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Oral Pathology

Author : Dr.Manjul Tiwari, MDS,

Abstract
Over the last two decades, immnocyto chemistry has had an immense impact on the practice of diagnostic pathology. It relies on the binding of immunoglobulins to antigens present in tissues. The antigen-antibody reaction is revealed by an enzymatic reaction or the colour emission of a fluorochrome. Due to the specificity and sensitivity of this reaction, immunocytochemistry is an excellent alternative to more complex, expensive and time-consuming other laboratory procedures. If used carefully, it can be an excellent diagnostic tool. But unfortunately, because of lack of standardizations or other various pitfalls, many times false positive or false negative results can be obtained. Hence, the results obtained with immunocytochemistry should always be correlated with the clinical history and other clinicopathological data available before making a final diagnosis.

In this article ,I have tried to focus on the basics of the immunocytochemistry and attempted to throw a light on the indications, advantages, complexities and limitations and the prognostic significance of this diagnostic tool.
Introduction
“The first step toward cure is to know what the disease is!” ------That means to diagnose the disease. ‘Diagnosis’ can be defined as the art of distinguishing one disease from another or the determination of the nature, location and causes of a disease.

Various diagnostic aids are implicated in the diagnosis of any pathologic lesion. But unfortunately, many lesions do not present a specific microscopic appearance or sometimes the tissue of origin of the histologically differentiated tumours cannot be identified. For these reasons, a definitive diagnosis can not always be rendered.

Although the microscope in the hands of a qualified pathologist is an irreplaceable diagnostic tool, its limitations must always be kept in mind. Fortunately, with the rapid advances now occurring in scientific techniques adaptable to microscopic diagnosis, such as histochemical techniques, fluorescent microscopy, microradiography, histoautoradiography, transmission and scanning electron microscopy and so forth, this sphere of diagnostic limitations is gradually shrinking1.

Since its inception, immunocytochemistry has gradually developed into a major diagnostic tool in diagnostic cellular pathology. In routinely processed paraffin wax embedded tissues the identification of specific or highly sensitive cellular epitopes, (which are usually protein in nature) with an antibody, an appropriate labeling system has made a significant impact on histological diagnosis2.

The form of histochemistry aims at disclosing the chemical behaviour of the tissue and the metabolic changes induced in it by external agents or conditions. The metabolic reaction which is tested is made to yield a coloured end product which can be seen at or very close to the site of the enzymes or other active groups responsible for the function studied .On the other hand ,it is concerned with the function rather than the stain3.

Hence, immunocytochemistry can be defined as a technique for identifying cellular or tissue constituents [antigens] by means of antigens – antibody interactions ,the site of antibody binding being identified either by direct labeling of the antibody, or by use of secondary labeling method.4 In other words ,immunocytochemistry is the application of immunologic principles and techniques to the study of cells and tissues.5 In some parts, of the world, the term ‘immunohistochemistry’ is employed when using tissue sections and ‘immunocytochemistry’ for cytological preparations. In United Kingdom and United States ‘immunocytochemistry’ is generally accepted as the term that covers the use of the technique on either type of sample.4
History
The origin of immunocytochemical technique lies in the pioneering work of Albert Coones in 1941, who first attempted to directly label antibody with flourescein isocyanate. Flourescein isocyanate was introduced as a label in 1958 and is still commonly employed as a label in fluorescence microscopy.6 But because of the greatest disadvantage of immunofluorescence that it is difficult to demonstrate the morphological detail of the labeled cells, it was later replaced by enzyme-labeling of antibodies.4

Nakane and Pierce in 1966, introduced counterstaining of labeled cells with traditional nuclear stains such as haematoxylin to improve morphology to be viewed in conjunction with the antigen demonstrated.7 Then Sternberger et al(1970) described the peroxidase-antiperoxidase (PAP) technique, Engvall and Perlman (1971) reported alkaline phosphatase labeling and Heggeness and Ash(1977) proposed Avidin-Biotin labeling.8,9,10 This latter technique was developed further using peroxidase labeling by Guesdon et al(1979) and Hsu et al(1981).11,12 Avidin- Biotin labeling especially with peroxidase is currently the most popular system used in diagnostic laboratories.

Neverthless with all the developments in antibodies and labeling systems, the main barrier to their use in histopathology was always formalin fixation and paraffin wax processing. During the early stages of development of immunocytochemistry in 1970s it was thought that routine processing methods destroyed some epitopes.4

Huang et al(1976) described the use of trypsin digestion on paraffin sections as a means of revealing some antigens otherwise masked by routine processing.13 However the greatest advance in retrieval came in the 1990s when Shi et al(1991) first described heat pre-treatment.14 The initial report shows use of microwave oven heating of dewaxed sections in a heavy metal solution as a means of recovering antigens. After that various other novel methods have been proposed which include-
  • Stainless steel pressure cooker 15,16
  • Autoclave antigen retrieval method 17
  • Use of ultrasound as a pre- embedding retrieval technique 18
  • Use of vegetable steamer 19

The various retrieval solutions that can be used include citrate buffer, Tris EDTA or Boric acid etc. Thus, growing use of antigen retrieval for immunocytochemistry has resulted in a large number of modifications of the method, especially the retrieval solutions and heating equipement. Many of these studies show that the major factors that govern the effectiveness of retrieval include the pH, volume of fluid, heating time and temperature.4


Basic Principle And Methods Of Immunocytochemistry
Immunocytochemical techniques are based on detecting antigens in cells and tissues. Antigens are mainly proteins, but also carbohydrates, nucleic acids, lipids and other compounds can act as antigens.20 These are highly specific topographical regions composed of a small number of amino acids or monosaccharide units and are known as ‘antigenic determinant groups’ or ‘epitopes’.

Antibodies belong to the class of serum proteins known as immunoglobulins. Of the five types of antibodies found in the blood, IgG is the commonest and most frequently used antibody for imunocytochemistry. The terminal regions of each arm vary in amino acid sequence and are known as ‘variable domains’. This variability of amino acids provides specificity for a particular epitope and enables the antibody to bind specifically to the antigen against which it was raised.4
The amino acid side-chains of the variable domain of the antibody form a cavity which is geometrically and chemically complementary to a single type of antigen epitope.21 The precise fit explains the high degree of specificity. The associated antigen and antibody are held together by a combination of hydrogen bonds, electrostatic forces and van der Waals’ forces.4
Two types of antibodies are used in immunocytochemistry-
  • Polyclonal antibodies and
  • Monoclonal antibodies
Polyclonal antibodies are multivalent, meaning that they contain antibodies for several regions of the antigen molecule, providing a strong detection capacity. However, polyclonal antibodies can cross react with antigens from different organisms.4,20
Monoclonal antibodies have the advantage of being highly specific for a single sequence of epitope of the antigen molecule. Because monoclonal antibodies will bind only to one site, fewer antibody molecules will bind to the antigen and be subsequently detected by the labeling method. However, with use of monoclonal antibodies, precaution has to be taken as prolonged fixation is more damaging to monoclonal antibodies than the polyclonal antibodies.20

Collection Of Samples
Immunocytochemistry is usually conducted on formalin fixed tissues( retrospective study can be carried out) and also on frozen samples.

Processing Of Fixed Samples
The unstained tissue sections are then subjected to the antigen unmasking or antigen retrieval to allow the antigens to be recognized by the antisera.The various systems to retrieve antigens are-
  • Detergents(Tween 20, Saponin) and chaotropic substances(guanidine, sodium isothiocyanate);
  • Enzymatic digestion(trypsin, proteinase K, proteinase E);
  • Heat induced antigen retrieval :
The heat based retrieval systems not only permit some antigens to be detected that otherwise go undetected, but also increase the sensitivity of some methods
allowing the antibody to be further diluted. 20

Signal-generating systems Visualization of the antigen-antibody reaction site depends on a signal-generating system which is conjugated to the antibody.
Three types of signal-generating systems are there :
  • Flurochromes, which are visualized by excitation with light of appropriate wavelength. Flurochromes are not popular on fixed and paraffin embedded samples due to antifluoresence, but are frequently used on frozen tissue sections. Fluorescein isothiocyanate, Rhodamine and Texas red are some common fluorochromes currently in use.
  • Enzymes, with their specific histochemical substrates and a variety of capturing chromogens can produce different coloured end products, usually brown, blue or red-that are visible by light microscopy. The enzymes commonly used are horseradish peroxidase (most common), alkaline phosphatase, glucose peroxidase and b-galactosidase.
Reaction with diaminobenzidine (DAB), results in a brown color and alkaline phosphatase is usually visualized in blue.22,23,24,25
  • Metals, such as colloidal gold, antigen-antibody reaction is detected with a developer that contains a reducing agent (usually hydroquinone) and a silver soloution (usually silver acetate). The sensitivity of this technique is similar to or higher than that of enzyme-based methods.4,20
Methods :
A direct, indirect or multiple layer method can be used in immunocytochemistry.
  • Traditional direct method : It is the simplest of the methods. It is a one step process with labeled primary antibody binding the antigen through F(ab) fragment.[Figure 1] It is frequently used for quick screening with fluorescent labeled antibodies and frozen sections.20 Its advantage is it is simple to use, but the main disadvantage is its low sensitivity than other methods.4
  • Indirect method : It relies on the addition of a second layer of antibody directed against the first layer ( which is specific for the antigen being detected). The antigen-binding fragment [F(ab)] of the second layer of antibodies will usually bind to the first layer through its Fc segment. In this case, it is the second layer of antibodies that is labeled, not the first one.[Figure 2] Its advantages are better sensitivity and its relatively inexpensive, but the disadvantage is it is more laborious.20
Multiple layer method – PAP – Peroxidase-antiperoxidase method [Figure 3] and the ABC (Avidin-Biotin Complex) method [Figure 4], are those commonly used in a diagnostic laboratory when working with formalin-fixed, paraffin-embedded sections.22,23,24,25,26 E.g. in Avidin-Biotin method , second layer of antibody is bionated and the third layer is a complex of avidin mixed with biotin that is mixed with a marker (enzyme, fluorochrome etc.).20 This method relies on the marked affinity of the glycoprotein avidin for biotin, a vitamin.4,20

Apart from these methods, the others are sometimes used are :
  • Immunogold silver staining technique27 [Figure 5]
  • Hapten labeling technique
  • Mirror Image Complementary Antibody labeling technique (MICA) etc.4

The immunologic techniques utilized are common to both, histology and cytology. Although immunologic methods are relatively widely followed in routine histologic laboratories, they are relatively in the initial stages in cytology laboratories. This is actually surprising because, alcohol fixation, which is common in cytology yields specimens that are usually optimal for immunologic procedures. In contrast, histology still relies largely on formaldehyde fixed and paraffin embedded material, a procedure which often results in epitope no longer being recognized by the antibody.

The choice of markers to be included in an immunologic panel of antibodies is of course critical. It is usually simple to tell benign from malignant cells even with only conventional cytology, but a precise diagnosis of tumour type can be more difficult and may not be possible in 10 to 15 % cases by routine methods.
Differentiation, or cell or tissue specific markers that are located either in cytoplasm or the membrane in normal cells and are retained in tumour cells seems to be helpful in further differential diagnosis of such cases.

Advantages of immunocytochemistry :
  • Immunocytochemistry provides independent objective differentiation or tissue specific information that can confirm or contradict the initial diagnosis of
    tumour type.
  • It can reveal the features of normal cytologic smear not previously recognized.
  • It is of particular value when the differential diagnosis between major tumour types is in doubt.26
  • A correct diagnosis of tumour type in metastatis may either indicate a primary tumour or narrow the search for an unknown primary with obvious benefits for the patient and reduction in cost for the hospital.
  • It also provides the cytopathologist/histopathologist with the opppurtunity for a continuing education based on most difficult and therefore the most informative cases.6

Complexities and Limitations :
Although helpful in many cases, immunocytochemistry will have its own restrictions in solving some cases of differential diagnosis for a variety of reasons. These include :
  • No appropriate marker may exist to solve a particular problem. E.g. Tumours with same IF content cannot be separated as no additional markers are available.
  • Double immunostaining may be necessary to resolve more than one type of cells in different parts of the tumour.
  • Expression of ‘ectopic’ markers by minor subpopulation of tumour cells occurs in some tumours. E.g. Keratins and neurofilaments in Ewings sarcoma.
  • A minor percentage of tumours can probably never be classified by immunocytochemistry because they exhibit signs of differentiation along
    more than one pathway.
  • It requires a good quality smear (for cytology) and appropriate fixation.26
  • Quality assurance programs for immunocytochemistry are necessary for diagnostic laboratories and for manufacturers.28
  • For increasing number of antigens that occur as member of multigene families, a better definition of reactivity profiles of monoclonal antibodies on each individual member of a multigene family is required.6

Indications for Immunocytochemistry :
After its introduction, immunocytochemistry has been applied with a starting success. Basically, the applications of the technique can be grouped into five main categories.
  • The demonstration of non hormonal oncodevelopmental tumour markers. E.g. Epithelial membrane antigen, carcinoembrionic antigen etc.
  • Identification of hormone containing cells and endocrine tumours. E.g. Gastrin, Calcitonin, Testosterone etc.
  • Identification of immunoglobulins in lymphoreticular and other diseases. E.g Alpha 1 antitrypsin and antilysozyme (as markers of macrophages and monocytes) etc.
  • Identification and localization of organisms. E.g. Hepatitis B antigen, Herpes Simplex, Vaccinia virus, HIV virus etc. Parasites include spirochetes, protozoa and helminthes.
  • Localization of enzymes. A variety of enzymes can be located with immunocytochemistry.2

The spectrum of diagnostic application of immunocytochemistry in pathologic conditions is very vast including a variety of lesions. Few of them are thyroid tumours, Pleural effusion for exclusion of malignancy, Breast tumours, Liver mass, Pancreas aspirates, Lung mass, Lymph node mass, renal tumours, adrenal tumours, colon and gastrointestinal tract tumours, prostrate tumours, melanomas, various infectious diseases caused by bacteria, viruses etc.20,26,28 In case of oral cavity, immunocytochemistry can be applied to both hard and soft tissue lesions.22,23,25,27,30

Prognostic Significance of Immunocytochemistry :
Immunocytochemical staining on histologic specimens or in fine needle aspirations can yield information relevant to grading of particular tumour types and this information may be of relevance in planning treatment, e.g. Proliferation markers like p53, Ki-67, p16 are extensively accepted as important biomarkers in diagnosis, prognosis and treatment of malignant and premalignant lesions. Higher expression of these markers indicate a more aggressive lesion.22,24,25 Some studies have indicated that cytoplasmic expression of p53 nuclear protein is a useful biological indicator of prognosis.29

Cytokeratin expression profile provides useful information on cell differentiation status and thus may help in predicting the prognosis of carcinomas.30 Vimentin co expression in breast and renal carcinomas indicate poor prognosis.31,32.33

Occurrence of increased number of S-100 positive cells in neuroblastomas and ganglioneuroblastomas indicates improved prognosis.34

Conclusion :
Although immunocytochemistry can guide the pathologist in diagnosis, it cannot be regarded as the sole mean for diagnosis. Also, it cannot be used routinely, as it is not economic. But it can certainly be used in cases of controversy or the lesions which cannot be diagnosed by conventional histopathology.
Growth in knowledge has reached a remarkable point, not even thought of, a few years ago. The new rapidly expanding knowledge in immunology and molecular biology gives justified hope, that even little understood, diseases will be deciphered in the future.

Bibliography:
  1. Shafer: A textbook of Oral Pathology. 4th edition.1993;pp597.
  2. Bancroft JD, Alan S: Theory and practice of histological techniques. 2nd edition. London: Churchill Livingstone. 1982; pp406-28.
  3. Wier DM: Handbook of experimental immunology. 1st edition.1967.
  4. Bancroft JD, Gamble M: Theory and practice of histological techniques. 5th edition. London: Churchill Livingstone. 2002; pp421-64.
  5. Ackerman VL: Ackerman’s surgical pathology. 7th edition. Volume I ;pp35-51.
  6. Bibbo M : Comprehensive Cytopathology. 2nd edition. Philadelphia: Pennsylvania. 1997;pp1033-74
  7. Nakane PK, Pierce GB. Enzyme labeled antibodies: preparation and localization of antigens. J of Histochem and Cytochem 1966;14:929-31.
  8. Sternberger LA, Hardy PH, Cuculis JJ, Meyer HG. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen - antibody complex (horse radish peroxidase-antiperoxidase) and its use in identification of spirochaetes. J of Histochem and Cytochem 1970;18:315.
  9. Engvall E, Perlman P. Enzyme linked immunosorbent assay [ELISA]. Qualitative assay of immunoglobulin G. Immunochem 1971:871-874.
  10. Heggeness MH, Ash JF. Use of the avidin-biotin complex for the localization of actin and myosin with fluorescence microscopy. J of Cell Biol 1977;73:783.
  11. Guesden JL, Terynck T, Avrameas S. The use of avidin –biotin interaction in immunoenzymatic techniques. J of Histochem and Cytochem 1979:1131-39.

References are available on request