Introduction

Of all the laboratory diagnostic procedures routinely used in human and veterinary medicine, the oldest technology is histopathological interpretation of biopsy tissue. Very little has changed in the mechanics of histopathology - in terms of tissue processing, standard or special histochemical staining and light microscopic evaluation for many decades. In the recent past, it was confidently predicted that routine histopathological diagnosis would fade away and be replaced by molecular analysis and computer interpretation of changes in a tissue biopsy - but such predictions were overly optimistic, and there is now recognition that 'steam-driven' light microscopic interpretation of biopsies will probably never be replaced by purely automated diagnosis. Although often considered more 'art' than 'science', subjective interpretation of tissue change remains a fundamental part of veterinary practice.

The process of histopathological examination has, however, not remained static, and a number of adjunct methods have been developed and applied in order to maximize the information gained from the biopsy procedure. This presentation will review these advances.

Immunohistochemistry

Although standard histopathology may not have changed much over the last two decades, during that same time period there has been a revolution in many aspects of immunohistochemical analysis.¹ In the 1980s, immunohistochemistry was rarely available outside a university setting, was expensive and time consuming, was restricted to immunofluorescence methodology, and was limited by the availability of commercial antisera to detection of immunoglobulins (IgG, IgM, IgA) or complement factor C3 (for dogs only).

Now, 30 years later, immunohistochemistry is routinely available through veterinary diagnostic pathology laboratories, is often fully automated so is relatively rapid and more cost effective, is generally performed by immunoperoxidase labelling, and benefits from an ever-increasing panel of dog- and cat-specific, or cross-reactive antisera.

Immunohistochemical Methodology

Immunohistochemistry is fundamentally defined as methodology that permits the identification of antigen within a tissue biopsy by the use of polyclonal or monoclonal antisera. Immunocytochemistry is the same procedure, but applied to cytological samples (cell monolayers) rather than tissue samples.

The first consideration in immunohistochemistry is sample type and collection. At one time, this methodology was only applicable to freshly collected and snap-frozen tissue. The other main disadvantage of frozen tissue immunohistochemistry is that there is relatively poor preservation of tissue architecture (as opposed to that with formalin-fixed samples), so interpretation is more difficult. However, the major advantage of this sample type is that a greater range of antisera will be capable of identifying antigens in frozen tissue which have not been damaged by the fixation process.

It has now become standard procedure to undertake immunohistochemistry on formalin-fixed tissue samples. The advantages of this sample type are clear: there is no specialized requirement for collection or processing the sample (although for some antibodies rapid processing after a minimum 24 hours in fixative provides better results), the same biopsy sample may be used for initial light microscopy and subsequent immunohistochemistry - thus directly relating lesions observed to antigen localization, retrospective studies may be performed on archived material, and there is excellent preservation of tissue microarchitecture. The disadvantage of fixed tissue is that the process of fixation can degrade or crosslink the target antigens, making them inaccessible to the detecting antibodies. This has necessitated the development of a range of 'antigen retrieval' techniques that can re-expose these antigens to enable antibody binding.

There are two fundamental means of visualizing the binding of antibody to antigen in tissue. Immunofluorescence microscopy entails conjugating the detecting antibody to a fluorochrome which will emit fluorescence when the section is subsequently examined with a fluorescence microscope that produces light of the correct wavelength. In a research setting, it is now possible to detect multiple antigens within a single tissue sample by the use of multicolour immunofluorescence.

The second modality is immunoperoxidase (or alkaline phosphatase) immunohistochemistry, in which the detecting antibody is conjugated to either of these two enzymes. The binding of antibody is subsequently demonstrated by application of an enzyme substrate which results in local colour change within the tissue section. The advantages of this method are that the tissue section can be lightly counterstained for examination of microarchitecture, that only a light microscope is required for examination, and that the sections can be mounted for permanent archiving.

Apart from the detection system, there are also numerous variations on the methodology that are designed to increase the sensitivity of the techniques. Described above is the 'direct' technique using a single antibody reagent directly conjugated to a marker. In the 'sandwich' technique, a multilayered effect is created whereby the original detecting antibody is itself bound by a second antibody which is conjugated to enzyme or fluorochrome. The advantage of this method is that more than one molecule of conjugated ('secondary') antibody may bind to a single detecting (or 'primary') antibody, thus increasing the sensitivity of the technique. A more complicated sandwich of primary, secondary, and tertiary (conjugated) antibodies is also possible to further enhance the result obtained. A range of other means of enhancing the reaction is also used. In the 'peroxidase anti-peroxidase' (PAP) technique, the primary antibody and secondary antibody (which is specific for peroxidase) are linked by a third antibody. In the ABC or streptavidin-biotin reactions, the primary antibody is conjugated to biotin, which in turn binds multiple molecules of streptavidin conjugated to peroxidase (streptavidin-biotin) or a complex of streptavidin with further molecules of biotin that are conjugated to peroxidase (ABC). The most recent innovation involves detecting the primary antibody with long polymers that are linked to the secondary antibody and carry multiple molecules of enzyme label.

As mentioned above, there is now a large panel of antisera that can be applied to canine and feline tissue for the detection of an array of molecules including:

 Surface markers that identify specific leukocyte populations

 Adhesion molecules expressed by vascular endothelium or epithelium

 Immunoglobulins (IgG and subclasses, IgM, IgA, IgE), complement C3 and some proinflammatory cytokines

 Neurological molecules (e.g., GFAP)

 Cytokeratins

 Cellular proliferation markers (e.g., Ki67, PNCA)

 Apoptosis markers (e.g., bcl-2, p53, c-Myc)

 Infectious agents (e.g., leishmania, FeLV, FIP)

 Cytokines and cytokine receptors

Applications of Immunohistochemistry

Detection of Immunoglobulin and Complement

Immunoglobulin and complement may be detected in biopsy tissue from dogs and cats with immune-mediated disease. There are limitations of this as a diagnostic procedure - not all samples for animals with histological evidence of an immune-mediated process will be positive, and immune complexes are notoriously difficult to demonstrate by this procedure. The WSAVA Renal Standardization Group recommends that immunofluorescence studies should be performed routinely on kidney biopsy samples from animals with suspected glomerulonephritis.²

Tumour Phenotyping

Increasingly, immunohistochemical phenotyping is used to support a light microscopic diagnosis of neoplasia. This technique is particularly valuable when the origin of an anaplastic tumour (e.g., sarcoma or carcinoma) is not clearly apparent using HE or histochemical stains for matrix tissue. Immunohistochemistry is also valuable in the case of poorly differentiated round cell tumours - and may be able to distinguish lymphoid from histiocytic neoplasms. Of greatest current use is the ability to distinguish between T- and B-cell lymphoma via the use of cross-reactive anti-CD3 and anti-CD79a reagents. There is evidence that canine multicentric T-cell lymphoma carries a poorer prognosis than B-cell lymphoma. The immunophenotypic characterization of feline alimentary lymphoma has prognostic and therapeutic significance.^3,4 Immunohistochemical evaluation of cellular proliferation markers (e.g., Ki67) has prognostic significance in canine mast cell tumours.⁵

Characterization of Inflammation

Although not often used diagnostically, immunohistochemistry has proven invaluable in research studies of the immunopathogenesis of numerous diseases of the dog and cat for detection of the phenotype of infiltrating inflammatory cells or the expression of tissue cytokines and cytokine receptors.⁶

Detection of Infectious Agents

The sensitivity of classical histochemical techniques for the detection of infectious agents in tissue biopsies (e.g., gram stain, periodic acid Schiff, ZN, Giemsa) is generally considered relatively poor. To that end, immunohistochemistry can be used to locate organisms that fail to stain histochemically.

Alternative Methodologies

Immunohistochemistry is likely to remain an important research and diagnostic tool for years to come. The range of antisera applicable to the technique should increase, and availability to general practitioners should grow. The next step in this area involves the application of molecular biology to tissue biopsy samples, and we are already seeing developments in this area. These advances will mean that it becomes possible to use fixed biopsy material for both light microscopic, immunohistochemical and molecular diagnosis.

The types of molecular applications include:

 The use of PCR for detection of infectious agents in tissue samples.

 The use of RT-PCR for quantification of gene transcription in a sample.

 The use of microarray technology for gene profiling within a sample.

 The use of clonality testing for confirmation of lymphoid neoplasia.

 The use of in-situ hybridization to localise gene expression to specific cells within a tissue sample or to detect infectious agents (often by fluorescence in-situ hybridization; FISH).^7,8

 The use of laser-capture dissection microscopy to excise small clusters of specific cells from a biopsy for molecular analysis.

References

1.  Ramos-Vara JA, Miller MA. When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry - the red, brown, and blue technique. Vet Pathol. 2014;51:42–87.

2.  Cianciolo RE, Brown CA, Mohr FC, et al. Pathologic evaluation of canine renal biopsies: methods for identifying features that differentiate immune-mediated glomerulonephritides from other categories of glomerular diseases. J Vet Intern Med. 2013;27:S10–S18.

3.  Moore PF, Rodriguez-Bertos A, Kass PH. Feline gastrointestinal lymphoma: mucosal architecture, immunophenotype, and molecular clonality. Vet Pathol. 2012;49:659–668.

4.  Barrs V, Beatty J. Feline alimentary lymphoma. 1. Classification, risk factors, clinical signs and non-invasive diagnostics. J Feline Med Surg. 2012;14:182–190.

5.  Scase TJ, Edwards D, Miller J, et al. Canine mast cell tumors: correlation of apoptosis and proliferation markers with prognosis. J Vet Intern Med. 2006;20:151–158.

6.  Krafft E, Lybaert P, Roels E, et al. Transforming growth factor beta 1 activation, storage, and signalling pathways in idiopathic pulmonary fibrosis in dogs. J Vet Intern Med. 2014;28:1666–1675.

7.  Manchester AC, Hill S, Sabatino B, et al. Association between granulomatous colitis in French bulldogs and invasive Escherichia coli and response to fluoroquinolone antimicrobials. J Vet Intern Med. 2013;27:56–61.

8.  Peters IR, Helps CR, Willi B, et al. Detection of feline haemoplasma species in experimental infections by in-situ hybridisation. Microb Pathog. 2011;50:94–99.