Direct immunofluorescence or immunoperoxidase labeling is used to detect a specific target antigen within a tissue biopsy (immunohistochemistry), or associated with individual cells on a cytological preparation (smear or cytospin) (immunocytochemistry).
The target antigen is detected by an antibody, and the binding of the antigen-antibody is demonstrated by an indicator system.
The indicator may be the emission of fluorescence when exposed to light of a particular wavelength (immunofluorescence), or deposition of color at the site of antibody binding following an enzyme-substrate reaction.
The indicator fluorochrome or enzyme may be chemically conjugated to the detecting antibody, or to a secondary antibody that binds in turn to the primary antibody in a 'sandwich' reaction.
The use of this 'sandwich' technique increases the sensitivity of the method, eg reduces the likelihood of picking up non-specific antibody binding, etc.
A range of other such 'amplification' techniques are available, eg the detecting antibody may be conjugated to biotin , which in turn binds to the avidin-linked enzyme.
Uses
Alone
These immunological detection methods have a wide range of applications in veterinary medicine, and these are limited only by the availability of suitable antibodies.
In infectious disease diagnosis – antibodies can be used to probe tissue biopsies for the presence of infectious agents that may not be visible at the light microscope level.
This methodology is applied to effect in the demonstration of viral infections in particular.
Antibodies to immunoglobulin (IgG , IgM, IgA) or to complement factor C3 may be used to detect the deposition of immune complexes or tissue-specific autoantibody within tissue biopsies.
This is most often performed for the confirmation of immune-mediated skin diseases , eg in pemphigus foliaceus IgG may be expected to localize within the interepithelial area of a skin biopsy.
A range of antibodies are available that can detect the presence of cell membrane or intracytoplasmic molecules of a variety of types. These are particularly useful in characterizing the nature of poorly differentiated tumors.
Antisera that can distinguish between T and B lymphocytes are now commonly employed to phenotype lymphoma .
In combination
Immunohistochemical or cytochemical investigations will normally be considered only subsequent to more standard diagnostic tests, eg a tissue biopsy will first be examined for histopathological changes consistent with infection, neoplasia or autoimmunity. Subsequently, the nature of these processes can be characterized immunologically.
Immunohistochemistry/cytochemistry may be used in parallel with other diagnostic procedures, eg in the investigation of an infectious disease, culture for the infectious agent (such as viral, bacterial and or fungal cultures), serology or PCR may be utilized.
The sample required for direct immunolabeling is either a tissue biopsy or cytological preparation.
Quantity of test material
The chance of detecting the target substance is increased if a large biopsy, or an increased number of biopsies, are taken.
Larger tissue biopsies make the immunolabeling process technically more easy to perform, but the method is applicable to even small samples, eg Tru-cut core biopsies of liver or kidney, endoscopic biopsies of intestinal mucosa. For the latter, it is recommended that 4-6 biopsy samples are obtained.
Similarly, more cellular cytological preparations increase the likelihood of a positive result.
Sample collection technique
The sample required is limited by the nature of the detecting antibodies.
Many monoclonal antibodies will not be able to detect antigens in formalin-fixed tissue, so require fresh tissue that is snap-frozen in a cryoprotectant gel for subsequent preparation of frozen sections cut with a cryostat.
This demanding sample handling procedure means that some immunolabeling techniques may only be available within research institutions. By contrast, other detecting antibodies are readily applicable to formalin-fixed tissue samples – including those used to confirm autoimmune skin disease, and to phenotype lymphoma. In this case, there is no need to take a special sample for immunolabeling.
A single formalin-fixed biopsy can be processed for standard histopathology, and then further serial sections can be taken from the wax-embedded tissue for immunohistochemistry.
There is no longer a place for 'special fixatives' such as Bouin's solution that were once recommended for immunohistology.
Samples for immunocytochemistry are prepared as for standard cytological examination, ie unstained, air-dried smears or cytospin preparations.
Quality control
Timing of test
Samples should be placed into an excess would define what is meant by 'an excess', eg 10 x the volume of the tissue itself of 10% neutral buffered formalin as soon as possible after collection. It is normally recommended that samples are no greater than 1 cm thickness.
These should be submitted to the laboratory and processed to wax embedded samples as soon as possible.
Sample storage
Once tissue is processed and wax-embedded, immunohistochemistry can be performed successfully on future occasions.
Retrospective studies have shown that immunohistochemistry can be positive on samples that are 20 years old, but the technique is more likely to be successful on samples under 5 years of age.
Sample transport
Samples may be submitted to the laboratory via standard postal services.
For formalin-fixed tissue, pre-treatment of sections is required to remove wax from the tissue.
For the immunoperoxidase technique, sections are pre-exposed to dilute hydrogen peroxide to exhaust endogenous peroxidase enzyme that may be present within the tissue.
Endogenous peroxidases may potentially react with the substrate that is used in the latter stages of the test. Additionally, fixed tissue sections are usually pre-treated to 'open' the tissue and 'expose antigenic sites' to increase the likelihood of the detecting antibody being able to bind to the relevant antigen ('antigen retrieval').
A range of such pre-treatment techniques are utilized, eg incubation in a trypsin or pronase solution, or incubation in citrate buffer with microwaving.
Cytological preparations and frozen tissue sections may be fixed in cold acetone before immunolabeling.
Following pre-treatment, the sections are then incubated with the detecting antibody. This occurs for variable time periods (from 30 minutes to 16 hours) and temperatures, depending upon the nature of the antibody.
In simple direct immunolabeling, the fluorochrome or enzyme are conjugated to the primary antibody.
Where a secondary antibody is used, a subsequent period of incubation is required after thorough washing of the tissue to remove unbound primary antibody.
The use of a secondary antibody necessitates a 'blocking step' using dilute serum of the animal species in which the secondary antibody was raised.
For direct immunofluorescence, the section is coverslipped under an aqueous mountant and examined with a fluorescence microscope. Fluorescence will be localized to the area of antibody deposition.
A range of fluorochromes are available that emit light at different wavelengths, but that most commonly used (fluorescein isothiocyanate or FITC) produces an apple-green fluorescence.
It is possible to probe sections for several different antigens using several different antibodies coupled to different fluorochromes, and thus build up a composite picture.
For direct immunoperoxidase, the final stage of the reaction involves incubation of the tissue with chromogenic substrate.
The most commonly used enzyme is peroxidase, and the most commonly used substrate-chromagen is peroxide with diaminobenzidine. This reaction produces a chocolate-brown deposit at the site of antibody binding within the tissue.
The section can be counterstained with haematoxylin to provide an indication of tissue morphology, and after mounting under DPX can be examined by standard light microscopy.
Control
The laboratory should include appropriate positive and negative controls in each test.
Availability
Not all laboratories will offer immunolabeling, but most will be able to refer material on to other laboratories.
Validity
Sensitivity
The direct immunolabeling method is a relatively sensitive technique, eg organisms that are not obvious in routine HE stained tissue sections may be identified by immunohistochemistry.
The sensitivity of the test can be increased by the use of amplification techniques such as the 'sandwich' of two antibodies, or the biotin-avidin method.
Specificity
The specificity will be related to the specificity of the detecting antibody.
In general, monoclonal antibodies are likely to give a more specific result than polyclonal antisera.
An antiserum known to cross-react with multiple species of microbe, will still have this limitation when applied to an immunohistochemical technique.
Predictive value
The success of the technique will have a number of limitations.
A higher success rate may be obtained by screening multiple biopsies or cytological preparations, by sampling from appropriate sites, by the use of particular antibodies that are optimal for use in this technique, and by the experience of the observer who interprets the test.
Technique (intrinsic) limitations
Direct immunolabeling is a subjective test, the result of which depends on the interpretation made by the observer.
A descriptive or 'yes/no' answer may be given.
Precision can be increased by requesting that the labeling is enumerated, eg the percentage of positively labeled cells within a tumor population may be determined to give a more objective result.
Fournel-Fleury C, Ponce F, Felman P, Blavier A, Bonnefort C, Chabanne L, Marchal T, Cadore J L, Goy-Thollot I, Ledieu D, Chernati I & Magnol J P (2002) Canine T-cell lymphomas: a morphological, immunological, and clinical study of 46 new cases. Vet Pathol39 , 92-109.
Koutinas A F, Polizopoulou S, Baumgaertner W, Lekkas S & Kontos V (2002) Relation of clinical signs to pathological changes in 19 cases of canine distemper encephalomyelitis. J Comp Pathol126 , 47-56.
German A J, Hall E J & Day M J (2001) Characterization of immune cell populations within the duodenal mucosa of dogs with enteropathies. JVIM15 , 14-25.
Waly N, Gruffydd-Jones T J, Stokes C R & Day MJ (2001) The distribution of leucocyte subsets in the small intestine of normal cats. J Comp Pathol124 , 172-182.
German A J, Hall E J, Moore P F, Ringler D J, Newman W & Day M J, (1999) Analysis of the distribution of lymphocytes expressing ab and gd T cell receptors and expression of mucosal addressin cell adhesion molecule-1 in the canine intestine. J CompPathol121 , 249-263.
Day M J, Hanlon L & Powell L M (1993) Immune-mediated skin disease in the dog and cat. J Comp Pathol109 , 395-407.
Zipfel W et al(1992) Demonstration of immunoglobulins and complement in canine and feline autoimmune and non-autoimmune skin diseases with the direct immunofluorescence and indirect immunoperoxidase method. ZentralblVeterinarmed A39 , 494-501.
Bradley G A & Mays M B (1990) Immunoperoxidase staining for the detection of autoantibodies in canine autoimmune skin disease; comparison to immunofluorescence results. Vet Immunol Immunopathol 26 ,105-13.
Other sources of information
Day M J (1999) Clinical Immunology of the Dog and Cat. Manson Publishing, London.
Vetstream contributor(s)
Professor Michael J Day BSc BVMS PhD FASM DipECVP MRCPath FRCVS , Division of Veterinary Pathology, Infection and Immunity, Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU, UK.
Dr Helen R Milner BVSc(Dist) PhD CertVR MRCVS , PO Box 8730, Riccarton, Christchurch, New Zealand.
It is normally recommended that samples are no greater than 1 cm thickness.