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Bone Marrow Evaluation

Bone Marrow Evaluation. Dr. Perry Bain. Basic Evaluation Steps. Visual examination. Low power microscopic evaluation. Marrow cellularity. Megakaryocytes. High power microscopic examination. Assessment of myeloid and erythroid cells. Assessment of other cell lines.

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Bone Marrow Evaluation

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  1. Bone Marrow Evaluation Dr. Perry Bain

  2. Basic Evaluation Steps • Visual examination. • Low power microscopic evaluation. • Marrow cellularity. • Megakaryocytes. • High power microscopic examination. • Assessment of myeloid and erythroid cells. • Assessment of other cell lines. • Calculation of M:E ratio.

  3. Quality of aspirate slides can be assessed visually. Good quality slides should contain blue/purple staining “particles,” or aggregates of bone marrow cells. Areas containing particles should be examined microscopically. Visual Inspection of Slide Particles

  4. Low Magnification (4-10x Objectives) • Evaluation of marrow particle cellularity. • Megakaryocytes: • Number • Maturity

  5. Evaluation of Particle Cellularity • Assessment cellularity of the bone marrow sample is required for cytologic interpretation. • Marrow cellularity can be estimated on a smear from an aspirate, but histopathologic examination of a core biopsy sample generally gives a more accurate assessment. • Cellularity is expressed as a percentage of the total marrow particle volume that consists of hematopoietic cells and stroma, rather than fat. Fat (or adipose) appears as clear, “bubble-like” areas within the blue to purple-staining particles.

  6. Bone Marrow Particles Fat (adipose) Both of the illustrated particles have >75% cellularity (<25% fat).

  7. Cell Types in Bone Marrow • Megakaryocytes. • Erythroid cells (RBC precursors). • Myeloid cells (granulocyte precursors). • Monocytes and macrophages (note that monocyte precursors appear morphologically similar to granulocyte precursors). • Lymphocytes and plasma cells. • Other cells (osteoclasts, stromal cells, etc.)

  8. Megakaryocytes • The number and maturity of megakaryocytes are evaluated. • The number of megakaryocytes are assessed subjectively (normal, increased, or decreased). • Mature megakaryocytes have abundant, granular cytoplasm. Immature megakaryocytes have less cytoplasm (higher N:C ratio) and cytoplasm is more basophilic & lacks granulation.

  9. Megakaryocytes Megakaryocytes Megakaryocyte numbers can be assessed at low power (4-10x)

  10. Mature Megakaryocytes

  11. Immature Megakaryocytes • Compared to mature cells, have: • Smaller size. • Higher nucleus: cytoplasm (N:C) ratio • Bluer (more basophilic) cytoplasm.

  12. Evaluation of Hematopoietic Cells • Evaluation of individual cells is performed at high magnification (40-100x objectives). • Cells should be examined in the areas surrounding bone marrow particles. • Within the particles, cells are typically too thick to be examined. • In areas distant from particles, blood contamination may skew cell counts, especially if the animal has a high peripheral leukocyte count.

  13. Erythrocyte and Granulocyte Maturation From: Meyer & Harvey: Veterinary Laboratory Medicine, 2nd Ed.

  14. Granulocyte Precursors • Blast cells (the earliest recognizable form) have an irregularly-shaped nuclei with prominent nucleoli and pale blue cytoplasm. • Promyelocytes (progranulocytes) are recognized by the appearance of primary (nonspecific) granules. • Secondary (specific) granules appear at the myelocyte stage, and the following stages are distinguished based on granule type (neutrophil, eosinophil, basophil), and nuclear morphology (oval=myelocyte, indented=metamyelocyte, band, segmented).

  15. Granulocyte Precursors Myeloblast Promyelocyte or progranulocyte Myelocyte Band Metamyelocytes

  16. Eosinophil and Basophil Precursors • Recognized by the appearance of secondary (specific) granules. • Can be distinguished at myelocyte stage and later. Eosinophils Basophil

  17. Erythrocyte Precursors • Rubriblasts (aka erythroblasts) have round nuclei with prominent nucleoli, and deeply basophilic cytoplasm. • In subsequent maturation nuclei become smaller and more condensed (darker), while cytoplasm becomes lighter and eventually stains reddish with hemoglobin production. • Nucleus is lost at transition from metarubricyte to polychromatophilic erythrocyte (reticulocyte).

  18. Erythrocyte Precursors Nucleolus Rubriblast Prorubricyte and rubricytes Metarubricytes, polychromatophilic cells, and mature erythrocytes

  19. Mitotic Figures • Cells may be “caught” in division on smear. • May appear as “fragmented” nucleus or as two nuclei. • May occur in any of the marrow cell lines.

  20. Other Cells Found in Bone Marrow • Lymphocytes and plasma cells • Macrophages and monocytes • Osteoclasts • Lysed cells • Mast cells • Endothelial cells and fibroblasts (stroma)

  21. Lymphocytes • Lymphocytes have round to oval, sometimes indented nuclei. • Cytoplasm is scant, and may appear only as a small “tag” on one side of the cell.

  22. Plasma Cells • These are antibody-producing B lymphocytes. • They resemble erythroid cells, but are distinguished by: • Abundant, deep blue cytoplasm. • Perinuclear Golgi zone. • Eccentrically-located nucleus. Golgi zone

  23. Macrophages • Macrophages are large cells with abundant cytoplasm. • They may have cytoplasmic vacuolation. • May contain phagocytized cellular material, hemosiderin, etc. A macrophage containing several phagocytized erythrocytes

  24. Osteoclasts • These cells resemble megakaryocytes in size and staining characteristics. • Have multiple, individual nuclei, rather than the single lobulated nucleus of the megakaryocyte.

  25. Lysed Cells • Preparation of bone marrow smears creates broken or lysed cells (sometimes called “smudge cells” or “basket cells”). • These cells should not be counted as part of a cell differential or M:E ratio. • Evaluation of marrow should be done in an area with few or no lysed cells if possible.

  26. Lysed Cells These are nuclei of cells that ruptured or lysed during sample collection or slide preparation.

  27. Estimation of Myeloid to Erythroid (M:E) Ratio • Compares the number of myeloid (granulocytic) and erythroid cells. • Determination of M:E ratio can be done by differential count (500 cells) or by subjective estimation. • Only nucleated cells are counted (i.e., mature erythrocytes are not counted). • Gives information about relative production of myeloid and erythroid cells. For instance, M:E ratio should be decreased with regenerative response to anemia, and increased with increased neutrophil production during inflammation.

  28. Estimation of M:E Ratio Myeloid cells This field contains 14 myeloid (granulocytic) cells, and 21 erythroid cells, for an M:E ratio of 0.67. (Note that multiple fields must be examined for an accurate M:E estimate.) Erythroid cells

  29. Normal M:E Ratios • Normal M:E ratio varies among species: • Dog: 0.75-2.53 • Cat: 1.21-2.16 • Horse: 0.50-1.50 • Cattle: 0.31-1.85

  30. End of Bone Marrow Evaluation

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