Macroscopic test v2.2 01/Apr/2018 RIKENMPP_012a_002 test 2 1 testSt test modification

The macroscopic test, which is performed with the naked eye during autopsy, yields morphological findings that serve as important criteria for a pathologic diagnosis. These pathologic findings from the macroscopic test are recorded in the form of images and detailed notes. To determine pathology, 16-26-week-old mice should be compared with a control group (16-26-week-old mice with the same genetic background) and affected organs should be examined in terms of pathological changes and incidence.

The macroscopic test should be performed by staff members who have completed autopsy training, including experimental zoology, anatomy, and pathology, and are qualified to make pathoanatomic diagnoses based on macroscopic observations. The staff members must fully understand the SOP prepared by the research team, follow the testing procedure, and record the results. Pathologic changes found during the macroscopic test and the organs that require further examination should be fixed in 10% buffered formalin and stored at a specified repository. Testing must follow the SOP and all records, including results of the macroscopic test and deviations from the SOP, must be kept. Macroscopic observation data for the organs must be recorded with consistent terminology, symbols, and expressions.

To prepare for autopsy, adequate equipment and clothing must be kept ready for the health and safety of the staff members performing the examination. The macroscopic test must be performed in an environment with air conditioning. In each examination area, laboratory coats, gloves, glasses, and masks must be worn at all times during the macroscopic test. If something unexpected happens during the macroscopic test, it must be quickly reported to the manager and recorded.

Refer to each item.

The validity and interpretation of results obtained from the macroscopic test are dependent on pathoanatomic knowledge, experience, and necropsy results. Thus, the staff should always consult with clinical pathologists to interpret the results.

The macroscopic test is performed with the naked eye following the SOP for Pathology (anatomic procedure). Moreover, macroscopic observation data of the organs (terminology, symbols, and expressions) must be consistent with the pathologic terminology. Measure and record the body weights of 16- to 26-week-old mice at autopsy. The macroscopic test involves examination of the organs with the naked eye based on the external pathoanatomy. Additionally, macroscopic observation data must be recorded using consistent pathologic terminology, symbols, and expressions. Findings obtained as a result of external periodic observations (body weight measurements, hematological analysis, clinical biochemical testing, breeding management) are subject to final confirmation by autopsy. Findings during autopsy are also recorded. a. Hair condition Determine the positions and characteristics of bristles, epilation, piloerection, and white hair. Also examine the mice for epilation spots and other findings (the presence of lacerations, eruption, erosion, ulcers, and tumor lesions). b. Skin condition Determine the skin color on the auricle, limbs, tail, and reproductive organs as well as the presence, morphology, sizes, and degrees of abrasion, laceration, incrustation, eruption, edema, and tumor lesions. (note 1) Palpation is used in addition to observation with the naked eye to avoid errors. c. Skeleton condition Examine the mice for curvature, stiff neck, head shape (bulging in the skull), the presence of tumor lesions, tail deformation, and swelling of the limbs, fingers, and toes. d. Conditions of the eyelids, nostrils, labia, anus, and reproductive organs Examine the mouse for the presence of excretion, anal prolapse (hernia), erosion, ulcers, and tumors. e. Condition of the eyes Examine the eye for size (small), cloudy cornea, abnormal projection, closure, symmetry of the abnormalities, and stage of the abnormalities. f. Other Observe the mouse for behavior (slow, hypersensitive), drop in body temperature, weakness, paralysis in the limbs, and other defects. g. Overall observations During periodic testing, the mice should be examined while performing palpation during the preparation for testing. Observations at autopsy are made either when the mice are anesthetized or after death. Examination of mouse organs and body cavities must follow the SOP for Pathology (anatomic procedure) to extirpate the organs and record the results. The abdominal, thoracic, and skull cavities are examined for the positions of the aforementioned organs. For example, there may be positional abnormalities due to inversion (heart, lung, aorta), defects (defect in one of a pair of organs), or hernias. Each organ has an inherent shape. Determine whether the shape of the organ differs from its inherent shape and record the results with the autopsy application. For example, multilobular morphology for the lungs, kidneys, and spleen, and horseshoe-shaped kidneys are typical prenatal abnormalities. The surfaces of most organs are covered with serous membrane, which is glossy and smooth (flat, smooth). Determine whether the organs have irregular surfaces (fine-granular, coarse-granular, rough, recessed, nodular protrusions). For example, kidneys affected by functional disorders (common kidney disorders, glomerulonephritis) and livers with fibrosis have markedly irregular surface structures that are granular or rough. Each organ has an inherent color. Determine whether the color of an organ differs from its inherent color and record the results with the autopsy application. For example, pronounced fat deposition in hepatocytes turns the liver to a whitish color. Additionally, hepatic or renal inflammation causes the large number of inflammatory cells (lymphocytes and neutrophils) to turn the liver or kidney completely or partially white. (note 1) Color brightness: light/dark (note 2) Color density: dense/light Each organ has an inherent size. Compare the size of each organ with that of a normal organ and record the results with the autopsy application. Common expressions used to describe larger and smaller organs are given below. Another way to express size is to use familiar objects that do not vary in size (e.g., large as a soybean, millet grain, rice grain, walnut, quail egg, or chicken egg). (note 1) Size should be expressed in terms of length, width, and height. (note 2) Consistently use mm or cm to express size. Examples of size expressions larger than normal size: hypertrophy smaller than normal size: atrophy For example, kidneys affected by acute functional disorders (common kidney disorders, glomerulonephritis) tend to be hypertrophic, whereas kidneys affected by chronic functional disorders tend to be atrophic. Weigh extirpated organs after rinsing off the blood in and on the organs (such as the heart). Animals should be sacrificed using the same technique and results should be recorded with the autopsy application. Methods for animal sacrifice should be the same for both the experimental and control groups at baseline and throughout the experimental period. For example, mouse hearts affected by cardiovascular disease (hypertrophy); livers with progressive tumors, and adipose tissues from mice with suspected abnormal lipid metabolism (obesity) tend to increase in weight. Organ hardness depends on the cells that make up the tissue. Compare the hardness of an organ with that of a normal organ using palpation and record the findings with the autopsy application. For example, fibrosis-a chronic change in the liver-tends to increase the hardness of a liver. Additionally, interstitial pneumonia and pulmonary fibrosis in the lung can increase the hardness for the organ. When recording the findings of the organ evaluations or results from the aforementioned a. to g. in sequence, abnormal specimens must be prepared from the organs (immersed in fixative) that were kept for tissue analysis. As fixation proceeds, however, organ specimens immersed in fixative often turn different colors than those originally observed and recorded. Therefore, it is important to obtain images of as many of the abnormal specimens as possible. Also, it is a common practice to show the image, specimen, and relevant findings together when explaining the results to a third party or asking an expert for their opinion. The abdominal, thoracic, and skull cavities are examined for the positions, shapes, colors, and sizes of the aforementioned organs. In addition, when exudate (body fluid, blood, urine) are found in the body cavities, the colors and amounts of the exudates should be recorded with the autopsy application. The colors should be examined when cutting the peritoneum of the abdominal cavity, the diaphragm of the thoracic cavity, and the skull or pachymeninx of the skull cavity. For the amount of exudate in the abdominal cavity, the peritoneum is pinched with forceps and cut about 1 cm along the midline; a syringe is then inserted to withdraw the exudates and determine the total amount. For the thoracic cavity, a similar procedure is followed when cutting the diaphragm. For the skull cavity, however, the skull is cut open in a small tray and the amount of exudate that flows out is measured. (note 1) When the peritoneum, diaphragm, or other organs are injured, they may bleed. This blood may mix with the ascites, pleural fluid, or cerebrospinal fluid resulting in errors when determining the exudate color. Therefore, care should be taken when performing the autopsy if ascites, pleural fluid, or cerebrospinal fluid is present. Examples of expressions used to describe body cavity fluids l Exudate in the abdominal cavity: referred to as ascites; observe the color, liquid qualities, and retained amount. l Exudate in the thoracic cavity: referred to as pleural fluid; observe the color, liquid qualities, and retained amount. l Exudate in the skull cavity: referred to as cerebrospinal fluid; observe the color, liquid qualities, and retained amount. Recording observations about tumor lesions is performed in a similar manner to the method used for organ observations. Observe the affected location, size, shape, number, color, hardness, and the sectioned surface structure of the tumors; record the findings with the autopsy application.

The examination age was revised to 16-26 weeks (1.3, 9.1.1). Some equipment were corrected. Blood sampling procedure from the abdominal vena cava was deleted. Method of recording observation results was revised (9.3.2, 9.3.4-10).