Bone marrow evaluation can be used as a complementary diagnostic tool for hematologic evaluation of animals in clinical veterinary practice and research laboratories. Indications for evaluation of bone marrow include pancytopenia; persistent unexplained nonregenerative anemia, leukopenia, or thrombocytopenia; suspected iron deficiency1,2; the presence of abnormal blood cells in the circulation; or other unexplained hematologic abnormalities.1,3 In such instances, evaluation of bone marrow can help determine whether cytopenias are the result of a bone marrow production problem (eg, aplastic bone marrow or erythroid hypoplasia) or a deficiency in bone marrow iron stores and help identify bone marrow myelodysplasia or neoplasia.1,4 Evaluation of blood and bone marrow is also an important component of safety and toxicity studies owing to the fact that the hematopoietic system is a potential target of toxicity following chemical exposure.2,4 Bone marrow is the major hematopoietic organ and, as such, is the site of intense cellular multiplication and maturation. It can be altered by drugs that affect specific hematopoietic cell types as well as drugs with nonspecific effects on cellular proliferation.2
Bone marrow can be evaluated either histologically or cytologically, with both techniques having strengths and weaknesses. Histologic evaluation allows for better assessment of overall bone marrow cellularity and megakaryocyte numbers and is more sensitive than cytologic evaluation for identification of focal changes such as necrosis.2,5,6 However, compared with histologic evaluation, cytologic evaluation allows for better assessment of individual cell morphology, which facilitates distinguishing between erythroid precursors and lymphocytes, identification of early hematopoietic precursors, and characterization of abnormal hematopoietic cells.2,5,6 Bone marrow is an extremely reactive organ that can rapidly expand blood cell production to meet the needs of peripheral tissues and maintain homeostasis of the hematologic system. Therefore, it is necessary to assess bone marrow samples in conjunction with the CBC of a peripheral blood sample that was collected at the same time as the marrow sample so that any apparent anomalies can be correlated with findings in the peripheral circulation.1
Accurate assessment of bone marrow in a clinical or laboratory setting requires the ability to compare the acquired results with those of healthy populations. The existing literature7,8 on rabbit bone marrow is based on data obtained > 60 years ago when rabbit research colonies were managed differently than they are now. Additionally, the investigators of those studies7,8 did not compare hemogram results with bone marrow cytologic findings or report the extent of stainable iron stores in rabbit bone marrow, which is important information for the evaluation of iron deficiency in clinically ill or research rabbits. The purpose of the study reported here was to provide contemporary preliminary guidelines for the morphological evaluation of rabbit bone marrow in conjunction with CBC results for healthy juvenile female New Zealand White rabbits (Oryctolagus cuniculus), the rabbit species most commonly used in biomedical research.3
Materials and Methods
Animals
The study was approved by the Cornell University Institutional Animal Care and Use Committee. Twenty-two juvenile female specific pathogen–free New Zealand White rabbitsa with ages ranging from 3 to 6 months were obtained opportunistically at the completion of existing terminal investigations. Those studies were anatomic in nature and did not affect the overall health or immune systems of the rabbits. All rabbits were housed, maintained, and monitored following standard operation procedure protocols for research rabbits.
Sample collection and analysis
All rabbits were sedated with dexmedetomidine (0.125 mg/kg, IM), ketamine (20 mg/kg, IM), and butorphanol (0.2 mg/kg, IM) for blood collection. Blood (approx 3 mL) was collected from an ear artery, placed into blood microcontainers containing EDTA as an anticoagulant, and submitted for a CBC, which was performed by an automated hematology analyzer.b Internal quality control for the hematology analyzer included analysis of a commercially available controlc at the beginning of each 8-hour work shift and use of a 12s rule to determine the acceptability of control values. External quality control for the hematology analyzer was performed quarterly by both the College of American Pathologists and the Veterinary Laboratory Association Quality Assurance Program. Blood smears were prepared and stained with a modified Wright stain, and a manual WBC differential count was performed by classification of 100 consecutive leukocytes by a board-certified veterinary clinical pathologist (DMWS).
Following blood sample collection, each rabbit was euthanized by IV administration of an overdose of pentobarbital. As soon as death was confirmed, an incision was made on the medial aspect of the proximal portion of a pelvic limb. Sharp dissection was used to expose the femur, and sterile rongeurs were used to create a window in the exposed femur for access to the bone marrow. Within 5 minutes after euthanasia, microscope slides containing bone marrow smears for cytologic evaluation were prepared by use of 5% bovine serum albumind and the paint brush method as described.9 Small bone fragments with intact marrow were collected for histologic evaluation and fixed in neutral-buffered 10% formalin for a minimum of 24 hours. After fixation, the marrow was carefully separated from the bone and processed routinely into paraffin blocks for histologic slide preparation. Histologic sections of bone marrow were stained with H&E stain for assessment of cellularity and megakaryocyte count and Prussian blue stain for evaluation of iron stores. A section of intestine with enterocyte iron accumulation was used as a positive control for bone marrow slides stained with Prussian blue stain.
The cytologic and histologic preparations of bone marrow were evaluated by the same clinical pathologist (DMWS) who evaluated the blood smears and performed the manual WBC differential counts. Because the cellularity of bone marrow is most accurately assessed histologically,1,2 cytologic evaluation involved only a semiquantitative assessment of cellularity at low power (magnification, 100X), with values between 25% and 75% considered adequate cellularity.1 For cytologic assessment, the number of megakaryocytes in 10 low-power fields containing bone marrow particles were counted. All megakaryocytes were counted in each field regardless of whether the cells were inside or outside of the bone marrow particle. The megakaryocyte count was reported as the mean number of megakaryocytes per low-power field. Maturation of megakaryocytes was assessed subjectively and was considered normal if the majority (> 50%) of the megakaryocyte lineage consisted of mature megakaryocytes (ie, cells with high nuclear ploidy and abundant pink granular cytoplasm) rather than promegakaryocytes (ie, small cells with 2 to 4 nuclei and dark blue cytoplasm).1 The slides were then evaluated at high power (magnification, 1,000X), and 1,500 nucleated cells were morphologically classified to determine the M:E ratio, percentages of cells at each stage of myeloid and erythroid maturation, and percentages of other cells (lymphocytes, plasma cells, and macrophages). The M:E ratio was calculated by dividing the number of all myeloid lineage cells (including mature cells) by the number of nucleated erythroid cells.
Classification of the maturation stages within the myeloid and erythroid lineages was based on previously described criteria.1,10 Within the erythroid lineage, rubriblasts were identified as cells with a high nuclear-to-cytoplasmic ratio, round nucleus, fine chromatin, ≥ 1 visible nucleoli, and dark blue cytoplasm (Figure 1). Prorubricytes had morphological features similar to that of rubriblasts but lacked nucleoli and had slightly coarser chromatin. Basophilic rubricytes were smaller than prorubricytes and had dark blue cytoplasm and chromatin condensed into distinct light and dark areas. Polychromatophilic rubricytes had red-blue cytoplasm and nuclear chromatin that was more condensed than that of basophilic rubricytes. Metarubricytes were identified as small cells with a dark pyknotic nucleus and red to red-blue cytoplasm. Within the myeloid lineage, myeloblasts were identified as cells with a high nuclear-to-cytoplasmic ratio, round to oval nucleus, fine chromatin, ≥ 1 visible nucleoli, and moderately basophilic cytoplasm that was not as dark as that of rubriblasts (Figure 2). Promyelocytes were cells that lacked nucleoli, had more abundant cytoplasm than myeloblasts, and had many magenta cytoplasmic primary granules. Heterophil myelocytes had a round to oval nucleus and many small dark pink cytoplasmic secondary granules. Those secondary granules were also evident in heterophilic metamyelocytes, band heterophils, and segmented heterophils, and those 3 stages were distinguished on the basis of nuclear shape. Metamyelocytes had a nucleus with an indentation that gave it a kidney bean shape. Band heterophils had a long U- or S-shaped nucleus with parallel sides, and segmented heterophils had a long nucleus with nonparallel sides containing irregular indentations and lobulations. If any area of the nucleus had a diameter that was less than two-thirds the diameter of any other area of the nucleus, the cell was classified as a segmented heterophil rather than as a band heterophil. Eosinophils and basophils in the early stages of development prior to the myelocyte stage (ie, early eosinophils and basophils) could not be distinguished from early heterophils. At the myelocyte stage, eosinophils and basophils had cytoplasmic secondary granules that allowed them to be distinguished from heterophils. The cytoplasmic secondary granules of eosinophils were larger and lighter pink, compared with those of heterophils, and the cytoplasmic secondary granules of basophils were purple. Eosinophils and basophils were enumerated as the percentage of nucleated cells in each of their respective lineages without regard to stage of maturation. Early monocyte precursors (monoblasts and promonocytes) could not be reliably distinguished from myeloblasts morphologically and were counted together with myeloblasts and reported as early myeloid precursors. If a cell of either erythroid or myeloid lineage had morphological features consistent with 2 successive stages of maturation, it was classified as the more mature of the 2 stages.
Representative photomicrographs of erythroid precursors in the bone marrow of healthy juvenile (3- to 6-month-old) female New Zealand White rabbits (Oryctolagus cuniculus). A—Rubriblast characterized by a high nuclear-to-cytoplasmic-ratio and the presence of a round nucleus, fine chromatin, ≥ 1 visible nucleoli (arrow), and dark blue cytoplasm. B—Prorubricyte with morphology similar to that of a rubriblast except it lacked nucleoli and had slightly coarser chromatin. C—Basophilic (arrowheads) and polychromatophilic (arrows) rubricytes; basophilic rubricytes were smaller than prorubricytes and had dark blue cytoplasm and chromatin condensed into distinct light and dark areas, whereas polychromatic rubricytes had red-blue cytoplasm and nuclear chromatin that was more condensed than that of basophilic rubricytes. D—Metarubricyte characterized by its small size and dark pyknotic nucleus and red to red-blue cytoplasm. Modified Wright stain; bar = 10 μm.
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Representative photomicrographs of erythroid precursors in the bone marrow of healthy juvenile (3- to 6-month-old) female New Zealand White rabbits (Oryctolagus cuniculus). A—Rubriblast characterized by a high nuclear-to-cytoplasmic-ratio and the presence of a round nucleus, fine chromatin, ≥ 1 visible nucleoli (arrow), and dark blue cytoplasm. B—Prorubricyte with morphology similar to that of a rubriblast except it lacked nucleoli and had slightly coarser chromatin. C—Basophilic (arrowheads) and polychromatophilic (arrows) rubricytes; basophilic rubricytes were smaller than prorubricytes and had dark blue cytoplasm and chromatin condensed into distinct light and dark areas, whereas polychromatic rubricytes had red-blue cytoplasm and nuclear chromatin that was more condensed than that of basophilic rubricytes. D—Metarubricyte characterized by its small size and dark pyknotic nucleus and red to red-blue cytoplasm. Modified Wright stain; bar = 10 μm.
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Representative photomicrographs of erythroid precursors in the bone marrow of healthy juvenile (3- to 6-month-old) female New Zealand White rabbits (Oryctolagus cuniculus). A—Rubriblast characterized by a high nuclear-to-cytoplasmic-ratio and the presence of a round nucleus, fine chromatin, ≥ 1 visible nucleoli (arrow), and dark blue cytoplasm. B—Prorubricyte with morphology similar to that of a rubriblast except it lacked nucleoli and had slightly coarser chromatin. C—Basophilic (arrowheads) and polychromatophilic (arrows) rubricytes; basophilic rubricytes were smaller than prorubricytes and had dark blue cytoplasm and chromatin condensed into distinct light and dark areas, whereas polychromatic rubricytes had red-blue cytoplasm and nuclear chromatin that was more condensed than that of basophilic rubricytes. D—Metarubricyte characterized by its small size and dark pyknotic nucleus and red to red-blue cytoplasm. Modified Wright stain; bar = 10 μm.
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Representative photomicrographs of myeloid precursors in the bone marrow of healthy juvenile (3- to 6-month-old) female New Zealand White rabbits. A—Myeloblast characterized by a high nuclear-to-cytoplasmic ratio, round to oval nucleus, fine chromatin, ≥ 1 visible nucleoli (arrow), and moderately basophilic cytoplasm that was not as dark as that of a rubriblast. B—Promyelocytes characterized by the absence of nucleoli and presence of abundant cytoplasm and numerous small magenta cytoplasmic primary granules. C—Heterophil myelocyte characterized by the presence of a round to oval nucleus and numerous small dark pink cytoplasmic secondary granules. D—Heterophil metamyelocyte characterized by the presence of a kidney-bean shaped nucleus and numerous small dark pink cytoplasmic secondary granules. E—Band heterophil characterized by the presence of a U-shaped nucleus and numerous small dark pink cytoplasmic secondary granules. F—Mature or segmented heterophil characterized by the presence of a long nucleus with nonparallel sides containing irregular indentations and lobulations and numerous small dark pink cytoplasmic secondary granules. G—Eosinophil myelocyte characterized by the presence of cytoplasmic secondary granules that are larger and lighter pink than those of heterophils. H—Basophil metamyelocyte characterized by the presence of purple cytoplasmic secondary granules. Modified Wright stain; bar = 10 μm.
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Representative photomicrographs of myeloid precursors in the bone marrow of healthy juvenile (3- to 6-month-old) female New Zealand White rabbits. A—Myeloblast characterized by a high nuclear-to-cytoplasmic ratio, round to oval nucleus, fine chromatin, ≥ 1 visible nucleoli (arrow), and moderately basophilic cytoplasm that was not as dark as that of a rubriblast. B—Promyelocytes characterized by the absence of nucleoli and presence of abundant cytoplasm and numerous small magenta cytoplasmic primary granules. C—Heterophil myelocyte characterized by the presence of a round to oval nucleus and numerous small dark pink cytoplasmic secondary granules. D—Heterophil metamyelocyte characterized by the presence of a kidney-bean shaped nucleus and numerous small dark pink cytoplasmic secondary granules. E—Band heterophil characterized by the presence of a U-shaped nucleus and numerous small dark pink cytoplasmic secondary granules. F—Mature or segmented heterophil characterized by the presence of a long nucleus with nonparallel sides containing irregular indentations and lobulations and numerous small dark pink cytoplasmic secondary granules. G—Eosinophil myelocyte characterized by the presence of cytoplasmic secondary granules that are larger and lighter pink than those of heterophils. H—Basophil metamyelocyte characterized by the presence of purple cytoplasmic secondary granules. Modified Wright stain; bar = 10 μm.
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Representative photomicrographs of myeloid precursors in the bone marrow of healthy juvenile (3- to 6-month-old) female New Zealand White rabbits. A—Myeloblast characterized by a high nuclear-to-cytoplasmic ratio, round to oval nucleus, fine chromatin, ≥ 1 visible nucleoli (arrow), and moderately basophilic cytoplasm that was not as dark as that of a rubriblast. B—Promyelocytes characterized by the absence of nucleoli and presence of abundant cytoplasm and numerous small magenta cytoplasmic primary granules. C—Heterophil myelocyte characterized by the presence of a round to oval nucleus and numerous small dark pink cytoplasmic secondary granules. D—Heterophil metamyelocyte characterized by the presence of a kidney-bean shaped nucleus and numerous small dark pink cytoplasmic secondary granules. E—Band heterophil characterized by the presence of a U-shaped nucleus and numerous small dark pink cytoplasmic secondary granules. F—Mature or segmented heterophil characterized by the presence of a long nucleus with nonparallel sides containing irregular indentations and lobulations and numerous small dark pink cytoplasmic secondary granules. G—Eosinophil myelocyte characterized by the presence of cytoplasmic secondary granules that are larger and lighter pink than those of heterophils. H—Basophil metamyelocyte characterized by the presence of purple cytoplasmic secondary granules. Modified Wright stain; bar = 10 μm.
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Histologic evaluation included assessment of bone marrow cellularity and iron stores and a megakaryocyte count. Cellularity of the marrow was assessed on an H&E-stained section at low power by visual estimation of the percentage of marrow area occupied by cells versus fat and was reported in 10% increments. The number of megakaryocytes in 10 hpfs (magnification, 400X) were counted and reported as the mean number of megakaryocytes per hpf. Bone marrow iron stores were visually assessed on sections stained with Prussian blue stain as described.11
Statistical analysis
Descriptive statistics were generated. Spearman rank correlation (ρ) was used to assess the respective correlations between the M:E ratio and the following CBC variables: Hct, hemoglobin concentration, RBC count, total WBC count, absolute heterophil count, and absolute lymphocyte count. Pearson correlation (r) was used to assess the correlation between the automated and manual WBC differential counts. The Mann-Whitney test was used to compare the M:E ratio between rabbits with WBC counts below the published reference range (ie, leukopenia) and those with WBC counts within the published reference range. All analyses were performed with statistical software,e and values of P < 0.05 were considered significant.
Results
All rabbits evaluated in the study were considered healthy on the basis of demeanor, appetite, and absence of notable abnormalities on physical examination. Because of the small study population (22 rabbits), reference ranges were not calculated. Descriptive statistics for CBC variables were summarized (Table 1). All CBC variables were within the published reference ranges6 for all rabbits except 4. Each of those 4 rabbits had leukopenia (WBC count < 5.2 × 109 WBCs/L), with WBC counts ranging from 3.6 × 109 WBCs/L to 4.4 × 109 WBCs/L. Because those 4 rabbits appeared otherwise healthy and no other abnormalities were detected on the CBC, it was unclear whether the WBC counts were aberrations or reflective of a diseased state; therefore, descriptive statistics were generated with and without those 4 rabbits included in the dataset. There was a significant (P < 0.001 for all comparisons) strong positive correlation between the manual and automated heterophil (r = 0.98), lymphocyte (r = 0.95), and basophil (r = 0.79) counts and a moderate correlation between the manual and automated monocyte counts (r = 0.50; P = 0.049). Too few eosinophils were present for comparison to determine the correlation between the manual and automated counts.
Descriptive statistics for automated CBC results and manual WBC differential counts for 22 juvenile (3- to 6-month-old) female New Zealand White rabbits (Oryctolagus cuniculus).
| All 22 rabbits | 18 rabbits without leukopenia | |||
|---|---|---|---|---|
| Variable | Median (range) | SD | Median (range) | SD |
| Hct (L/L) | 0.38 (0.350–0.410) | 0.017 | 38.0 (38.0–35.0) | 1.6 |
| RBC (× 1012 RBCs/L) | 6.0 (5.1–6.6) | 0.4 | 6.0 (5.1 6.6) | 0.4 |
| Hemoglobin (g/dL) | 12.8 (11.7–14.0) | 0.6 | 12.8 (11.7–3.4) | 0.5 |
| Mean corpuscular volume (fL) | 64 (60–72) | 3 | 64.5 (60.0–72.0) | 3.1 |
| Mean corpuscular hemoglobin (pg) | 21 (20–24) | 1.1 | 21.0 (20.0–24.0) | 1.2 |
| Mean corpuscular hemoglobin content (g/dL) | 33.5 (32–35) | 0.7 | 33.0 (32.0–34.0) | 0.7 |
| RBC distribution width (L/L) | 13.7 (12.4–15.0) | 0.7 | 13.8 (12.8–15.0) | 0.7 |
| WBC (× 109 WBCs/L) | 6.3 (3.6–7.2) | 1.0 | 6.3 (5.3–7.2) | 0.6 |
| Segmented heterophils (× 109 cells/L) | 1.2 (0.8–2.9) | 0.5 | 1.1 (0.8–2.9) | 0.6 |
| Lymphocytes (× 109 cells/L) | 4.1 (1.7–5.2) | 1.0 | 4.4 (3.1–5.2) | 0.6 |
| Monocytes (× 109 cells/L) | 0.2 (0.1–0.6) | 0.1 | 0.2 (0.1–0.6) | 0.1 |
| Eosinophils (× 109 cells/L) | 0.1 (0.1–0.1) | 0 | 0.1 (0.1–0.1) | 0 |
| Basophils (× 109 cells/L) | 0.4 (0.1–0.6) | 0.1 | 0.3 (0.1–0.6) | 0.1 |
| Platelets (× 109 cells/L) | 297.0 (160.0–462.0) | 72.7 | 306 (160.0–462.0) | 77.7 |
For each rabbit, a manual WBC differential count was performed by classification of 100 consecutive leukocytes on a blood smear stained with a modified Wright stain by a board-certified veterinary clinical pathologist (DMWS). Four rabbits had leukopenia (WBC count, < 5.2 × 109 WBCs/L) despite the fact that they otherwise appeared healthy; the WBC count for those 4 rabbits ranged from 3.6 × 109 WBCs/L to 4.4 × 109 WBCs/L. Because it was unknown whether those 4 rabbits were outliers, results are reported with and without those 4 rabbits included in the dataset.
For all but 1 rabbit, cytologic evaluation revealed that the bone marrow sample quality and cellularity were both acceptable, and the prepared slides contained many identifiable bone marrow particles of adequate cellularity (cell-to-fat ratio, 25% to 75%). The slides for the rabbit that was the exception had only a few bone marrow particles, which was attributed to a sample preparation problem because the CBC results for that rabbit were all within the respective reference ranges. Despite the paucity of bone marrow particles on the slides prepared for that rabbit, the background contained many individual hematopoietic cells, and the cellularity was considered adequate.
Cytologic results indicated balanced maturation (ie, more mature or late stage precursors than immature or early stage precursors) of megakaryocytes (Table 2) as well as cells of the erythroid and myeloid lineages (Table 3). The bone marrow of most rabbits had few granulocyte precursors, metamyelocytes, and myelocytes that were considerably larger than the other myeloid cells, with diameters 2 to 3 times that of mature heterophils (Figure 3). Although that phenomenon was identified in the heterophil, eosinophil, and basophil lineages, it was most notable in the heterophil lineage. However, cells of the heterophil lineage outnumbered the cells of the eosinophil and basophil lineages, which might have made them easier to find and identify. Mast cells (typically < 1/slide) were rarely identified in some of the samples.
Representative photomicrographs of cytologic bone marrow smears obtained from a healthy juvenile female New Zealand White rabbit that depict large granulocyte precursor cells such as a band heterophil (*), heterophilic metamyelocytes (†), heterophilic myelocyte (‡), eosinophil myelocyte (long black arrow), basophil metamyelocyte (short black arrow), and band basophil (black arrowhead). Notice the diameter of the myeloid precursor cells are 2 to 3 times that of mature heterophils (red arrows). Modified Wright stain; bar = 20 μm (A and B) or 10 μm (C, D, and E).
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Representative photomicrographs of cytologic bone marrow smears obtained from a healthy juvenile female New Zealand White rabbit that depict large granulocyte precursor cells such as a band heterophil (*), heterophilic metamyelocytes (†), heterophilic myelocyte (‡), eosinophil myelocyte (long black arrow), basophil metamyelocyte (short black arrow), and band basophil (black arrowhead). Notice the diameter of the myeloid precursor cells are 2 to 3 times that of mature heterophils (red arrows). Modified Wright stain; bar = 20 μm (A and B) or 10 μm (C, D, and E).
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Representative photomicrographs of cytologic bone marrow smears obtained from a healthy juvenile female New Zealand White rabbit that depict large granulocyte precursor cells such as a band heterophil (*), heterophilic metamyelocytes (†), heterophilic myelocyte (‡), eosinophil myelocyte (long black arrow), basophil metamyelocyte (short black arrow), and band basophil (black arrowhead). Notice the diameter of the myeloid precursor cells are 2 to 3 times that of mature heterophils (red arrows). Modified Wright stain; bar = 20 μm (A and B) or 10 μm (C, D, and E).
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Descriptive statistics for bone marrow cytologic results for the rabbits of Table 1.
| All 22 rabbits | 18 rabbits without leukopenia | |||
|---|---|---|---|---|
| Variable | Mean (SD) | Median (range) | Mean (SD) | Median (range) |
| M:E ratio | 0.8 (0.3) | 0.7 (0.4–1.4) | 0.7 (0.2) | 0.7 (0.4–1.2) |
| Megakaryocytes/lpf* | 3.0 (0.9) | 2.8 (1.0–5.2) | 3.0 (1.0) | 2.9 (1.0–5.2) |
| Lymphocytes (%)† | 12.8 (5.1) | 11.5 (4.8–23.0) | 12.6 (5.0) | 11.3 (4.8–23.0) |
| Plasma cells (%)† | 0.2 (0.2) | 0.1 (0–0.8) | 0.1 (0.2) | 0.1 (0–0.4) |
| Macrophages (%)† | 0.1 (0.2) | 0 (0–0.6) | 0.1 (0.2) | 0 (0–0.6) |
Mean number of megakaryocytes per lpf containing bone marrow particles.
Percentage of total nucleated cells in the cytologic specimen. lpf = Low-power field (magnification, 100X).
See Table 1 for remainder of key.
Descriptive statistics for erythroid and myeloid cell lineages in the bone marrow of the rabbits of Table 1 as determined by cytologic evaluation.
| All 22 rabbits | 18 rabbits without leukopenia | |||
|---|---|---|---|---|
| Lineage | Cell type | Median (range) | SD | Median (range) |
| Erythroid | Rubriblasts | 0.7 (0.0–1.6) | 0.4 | 0.6 (0.0–1.6) |
| Prorubricytes | 2.0 (0.8–4.5) | 0.9 | 2.1 (0.8–4.5) | |
| Basophilic rubricytes | 11.3 (8.6–20.4) | 2.9 | 12.1 (8.6–20.4) | |
| Polychromatophilic rubricytes | 38.9 (31.1–44.4) | 3.7 | 38.6 (31.1–44.4) | |
| Metarubricytes | 46.8 (39.6–54.8) | 4.5 | 46.7 (39.6–53.6) | |
| Myeloid | Early myeloid precursors* | 4.3 (1.4–8.8) | 1.9 | 4.2 (1.4–8.8) |
| Promyelocytes | 1.0 (0.4–3.0) | 0.6 | 1.0 (0.6–3.0) | |
| Heterophil myelocytes | 5.6 (2.3–9.2) | 1.8 | 5.7 (3.6–9.2) | |
| Heterophil metamyelocytes | 5.6 (3.2–10.2) | 1.9 | 6.1 (4.0–10.2) | |
| Heterophil bands | 27.0 (20.8–36.8) | 4.8 | 28.1 (20.8–36.8) | |
| Segmented heterophils | 49.6 (33.8–58.6) | 6.7 | 48.7 (33.8–57.4) | |
| Eosinophils† | 3.3 (1.2–5.6) | 1.3 | 3.3 (1.2–5.6) | |
| Basophils† | 3.1 (0.8–6.0) | 1.5 | 3.1 (0.8–6.0) | |
Values represent the percentage of cells within the given cell lineage.
Includes myeloblasts, monoblasts, and promonocytes.
Includes all stages.
See Table 1 for remainder of key.
The M:E ratio ranged from 0.4 to 0.7 for the 4 rabbits with leukopenia and from 0.4 to 1.4 for the remaining 18 rabbits without leukopenia. However, the number of rabbits with leukopenia was too small for the M:E ratio to be statistically compared between rabbits with and without leukopenia. The M:E ratio was not significantly correlated with any of the CBC variables evaluated (Hct, hemoglobin concentration, RBC count, total WBC count, absolute heterophil count, and absolute lymphocyte count).
Histologic evaluation of the bone marrow specimens revealed that the cellularity ranged from 30% to 50% (Figure 4). There were 2.1 to 7.7 megakaryocytes/hpf (median, 4.6 megakaryocytes/hpf). Iron stores were not visually detectable on histologic sections of bone marrow that were stained with H&E stain or on cytologic smears of bone marrow that were stained with modified Wright stain. Evaluation of histologic sections of bone marrow that were stained with Prussian blue stain confirmed that stainable iron was not present in the bone marrow of any of the rabbits.
Representative photomicrographs of bone marrow specimens from a healthy juvenile female New Zealand White rabbit that were obtained for histologic (A and B) and cytologic (C) evaluation. A—The cellularity of the bone marrow is approximately 50%; a few megakaryocytes are seen (arrows). H&E stain; bar = 100 μm. B—Section of bone marrow stained with Prussian blue stain for detection of stainable iron stores; notice that no iron stores are visible. Bar = 100 μm. C—Cytologic smear that contains a bone marrow particle of adequate cellularity (defined as 25% to 75%) and a megakaryocyte (arrow). Modified Wright stain; bar = 100 μm.
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Representative photomicrographs of bone marrow specimens from a healthy juvenile female New Zealand White rabbit that were obtained for histologic (A and B) and cytologic (C) evaluation. A—The cellularity of the bone marrow is approximately 50%; a few megakaryocytes are seen (arrows). H&E stain; bar = 100 μm. B—Section of bone marrow stained with Prussian blue stain for detection of stainable iron stores; notice that no iron stores are visible. Bar = 100 μm. C—Cytologic smear that contains a bone marrow particle of adequate cellularity (defined as 25% to 75%) and a megakaryocyte (arrow). Modified Wright stain; bar = 100 μm.
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Representative photomicrographs of bone marrow specimens from a healthy juvenile female New Zealand White rabbit that were obtained for histologic (A and B) and cytologic (C) evaluation. A—The cellularity of the bone marrow is approximately 50%; a few megakaryocytes are seen (arrows). H&E stain; bar = 100 μm. B—Section of bone marrow stained with Prussian blue stain for detection of stainable iron stores; notice that no iron stores are visible. Bar = 100 μm. C—Cytologic smear that contains a bone marrow particle of adequate cellularity (defined as 25% to 75%) and a megakaryocyte (arrow). Modified Wright stain; bar = 100 μm.
Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.910
Discussion
Rabbits are commonly used in research settings and are also common companion animals. Bone marrow examination is an important component of toxicity studies in research rabbits and hematologic disease evaluation in companion rabbits. Accurate assessment of bone marrow in those situations or animals is dependent on the availability of bone marrow data for healthy rabbits. Results of the present study can be used as preliminary guidelines for bone marrow findings for clinically normal rabbits and provide an update to commonly cited references7,8 that are > 60 years old. Unique to the present study was the quantification of bone marrow cellularity and stainable iron stores and correlation of bone marrow findings with CBC results. Additionally, photomicrographs of erythroid and myeloid precursor cells were provided to supplement those currently available.10
The median M:E ratio in the present study (0.7; range, 0.4 to 1.4) was slightly lower than the M:E ratio (1.0) reported by investigators of a 1957 study.8 That disparity might be attributable to the small sample sizes in both the present and previous8 studies, gradual changes in the bone marrow cell population of rabbits over decades, sex differences (the sex of the rabbits in the other study8 was not reported), or differences in antigenic stimulation. The rabbits of the present study were specific pathogen–free rabbits and housed in a clean controlled environment. Although housing was not specified in the previous study,8 we presume that sanitization and air filtering requirements for rabbit research colonies 6 decades ago were less strict than they are today. It is also reasonable to suppose that the level of antigenic stimulation for the rabbits of that other study8 was greater than that for the rabbits of the present study, which resulted in an increase in inflammatory (myeloid) cell numbers, thereby increasing the relative M:E ratio. In the present study, the M:E ratios for the 4 rabbits with leukopenia were less than or equal to the median M:E ratio for the study population. That finding suggested that the inflammatory stimuli for those 4 rabbits might have been less than the inflammatory stimuli for the other study rabbits. However, some rabbits without leukopenia had M:E ratios that overlapped with those for the 4 rabbits with leukopenia. Moreover, if a decrease in inflammatory stimuli was the reason that the median M:E ratio for the rabbits of the present study was slightly lower than that of the previous study,8 then the bone marrow M:E ratio should have been positively correlated with the total WBC count or absolute heterophil count. Instead, results indicated that the M:E ratio was not significantly correlated with any CBC variable. Therefore, factors other than a decrease in inflammatory stimuli might be responsible for the slightly decreased M:E ratio (relative to the M:E ratio reported in a previous study8) for the rabbits of the present study
The present study also provided information regarding bone marrow lymphocytes, plasma cells, macrophages, and iron stores in rabbits. The percentage of bone marrow lymphocytes for the rabbits of this study was similar to that for cats and other small mammals such as guinea pigs and rats (< 20%) and was often higher than that for other species (< 10 %).1,6 The percentages of plasma cells and macrophages were similar to expected findings in other species.1 The bone marrow of most domestic species, except cats, contains detectable amounts of stainable iron.1 Similar to cats, stainable iron was not detected in the bone marrow of the rabbits of this study despite the fact that bone marrow specimens were stained with 3 stains (modified Wright, H&E, and Prussian blue stains).
An atypical finding in the bone marrow of the rabbits of the present study was the low numbers of large band heterophils, metamyelocytes, and myelocytes that were 2 to 3 times the diameter of mature heterophils. In most species, giant granulocyte precursors are considered evidence of dysgranulopoiesis.1 To our knowledge, giant myeloid precursors have not been described in rabbits. However, review of photomicrographs of bone marrow from healthy rabbits published in a hematology atlas10 revealed that low numbers of giant heterophil precursors were present, which suggested that low numbers of those cells are normally present in the bone marrow of rabbits and should not be interpreted as evidence of dysgranulopoiesis.
The present study was limited by the fact that all rabbits evaluated were juvenile (3- to 6-month-old) females. Results of other studies7,8 involving rabbits ≤ 6 months old indicate that most changes in bone marrow composition occur during the first month after birth. However, it is unknown whether sex or age affects the cellular composition of bone marrow in rabbits. Additionally, the rabbits of this study were housed in a clean environment, and it is unclear how exposure of rabbits to an environment with a presumptively low level of antigenic stimulation affects bone marrow myelopoiesis. Consequently, it is likely the results of the present study will be most applicable when used for comparison purposes during the evaluation of bone marrow for rabbits in research settings that have similar signalment and are housed in similar environments as the rabbits of this study.
Results of the present study provided contemporary preliminary guidelines for the evaluation of bone marrow in healthy laboratory rabbits. The information provided included the M:E ratio, proportions of cells at various stages of erythroid and myeloid maturation, and megakaryocyte, lymphocyte, and plasma cell counts. Additionally, this study resulted in the generation of photomicrographs of erythroid and myeloid cells as well as information regarding cellularity, macrophage count, and stainable iron stores in rabbit bone marrow that has not been previously reported. The study population consisted of only juvenile (3- to 6-month-old) female New Zealand White rabbits; therefore, the results are most applicable for comparison with rabbits in research settings.
ABBREVIATIONS
| M:E ratio | Myeloid-to-erythroid ratio |
Footnotes
Rabbits tested free of the following pathogens prior to purchase from the vendor: reovirus type 3, lymphocytic choriomeningitis virus, parainfluenzavirus types 1 and 2, rotavirus, rabbit hemorrhagic disease virus, Bordetella bronchiseptica, Helicobacter spp, Lawsonia spp, Pasteurella spp including Pasteurella multocida, Salmonella spp, Treponema spp, Clostridium piliforme, cilia-associated respiratory bacillus, Pseudomonas aeruginosa, Cheyletiella parasitovorax, Leptostylus gibbus, Psoroptes cuniculi, Passalurus ambiguus, and Eimeria spp including Eimeria stiedae and Eimeria cuniculi. Charles River Laboratories, Saint Constant, QC, Canada.
ADVIA 2120 Hematology Analyzer, Siemens Healthcare, Erlangen, Germany.
3-in-1 Testpoint Hematology Control, Siemens, Tarrytown, NY.
Sigma-Aldrich Corp, St Louis, Mo.
MedCalc, version 14.12.1, Ostend, Belgium.
References
1. Harvey J W. Veterinary hematology: a diagnostic guide and color atlas. St Louis: Elsevier Saunders, 2012; 234–258.
2. Reagan WJ, Irizarry-Rovira A, Poitout-Belissent F, et al. Best practices for evaluation of bone marrow in nonclinical toxicity studies. Toxicol Pathol 2011; 39:435–448.
3. Marshall KL. Rabbit hematology. Vet Clin North Am Exot Anim Pract 2008; 11:551–567.
4. Travlos GS. Histopathology of bone marrow. Toxicol Pathol 2006; 34:566–598.
5. Ryan DH, Felgar RE. Examination of the marrow. In: Lichtman MA, Beutler E, Kipps TJ, et al, eds. Williams hematology. 7th ed. New York: McGraw-Hill, 2006; 21–31.
6. Weiss DJ, Wardrop KJ. Schalm's veterinary hematology. 6th ed. Ames, Iowa: Wiley-Blackwell, 2010; 862–869, 1039–1046.
7. Sabin FR, Miller FR, Smithburn KC, et al. Changes in the bone marrow and blood cells or developing rabbits. J Exp Med 1936; 64:97–120.
8. Dikovinova NV. Absolute number of cells in bone marrow and myelograms of normal rabbits. Biull Eksp Biol Med 1957; 44:102–105.
9. Bolliger AP. Cytologic evaluation of bone marrow in rats: indications, methods, and normal morphology. Vet Clin Pathol 2004; 33:58–67.
10. Sanderson JH, Phillips CE. An atlas of laboratory animal hematology. London: Oxford University Press, 1982; 274–307.
11. Phiri KS, Calis JC, Kachala D, et al. Improved method for assessing iron stores in the bone marrow. J Clin Pathol 2009; 62:685–689.
