Comparison of Microwave Treatment and Pressure Cooker Methods for Antigen Retrieval Techniques in Immunohistochemistry

Background: Immunohistochemistry (IHC) is a method which is capable of detecting antigens or antibodies on the cells. The antigen antibody complex formed as a result of this reaction can be visualized at a light microscopic level. The main procedure involves application of a primary antibody which is targeted against a selected tissue antigen. Immunohistochemistry implements various polyclonal and monoclonal antibodies which help to identify the tissue location of the disease. Antigen retrieval (AR) is a key step in IHC which can be done by two methods: 1] Pressure cooker treatment 2] Microwave treatment method. This study aims to compare antigen retrieval efficacy and quality of immunohistochemical sections obtained by these two methods. Methodology: This prospective analytical study will be conducted in the histopathology and immunohistochemistry divisions of department of Pathology, J.N.M.C. Wardha. The study will evaluate immunohistochemical sections during which antigen retrieval would be done either by microwave treatment method or autoclave method in 30 cases. All the cases will be divided in two groups. For Group 1 antigen retrieval will be performed by microwave treatment method. For Group 2 antigen retrieval will be done by autoclave method. Qualitative evaluation and comparison of the sections obtained by these 2 methods will be done. Statistical evaluation will be Study Protocol Jagtap et al.; JPRI, 33(61B): 395-401, 2021; Article no.JPRI.81039 396 done by SPSS software version 22.0, Prism 6.0 analysis software. Chi square test will be used for comparison. Results: A significant difference is expected in the between the efficacy of antigen retrieval by pressure cooker method and microwave treatment. Conclusion: Conclusions will be drawn on careful analysis of results.


INTRODUCTION
Immunohistochemistry and Immunohistochemical Stains: Immunohistochemistry (IHC) is a method which is capable of detecting antigens or antibodies on the cells [1]. The antigen antibody complex formed as a result of this reaction can be visualized at a light microscopic level. The procedure was conceptualized and first implemented by Albert Coons in 1941 [2]. The main procedure involves application of a primary antibody which is targeted against a selected tissue antigen. Immunohistochemistry implements various polyclonal and monoclonal antibodies which help to identify the tissue location of the disease [3].
Immunohistochemistry is widely used for the subtyping of various malignancies because certain antigens are abnormally expressed in certain cancers [4]. IHC utilizes biopsies, which are then processed further into sections. After this, the sections are incubated with primary and/ or secondary antibodies [5]. The antigen antibody complexes which are formed as a result of the reaction get localized either in the cytoplasm, nucleus or the cell membrane. The location where this reaction takes place (nucleus, cytoplasm and membrane) has a wide range of clinical implications in the management of various disorders. Special reagents like fluorescein and colloidal gold are the various markers used for visualization of these antigen antibody reactions [6].
The basic steps in Immunohistochemistry are as follows: 1. Tissue preparation 2. De-paraffinization 3. Inactivation of non specific elements 4. Antigen retrieval 5. Blocking of endogenous peroxidase 6. Primary antibody incubation 7. Secondary antibody incubation 8. Application of counter stain (Haematoxylin) 9. Application of DAB (di-amino benzidine) substrate 10. Mounting by a suitable agent 11. Microscopic examination [7] The aim of this study is to compare the quality of sections stained by immunohistochemistry with two different methods of antigen retrieval: the microwave method and the autoclave method.

Aim:
To know the concept of antigen retrieval in immunohistochemistry and compare the efficacy of antigen retrieval performed by microwave treatment thereupon of autoclave method.

Objectives:
1. To evaluate immunohistochemical sections subjected to antigen retrieval by microwave treatment method.
2. To assess immunohistochemical sections subjected to antigen retrieval by autoclave method.
3. To match the morphology of the immunohistochemical sections during which antigen retrieval has been done by microwave treatment method thereupon of immunohistochemical sections during which antigen retrieval (AR) has been administered by autoclave method. 4. To assess which method of antigen retrieval is best fitted to obtaining top quality immunohistochemical sections in routine histopathological examination.

MATERIALS AND METHODS
The present study is of prospective and analytical type and will be conducted for duration of two years in the histopathology and immunohistochemistry divisions of department of Pathology, J.N.M.C. The study will evaluate immunohistochemical sections during which antigen retrieval would be done either by microwave treatment method or autoclave method in 30 cases. All the cases are going to be divided in two groups: • Group 1 during which antigen retrieval has been performed by microwave treatment method [8].
• Group 2 during which antigen retrieval has been administered by autoclave method.

Inclusion criteria:
All the cases where the paraffin embedded tissue blocks are available are going to be included within the study.

Exclusion criteria:
Cases where the paraffin embedded tissue blocks aren't available are going to be excluded from the study.

Approach to the present study:
• Approval from I.E.C.
• Duly informed consent (if needed). • Selection of appropriate paraffin embedded tissue blocks with adequate tissue (either a tumor mass or normal tissue) for immunohistochemistry. • Antigen retrieval with either microwave treatment method or pressure cooker method. • Immunohistochemical staining by standard protocol. • Microscopic examination of immunohistochemicaly stained sections. • Comparison of morphology of the sections (AR by microwave treatment method) with those in which AR has been done by pressure cooker method.

Materials required:
 Automated tissue processor-Histokinette (Leica™ TP 1020).  Paraffin wax.  Induction plate for heating purpose (for preparing liquid paraffin wax for embedding). Pressure cooker method of antigen retrieval: Two liters of retrieval solution are placed in a stainless steel pressure cooker which has a capacity of 6 liters with an operating pressure of 103kPa which is kept to boil on a hotplate (1.5kW)

Immunohistochemistry staining protocol:
Principle: High quality nuclear, cytoplasmic and membrane staining with minimum background staining is facilitated by the Polymer-HRP detection system especially in tissues rich in endogenous biotin. Sections are treated with primary antibody which will bind the antigen. The secondary antibody is integrated along with a suitable polymer. This complex is again integrated with a suitable enzyme marker. After this, a suitable substrate is added. The substrate is acted upon by an enzyme. This process leads to a color reaction. The color of the reaction is attributed to DAB chromogen substrate.

Interpretation:
The antigen antibody reaction is visible as a colored complex which is localized either to the membrane, cytoplasm or the nucleus.

INTERPRETATION OF RESULTS
The results will be examined by 3 observers and will be assessed as follows: Statistical Analysis: Will be done by SPSS software version 22.0, Prism 6.0 analysis software. Chi square test will be used for comparison.

OBSERVATIONS AND RESULTS
The data collected from observations will be analyzed and then compared with similar research. The data collected will be tabulated in a master chart and. The efficacy of antigen retrieval carried out either by pressure cooker method or microwave treatment will be compared. The quality of immunohistochemical sections will be compared and appropriate conclusions will be drawn out.

DISCUSSION
Norton AJ et al.
[8] in their study explored the advantages of microwave based antigen retrieval for diagnostic immunohistochemistry. An alternative method of antigen retrieval, heat mediated antigen retrieval was also investigated. It was observed that heating the sections in 0.01 M citrate buffer (pH 6.0) gave similar results as those obtained by microwave based antigen retrieval. Added benefits like increasing the speed of treatment, results reproducibility with large slide batches and the power to implement the use of metal slide racks were observed.
In 1991, two autonomous gatherings revealed that exposure of routine areas to high temperatures in an watery medium could improve the location of antigens by defeating the concealing effect of formal infixations. Since at that point, articles managing the problem of AR in routine segments have been flourishing in the writing, in light of warming or presentation to a strong alkali or corrosive. At present, HBAR strategies are day by day used in histopathology labs, as they have been found to be more effective and all the more effectively pertinent than other AR frameworks. Nonetheless, the fluids and warmth sourcesemployed differ from site to site. this doesn't favour standardization in immunohistochemistry, which is felt to be significant by numerous scientists and open institutions. Even the components by which these new techniques are so effective in antigen exposing are still unclear. Cattoretti and Suurmeijer suggested that 'heat and hydrolysis may both denaturate and break the tissue proteins at or close to the connections made by the formalin between neighboring amino acids' and thought it 'conceiv-capable that selfgathering of unfurled protein chains with subsequent reclamation of antigenic destinations happens when the retrieval arrangement is permitted to cool'. Shi et al. have stressed that the pH of the recovery arrangement is an important cofactor for certain antigens. Morgan et al. have revealed that tight complexing of calcium ions or other divalent metal cations with proteins during formaldehyde fixation can be answerable for masking certain antigens; along these lines, the chelation or precipitation of these particles can speak to a basic advance in salt-mediated AR. The extreme high temperatures may be expected to provide sufficient vitality to deliver the calcium particles and divalent metal cations from the pen like buildings that they form with proteins. A Number of related studies were reviewed [12][13][14][15][16][17]. The present investigation gives further help to the usefulness of HBAR and gives new data in order to change this 'kitchen' approach into a more standardized apparatus [18][19][20][21][22][23][24][25][26][27][28][29][30].

CONCLUSION
Appropriate conclusions will be drawn from the findings in observations and discussion.

CONSENT
Consent will be taken from the patients participating within the study.