Participants Identification No. % Evaluation. Mitotic figure Educational Erythrocyte precursor, abnormal/
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1 Cell Identification BMD-09 Participants Identification No. % Evaluation Mitotic figure Educational Erythrocyte precursor, abnormal/ Educational dysplastic nuclear features Erythrocyte precursor with megaloblastic changes/maturation Educational The arrowed objects are mitotic figures and were correctly identified by 96.7% of participants. Mitotic figures vary in size and shape. Mitotic figures contain the cytoplasmic characteristics such as color and granulation of the resting cell. During mitosis, the nucleus appears as a dark irregular mass, taking on a variety of shapes based on the phase of mitosis. In metaphase, the individual chromosomes are visible and migrate toward opposite poles. In anaphase or telophase, two separate sets of chromosomes forming two daughter cells may be seen. Typically only a small number of mitotic figures are seen in a bone marrow aspirate smear slide. 3
2 Participants Identification No. % Evaluation BMD-10 Erythrocyte precursor, abnormal/ Educational dysplastic nuclear features Erythrocyte precursor, normal Educational Erythrocyte precursor with megaloblastic changes/maturation Educational The arrowed cells are dysplastic erythrocytes and were correctly identified by 93.4% of participants. Erythroid dysplasia is typically denoted by changes in the chromatin and nucleus of erythroid precursors. Normally, erythroid precursors through all stages of maturation, have a well-defined round nucleus and as the cell matures, the chromatin appropriately becomes increasingly dense and hyperchromatic. In contrast, atypical/dysplastic erythroid changes include nuclear budding which appears as nuclear lobulation or rosette formation as seen in this case. Additional dysplastic features include internuclear bridging by thin strands of chromatin, multinucleation and megaloblastic changes. Dysplastic changes are best appreciated in the later stages of erythroid maturation. Dysplastic cytoplasmic changes seen in the erythroid series include ring sideroblasts, cytoplasmic vacuolization and periodic acid-schiff positivity. 4
3 Participants Identification No. % Evaluation BMD-11 Neutrophil, giant band or giant Educational metamyelocyte Neutrophil, segmented or band Educational Neutrophil, metamyelocyte Educational The arrowed cell is a giant band and was correctly identified by 73.4% of participants. Giant bands are cells that are at least 1.5 times bigger than a normal band (15 to 20µm). Compare the size of the arrowed cell to the normal mature neutrophil immediately to its right. Giant bands are typically seen in the neutrophil series and the nucleus is indented to more than half the nuclear width and fold into bizarre C or S shapes. The chromatin may be loosely clumped or less condensed when compared to a normal band. The cytoplasm is typically similar to the normal counterpart, appearing pink with lilac granules. These precursors are a result of megaloblastic hematopoiesis indicated by loss of nuclear and cytoplasmic maturation synchrony. 5
4 Participants Identification No. % Evaluation BMD-12 Neutrophil, metamyelocyte Educational Neutrophil, myelocyte Educational Neutrophil with dysplastic nulceus and/or hypogranular cytoplasm Educational The arrowed cell is a metamyelocyte and was correctly identified by 88.8% of participants. Metamyelocytes are 10-18µm in size with abundant pink cytoplasm with numerous specific, fine lilac colored granules and only few residual primary granules. The nucleus is indented to less than half of the potential round nucleus and thus appears kidney shaped. The chromatin is clumped with no distinct nucleoli seen. The metamyelocyte stage is the first post-mitotic stage in the myeloid precursor development. Metamyelocytes compose 15-20% of nucleated cells in the bone marrow and may be seen in the blood in response to stress. 6
5 Participants Identification No. % Evaluation BMD-13 Macrophage containing hemosiderin Educational Macrophage (histiocyte) Educational Histiocyte, sea blue Educational Macrophage with phagocytized cells Educational The arrowed cells are macrophage with hemosiderin, also known as a siderophage and was correctly identified by 71.4% of participants. Macrophages containing hemosiderin are 20-80µm in size and have a low N:C ratio. The nuclei are round to indented and the cell usually contains a single nucleus. The cytoplasm is abundant, typically containing numerous dark blue-black coarse granules of variable size, seen in clumps. This is in contrast to the less coarse yet variably sized blue or bluish-green cytoplasmic globules present in sea blue histiocytes. Hemosiderm is a byproduct of the breakdown of hemoglobin from phagocytized erythrocytes. On Prussian blue iron staining, which is performed on most bone marrow specimens, the granules stain blue, differentiating them from other pigmented cytoplasmic granules such as melanin and ceroid pigment present in seablue histiocytes. Increased numbers of siderophages in the bone marrow may be seen in patients with a chronic blood transfusion history or post-chemotherapy treatment, as seen in the current case. Kathryn Rizzo, DO, PhD, FCAP Hematology and Clinical Microscopy Resource Committee 7
6 BMD-14 Bone Marrow Interpretation Questions 1. Which of the following is not a feature of dysplasia? Response No. % A. Nuclear budding in a RBC precursor cell B. Hypogranulated neutrophil cytoplasm C. Multi-lobated megakaryocytic nucleus D. Pseudo-Pelger-Huet neutrophil nuclei Intended Response: C Multi-lobated megakaryocytic nucleus Normally, as the megakaryocyte matures the cell does not divide; however the nucleus continues to undergo nuclear replication which causes the nucleus to contain several lobes. Thus answer C is a normal finding. Answers A, B and D are incorrect as they are all features of dysplasia. 2. Which of the following statements is true? Response No. % A. Increased numbers of macrophages may be seen in regenerating postchemotherapy bone marrow specimen. B. It is not necessary to quantitate the number of blasts in a post-chemotherapy bone marrow specimen. C. Dysplastic features in erythrocytes are never seen post-chemotherapy treatment. D. Bone marrow biopsy and morphological evaluation is of little utility in the posttherapy setting and is thus not performed Intended Response: A Increased numbers of macrophages may be seen in regenerating postchemotherapy bone marrow specimen. The correct answer is A. Increased numbers of macrophages are a common finding in post therapy specimens. The macrophages may have foamy cytoplasm, contain ingested debris, or be hemosiderin laden especially if the patient has had blood transfusions. Answer B is incorrect as it is important to quantitate the blast count, especially in these specimens, in order to evaluate for morphological evidence of residual acute leukemia and therapy efficacy. Answer C is incorrect as dyserythropoiesis is a common finding in post-therapy marrow specimens. Answer D is incorrect for reasons indicated in answer B and for evaluation of bone marrow regeneration posttherapy/bone marrow transplant. 8
7 3. Which of the following statements is false? Response No. % A. For evaluation of acute myeloid leukemia, promonocytes are considered blast equivalents and should be counted separately from mature monocytes. B. For evaluation of acute myeloid leukemia, atypical mature monocytes are considered blast equivalents. C. Acute myeloid leukemias may contain specific cytogenetic aberrations which may be detected in patients with minimal residual disease. D. Acute promyelocytic leukemia is composed of neoplastic promyelocytes which are considered blast equivalents Intended Response: B For evaluation of acute myeloid leukemia, atypical mature monocytes are considered blast equivalents Mature monocytes are not considered blast equivalents unlike answers A and D which are true statements. Answer C is true and thus cytogenetic/fish analysis is often performed on bone marrow specimens. 4. Which of the following statements best describes post-chemotherapy treated bone marrow? Response No. % A. There is a marked increase in hematopoietic cells compared with fat cells B. Transient therapy related dysplasia may be seen in erythroid and myeloid cells. C. Peripheral blood cytosis (ie, erythrocytosis) is common during chemotherapy treatment. D. Increased numbers of hematogones are never seen in post-chemotherapy marrows of children. - - Intended Response: B Transient therapy related dysplasia may be seen in erythroid and myeloid cells Dysplasia is often seen and is usually a transient finding, although it is important to note, especially if the dysplastic changes persist in subsequent evaluations. Answer A is incorrect as typically immediate post-therapy marrows show a decrease in hematopoietic cells. Answer C is incorrect as typically the peripheral blood would demonstrate cytopenias due to decreased marrow hematopoiesis. Answer D is incorrect as increased hematogones are a common finding in post-chemotherapy marrows of children and may be difficult to differentiate from residual lymphoblasts in patients with acute lymphoblastic leukemia. Kathryn Rizzo, DO, PhD, FCAP Hematology and Clinical Microscopy Resource Committee 9
8 The current case is a bone marrow aspirate smear from a 58 year old man with a history of acute myeloid leukemia status post induction chemotherapy. According to the 2008 WHO classification of tumours of hematopoietic and lymphoid tissue, acute myeloid leukemia is characterized by greater than or equal to 20% blasts or blast equivalents. Certain cases with less than 20% blasts can be classified as acute myeloid leukemia when specific cytogenetic abnormalities are present as well as cases of acute erythroid leukemia. This patient has a history of acute myeloid leukemia with monocytic differentiation. Monocytic differentiation may be noted morphologically, immunophenotypically or cytochemically. Morphologically the cells may show a range of monocytic maturation stages including monoblast, promonocyte and mature monocyte stage. According to the 2008 WHO classification, promonocytes are considered blast equivalents and should be designated separately from mature monocytes when performing a bone marrow differential. Promonocytes would be counted along with the monoblasts and myeloblasts in determining total blast count. This is similar to acute promyelocytic leukemia where the abnormal promyelocytes are considered blast equivalents and counted as such. There is morphological overlap in the various stages of monocytic differentiation and definitive stage designation can at times be difficult. Monoblasts are typically large and have the typical myeloblast morphology or may have a moderate amount of cytoplasm. The cytoplasm ranges from light to dark blue, may contain vacuoles and/or fine granules, and may have cytoplasmic blebbing. The nucleus is typically round with lacy chromatin and distinct nucleoli. Promonocytes have a moderate amount of cytoplasm which typically contains fine granules. The nucleus has a similar fine chromatin pattern; however, it may have a delicate folded or grooved appearance and nucleoli are indistinct to absent. Abnormal monocytes may be difficult to differentiate from promonocytes due to their more immature morphology. However, the chromatin appears coarser and more condensed, their folds are more defined by a more distinct nuclear membrane, and their cytoplasm is more granulated. A B C FIGURE 1. MONOCYTIC LINEAGE: A. Monoblast with round nuclei, lacy chromatin and indistinct nucleoli. B: Promonocytes with delicate nuclear folds and moderate amount of cytoplasm. C: Monocyte with lobulated nuclei and coarse chromatin. In the current 2008 WHO classification, several categories of acute myeloid leukemia may contain monocytic differentiation. Thus, correlating cytogenetic and FISH analysis with morphology is essential in subclassifying a newly diagnosed acute myeloid leukemia. Acute myeloid leukemia with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 is one type of leukemia with a distinctive morphological appearance. A proportion of the cells in this type of leukemia typically are of monocytic lineage. However a striking characteristic of this type of leukemia are the eosinophilic precursors, typically the eosinophilic promyelocytes and myelocytes, which contain 10
9 abnormal, purple-violet basophilic colored granules. Acute myeloid leukemias containing translocations of the MLL gene in the 11q23 region commonly present with monocytic/monoblastic morphology. There are a number of translocation partners with the MLL gene and thus the acute myeloid leukemia is designated based on a balanced translocation partner. Acute myeloid leukemia with t(6;9)(p23;q34); DEK-NUP214 is associated with basophilia and multilineage dysplasia and may also contain monocytic features. In patients with specific genetic aberrations, cytogenetic/fish analysis may detect minimal residual disease in post-chemotherapy treated bone marrow specimens. Frequently, bone marrow specimens are also analyzed for specific gene mutations. Acute myeloid leukemias with mutation of the NPM1 gene show a strong association with acute myeloid leukemias with monocytic differentiation. However, mutations in the NPM1 gene are not restricted to this subtype and are also seen in acute myeloid leukemias without monocytic differentiation. The current aspirate smear is a specimen which is status post induction chemotherapy. It is important to document on a differential count, the number of residual blasts/blast equivalents. This number will indicate morphologically, the presence of residual leukemia and the effectiveness of the chemotherapy regimen. Post-chemotherapy aspirate specimens show a number of characteristic features. Initially the specimen will be hypocellular, containing spicules composed largely of fat. Due to the depletion of the bone marrow, peripheral blood cytopenias such as anemia, leukopenia and thrombocytopenia are observed. In marrow specimens showing appropriate response to therapy, residual cellular components in the bone marrow consist largely of plasma cells, small lymphocytes, mast cells and macrophages. Macrophages may have foamy cytoplasm, cytoplasm containing debris, or cytoplasm containing hemosiderin (also known as siderophages). Reconstitution of the marrow then ensues and clustering of immature hematopoietic precursors, as well as megakaryocytes, are seen. The erythroid lineage is typically the first lineage to regenerate followed by the myeloid series and then the megakaryocytic series. Dyspoiesis in the erythroid and myeloid lineages are common and transiently observed during this reconstitution time. (FIGURE 2) Other cellular elements such as hematogones (especially in pediatric marrows) and macrophages/ siderophages are also readily apparent. A B C FIGURE 2. POST-CHEMOTHERAPY TRANSIENT CELLULAR CHANGES. A,B: The above changes are examples of dyspoiesis seen in the erythroid cell lineages. Dyspoiesis is denoted by nuclear irregularity and budding. C: This is an example of a giant band with nuclear and cytoplasmic maturation dyssynchrony. Kathryn Rizzo, DO, PhD Hematology and Clinical Microscopy Resource Committee 11
10 REFERENCES 1. Reichard K. Post-therapy bone marrow findings. In: Foucar K, Reichard K, Czuchlewski D. Bone Marrow Pathology, 3 rd ed. vol. 2. Chicago, IL: ASCP press; Arber D.A, et al. Acute myeloid leukaemia with recurrent genetic abnormalities. In: Swerdlow SH, ed. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4 th ed. Lyon, France: IARC Press; Goasguen JE, Bennett JM, Bain BJ, et al. International Working Group on Morphology of Myelodysplastic Syndrome. Morphologic evaluation of monocytes and their precursors. Haemoatologica. 2009;94(7):
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