Effect of tissue processing on assessment of endoscopic intestinal biopsies in dogs and cats.

BACKGROUND: Prior studies failed to detect significant association between hypoalbuminemia and small intestinal lesions. HYPOTHESIS: Use of pictorial templates will enhance consistency of interpathologist interpretation and identification of intestinal lesions associated with hypoalbuminemia. ANIMALS: Tissues from 62 dogs and 25 cats examined as clinical cases at 7 referral veterinary practices in 4 countries. METHODS: Retrospective, observational study. Histopathology slides from sequential cases undergoing endoscopic biopsy were examined by 4 pathologists by pictorial templates. Changes for 9 microscopic features were recorded as normal, mild, moderate or severe, and 2- and 4-point scales were tested for consistency of interpretation. Logistic regression models determined odds ratios (OR) of histologic lesions being associated with hypoalbuminemia while kappa statistics determined agreement between pathologists on histologic lesions. RESULTS: There was poor agreement (kappa = -0.013 to 0.3) between pathologists, and institution of origin of slides had effect (kappa = 1.0 for 3 of 4 lesions on slides from Institution 5) on agreement between pathologists on selected histologic features. Using 2 point as opposed to 4-point grading scale increased agreement between pathologists (maximum kappa = 0.69 using 4-point scale versus maximum kappa = 1.0 using 2-point scale). Significant association (P = .019- .04; 95% OR = 3.14-10.84) between lacteal dilation and hypoalbuminemia was found by 3 pathologists. CONCLUSIONS AND CLINICAL IMPORTANCE: Substantial inconsistency between pathologists remains despite use of pictorial template because of differences in slide processing. Distinguishing between mild and moderate lesions might be important source of the disagreement among pathologists.

Effect of sample quality on the sensitivity of endoscopic biopsy for detecting gastric and duodenal lesions in dogs and cats.

BACKGROUND: The quality of histopathology slides of endoscopic biopsies from different laboratories varies, but the effect of biopsy quality on outcome is unknown. HYPOTHESIS: The ability to demonstrate a histologic lesion in the stomach or duodenum of a dog or cat is affected by the quality of endoscopic biopsy samples submitted. More endoscopic samples are needed to find a lesion in poor-quality tissue specimens. ANIMALS: Tissues from 99 dogs and 51 cats were examined as clinical cases at 8 veterinary institutions or practices in 5 countries. METHODS: Histopathology slides from sequential cases that underwent endoscopic biopsy were submitted by participating institutions. Quality of the histologic section of tissue (inadequate, marginal, adequate), type of lesion (lymphangiectasia, crypt lesion, villus blunting, cellular infiltrate), and severity of lesion (normal, mild, moderate, severe) were determined. Sensitivity of different quality tissue samples for finding different lesions was determined. RESULTS: Fewer samples were required from dogs for diagnosis as the quality of the sample improved from inadequate to marginal to adequate. Duodenal lesions in cats displayed the same trend except for moderate duodenal infiltrates for which quality of tissue sample made no difference. Gastric lesions in dogs and mild gastric lesions in cats had the same trend, whereas the number of tissue samples needed to diagnose moderately severe gastric lesions in cats was not affected by the quality of tissue sample. CONCLUSIONS AND CLINICAL IMPORTANCE: The quality of endoscopically obtained tissue samples has a profound effect on their sensitivity for identifying certain lesions, and there are differences between biopsies of canine and feline tissues.

Dynamic measurement of intraocular pressure using a mechanical model of the human eye.

Heightened intraocular pressure (IOP) is not always indicative of glaucoma, but is an important risk factor for the progression of certain types of eye damage not obviously felt by the sufferer. A number of measurement systems have been devised in the past to measure the IOP by applying force or pressure to the cornea, but past studies have shown that the cornea thickness and its curvature have a significant effect on measurements which may ultimately lead to clinical misdiagnosis. A cyclic strain controlled dynamic probing measurement system has been developed using an indenter of diameter 3.06 mm operating at actuation frequencies of between 0.1 Hz and 4 Hz and displacements up to 1 mm. The cyclic strain is actuated by a linear stage with a load cell and indenter coupled in series. The load cell records the resultant cyclic force where the dynamic modulus is expressed as amplitude ratio and phase lag. The mechanical eye model consists of a silicone membrane that can be varied in thickness and it is distended hydraulically to simulate a range of IOP. A pressure sensor measures the dynamic IOP within the system which will be compared against the dynamic modulus. The relationship between the mechanical properties and the physical properties of the membrane will be established in order to develop a probe which can be used clinically taking into account the effects of corneal stiffness and hydraulic behaviour of the eye. The preliminary study reported here a significant increase in amplitude ratio and mean ratio with increasing the frequency similar to the behaviour found in biological materials and gelatin.

Assessing the quality of biological tissues using structure-property relationships: macro-scale tests on engineered phantoms.

The overall aim of this work is to determine the quality of biological tissues based on the relationship between the dynamic mechanical properties and their histology. Two sets of rigs have been developed for dynamic mechanical measurement, one for micro-scale testing and the other for macro-scale testing. Preliminary results using the macro-scale measurement system only are reported here. This system uses strain-controlled cyclic probing actuated by a linear stepper motor operating at actuation frequencies between 0.5Hz and 20Hz. A 1mm diameter indenter probes the specimen up to a displacement of 0.2mm and a load cell measures the resultant cyclic force. A series of tissue mimics were prepared using various formulations of gelatin and safflower oil and preliminary tests carried out to determine a suitable range of experimental variables and to establish the repeatability of the tests. The dynamic mechanical properties are expressed as amplitude ratio, phase difference and mean ratios of stress and strain, and the behaviour of these measurands with actuation frequency, mean strain and strain amplitude was observed. Results consistent with the literature were found which form a foundation for measurements on collagen-lipid biological tissues.

Micro-engineered remote palpation device for assessing tissue compliance.

This paper concerns the operation of the actuator for a prototype micro-engineered mechanical palpation device for deployment via a cystoscope to measure the dynamic mechanical properties of the prostate gland in vivo. The subassembly consists of a 400x200 microm silicon (Si) piston manufactured using deep reactive ion etching (DRIE) housed within an anodically bonded glass-Si-glass sandwiched housing. The micro-channel on the Si layer was formed by powder blasting and contains the micro-piston with one end pointing to the side of the housing and the other facing a via hole leading to a capillary tube. The opening on the side of the housing was sealed by a 5 microm thick silicone membrane which acts to retain the micro-piston and act as a return spring. A 320 microm diameter capillary forms the connection between the micro-channel and a micro-syringe which is operated by a programmable syringe pump to produce a reciprocating action. A pressure sensor is connected along the capillary tube to measure the dynamic pressure within the system. The micro-piston has already been used, separately actuated to measure the dynamic mechanical properties of known viscoelastic materials and prostate tissue. The purpose of the present work is to assess the functionality of the actuator assembly.

Evaluation of disease activity markers in dogs with idiopathic inflammatory bowel disease.

OBJECTIVES: To evaluate the clinical utility of serum tumour necrosis factor-alpha, C-reactive protein and microalbuminuria as disease activity markers in canine idiopathic inflammatory bowel disease. METHODS: Dogs with chronic gastrointestinal disease for which no underlying cause could be identified were considered to have idiopathic inflammatory bowel disease and were included in the study. Serum tumour necrosis factor-alpha was assessed using a canine-specific ELISA, C-reactive protein by immunoturbidometric assay and quantitative microalbuminuria was analysed using a monoclonal antibody directed against canine albumin. The canine inflammatory bowel disease activity index and histopathologic grade were used to assess disease severity; biologic markers were then compared with the canine inflammatory bowel disease activity index and histopathologic grade. RESULTS: Sixteen dogs were included in the study. C-reactive protein level was mildly elevated in 15 dogs. Microalbuminuria was elevated in two of 15 dogs, and tumour necrosis factor-alpha was not detected in any dog tested. No correlation was found between the canine inflammatory bowel disease activity index and C-reactive protein or microalbuminuria or between histopathologic grade and C-reactive protein or microalbuminuria. There was no correlation between histopathologic grade and the canine inflammatory bowel disease activity index. CLINICAL SIGNIFICANCE: Although only a small number of dogs were evaluated, this study does not support the use of serum tumour necrosis factor-alpha measured by canine-specific ELISA or microalbuminuria in the evaluation of disease activity in dogs with idiopathic inflammatory bowel disease. Although mildly elevated in most dogs, C-reactive protein did not reflect disease severity as assessed by the canine inflammatory bowel disease activity index or histopathologic grade.

Multi-scale techniques of measuring the dynamic properties of biological tissues.

The overall aim of this study is to establish relationships between the histology of biological tissues and their mechanical properties, especially their dynamic modulus. The emphasis is on the physical scale of the probe used in relation to the size of the histological features in the material of interest. The selection of a model system (connective tissue) is described along with a corresponding gelatin-based system in which the make-up of the material can be "engineered" reproducibly. Next three experimental rigs are described with regard to their suitability for making measurements at a variety of scales; the micro-scale, the meso-scale and the macro-scale. Finally, some preliminary measurements on the micro-scale rig are presented and preliminary conclusions drawn on the methodology.

Diagnostic value of cytology of bronchoalveolar fluid for lung diseases of sheep.

Bronchoalveolar lavage fluid was collected postmortem from the lungs of 113 sheep, and total and differential cell counts were analysed in relation to the presence of gross and microscopic lung pathology. The diffuse lung diseases, maedi and adenomatosis, were both characterised by an increase in overall cellularity and by increases in the percentages of lymphocytes and neutrophils, respectively. Focal parasitic lung disease was characterised by an increase in the percentage of eosinophils and mast cells. Consolidated lung lesions were characterised by a slight increase in cellularity but no change in the differential cell profile. In regions of parasitised and consolidated lungs without lesions the differential cell profile was consistent with focal lung pathology, although the slight increase in cellularity observed in the consolidated regions was not observed in the regions without lesions. A decision tree was developed to facilitate the interpretation and indicate the likely predictive capacity of the differential cytology of the fluid.

Immunohistochemical detection of canine leucocyte antigens by specific monoclonal antibodies in canine normal tissues.

This report describes the immunocytochemical detection and distribution of different canine leucocytes antigens (Thy-1, CD5, CD4, CD8, MHC-II and B-cell antigen) by means of monoclonal antibodies (mAbs) in frozen tissue sections of lymphoid organs of the dog. CD5 is expressed exclusively by T lymphocytes. Thy-1 is expressed by T-lymphocytes and in a variety of non-lymphoid tissues. The mAb against canine MHC-II could detect B and T lymphocytes, macrophages, fibroblasts and antigen presenting cells. The CD4 antigen is expressed on many lymphocytes of the T-dependent areas of lymph organs and is also expressed by certain antigen presenting cells. CD8 is expressed in a minor proportion of T cells. The mAb against B cells detected lymphocytes in lymphoid follicles and epithelial cells of the thymic medulla.

Early innate and longer-term adaptive cutaneous immunoinflammatory responses during primary infestation with the sheep scab mite, Psoroptes ovis.

Clinical observation has indicated that Psoroptes ovis mites provoke cutaneous inflammation within hours of experimental infestation, but the nature of this reaction has not been described. After infestation of naive sheep with ovigerous P. ovis mites, significant influxes of eosinophils (P<0.004) and neutrophils (P<0.001) were detected within 24 h. A significant (P<0.001) increase in mast cell numbers was observed by 96 h post-infestation. In addition, marked degenerative and proliferative epidermal lesions were evident 24 and 96 h, respectively, after infestation. The influence of the later, adaptive response on the cellular infiltrate at the advancing margin of the lesion and the original site of infestation was also monitored. Mast cell numbers were greatest at 21 days while recruitment of eosinophils and neutrophils was maximal 63 days after infestation. Lesional severity was particularly pronounced from 42 to 63 days after infestation, but significant resolution had occurred by 84 days. Pathological changes at the advancing margin of the lesion were more severe than at the initial site of infestation, and this was reflected by the numbers of mites present. These data suggest that P. ovis elicits an early innate cutaneous response that is subsequently augmented by the development of an adaptive immune response, the intensity of which corresponds to the local population density of mites.

Immunohistochemical localisation of cytokeratin and vimentin intermediate filament proteins in canine mammary tumours.

Monoclonal antibodies to human stratifying keratin (StK), simple keratin (SK), broad spectrum keratin (BSK) and vimentin were applied to 47 canine mammary tumours (three benign hyperplasia, 26 benign mammary tumours, one malignant mixed tumour, two malignant complex tumours, seven lobular carcinomas, three papillary carcinomas and five squamous cell carcinomas). In benign hyperplasia the SK antibody reacted with acinar and duct epithelial cells; the StK and BSK antibodies reacted strongly with duct epithelial cells in the canine mammary tumours expressed mainly keratins of stratified epithelia rather than those of simple epithelia. Myoepithelial cells in complex and mixed tumours can express StK and vimentin. The finding might be helpful in the assessment of complex tumours where stromal reactions may be confused with myoepithelial neoplastic proliferation. Stromal mesenchymal tissues, including precartilage in complex tumours, and clearly differentiated myoepithelial cells always stained positively for vimentin. Occasional carcinoma cells stained positively for vimentin.

The effect of tamoxifen on tumours induced by cells from a canine mammary carcinoma cell line in athymic nude mice.

Tumours were induced in CBA athymic nude mice by subcutaneous injection of REM 134 cells. These cells were from a continuous line derived from a canine mammary carcinoma and have no detectable oestrogen receptors. In vitro, the anti-oestrogen tamoxifen was growth inhibitory at a concentration of 10(-6) M and adding 10(-8) M oestradiol-17 beta did not reverse this effect. The relative rate of growth of the tumours induced by the cultured cells was the same in male and female mice. Oral tamoxifen at a dose of 1 mg mouse-1 week-1 suppressed the early growth rate significantly and to the same extent in male and female mice; conversely subcutaneous tamoxifen at 2 mg mouse-1 week-1 had only a transient early effect. These results argue against tamoxifen acting solely as an antagonist for oestrogen.

Characteristics of a feline mammary carcinoma cell line.

The establishment of an epithelial cell line, JM, from a feline mammary adenocarcinoma is described. In vitro, the morphological and cultural properties of these cells, their surface features such as lectin binding and their response to hormones were ascertained. In vivo, they are tumorigenic in athymic nude mice inducing tumour nodules composed of epithelia-like cells.

Evaluation of B and T lymphocytes and plasma cells in colonic mucosa from healthy dogs and from dogs with inflammatory bowel disease.

The aim of this study was to investigate the subpopulations of lymphocytes in the colonic mucosa of healthy dogs and dogs with inflammatory bowel disease (IBD). Fourteen normal dogs and 13 dogs with IBD were examined. Endoscopic biopsy specimens of colonic mucosa from each dog were stained specifically for pan T lymphocytes (CD3) and pan B lymphocytes (CD79a), and for plasma cells with methyl green pyronin (MGP) stain. Cells were counted by means of a grid and statistical analysis was performed on the data collected. B and T lymphocytes were also counted in the glandular epithelium of normal dogs and dogs with IBD and the normal and abnormal groups compared statistically. Healthy dogs had significantly lower numbers of T cells in the lamina propria and glandular epithelium and significantly lower numbers of B cells in the lamina propria. Significant group differences for plasma cells were not evident. Our results indicate that in IBD a chronic cellular immune reaction is present in the diseased gut involving increased numbers of B and T lymphocytes.

Identification of CD45 (leucocyte common antigen) in the domestic cat.

Feline CD45 (leucocyte common antigen, LCA) was characterised by using the mouse monoclonal antibody, WC45a. Its specificity was established on the basis of its reactivity with different lymphoid tissues, as judged by flow cytometry and immunohistochemistry, and of the molecular weight of its antigen in different tissues. It reacted with all the leucocyte populations tested including T and B lymphocytes, granulocytes and monocytes. By immunohistology it reacted only with leucocytes and not with endothelia, epithelia or connective tissue. It precipitated cell surface polypeptides, of M(r) 180,000 to 220,000 from lymph node cells, and therefore apparently recognised an epitope represented on most of the isoforms of the CD45 family.

Variable expression of major histocompatibility complex class II in the domestic cat.

This paper describes the characterisation of six independently produced monoclonal antibodies (mAbs) specific for non-polymorphic determinants of feline major histocompatibility complex (MHC) class II. One mAb is an anti-sheep class II which cross-reacts with the cat and five have been produced in response to immunisation with purified feline immunodeficiency virus. Despite their independent source all the mAbs have identical reactivities, immunoprecipitating two complex groups of polypeptides of M(r) 33 to 36.000 (MHC class II alpha chains) and M(r) 28 to 31,000 (MHC class II beta chains). Immunoblot analysis showed them to be beta chain-specific. One and two-dimensional electrophoresis revealed the complexity of feline class II mass and charge and implied the expression of multiple class II loci in the cat. Furthermore, it was demonstrated that distinct cell populations expressed a distinct range of class II variants. This suggesting either the differential expression or the distinct post-translational modification of lymphocytes from different sites. The mAbs have also been used for the detailed examination of the cellular distribution and tissue localisation of MHC class II in the cat.

Diagnostic value of exfoliative cytology of body fluids in dogs and cats.

A method for collecting body fluids for cytological examination and their subsequent preparation and staining is described. The results of the cytological examination of 80 dogs and 26 cats over a period of three years are reported, indicating the number and types of condition which can be diagnosed, together with the clinicopathological correlations.

Diagnostic value of tissue biopsy in gastrointestinal and liver disease.

Methods are described for the endoscopic examination and biopsy of the gastrointestinal tract and liver of the dog, cat, ox and horse. The results of the examination of 41 biopsy samples are shown, followed by a discussion of the value of biopsy in the diagnosis of diseases of the digestive tract.

Antigens associated with canine spontaneous mammary carcinoma.

Soluble proteins were prepared from canine spontaneous mammary carcinoma (CSMC) cells by extraction with 3M KCl or limited hydrolysis of isolated cell membranes with papain. These preparations were assayed for tumour-associated antigens by inhibition of fully absorbed rabbit antisera. Organ-specific reactivity was demonstrated and antigenic specificity indicated the possible involvement of fetal components. Exclusion chromatography showed that antigenic activity was associated with macromolecules of different sizes in each of the protein preparations which allowed partial purification of tumour-associated antigens. Antigens were also prepared from circulating immune complexes in the serum of tumour-bearing animals. These studies provide a basis for the development of serological markers of malignancy in CSMC.