Brian J. Chelack

Technical considerations for developing enzyme immunohistochemical staining procedures on formalin-fixed paraffin-embedded tissues for diagnostic pathology.

Immunohistochemical staining is the detection of Techniques in which the primary antibodies specific antigens in tissue sections through the use of specific for the antigen of interest are labeled with an enzyme antibodies that are labeled so that the sites of antibody are termed direct methods (Fig. la). In direct immuattachment become microscopically visible. Immu- nostains, the antiserum is incubated on the tissue folnohistochemistry has been used for diagnosis of dis- lowed by addition of an enzyme substrate that causes eases associated with autoantibodies, tumors, immune the deposition of an insoluble colored reaction product complex deposition, and for the detection of micro- at the sites of antibody binding in the tissues. This organisms causing infectious diseases. 9,18,26,31 Until recently, most immunohistochemical stains have used fluorescein dye-labeled antibodies on fresh or frozen tissue specimens. 10 These methods produce a labile stain that is visible only with an ultraviolet microscope. Enzyme-labeled antibodies and methods applicable to tissues fixed in standard fixatives such as formalin have been more recently described. 16,24 These techniques have added a new dimension to diagnostic pathology by creating permanent stains demonstrating the distribution of antigens visible with ordinary light microscopy. This paper summarizes some of the techniques, the technical problems, and the limitations of the application of enzyme immunohistochemical stains on fixed tissues in diagnostic veterinary pathology. Recently, automated systems for performing immunohistochemical stains have been developed, 7 and the use of 1 of these systems is described. Immunohistochemical staining methods

Suggested guidelines for immunohistochemical techniques in veterinary diagnostic laboratories.

This document is the consensus of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However, neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.

Morphine enhances adenosine release from the in vivo rat chemical cortex

Morphine (1 and 5 mg/kg), administered intravenously, increased the rate of efflux of purines from intact rat cerebral cortices prelabelled with 3H-adenosine. Naloxone antagonized morphines action. It is suggested that the depressant actions of morphine on transmission in the central nervous system may be related to this enhancement of extracellular levels of adenosine and the adenine nucleotides.

The effect of morphine on purine and acetylcholine release from rat cerebral cortex: Evidence for a purinergic component in morphine's action

Morphine enhances the release of adenosine and its metabolites from the rat cerebral cortex and inhibits the release of acetylcholine. Naloxone antagoinizes the effects of morphine on both purine and acetylcholine release. The adenosine antagonists, caffeine and theophylline, reduce morphines effects on acetylcholine release, and at the same time increase the spontaneous release of acetylcholine. It is suggested that morphine, acting at a naloxone-sensitive site, enhances the level of extracellular adenosine, which in turn inhibits the release of acetylcholine, and that some of morphines actions are mediated by a purinergic step.

Application of a Direct Flow Cytometric Erythrocyte Immunofluorescence Assay in Dogs with Immune-Mediated Hemolytic Anemia and Comparison to the Direct Antiglobulin Test

A direct flow cytometric erythrocyte immunofluorescence assay (FC) was developed and compared with the direct antiglobulin test (DAT) for detection of erythrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in dogs with immune-mediated hemolytic anemia (IMHA). Tests were performed on erythrocytes from 13 healthy nonanemic dogs and from 13 anemic dogs with IMHA. The FC and DAT were negative for erythrocyte-bound immunoglobulin in all healthy dogs. The FC was negative for erythrocyte-bound C3 in 12 healthy dogs and positive in 1 healthy dog, and the DAT was negative for C3 in all healthy dogs. Of the 13 IMHA dogs tested for erythrocyte-bound IgG, 12 were positive using the FC and 7 were positive using the DAT. Sensitivity for the detection of erythrocyte-bound IgG in the 26 dogs was 92% for FC and 53% for DAT. Specificity for detection of erythrocyte bound IgG for FC and DAT was 100%. The addition of IgM and/ or C3 did not increase the sensitivity for FC or DAT. In this group of dogs, the FC provided a more rapid, cost-effective, sensitive, objective method to quantitate erythrocyte-bound immunoglobulin and/or complement compared with the currently used DAT.

Pathology of Newcastle disease in double-crested cormorants from Saskatchewan, with comparison of diagnostic methods.

Newcastle disease (ND) in juvenile double-crested cormorants (Phalacrocorax auritus) occurred several times since 1975, but there are relatively few studies on its pathology and diagnosis. In order to describe the distribution of Newcastle disease virus (NDV) and associated lesions in cormorants with ND and to compare diagnostic methods, 25 cormorants with nervous signs from a ND epizootic in Saskatchewan in 1995 (NDE cormorants) were compared with 18 negative control cormorants. Tissues of these birds were examined by necropsy, histology, virus isolation, immunohistochemistry, serology, and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. The NDE cormorants had a characteristic non-suppurative encephalomyelitis, with a significantly higher prevalence of neuronal necrosis, gliosis, perivascular infiltration with mononuclear cells, and endothelial hypertrophy than control cormorants. These lesions were found more frequently in the cerebellum and brain stem than in other parts of the central nervous system. Immunohistochemically, NDV antigen was limited to neurons, glial and endothelial cells in the central nervous system, and to tubular epithelial cells in the kidney. Newcastle disease virus was isolated with the highest prevalence (4/5) and the highest concentration (104.8 ELD50/g) from the kidney. The virus isolates often did not agglutinate erythrocytes in the standard hemagglutination test; the presence of NDV was confirmed by use of an indirect immunoperoxidase assay. By RT-PCR, NDV was detected in kidney and jejunum of a NDE cormorant. There was no significant difference between sensitivity of histology, virus isolation, and serology for detecting ND in NDE cormorants.

Immunohistochemical detection of canine distemper virus in haired skin, nasal mucosa, and footpad epithelium: a method for antemortem diagnosis of infection.

A reliable antemortem diagnostic method is needed for determining infection with canine distemper virus (CDV). The utility of immunohistochemical detection of CDV antigen was examined was examined for samples of nasal and footpad epithelium and haired skin in dogs with and without detectable CDV antigen in the lung and/or brain. Tissues from 57 dogs at risk of CDV infection were tested. Viral antigen was found in the lung and/or brain of 28 dogs. Among these dogs, viral antigen was demonstrated in the epithelial cells of the nasal mucosa in 24 of 27 dogs, in the footpad epithelium in 24 of 26 dogs, and in the haired skin of the dorsal neck in 26 of 27 dogs. Among the 29 dogs without CDV antigen in either the lung or brain, 1 dog had positive staining for viral antigen in the skin and nasal mucosa. Biopsies of haired skin of the dorsal neck, which is relatively simple to sample, can be used for antemortem immunohistochemical testing for acute and subacute infection with CDV.

Altered Immunohistochemical Staining for Desmoglein in Skin Biopsies in Canine Pemphigus Foliaceus

In this study, 50 cases of canine pemphigus foliaceus and 49 cases of canine superficial pyoderma were examined by immunohistochemical staining for patterns of desmoglein expression. In 31/50 (62%) of pemphigus foliaceus cases, there was an altered staining pattern for desmoglein consisting of distinct clumped deposits at the periphery of keratinocytes and/or dark cytoplasmic staining of acantholytic cells (consistent with internalization of desmoglein). In contrast, desmoglein staining in biopsies from cases of superficial pyoderma was diffusely pale without evidence for clumping or distinct internalization. This study demonstrates that epidermal desmoglein expression is altered in some cases of pemphigus foliaceus in dogs and suggests that immunohistochemical staining for this protein may be useful in diagnosis.