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PubMed2004

Further assessment of the size, shape and surface features of superficial cells of the bovine corneal epithelium, using scanning electron microscopy.

PURPOSE: To qualitatively and quantitatively assess the size and shape of the surface cells of the bovine corneal epithelium as assessed by scanning electron microscopy. METHODS: After quality-control measures on recent postmortem bovine eyes obtained from a slaughterhouse, the ocular surface was aggressively washed with 0.97% saline and then fixed for scanning electron microscopy (SEM) by a dropwise application, in 37 degrees C moist chamber, of a 2% glutaraldehyde solution (in 80 mM cacodylate, pH 7.2 to 7.4). A set of images were taken from the central region of the surface of the corneal epithelium. The cell areas, perimeter and dimensions of 25 to 30 cells from 18 different samples were measured with a digitiser pad or rule, and number of bordering cells (sides) counted manually. Cell shape was assessed by comparing the longer and shorter dimensions (LS ratio) and calculation of the figure coefficient (FC, 4PiA/P2 ). RESULTS: With the washing protocol, surface cells were evident that displayed a range of electron reflexes (light, medium and dark) and that were decorated with microplicae and some crater-like features. These features were generally not evident if the surface was only rinsed with saline. The cell surface areas ranged from 58 to 6444 microm2 (mean 1748 +/- 1169 microm2), and increased as the number of sides increases. Cells with a higher number of sides (n=10) tended to be more rounded (LS ratio averaging 1.24) while cells with small number of sides (n=3) were more elongated (LS ratio 1.94). Shape (FC) values ranged from 0.379 to 0.967, and increased as the number of cell sides increases. Assessment of cell shape in relation to the number of cell sides identified only a small proportion of surface cells with unusually low FC values. CONCLUSIONS: With an appropriate preparative procedure (aggressive washing with saline), bovine corneal epithelial surface (squamous) cells can be shown to have a similar appearance by SEM to that previously observed for rabbit and human corneas. The cell sizes are however substantially greater than those seen in rabbits, presumably because of the much larger corneal diameter (c. 28 mm vs. 13 mm). Based on detailed analysis of cell size and shape, the results indicate that the squamous cells have reasonably predictable characteristics.

Animals↗
PubMed2004

Lung inflation with direct injection of agarose: a technique for simultaneous molecular and morphometric measurements.

The usual methods for preparing lungs for morphologic study involve the instillation of fixatives that modifyproteins and RNA such that the tissue is unsuitable for molecular studies. To develop a technique suitable for molecular studies, pieces of adult rat lungs were infiltrated with agarose, glutaraldehyde, or paraformaldehyde and the consistency of alveolar inflation was compared to lungs inflated with 10% formalin. Only direct injection with 1% agarose resulted in comparable inflation of lung tissue and preserved RNA and protein. Thus, this technique enables simultaneous molecular and morphometric analysis of the lung on small pieces of lung tissue in heterogeneous lung diseases.

Animals↗
PubMed1999

Disparate surgical margin lengths of colorectal resection specimens between in vivo and in vitro measurements. The effects of surgical resection and formalin fixation on organ shrinkage.

We noticed almost routine disparate results in margin lengths when colorectal specimens are measured in vivo by the surgeon and in vitro by the pathologist. We studied 26 sigmoid and rectum specimens to document the amount of organ shrinkage after surgical removal and fixation. Each specimen had a 5.0-cm segment at each end of the specimen marked by serosal sutures before vascular devitalization. The segments were measured after the specimen sat unfixed for 10 to 20 minutes and after 12 to 18 hours of formalin fixation. The segments shrank to a median length of 3.0 cm (40% of the in vivo length) after 10 to 20 minutes and an additional 0.85 cm, to a median length of 2.15 cm, after fixation. Overall after fixation, the segments shrank 57% of the in vivo length. Approximately 70% of the shrinkage occurred during the first 10 to 20 minutes after removal, and the remaining 30% occurred after fixation. For optimal accuracy, margin distance must be obtained immediately after surgical removal. Once the specimen has been removed for several minutes, the difference between unfixed and fixed margin lengths is 30%. A correction factor of approximately 2x should be applied when interpreting the margin length.

Adenocarcinoma↗
PubMed2005

Effects of delayed or prolonged fixation on immunohistochemical detection of bovine viral diarrhea virus type I in skin of two persistently infected calves.

The effects of delayed or prolonged fixation on immunohistochemical detection of bovine viral diarrhea virus (BVDV) antigen were evaluated in skin. Ear-notch specimens from 2 calves persistently infected with BVDV type 1 were handled in 1 of 3 ways: 1) fixed in formalin promptly and processed for immunohistochemistry (IHC) after 3-176 days; 2) held at 3-4degreesC in plastic bags up to 10 days, then fixed in formalin for 2-5 days before processing; or 3) exposed to room air and temperature for 1-5 days before formalin fixation. Immunohistochemical staining intensity was evaluated without the knowledge of specimen handling. Staining of specimens that had been promptly fixed in formalin was moderate to strong at all fixation periods through 36 days, weak or no staining was evident in specimens fixed for 176 days. Refrigerated specimens typically had moderate to strong immunohistochemical staining. Even after 10 days of refrigeration before fixation, all immunohistochemical reactions were positive. However, no immunohistochemical staining was detected in any specimen that was exposed to room air. Results indicate that prompt formalin fixation is optimal for BVDV IHC. Samples can be held in formalin at least 36 days, without loss of reactivity. A 1-day delay in fixation caused no loss of reactivity, provided the specimen was refrigerated and protected from desiccation.

Animals↗