Читать книгу Pathology of Genetically Engineered and Other Mutant Mice - Группа авторов - Страница 89

Many mouse developmental pathology assessments are limited to anatomic pathology evaluation of macroscopic and microscopic defects. Clinical pathology analysis for mouse developmental pathology experiments usually is confined to hematology (in whole blood), clinical chemistry (in serum), or organ cytology. These samples are collected due to the common role of cardiovascular dysfunction and hematopoietic defects in developmental lethal phenotypes [8, 77]. Common tests performed for mouse developmental pathology projects, since they need only a small blood sample, are an automated complete blood count (CBC) and a limited chemistry battery (albumin, blood urea nitrogen, calcium, glucose, phosphorus, total protein).

Blood collection for developing mice typically is undertaken as a terminal procedure since embryos and neonates have a very small blood volume. Common sampling options are decapitation and cardiac puncture [58, 77]. Blood is harvested into standard collection tubes (e.g. Microtainer^®). Greater blood volumes may be obtained from outbred mouse stocks relative to smaller inbred strains. In our experience, enough blood may be harvested from a single neonatal mouse (1.5 g or larger in weight) to permit either a CBC or a serum chemistry battery, but not both. Alternatively, collection of blood in a tube containing potassium or sodium ethylenediaminetetraacetic acid (EDTA) as an anti‐coagulant may allow measurement of both a CBC (from whole blood) and a very few chemistry values (from plasma obtained by centrifugation of the sample). For small subjects (embryos from GD15 to term), sample volumes typically need to be extended either by mixing whole blood with an equal volume of physiologic saline (pH 7.4) or by pooling blood or plasma samples from mice that have an identical genotype and/or that received the same treatment. In our experience, pooling blood from two (at GD18.5) to four (at GD15.0) embryos will provide enough sample for either a CBC or a chemistry battery, but not both.

When investigating some lethal phenotypes, cytological assessment of isolated cells may be necessary to define the pathogenesis [58]. Cytological evaluations permit assessment of cellular and subcellular features at high resolution. Techniques such as smears for blood and bone marrow and either squash or touch preparations for organs with dense parenchyma like liver, spleen, and thymus are particularly useful in this regard. In general, results from cytological methods should be interpreted along with findings from conventional histopathological evaluation so that both tissue organization and individual cell characteristics can be assessed. For most embryo phenotyping projects, microscopic examination of tissue sections is used for the initial screen for both cell and tissue lesions, while cytological methods are employed as a secondary procedure when warranted to better understand the nature of the changes first identified by histopathologic analysis of cells within the context of their usual tissue organization.