What Is Immunohistochemistry?

Historical Perspective

 1941--First use of antibodies to detect infectious disease in tissues. Coons used fluorescent labelled antibodies to detect Pneumococcus in lung tissue.

 Additional methods developed over time:

 Enzyme-linked antibody (an ELISA test in tissue) increased routine use (didn't need a fluorescent scope to read the reaction)

 Monoclonal antibodies--increased specificity

 Amplification methods--increased sensitivity

 New reagents decrease background and increase use in veterinary species

 Now a standard test in many veterinary reference laboratories.

Methodology

The technique is based on the specificity of antibodies for their antigens:

 Identify an antigen that is specific for a tissue or cell type or an infectious agent

 Find an antibody that is specific for that single antigen (often a protein)

 The higher the specificity of the antibody, the more useful the test is for diagnosis

Multiple steps are used to prepare the tissue (especially if the tissue is fixed in formalin) and decrease non-specific (background) staining. For example:

 Antigen retrieval for formalin-fixed tissue (not necessary with frozen sections)

 'Squelching' of background endogenous biotin binding

 Non-specific protein binding

Next steps are:

 Specific (primary) antibody application

 Detection of primary antibody on tissue (direct and indirect methods are summarised in Figure 1)

 Biotin-labeled primary antibody (direct) or secondary antibody linked to biotin (indirect)

 Streptavidin linked to enzyme (often horseradish peroxidase)

 Expose enzyme to substrate that develops a visible colour in the tissue

 Specific methods include:

 Avidin-biotin complex (ABC) method (Figure 2)

 Labelled streptavidin-biotin (LSAB) (Figure 3)

 Chain polymer-conjugated technology (EnVision+/PO) (Figure 4)

Click on the image to see a larger view

	Figure 1. Direct and indirect immunohistochemistry.
	Figure 2. Avidin-biotin complex (ABC) immunohistochemistry.
	Figure 3. Labelled streptavidin-biotin (LSAB) immunohistochemistry.
	Figure 4. Enzyme-labelled polymer-conjugated immunohistochemistry.

What Are The Issues Faced By The Use Of Immunohistochemistry?

 Endogenous biotin background:

 Biotin methods are not very useful for most avian tissues--lots of biotin in most tissues

 Liver of all species has very high biotin

 Must use avidin/biotin block for these to work in biotin methods

 Best to use the newer polymer-conjugated technology--biotin is not a component

 Cross-reactivity:

 Different proteins/tissues share the same epitope of the target tissue antigen, e.g., LPS, CD79a (smooth muscle, B lymphocytes)

 Non-specific cross-reactivity of antibodies with similar or different epitopes on different antigens, i.e., low-affinity antibodies

 Cross-reactivity can be produced/modified by antigen retrieval methods

 Quality control--all laboratory testing must be done with appropriate controls:

 Same species and tissue as that being tested

 Processed under similar conditions: fixation time can be essential!! Over-fixation can decrease staining.

 Sufficient but not excessive amount of antigen

 Each laboratory must standardise!

Why Do Immunohistochemistry (What Does It Tell You?)

Immunohistochemistry (in most cases) must be done in the context of routine histopathology. Except for specific tests for infectious disease agents, it is not a stand-alone test.

What Are Its Uses?

 To provide a more definitive diagnosis

 To assist in prognosis

 As a research tool to understand better neoplasia and its many faces

Is It Worth the Cost?

How much is the increased diagnostic certainty worth to the patient/client? IHC may increase the diagnostic certainty to 90-100% (H&E: 70%; IHC: 100% (best case scenario)). What is that 30% worth?

Costs range considerably between laboratories. Multiple stains may be needed to fully evaluate a tumour (four-stain panel costs US$95-150).

 Will knowing the identity of the tumour significantly affect outcome for the patient?

 Does knowing the specific tumour type predict therapy that can significantly increase the patient's life expectancy?

 Does confirming a specific infectious disease have impact on other contact animals?

Examples of Specific Immunohistochemical Stains

Neoplasia

 Vascular tumours--endothelial cells:

 Factor VIII rAG--often associated with significant background in haemorrhagic masses (stains VW/Factor VIII in the extravascular tissue)

 CD 31 (PECAM-1) somewhat easier than Factor VIII rAg. Expressed by the majority of vascular tumours

 Muscle tumours. Desmin:

 Expressed by a majority of muscle tumours

 May be present in: myofibromas, MPNST, rhabdoid tumours, primitive neuroectodermal tumours

 Carcinoma/adenocarcinoma. Cytokeratins (AE1/AE3)

 Sarcoma. Vimentin is a universal marker for soft tissue tumours

Infectious Disease

 How many positive cells make a positive?

 Interpretation of immunohistochemistry results has to be done in conjunction with the clinicopathological data available

 The presence of a particular antigen indicates the existence of an infectious organism: Its significance has to be interpreted looking at the whole picture. Examples:

 Toxoplasmosis

 Neosporosis

 Canine distemper

 Chlamydophilosis (chlamydiosis)

 Aspergillosis

 Coccidioidomycosis

 Numerous others

References

1.  Haines DM, West KH. Immunohistochemistry: forging the links between immunology and pathology. Veterinary Immunology and Immunopathology 2005; 108(1-2): 151-156.

2.  Ramos-Vara JA. Technical aspects of immunohistochemistry. Veterinary Pathology 2005; 42(4): 405-426.

3.  Rhind SM. Veterinary oncological pathology--current and future perspectives. The Veterinary Journal 2002; 163(1): 7-18.