Mary M. Christopher, DVM, PhD, DACVP, DECVCP
University of California Davis, Department of Pathology, Microbiology, and Immunology School of Veterinary Medicine
Davis, CA, USA
Unlike blood smear evaluation, the evaluation of bone marrow is limited to those clinical situations in which hematologic or other abnormalities already have been identified and require further characterization or investigation. In other words, a bone marrow evaluation is not a baseline test, but rather a confirmatory test used to rule in or rule out specific hypotheses. In this presentation, I will review the indications for bone marrow evaluation, provide useful hints for preparing high quality bone marrow smear preparations, describe the major components of bone marrow microscopic evaluation, and give examples of abnormal bone marrow findings.
Indications for Bone Marrow Evaluation
There are many indications for the evaluation of bone marrow in dogs and cats. These can be grouped into the investigation of unexplained cytopenias, investigation of atypical cells in the peripheral blood, and investigation of hemic neoplasia.
1. Investigation of unexplained cytopenias
a. Unexplained nonregenerative anemia, especially when persistent and progressive.
b. Unexplained leukopenia (neutropenia), especially when a left shift is not apparent.
c. Unexplained thrombocytopenia, to rule out decreased production as a causative mechanism. Also, bone marrow smears are needed for the antimegakaryocyte antibody test.
d. Bicytopenia or pancytopenia.
2. Investigation of atypical cells in the peripheral blood
a. Inappropriate NRBC in peripheral blood (i.e., in the absence of a markedly responsive anemia).
b. Immature hematopoietic cells (blasts) in the blood-atypical or immature hemic cells raise the possibility of hematopoietic neoplasia. Bone marrow evaluation is necessary to determine the source and number of these cells in the tissue.
c. Atypical RBC (e.g., basophilic stippling, multiple Howell-Jolly bodies), WBC, or platelet morphology.
d. Persistent, unexplained marked increases in RBC, WBC, or platelet numbers in the peripheral blood.
e. Evaluation of infectious diseases such as leishmaniosis or histoplasmosis.
3. Investigation of hemic neoplasia
a. Differentiation, diagnosis, and staging of leukemias and lymphomas.
b. Diagnosis and staging of other neoplasias, including histiocytic neoplasia, multiple myeloma, mast cell neoplasia, and metastatic carcinoma.
c. Immunophenotyping hemic neoplasia. Neoplastic populations of hemic cells in the bone marrow can be stained using immunocytochemical markers to differentiate B-lymphocytes, T-lymphocytes, histiocytes, myeloid cells, etc.
d. Unexplained hypercalcemia or fever of unknown origin. These may occur as paraneoplastic syndromes secondary to lymphoid neoplasia or other neoplasms affecting the bone marrow.
Bone Marrow Sample Preparation
Bone marrow evaluation consists of 3 sample components: 1) bone marrow aspirate; 2) bone marrow core biopsy; and 3) peripheral blood. When a high-quality bone marrow aspirate is obtained, and depending on the reasons for doing the bone marrow examination, sometimes an aspirate (without a core) is sufficient for evaluation. Peripheral blood (i.e., a CBC) ALWAYS is needed for accurate interpretation of bone marrow findings.
In dogs and cats, bone marrow can be aspirated using sterile technique from the iliac crest, femur, or humerus using a bone marrow aspiration needle and a syringe. A 2% EDTA solution (sterile) should be used to prevent clotting of the sample and to facilitate the preparation of smears. The syringe should be flushed with the EDTA solution, retaining no more than 0.1 ml per 1.0 ml of marrow.
Alternatively, marrow samples may be placed into an EDTA tube from which excess EDTA has been shaken out. This method may result in sample clotting if there has been a lag in the aspiration process. Excessive EDTA can result in poor staining quality and nuclear artifacts.
Up to 1-2 mL of bone marrow may be aspirated. The anticoagulated sample should be placed in a plastic Petri dish. The "spicules" or "unit particles" of bone marrow may be visible as glistening fat particles suspended in blood. Tilt the Petri dish so free blood flows to the side, leaving particles visible on the bottom of the plate. Using a microhematocrit tube, carefully pick up several marrow particles using capillary action. Transfer the particles to a clean glass microscope slide and tap the tube gently to let them flow onto the center of the slide. Place a second clean glass slide directly over the first (longitudinally), allowing the bone marrow to spread. Gently pull the top slide off the bottom slide, lengthwise, without exerting pressure on the slide. This should result in a central, oval-shaped monolayer of bone marrow cells surrounded by peripheral blood. The central area of the smear typically is rich in unit particles.
Smears should be air dried quickly. Prepare as many smears as possible with the available marrow. Smears may be sent unstained to a laboratory, or may be stained using a routine hematologic stain. Because bone marrow smears are thicker than ordinary blood smears, longer staining time or double-staining is required for adequate stain quality. Leave several smears unstained for possible future use for immunophenotyping or special stains.
A peripheral blood sample should be obtained on the same day as marrow collection. This is essential because rapid changes can occur in peripheral blood counts and accurate interpretation of cells in the marrow require knowledge of CBC results.
Microscopic Evaluation of Bone Marrow
Microscopic evaluation of bone marrow involves scanning at low magnification (X10), closer examination at X40, and high-magnification examination of individual cell types (X100). Bone marrow evaluation includes assessment of the following components:
1. Overall sample quality
2. Unit particle cellularity (hypocellular, normocellular, hypercellular). Cellularity is assessed in relation to the amount of adipose tissue in the particles and can be estimated as a percentage, with 30 to 70% fat being normal (with a normal CBC). Because the amount of active marrow decreases with age, interpretation of cellularity must take into account the patient's age. A good quality sample is important for differentiating true hypocellularity from an inadequate sample.
3. Megakaryocytes. These large cells are qualitatively assessed for number (adequate/ inadequate), maturation (mature forms should exceed immature forms in normal marrow), and morphology. Megakaryocytes often are associated with unit particles.
4. The myeloid:erythroid (M:E) ratio. This may be obtained by a rough visual estimate or can be obtained by performing a 200-or 500-cell differential count. Normal M:E ratios in dogs and cats ranges from about 1:1 to 2:1. Major shifts in the M:E ratio may indicate absolute or relative changes in the proportion of myeloid and erythroid cells.
5. Maturation index. The proportions of proliferating vs nonproliferating cells can be estimated or counted for calculation of an erythroid maturation index, myeloid maturation index, and overall maturation index. Maturation indices are sometimes useful for semi-quantifying left or right shifts in hematopoietic cell maturation.
6. Erythroid cell line. Evaluated for numbers, maturation, morphology.
7. Myeloid cell line. Evaluated for numbers, maturation, morphology.
8. Lymphocytes and plasma cells. Bone marrow is not an important site of lymphocyte production, however, lymphocytes may reside in the marrow. In cats, up to 18% of nucleated cells in the bone marrow may be small lymphocytes.
9. Monocytic/macrophage cell line. Immature monocytoid cells are not easily distinguished from other myeloid cells unless there is increased production in this cell line, as might occur when there is an increased need for phagocytes. Increased macrophages may indicate bone marrow inflammation, necrosis, or increased phagocytic activity.
10. Hemosiderin. The bone marrow is an important site of iron storage, for use in hemoglobin synthesis. Storage iron usually is visible in dogs but not in cats. In some disease conditions, storage iron increases in quantity, even in cats. A special stain (Prussian blue) can be used to more accurately evaluate the quantity of storage iron.
Interpretation of Abnormal Bone Marrow Findings
Normoplasia (normal production), hyperplasia (increased production), hypoplasia (decreased production), and dysplasia (abnormal maturation) in the erythroid, myeloid, or megakaryocytic cell lines must be interpreted in light of peripheral blood findings. Hypoplasia in the face of peripheral cytopenia suggests that primary bone marrow disease is causing decreased production.
More than 30% immature blast cells in the marrow are indicative of hemic neoplasia. Additional tests, such as immunocytochemistry and cytochemistry, may be needed to differentiate primary leukemia from lymphoma, to identify the cell origin of the neoplastic cells (i.e., to differentiate lymphoproliferative from myeloproliferative disease), and to characterize the neoplasia (i.e., B-cell or T-cell lymphoma). The number and accuracy of phenotypic markers has increased rapidly in the past few years, providing better diagnosis and prognosis of hemic neoplasia in dogs and cats.
Potential immune-mediated disease may be further evaluated using an antimegakaryocyte antibody test. Although not specific, the test has high sensitivity.
Plasmacytosis may be seen in immune-mediated disease or chronic inflammatory disorders. Macrophage hyperplasia may be observed in systemic inflammatory disease, immune-mediated diseases, and in histiocytic neoplasia. Increased storage iron is common in chronic inflammatory disease and other chronic diseases, including neoplasia. Iron deficiency should be confirmed by determination of serum iron concentration.