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Changes in bone marrow cellularity can be an indicator of systemic toxicity and, therefore, bone marrow should be included in the battery of tissues examined for enhanced histopathology. However, the majority of changes in the bone marrow that are observed in toxicology studies are a response to hematological changes or lesions elsewhere in the body. For this reason, a consideration of all tissue changes in the body is required in order to differentiate toxic effects versus physiological responses in the bone marrow. While enhanced histopathology involves evaluation of the separate compartments in each lymphoid organ using descriptive rather than interpretive terminology, bone marrow is unique in that it lacks specific compartments. Furthermore, identification of erythroid, myeloid, megakaryocytic, and stromal cells, plus adipose tissue and hemosiderin-laden macrophages, can be accomplished from conventional H&E-stained sections, but conclusive identification of lymphoid lineage cells is not likely. This limits the extent of initial enhanced histopathology on bone marrow and argues for the use of cytological preparations for more comprehensive assessment of potential immunomodulatory effects.
The bone marrow is the largest primary lymphoid organ and is one location of antigen-independent lymphocyte development. It is also a secondary lymphoid organ because terminal antigen-induced lymphoid cell differentiation occurs within its microenvironment (Tavassoli and Yoffey, 1983). The regenerative capacity of most peripheral lymphoid organs depends on the pluripotent progenitor cells in the bone marrow. Changes in bone marrow cellularity can be an indicator of systemic toxicity and, therefore, bone marrow should be included in the battery of tissues examined for enhanced histopathology of the immune system. In fact, many compounds that target the bone marrow have been associated with profound alterations in immune function (Irons, 1985). Comparison of the cellular changes observed in the bone marrow should always be compared with the complete blood count. In any toxicologic study, the bone marrow from the treated animals should be compared with age- and sex-matched control animals due to the variation in normal cytological features that can be seen between sexes, strains and species of animals.
The majority of bone marrow changes that are observed in toxicological studies are the physiological responses of the bone marrow to hematological changes or lesions elsewhere in the body. By implication and because hematopoietic and lymphopoietic lineages share a common progenitor stem cell, it is reasonable to anticipate that systemic toxicity could affect multiple cell lineages. For this reason, a consideration of the health of the animal as well as all tissue changes in the body is required in order to differentiate primary (direct toxic effect) versus secondary (physiological response) effects on the bone marrow. The article by Travlos (2006) may be referred to for more detailed information on the normal structure, function and histology of the bone marrow.
Bone marrow is typically collected from the sternum, vertebrae or femur and can be processed for cellularity assessment by preparing conventionally fixed, paraffin-embedded sections of decalcified bone or isolated marrow casts or by preparing bone marrow smears. Since unequivocal identification of lymphoid cells in H&E-stained sections is problematic, one approach is to prepare bone marrow smears at the time of necropsy and, if there are cellular changes detected during examination of the H&E-stained tissue, quantitative and qualitative evaluation of Romanowsky stained bone marrow smears could be performed. Also, bone marrow cytomorphology can be difficult to assess on 5-micron tissue sections and, therefore, 3-micron sections might be preferable. Romanowsky stains might be more useful than H&E on these thinner sections. Bone marrow smears give the highest quality of cytological details with respect to cell morphology and maturation sequence.
The evaluation of H&E-stained tissue sections can be used as an initial screening test for enhanced histopathology of the bone marrow. This evaluation would include an estimate of cellular density and a myeloid/erythroid (M:E) ratio. An increase or decrease in the numbers of megakaryocytes, adipocytes, stromal cells and amount of hemosiderin should be noted. Moreover, an indication of the presence and severity of necrosis, hemorrhage, fibrosis, granulomas, neoplasia, etc. should also be noted. Although the best indicator of immunomodulation would be a change in the lymphocyte population, lymphoid lineage cells are difficult to distinguish from many of the other nucleated cells in the H&E-stained bone marrow. If the M:E ratio appears to be altered, then a differential count on a bone marrow smear could be used to determine which cell line was altered and to give a quantitative assessment of that alteration. Subsequently, flow cytometry could be done to provide quantitative and immunophenotyping information about hematopoietic and lymphopoietic cell populations, although this would probably require subsequent studies to permit appropriate collection of samples.
An example of a checklist that can aid the pathologist during evaluation of the bone marrow is given in Table 1. This table is intended to be an example of a guideline that the pathologist can use during histological evaluation rather than a format for reporting lesions. The diagnoses listed in this table are descriptive rather than interpretive, consistent with the STP position paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System (Haley et al., 2005).
The M:E ratio is a comparison of relative proportions of granulocytic and erythrocytic cells. In a cytologic preparation, it does not include lymphocytes. Since lymphoid cells are not readily distinguished from other nucleated cells in H&E-stained bone marrow, estimation of an M:E ratio from H&E-stained histologic sections would probably include both myeloid and lymphoid cells in the myeloid component. The M:E ratio generally has a mild myeloid predominance in most species. A change in the M:E ratio may be due to either the myeloid or erythroid cell lines. Comparison of the M:E ratio derived from the H&E-stained section with the CBC may provide additional useful information for distinguishing which cell type is increased or decreased. It has been reported that the circulating lymphocyte count is not influenced by, nor does it reflect, changes in the lymphoid cell lineage in the bone marrow (Yoffey and Courtice, 1970). Obvious exceptions would include severe bone marrow cellular loss as occurs following exposure to cytostatic agents and increases in bone marrow and peripheral blood lymphocytes in lymphomas.
The maturation index is the ratio between the number of proliferative phase cells to the number of maturation phase cells in the bone marrow and in mammals this number is typically 1:4 (Valli et al., 2002). Determination of this index is not considered a component of enhanced histopathology and would only be performed to more clearly define the nature of a hematopoietic defect. Careful examination of marrow histology and cytology must be done first in order to determine if there is an altered pattern of cellular maturation. Differential counts of cells in marrow smears could then be performed in order to better define the nature of the hematopoietic abnormality. For the myeloid series, the proliferative phase cells are the myeloblasts, promyelocytes, myelocytes and metamyelocytes, whereas the mature phase cells are the band and segmented neutrophils. For the erythroid series, the proliferative phase cells are the erythroblasts, prorubricytes, and rubricytes, whereas the mature phase cells are the metarubricytes. See Valli et al. (2002) for tables of normal differential counts and maturation indices for the Sprague–Dawley rat and for a summary of adverse marrow changes, including asynchronous maturation and dysplasia.
This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.