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Immunohistochemistry (IHC)

ISSN 2398-2950


Synonym(s): Immunocytochemistry; immunofluorescence; immunoperoxidase

Overview

  • Immunohistochemistry (IHC) is a molecular technique which is widely used within veterinary diagnostic laboratories.
  • The technique involves the detection of a particular antigen within a section of tissue via the use of antibodies which bind specifically to that antigen.
  • A similar process can also be used on a cytological sample (eg smear or cytospin) when it is referred to as immunocytochemistry.
  • The antigen-antibody interaction can be visualized via different means such as a fluorescent dye or an enzymatic color change reaction.
  • If the antibody is conjugated to an enzyme such as peroxidase, this enzyme can then catalyze a color-producing reaction (known as immunoperoxidise staining).
  • Alternatively, the antibody can be labelled with a fluorophore and the interaction visualized via a fluorescence microscope (immunofluorescence).

Uses

Alone

  • IHC and similar techniques will normally only be used in conjunction with more routine tests such as histopathology (including examination of haematoxylin and eosin (HE)-stained sections) and may be used in parallel with other diagnostic tests such as PCR PCR (Polymerase chain reaction), microbial culture and serology where applicable.
  • One use of IHC is the detection and/or confirmation of infectious agents within tissues, including some not visible in routine HE-stained sections  Immunohistochemistry: leishmaniosis  Immunohistochemistry: leishmaniosis (HE stain) . This technique will detect both viable and non-viable organisms (unlike microbial culture), and is especially useful when those organisms are present in only low numbers. The technique also allows for the localization of organisms within suspicious lesions (see Table 1).
     
    Table 1. Examples of infectious agents in dogs and cats detectable using immunohistochemistry
    Canine adenovirus Feline calicivirus Leptospira
    Canine coronavirus Feline herpesvirus Neospora caninum
    Canine distemper virus Feline coronavirus Toxoplasma gondii
    Canine parvovirus Feline leukemia virus Rabies
    Helicobacter Feline parvovirus (panleukopenia)  
    Anaplasma phagocytophilum Borna disease virus  
    Leishmania    
  • IHC can also be used to confirm diagnosis of immune-mediated disease. Antibodies to immunoglobulins or to complement factor may be used to detect the deposition of immune complexes or autoantibodies within specific sites in tissues, eg pemphigus foliaceus or immune-mediated glomerulonephritis.
  • More definitive diagnosis of a poorly differentiated tumor  Immunohistochemistry: HE carcinoma  Immunohistochemistry: pan-cytokeratin marker (epithelial cells) may be aided by the use of IHC to detect cell markers of varying types, to try and establish the neoplastic cell type (see Table 2)
     
    Target antigen Cells stained Uses
    Table 2. Examples of immunohistochemical markers for differentiating neoplasms
    CD3 T-lymphocytes Neoplastic vs hyperplastic lymphoid tissues
    Round cell tumor differentiation
    (T-cell lymphoma)
    Immunophenotyping of lymphoma
    CD79a
    (CD20, PAX5, CD45)
    B-lymphocytes Neoplastic vs hyperplastic lymphoid tissues
    Round cell tumor differentiation
    (B-cell lymphoma, plasmacytoma, myelomas)
    Immunophenotyping of lymphoma
    CD18 Histiocytes (leukocytes) Round cell tumor differentiation
    Chromagranin Neuroendocrine cells Neuroendocrine tumors
    Cytokeratin (pan-) Epithelial cellse Carcinomas
    c-KIT (CD117) Haematopoietic stem cells Mast cell tumors (differentiation, prognostication)
    Gastrointestinal stromal tumors (GIST)
    Desmin Muscle (skeletal, smooth, cardiac) Rhabdomyosarcomas, some mesotheliomas
    Fascin Dendritic cells, histiocytes Histiocytic tumors
    Glial fibrillary acid protein (GFAP) Glial cells Tumors of glial origin
    (astrocytes, some ependymal cells, Schwann cells)
    Glucagon Peptide pancreatic hormone Tumors of pancreatic islet origin
    Insulin Peptide pancreatic hormone Tumors of pancreatic islet origin
    Kappa/lambda Immunoglobulin light chains Plasma cell tumor
    Inflammatory verses neoplastic
    Lysozyme Granulocytes, monocytes, macrophages Enzyme in neutrophils and macrophages
    MAC387 Tissue histiocytes Myeloid/histiocyte antigen
    Mast cell tryptase Mast cells Mast cell tumors (tumor differentiation
    MelanA Melanocytes Melanoma (tumor differentiation)
    MUM1 Plasma cells (some B-cells) Round cell tumor differentiation
    (B cell lymphoma, plasmacytoma, myelomas)
    PNL2 Melanocytes Melanoma, clear cell sarcoma
    S100 Melanocytes, some dendritic cells, macrophages, Schwann cells Melanoma, histiocytoma, clear cell sarcoma,
    peripheral nerve sheath tumor (others)
    Smooth muscle actin (SMA) Smooth muscle Smooth muscle tumors (leiomyoma, leiomyosarcoma)
    Synaptophysin Neuroendocrine cells Neuroendocrine tumors
    Vimentin Mesenchymal cells Sarcoma, lymphoid cells
    von Willebrand factor (factor VIII) Endothelial cells Vascular tumors (haemangiosarcoma)
  • IHC is used in the immunophenotyping of lymphomas Lymphoma  as B-cell (CD79a positive), T-cell (CD3 positive) or occasionally cell (also known as non-B-/non-T-cell; CD3 and CD79a negative), thus providing the clinician with further prognostic information Immunohistochemistry: B-lymphocytes (CD79a)  Immunohistochemistry: T-lymphocytes (CD3) . In dogs, the immunophenotype of a lymphoma has an influence on expected patient survival, with the T-cell phenotype associated with significantly shorter recurrence-free intervals. B-cell tumors tend to be more chemosensitive, leading to better results with chemotherapy.
  • IHC is used to assess proliferation markers such as Ki67 and MCM7 (minichromosome maintenance protein 7) within neoplastic cell populations. Ki67 is a nuclear protein expressed in all active phases of the cell cycle and the relative number of Ki67 positive cells indicates the growth fraction within a given tumor cell population. The most well-known example of the use of Ki67 is in canine mast cell tumors and melanomas. MCM7 is another proliferation marker used for prognostication of canine mast cell tumors and is also detected by IHC.
  • IHC is used to detect the expression of KIT, a tyrosine kinase receptor normally present on the cell surface (ie membrane-associated). Changes in KIT-staining patterns (from membrane associated to cytoplasmic) within canine mast cell tumors have also been used as a prognostic test.
  • IHC is a major tool in the field of molecular biology and is therefore used in a wide range of research, eg there are various markers of apoptosis detected via IHC.

Sampling

Source of test material

  • Routine tissue biopsies, ie formalin-fixed, paraffin-embedded (FFPE) or cytological preparations (unstained, air-dried smears or cytospin preparations).
  • As for routine histopathology, samples should be immediately placed in 10% neutral buffered formalin at a ratio of 10:1 volume of formalin to volume of tissue.
  • Fixation is optimized if tissue samples are no more than 1 cm thickness.
  • Once processed to paraffin blocks at the laboratory, samples can be utilized at any time for IHC, up to many years later.
  • Some laboratories can perform testing of fresh tissues which have been snap-frozen in a cryoprotectant gel, however, these sample types require specialized equipment such as cryostats.

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.

Tests

Methodologies

  • Depending on the sample and the antibody used, some cases will require antigen retrieval pre-treatments prior to the test itself. This is because the fixation and processing of tissues results in cross-linking of some of the proteins in the sample. If extensive, this cross-linking can make the antigen of interest unrecognizable by the antibody.
  • There are various types of antigen retrieval methods including those utilizing heat from a water bath, pressure cooker, steamer or microwave (known as heat-induced epitope retrieval or HIER), enzymatic treatments such as trypsin, or various combinations.
  • IHC involves several steps, the number of which depends on the precise technique used.
  • Regardless, the first step involves blocking of non-specific background staining, often using substances such as skimmed milk or normal serum.
  • Background staining can occur for a number of reasons, including inadequate or delayed fixation of samples (eg in the center of large tissue blocks), or endogenous enzyme activity or biotin within certain tissues.
  • Sections are thoroughly washed between each step in the process; to remove any unbound antibodies or other chemicals.

Direct method

  • A labelled antibody is used which directly detects the antigen within the tissues without the need for any further steps.
  • This technique is the shortest and simplest to perform.
  • However it is relatively insensitive compared to the indirect method.

Indirect method

  • An unlabelled primary antibody is used to detect the antigen within the tissues.
  • This primary antibody is then itself detected by labelled secondary antibody (directed against the species and isotype of the primary antibody).
  • This results in amplification of the signal and thus increased sensitivity of the test.
  • Some other methods involve a third layer; eg in the ABC method the secondary antibody is conjugated to biotin, which then in turn forms a complex with avidin and peroxidase.
  • For those methods using immunoperoxidase, the final step involves incubation of the sample with the chromogenic substrate before visualization by routine light microscopy.
  • The peroxidase catalyzes a color-change reaction; most commonly the substrate-chromagen used is peroxide with diaminobenzidine (DAB), which produces a brown deposit in the tissue sections.
  • By using different colored labels (either color-change or fluorochromes) more than one antigen can be detected in the same tissue section at the same time, which may allow for co-localization of different antigens.

Controls

  • Positive and negative controls are always performed.
  • Positive controls test the protocol is carried out correctly (positive control).
  • These may include internal positive control, ie tissue components within the sample itself which would be expected to stain positive with the particular antibody used. For example immunostaining of a skin biopsy for epithelial cell markers would use the epidermis as the internal positive control.
  • External positive controls use another tissue sample which would be expected to stain positive with the particular antibody used, ie a known previously positive-staining tissue.
  • Negative controls test the specificity of any antibody used. Negative controls can either consist of omitting the primary antibody or replacing the primary antibody with an antibody that should not bind to any components of the tissue, in which case no positive staining should be present. This aids differentiation between non-specific and specific staining of cells.

Validity

Sensitivity

  • The sensitivity of this type of assay is typically high due to amplification of the signal, since multiple antibodies can potentially bind to each antigen.
  • This sensitivity can often be increased depending on the precise technique used.
  • In situ visualization: staining is visualized in situ which is important as it allows correlation of the antigen, be it a cell surface marker or an infectious agent, with a specific cell population and/or lesion.
  • Utilization of biopsy tissues: as the technique uses FFPE tissues, the same tissue sample can be used first for routine HE-staining followed by IHC staining if required, without the need for further sampling of the patient.

Specificity

  • The staining is highly specific ot the target antigen, depending somewhat on the type of antibody used. Monoclonal antibodies are likely to give a more specific result than polyclonal ones.

Technique intrinsic limitations

  • False negative results: eg if an infectious agent is present in very low numbers within a tissue sample, then it simply may not be present within the particular section examined. Neoplastic cell populations may sometimes lose expression of the expected cell markers during the process of malignant transformation.

Technician extrinsic limitations

  • Non-specific labeling may obscure the specific staining if severe.
  • Non-specific labeling may confuse an inexperienced observer.

Result Data

Further Reading

Publications

Refereed papers

  • Recent references from VetMedResource and PubMed.
  • Berlato D et al(2012) Evaluation of minichromosome maintenance protein 7 as a prognostic marker in canine cutaneous mass cell tumours. Vet Comp Oncol 10 (2), 125-142 PubMed.
  • Giantin M et al (2012) c-KIT messenger RNA and protein expression and mutations in canine cutaneous mast cell tumours: correlations with post-surgical prognosis. J Vet Diag Invest 24 (1), 116-126 PubMed.
  • Bergin I L et al (2011) Prognostic evaluation of Ki67 threshold value in canine oral melanoma. Vet Pathol 48 (1), 41-53 PubMed.
  • Marconato L, Stefanello D, Valenti P, Bonfanti U et al (2011) Predictors of long-term survival in dogs with high-grade multicentric lymphoma. JAVMA 238 (4), 480-485 PubMed.
  • Maglennon G A et al(2008) Association of Ki67 index with prognosis for intermediate-grade canine cutaneous mast cell tumours. Vet Comp Oncol 6(4), 268-274.
  • Webster J D et al (2008) Evaluation of prognostic markers for canine mast cell tumours treated with vinblastine and prednisone. BMC Vet Res 4, 32 PubMed.
  • Webster J D et al (2008) Cellular proliferation in canine cutaneous mast cell tumours: associations with c-KIT and its role in prognostication. Vet Pathol 44 (3), 98-308 PubMed.
  • Scase T J et al (2006) Canine mast cell tumours: correlation of apoptosis and proliferation markers with prognosis. JVIM 20, 151-158 PubMed.
  • Kiupel M et al(2004) The use of KIT and tryptase expression patterns as prognostic tools for canine cutaneous mast cell tumors. Vet Pathol 42(4), 371-377 PubMed.
  • Fournel-Fleury C, Ponce F, Felman P et al (2002) Canine T-cell lymphomas: a morphological, immunological, and clinical study of 46 new cases. Vet Pathol 39, 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 Pathol 126, 47-56.
  • Dobson J M et al (2001) Prognostic variables in canine multicentric lymphosarcoma. JSAP 42 (8), 377-384 PubMed.
  • German A J, Hall E J & Day M J (2001) Characterization of immune cell populations within the duodenal mucosa of dogs with enteropathies. JVIM 15, 14-25.
  • Waly N, Gruffydd-Jones T J, Stokes C R & Day M J (2001) The distribution of leucocyte subsets in the small intestine of normal cats. J Comp Pathol 124, 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 CompPathol 121, 249-263.
  • Day M J, Hanlon L & Powell L M (1993) Immune-mediated skin disease in the dog and catJ Comp Pathol 109, 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 A 39, 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, UK.