotic figure 233 96.7 Educational Erythrocyte precursor, abnormal/ 4 1.7 Educational dysplastic nuclear features Erythrocyte precursor
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