Application of multiple labels IHC in research and pathological diagnosis
Employing enzymes or fluorescent dyes as markers for particular antigens that are limited to a particular area of a tissue, depending on the antigen-antibody reaction is known as IHC or Immunohistochemistry, which is also used as an effective research tool (1). In medical diagnosis and research, IHC is considered as a vital process (2). In order to locate the biomarkers and the expression of proteins at different tissue parts, immunohistochemistry is extensively used, which makes it a common technique in laboratory research. Locating the abnormal cells, such as the cancerous cells, is another major application of IHC. In cancer treatment, immunohistochemistry can work as prognostic markers. It can be used for oncogene identification, identification of tumor suppressor genes and enzymes, and can also be used as tumor cell proliferation markers (3). Other applications of IHC include diagnosis of conditions, drug development and laboratory based research. In diagnosis of cancer, IHC can be employed to confirm the nature of the tumor, that is, benign or malignant; to find out the level and stage of the tumor, and also the source of metastasis. IHC is used for assessing the effectiveness of the drugs by examining control or action of the target disease (4). IHC can also be used to identify infectious agents present within the tissue, by applying particular antibodies against DNA or RNA of the microbes. In genetic studies, IHC is employed to pin point the action of a number of products produced by the genes. IHC can also be used in many other arenas of laboratory based research and pathological diagnosis, like neurodegenerative conditions, diseases of muscles, brain trauma or tumors with undefined histogenesis (5). The IHC staining method is studied under three classes: the sequential staining technique, simultaneous staining and the multi-step staining. Each of these methods has their own strengths and weaknesses.
The multi-step staining includes finding two or even more targets on a single section of tissue. Hence, it enables to get more information from each of the slides. Multiple staining has a short turnaround time, as compared to single staining. For staining two or even more number of antigens in a single section of tissue, immunofluorescence can be employed by using two separate dyes. It can also be achieved through peroxide conjugated antibodies made with separate chromogen substrates, so that they create differently colored end products. However, the objective of this study is to examine application of multiple label immunohistochemistry in pathological diagnosis and laboratory research.
As per Arvydas et al (6), biological as well as clinical activities of tumors can be detected by using multiple combined immunohistochemistry. The process included 109 numbers of breast ductal carcinoma tissue samples and used 10 immunohistochemistry markers. To get accurate outcomes, multiple immunohistochemistry markers were measured through digital image analysis, which is considered as an effective tool for examination. The process was also supported by publications of other researchers, such as Anthony et al (7), who proposed that weaknesses of visual scoring done by pathologists can be covered up by using automatic IHC measurements. In the staining range, the measurements provided by automatic IHC are highly precise, as compared to the weak visual scoring process followed by pathologists. The automated IHC also offers a continual data set. According to Arvydas et al (6), they observed a reverse relationship between HER2 and expression of progesterone receptors within the tumors that are hormone receptor positive. They also noticed an inverse relationship between Ki67 and estrogen receptors which can be helpful for getting differential information through expression of progesterone receptors and estrogen receptors. As per the findings of Gown AM (2008) (8), in case of cancer of the breast, IHC detection of the presence of estrogen receptors is a subtle prognostic marker. However, in case of the therapy response, the same works as a strong forecasting marker. Gown AM (8) also reported that study of estrogen receptors and progesterone receptors expression, together with IHC identification of progesterone receptor expression was authenticated through clinical studies.
As reported by Gian et al (9), multiple IHC carried out simultaneously in lung cancer, which involves non-Small cells, enhances precision of diagnosis, particularly in tissue sections that are small. In the article, precision of diagnosis, economy, viability, as well as the prognostic impacts of simultaneous multi IHC markers that spares the tissues in sub differentiation of lung cancer of non-small cell type in tissues of 265 NSCLC patients. Researchers reached the inference that IHC detection of lung cancer that involves non-small cells by employing concurrent multi IHC markers for evaluation of differentiation in lineage, as well as for the neuroendocrine carcinoma of large cells assists in precise and authentic tissue dependent diagnosis. This strength of simultaneous staining was approved by Chaubert P et al (10), who mentioned that the process takes less time, because it supports mixing of reagents. However, the process also has some drawbacks, which primarily involves accessibility of proper primary antibodies and cross-reactions (10).
As reported by Katia R et al (11), they employed multiple labels IHC in clinical as well as prostate cancer diagnosis. Research had the aim to diagnose a typical gland in biopsies of prostate and detection of cancer in re-biopsy. According to Katia R et al (11), antibiotics are often used for identifying basal cells to shun re-biopsies. The study reached the result that immunohistochemistry did not result in irrelevant tumor diagnosis. This was demonstrated by the fact that there was no difference in the pathological stage or in positive surgical limits in men subjected to radical prostatectomies. Hence, it was suggested from the outcomes that this process can be used in practice for borderline biopsy, as well as typical cases of glands. However, α-methylacyl-CoA racemase is generally used as a marker in practice. However, as stated by Brimo F and Epstein JI (12), the CoA racemase demonstrates over expression in the cytoplasm, in case of adenocarcinoma affected glands of prostate.
The study by Brimo F and Epstein JI (12) suggested 34BE12 and p63 as the most efficient markers for labelling urothelial carcinoma. The study highlighted that 34BE12 staining is normally robust and non-focal. It is capable to stain poorly segregated adenocarcinoma. However, is not very reliable and is limited to a very small number of cells (13). Therefore, in case of a tumor that is not well differentiated, negativity for markers of prostate are focal and patchy. 34BE12 staining is not suggested, which can be considered as a conclusive diagnosis for urothelial origin.
Elinor Burke et al (14) reported that double labeling IHC for O6-methylguanine-DNA-methyltransferase and the mixture is a speedy and authentic process for elements that are not tumor based, for the purpose of inferring the status of methylation in gliobla stoma and other basic types of brain tumors. The objective of the study was to establish a guideline for O6-methylguanine-DNA-methyltransferase IHC, with the help of molecular genetics based studies of tumor methylation. As per Christmann M et al (15), the expression level of MGMT can offer substantial information on the susceptibility of cancer and the level of response to different therapies. Elinor Burke et al (14) studied 40 primary brain tumor samples, including 30 glioblastomas and 10 oligodendroglial tumors. They used double labelling IHC for both cocktail and MGMT of the antigens that were non-tumor (CD34, CD45 and CD68). Finally, they assessed the outcomes with single label IHC for MGMT and probe multiplication which is methylation specific and dependent on multiplex ligation. The researchers inferred that applying double labelling IHC for both MGMT and Cocktail of elements that are non-tumorous is a speedy and dependable process for determining if the nuclei of the tumor cells are MGMT negative or positive, at a glance.
Employment of an enhanced double IHC staining process was first reported by Tao Q et al (16) in 1994. They informed about using paraffin wax, along with cryostate sections, mixing with immunofluorescence that is not direct and APAAP or alkaline phosphatase anti-alkaline phosphatase. The research was conducted to find out the two separate antigens in the same section of tissue, by applying grouping processes of immunoenzymes and immunoflourescnce. The study suggested that in combination application of immunofluorescence and immunoenzymes can cover up for the drawbacks of double immunofluorescence and double immunoenzyme processes, particularly when used in paraffin wax. At the same time, one thing that should be kept in mind is that using the two processes in combination is quite time intensive.
David Robertson et al (17) reported application of multiple immunofluorescence labelling in FFPE (formalin-fixed paraffin-embedded) tissue. An optimized process for high resolution immunofluorescence labelling of FFPE tissues was used. It includes a combination of the three: confocal laser scanning microscopy, antigen retrieval and indirect immunofluorescence. Utility of their process was demonstrated by employing immunofluorescence labelling of human kidney, breast and a tissue microarray of aggressive breast cancers in human. They established that the stained slides can be kept for a long term at -20 degree Celsius temperature. In case they are needed to be stored for short term before the images are collected, a temperature of 4 degree Celsius can be sufficient. As stated by David Robertson et al (17), the process will help the researchers to find out more than one antibody on a single section of the tissue, to find out specific population of cells or to study several biomarkers in a small amount of sample, and define cellular heterogeneity in samples of tissues with better precision.
As informed by David Y et al (18), in several histopathological labs, double labeling immunoenzyme has been applied for comparing the patterns of expression of the paired antigenic markers. However, the process also has its limitations. It is time intensive and is not able to detect more than one antigen present in the same cell, because of its cross-reactive nature. M V Macville et al (19) also expressed similar views about using double label immunoenzyme for the targets. He noted that for co-localized targets, identifying single colors is not actually simple. In case triple-staining is used, it will be even tougher to identify the colors. Hence, according to Van der loos CM (20), specialized imaging techniques might be used in these situations. In addition to that, it is argued by David Y et al (18) that for antigen markers in regular paraffin embedded tissues, using double labeling immunofluorescence is more beneficial, as it supports viewing of two markers within the same cell, and it is also comparatively speedy.
REFERENCES:
1. Miller J. When Tissue Antigens and Antibodies get along.Vet Pathol. 2014
2. Xiao C. Double Staining Immunohistochemistry. N Am J Med Sci.2(5); 2010
3. Garcia CF SS. Best practices in contemporary diagnostic immunohistochemistry: panel approach to hematolymphoid proliferations. Arch Pathol Lab Med. 133(5); 2009
4. Mohan. H. Essential pathology for dental students. 3rd edition. New Delhi: Jaypee Brothers; 2005
5. Jeyapradha D. Applications of immunohistochemistry. J Pharm Bioallied Sci.; 2012
6. Arvydas L. Immunohistochemistry profiles of breast ductal carcinoma: factor analysis of digital image analysis data.Diagn Pathol; 2012
7. Anthony E R. Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagnostic Pathology; 2012
8. Grown AM. Current issues in ER and HER2 testing by IHC in breast cancer. Mod Pathol. ;2008
9. Gian Kayser. Simultaneous Multi-Antibody Staining in Non-Small Cell Lung Cancer Strengthens Diagnostic Accuracy Especially in Small Tissue Samples. PLoS One; 2013
10. Chaubert P et al. Immunoenzyme Multiple Staining Methods. Modern Pathology; 1997
11. Leite KR. The Use of Immunohistochemistry for Diagnosis of Prostate Cancer. International Braz J Urol.
12. Brimo F EJ. Immunohistochemical pitfalls in prostate pathology. Hum Pathol; 2012
13. Chuang AY. Immunohistochemical differentiation of high-grade prostate carcinoma from urothelial carcinoma. Am J Surg Pathol;
14. Elinor Burke. Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours. ; Acta Neuropathol Commun; 2013
15. Christmann M. Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: enzyme activity, promoter methylation and immunohistochemistry. Biochim Biophys Acta.; 2011
16. Tao Q. Improved double immunohistochemical staining method for cryostat and paraffin wax sections, combining alkaline phosphatase anti-alkaline phosphatase and indirect immunofluorescence. ; J Clin Pathol; 1994
17. David Robertson. Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. BMC Cell Biology; 2008
18. David Y. Mason. Double immunofluorescence labelling of routinely processed paraffin sections. ; J Pathol.; 2000
19. MacvilleM V . Spectral imaging of multi-color chromogenic dyes in pathological specimens. ; Analytical Cellular Pathology ; 2001
20. CM. vdL. Multiple immunoenzyme staining: methods and visualizations for the observation with spectral imaging. ; J Histochem Cytochem; 2008
