|Year : 2008 | Volume
| Issue : 1 | Page : 2-11
|Immunohistochemistry in surgical pathology practice: A current perspective of a simple, powerful, yet complex, tool
Nirmala Ajit Jambhekar, Anshuman C Chaturvedi, Bhulaxmi Prakash Madur
Department of Pathology, Tata Memorial Hospital, Mumbai, Maharashtra, India
Click here for correspondence address and email
| Abstract|| |
Immunohistochemistry (IHC) is a powerful tool in the surgical pathologists' armamentarium. The requests for IHC and the list of monoclonal antibodies have increased tremendously in the past decade. Issues concerning technical reproducibility, uniformity of interpretation, inter-laboratory comparability, and quality assurance are assuming greater importance due to the increased availability of IHC and its impact on diagnosis and therapy. An attempt has been made to give a current perspective of this simple and yet, in some aspects, a complex tool.
Keywords: Immunohistochemistry, monoclonal antibodies, surgical pathology, tumors
|How to cite this article:|
Jambhekar NA, Chaturvedi AC, Madur BP. Immunohistochemistry in surgical pathology practice: A current perspective of a simple, powerful, yet complex, tool. Indian J Pathol Microbiol 2008;51:2-11
|How to cite this URL:|
Jambhekar NA, Chaturvedi AC, Madur BP. Immunohistochemistry in surgical pathology practice: A current perspective of a simple, powerful, yet complex, tool. Indian J Pathol Microbiol [serial online] 2008 [cited 2015 May 28];51:2-11. Available from: http://www.ijpmonline.org/text.asp?2008/51/1/2/40382
| Introduction|| |
Immunologic methods for diagnostic use were first described by Coons and Jones, using immunofluorescent technique for the detection of bacteria.  However, it was in the late 1970s that the discovery of monoclonal antibodies gave the much needed impetus for ushering objectivity in surgical pathology diagnosis. Initially begun as a diagnostic tool, immunohistochemistry (IHC) today has far surpassed its initial expectations. Newer bio-molecules which have a role in prognostication or which form the basis of justification of expensive targeted therapy have increased the demands from surgical pathology services.  Today we are indeed in an era of "translational crossroads for biomarkers," and IHC will remain center stage in the demonstration of newer monoclonal antibodies. 
It is not infrequent to read surgical pathology reports with a footnote stating "IHC confirmation is necessary," almost as a disclaimer to the diagnosis being offered. Ever so often, clinical colleagues directly refer cases requesting IHC tests. In either situation, sometimes, IHC may be irrelevant. This happens due to a prevailing belief in some quarters that IHC is akin to any biochemical test. To some extent this is true, because IHC is slide-based chemistry; but the similarity unfortunately ends there; the vital difference being IHC is not a machine-generated value. The variables involved in the generation of the IHC result are far too many; and unlike biochemistry tests, the concept of control samples and "reference range" is complex. Even after all technical and methodological issues are sorted out, the final IHC interpretation is largely subjective. Issues of major concern today are those related to ushering in objectivity in interpretation, reproducibility, inter-laboratory comparability, and quality assurance. This article is a broad overview on IHC in the current context.
The following format has been applied to the ensuing text:
- Generation of IHC result
- Recent advances and future directions
- Quality assurance in immunohisto- chemistry
- Information sources
For a more elaborate understanding of IHC and also for the theoretical and technical aspects, the reader is requested to refer and study standard texts.
| Generation of IHC Result|| |
Several factors influence the final IHC result, and no two tissue samples react in an identical way. Pre-analytical, analytical, and post-analytical factors all contribute to this variation.
(a) Pre-analytical factors pertain to generation of the sample at source. This includes the time taken to remove the tissue at surgery, the ensuing ischemia, the interval between surgical resection and fixation, the type of fixative, the length of fixation, the dimensions of the tissue, prior frozen section, etc.
(b) Analytical factors pertain to laboratory procedures. A laboratory SOP (standard operating procedure) manual ensures reproducibility, comparability, and evaluation. It is useful to identify and fix problematic areas with precision. The IHC technique includes the following steps:
- De-paraffinization of tissue sections taken on polylysine -coated slides (or else the aqueous solutions do not penetrate).
- Quenching of endogenous enzymes (which otherwise react with IHC reagents giving false-positive results). Alkaline phosphatases, peroxidases, and biotin are examples of these endogenous enzymes. This is usually done by 3% H 2 O 2 or with free avidin.
- Antigen retrieval as elaborated in the ensuing text.
- Blocking of nonspecific binding sites.
- Binding primary antibody.
- Binding with biotinylated secondary antibody.
- Detection methods using peroxidase-antiperoxidase methods, avidin-biotin conjugates, peroxidases complexes, or the more recently widely used polymer-labeling two-step method.
- Addition of chromogen substrate, usually DAB.
- Counterstaining, dehydrating, and cover-slipping the slide. 
Antigen retrieval (AR) methods are necessary because formalin fixation masks epitopes, and this procedure increases accessibility to the antigens.  The AR technique with its numerous modifications includes using the water bath, autoclaving, microwave heating, the pressure cooker treatment, etc. ,,, The high temperatures or, in the microwave, the molecular movement apparently contributes most directly to the reversal of formalin-based loss of antigenicity.  Of the several methods, pressure cooking and autoclaving are considered to be more effective than microwaving, due to the more uniform heating and higher temperatures achieved.  The use of nondeleterious aqueous media, retrieval fluids with an alkaline pH, and EDTA (greater capacity for calcium chelation compared to citrate and other types of salts) have also been found to be more effective.  However, heat-mediated antigen retrieval methods (HMAR) are not necessarily suitable for all antigens. To quote an expert, "HMAR is a powerful and versatile method and it is essential for recovering the antigenicity of nuclear antigens (such as Ki-67, ER and PR) and certain low-density surface antigens (such as CD4 on T cells and CD5 on B cells), in addition to its value in increasing or enhancing the antigenicity of a wide variety of surface antigens (including CD20, CD3 and CD8); but however, it is not valuable for high-density antigens such as over-expressed Her-2 in breast cancer."  All AR methods have their limitations, and excessive or reduced temperatures may produce negative results, stressing the importance of adhering to the procedures.  Thus antigen retrieval is indeed a complex task.  Each laboratory needs to carefully fine-tune this vital aspect of the IHC recipe.
Between rabbit polyclonal antibodies and mouse monoclonal antibodies, the former have the advantage of sensitivity but nonspecific staining is known; whereas the latter confer greater specificity but the immunoreactivity is sometimes weak. Monoclonal antibodies from rabbit hybridomas offer the promise of both high specificity and high sensitivity.  The ever-expanding list of monoclonal antibodies is available on websites.
The number of antibodies an IHC laboratory would want to invest in would depend on the type of pathology practice, the demands of the clinicians being serviced, and the cost considerations. At a very modest estimate, it would be advantageous to have the following antibodies in a routine surgical pathology IHC laboratory: (i) panel for carcinomas - CK, EMA, CEA; (ii) panel for differentiating metastatic carcinomas - CK7, CK20; (iii) panel for lymphomas - LCA, CD20, CD3, Bcl 2, CD30, CD5, CD23, Alk-1; (iv) panel for malignant round cell tumors [CD99 (MIC2), Desmin, Myoglobin] and for neuroendocrine tumors (NSE, Chromogranin, Synaptophysin); (v) panel for melanomas - S-100, HMB-45; (vi) panel for germ cell tumors - AFP, B-HCG; (vii) panel for CNS tumors - GFAP, MIB1, S-100; (viii) panel for mesotheliomas - HBME1, Calretinin; (ix) panel for IHC tests related to therapy and prognosis - ER, PR, Cerb-2 in breast carcinoma, C-kit in case of spindle cell tumors suspected to be gastrointestinal stromal tumor (GIST), and CD20 in case of B-cell non-hodgkin lymphoma (NHL).
(c) Post-analytical evaluation is the crucial component of the IHC test. Interpreting immunostains as merely positive or negative without appreciating the staining pattern is inappropriate.  The IHC reactions need to be interpreted not only in the cells or the structure which is being sought, but the positive cells should also reveal the correct pattern of staining (membranous, cytoplasmic, nuclear; diffuse, dot-like or perinuclear). The number of cells showing immunoreactivity (percentage) and the intensity of staining make up the remaining variables. All these features have a bearing on the final interpretation. To cite a few examples, the CD20, CD99 (MIC2), EMA, and C-erB-2 (HER-2/neu) stains are membrane stains [Figure - 1]. The hormone receptors ER, PR, and nuclear proliferation markers such as Mib1 are nuclear stains. Sometimes the staining pattern of a single stain could be different in different diagnostic contexts. For example, CD3, which is a T cell marker, reveals cytoplasmic staining of precursor T-cell neoplasms; whereas the peripheral T-cell neoplasms show membranous staining. 
Unexpected immunoreactivity should be viewed critically and interpreted with caution. A false-positive result due to endogenous biotin present in both normal and neoplastic tissues is known. The false-positive staining appears as a dull brown granular or fluffy staining pattern. Meticulous examination of tissue-specific negative controls has been recommended to avoid an erroneous interpretation.  A negative IHC result in general has less value, and lack of immmunoreactivity per se is not sufficient evidence, either to support or to negate a good hematoxyline and eosine (H and E) diagnosis.
Immunohistochemistry results dubbed as "nonspecific reactivity," cross reactivity, or aberrant expression of antibodies are well recognized. The original dogmatic belief implying lineage specificity to antibodies is no longer valid. A highly cited paper in histopathology documenting expression of cytokeratin in over 50% cases of normal, benign, and malignant smooth muscle tissues challenged the concept that cytokeratin expression was a completely reliable marker of epithelial differentiation.  The practical implications of such aberrant or unexpected IHC reactions in diagnostic surgical pathology are significant.  Over the years, increasing evidence of such expression in diverse histological entities has accumulated. To cite an example, CD30 is positive in anaplastic large cell lymphoma (ALCL) and Hodgkin's disease; it is also positive in embryonal carcinoma, seminomas, mesenchymal tumors, decidual cells, lipoblasts, myoepithelial cells, reactive and neoplastic vascular lesions, mesotheliomas, and macrophages.
The ultimate step of IHC involves integration of the IHC result in the correct clinical setting. In the absence of an expected IHC result, there is a tendency to fault the technique, or the potency of the antibody. Assuming that proper pre-analytical and analytical factors have been adhered to, the final label in case of a diagnostic dilemma is not merely at the mercy of the IHC result; rather, it is a function of a well-knit integration of all available clinical, routine H and E, and the IHC observations. An inter-laboratory trial involving 172 pathologists and 3,526 immunostains has aptly highlighted this. The multivariate model of this study, wherein each step of the diagnostic pathway was introduced, revealed that the only factors independently predictive of the correct final diagnosis were the correct tentative diagnosis, the interpretation of the IHC stains, and the conclusions drawn from the IHC stain. Neither antibody selection nor quality of immunostain correlated independently to the correct final diagnosis. Thus a definitive diagnosis could not be rendered if the morphological tentative diagnosis was incorrect or not included in the differential diagnosis. 
Application of IHC in routine settings: In a routine surgical pathology service, the maximum utility of IHC is in distinguishing carcinoma from lymphoma [Figure - 2] and melanoma [Figure - 3] and also in the work-up of hematolymphoid neoplasms [Figure - 4],[Figure - 5]. The following text comprises some comments as alluded in standard textbooks and publications on the frequently used IHC antibodies in hematolymphoid neoplasms. , CD20 is a B-cell marker which shows membrane staining, CD3 is a T-cell marker, CD21/CD35 stain follicular dendritic cells and are useful to demonstrate germinal centers within lymphoid proliferations; CD5 is used to detect B-cell CLL/SLL and mantle cell lymphomas. CD23 is a marker for B-CLL/SLL, whereas mantle cell lymphoma is negative, the later shows immunoreactivity to Cyclin D1, a nuclear protein. Hence CD23 is a useful marker in differentiating CD5-positive small lymphoid disorders. A useful antibody is Alk-1, which is specific for anaplastic large cell lymphomas (ALCL). ALK-positive ALCL tumors are usually CD30 positive [Figure - 4] and focally EMA positive. Bcl-2 is particularly useful to distinguish reactive follicles (negative) from follicles of follicular NHL (positive), and it is also positive in several other NHLs.  CD10 is positive in Burkitts lymphoma. CD15 and CD30 are positive in Hodgkin's disease [Figure - 5]. CD45 is found on all leukocytes, and it is useful in identifying most lymphomas. CD99 (MIC2) is positive in lymphoblastic leukemia-lymphoma. Terminal deoxynucleotidyl transferase (Tdt) is a sensitive marker for lymphoblastic lymphoma/leukemia of both B and T cell lineage.  Immunohistochemical staining has also become an integral part of the diagnostic work-up of bone marrow trephine biopsies, and procedural modifications may be necessary due to the alternative fixatives and decalcification procedures used for bone marrow biopsies. 
The second major workload of an IHC laboratory comprises breast tumors. The diagnostic dilemmas posed by papillary and intraductal proliferations, low-grade carcinomas, radial scars, sclerosing adenosis, tubular carcinomas, and lobular carcinomas have been addressed utilizing various IHC markers to demonstrate myoepithelial cells (actin, smooth muscle myosin heavy chain, p63, calponin) and by the loss of E-cadherin in lobular carcinoma as opposed to ductal carcinoma. ,, The assessment of ER/PR and Cerb B2/HER-2 status and its incorporation in the report of a breast cancer case has now become a standard practice. Immunoscoring is based on a combination of the intensity of staining (nil, weak, intermediate, or strong) and the percentage of positive cells to quantify the ER-PR results.  A maximum score would indicate intense staining of all the cells [Figure - 6]. Different scoring systems such as H-score, the Quick score, and the Allred score have been employed. ,, Interpretative nonconcordance of ER-PR immunostains as a result of diverse reagents, diverse detection systems, and diverse scoring systems has been a much-debated issue. Of late, the importance of scoring has been undermined. The statement by the National Institutes of Health recommends any nuclear ER immunostaining (weak and in 1% to 10% of cells) as enough reason for initiation of anti-estrogen treatment. As regards estimation of Cerb B2 (HER-2/neu), two IHC methods are in use: the routine IHC HER-2/neu (Cerb-2) or the patented and FDA-approved "Hercep test." The latter is much more expensive. The scoring of Cerb-2 (HER-2/neu) is based on membrane immunostaining of invasive carcinoma cells. A score of 3+ shows strong, complete membrane staining in more than 10% of tumor cells [Figure - 7]. Weak-to-moderate but complete membrane staining in more than 10% of cells is scored 2+. Membrane staining seen only in part of the cell is scored as 1+. 
The diagnosis of malignant round cell tumors often necessitates IHC and forms the third most frequent request category after lymphomas and breast tumors. Antibodies to CD99/MIC 2, neuron-specific enolase, synaptophysin, S-100 protein, muscle-specific actin (MSA), Desmin, and the myoregulatory proteins MyoD1, and myogenin each have a role. CD99 reveals almost 95% sensitivity for a diagnosis of Ewing's sarcoma/PNET [Figure - 8].  But both CD99 and NSE lack specificity and have been demonstrated not only in ES/PNET but also in DSRCT and rhabdomyosarcoma. Synaptophysin is relatively a more specific marker for neuroendocrine differentiation.  Thus no single exclusive marker exists.
In certain situations such as metastatic carcinoma from unknown origin, the intelligent use of a minimum optimal panel is of immense value.  This has been highlighted by the judicious combination of CK7 and CK20 antibodies, which are expressed differently in different epithelia.  An undetected adenocarcinoma of the lung metastatic to a supraclavicular lymph node will be CK7 positive/CK20 negative; further application of more specific markers such as thyroid transcription factor and surfactant protein B helps to confirm metastasis from a lung carcinoma [Figure - 9].  The following CK7/CK20 immunoreactivity pattern is helpful: (i) CK7+/CK20+ - transitional cell carcinoma, pancreatic carcinoma, cholangiocarcinoma, gastric carcinoma, ovarian mucinous carcinoma; (ii) CK7+/CK20 - nonsmall cell carcinoma and adenocarcinoma lung, duct and lobular carcinoma of breast, nonmucinous ovarian carcinoma, endometrial adenocarcinoma, mesothelioma; (iii) CK7−/CK20+ - colorectal adenocarcinoma, merkel cell carcinoma; iv) CK7−/CK20− - small cell carcinoma and squamous carcinoma lung, prostate adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, adrenocortical carcinoma. 
Soft tissue neoplasms often have overlapping morphology. A broad practical approach to investigate the four common diagnostic settings, namely, round cell tumors, monomorphic spindle cell tumors, the epithelioid soft tissue tumors, and the pleomorphic spindle cell tumors, is useful.  The instability of soft tissue tumor cells with respect to differentiation has been highlighted by IHC, and no single stain is pathognomonic for any given condition, emphasizing thereby that routine H and E stain should still be regarded as the gold standard.  Typically, synovial sarcomas show, in addition to vimentin reactivity, conspicuous positivity to epithelial markers and also to Bcl 2 [Figure - 10]. Tumors of adipocytes, cartilage, and bone are best diagnosed by light microscopy. In the skeleton, IHC is useful in the differential diagnosis of primary from metastatic nonosseous tumors [Figure - 11] and in the categorization of small-round-blue-cell tumors.  Surgical pathologists toil to distinguish between mesothelioma and its mimics, but this exercise has been aptly summed up as "new tools, same dilemmas."  Immunohistochemistry has been used extensively to tell apart epithelioid mesotheliomas and carcinoma; however, no single antibody is able to differentiate reliably between these two tumors. , In case of central nervous system neoplasms, antibodies to glial fibrillary acidic protein (GFAP) - Synaptophysin, NSE, and S-100 protein - are valuable.  Meningiomas are EMA positive. Germ cell tumors show positivity for HCG and PLAP.  Pituitary adenomas need a combined morphologic and immunohistochemical diagnostic approach.  For the diagnosis of choroid plexus tumors, immmunoreactivity pattern of CK7, CK20, CK, VIM, and S100 protein is useful. , The ovary is a common site of metastatic tumor, and many metastatic adenocarcinomas mimic primary ovarian adenocarcinomas. The differential cytokeratin staining has been widely used as an aid to distinguish between a primary and secondary ovarian adenocarcinoma.  The neuroendocrine neoplasms include one group with epithelial lineage which is CK positive and a second group which shows neuroectodermal differentiation and is positive for neurofilament protein.  Testicular and paratesticular neoplasms can be investigated with placental-like alkaline phosphatase and alpha-fetoprotein, but newer markers such as OCT4 show almost 100% sensitivity and specificity for seminoma, embryonal carcinoma, and intratubular germ cell neoplasia, unclassified type.  In case of prostatic adenocarcinoma, P63 and high-molecular weight cytokeratin highlight the basal cells found in benign glands, and negative staining is most suspicious for adenocarcinoma. A recently recognized cytoplasmic protein AMACR has been found to be significantly up-regulated in (80-100%) prostate cancer. Thus negative staining for basal cell markers and positive staining for AMACR can help to recognize carcinomas. 
Application of IHC in prognostic and predictive settings: The prognostic markers include those that allow assessment of microscopic invasion of basal lamina, micro-metastasis to sentinel/regional nodes and bone marrow, hormone receptors (ER/PR), angiogenesis, a number of tumor-associated genes including p53, growth factor receptors and anti-metastasis genes and markers that predict response to therapy such as p-glycoprotein and c-erbB-2.  The breach in basement membrane by epithelial neoplasms constitutes invasion; and the chances of local invasion, destruction, and access to the lymphovascular channels increase, allowing for metastases. Antibodies to the basement membranes (type IV collagen and laminin) are used to study invasion.  Similarly endothelial markers (CD31 and CD34, Factor VIII-related protein, and Ulex europeaus lectin) assist in identification of lymphovascular spaces to ascertain tumor embolization within, particularly in cases showing retraction artifact of an invasive tumor. Micro-metastasis in lymph nodes and bone marrow can be recognized with immunomarkers, and IHC could correctly identify occult metastases in as many as 23% nodes previously declared node negative on routine examination. , The predictive markers have the ability to predict a definite response to a particular intervention and are therefore considered to be distinct from prognostic markers. The former are few. , Of these, hormone receptors (ER, PR) and C-erbB-2 in breast malignancies are currently the only predictive markers of note that are in routine clinical practice.
| Recent Advances and Future Directions|| |
Several recent developments emphasize the increasingly important role immunohistochemistry will play in the coming years. These include genogenic immunohistochemistry for diagnosis, search for proteins for targeted therapy, methods to develop better monoclonal antibodies with recombinant technology, "technician-free" automation of the IHC procedures, and "pathologist-free" microscopic image analysis technology for interpretation of high-throughput results.
"Genogenic immunohistochemistry" heralds a new era in immunohistochemistry, and identification of the underlying molecular changes by immunohistochemistry is being used both for diagnosis and therapy.  Markers to monitor drug resistance include P-glycoprotein, the product of the mdr gene (multidrug resistant); N-myc and tumor suppressor genes such as p 53; retinoblastoma susceptibility suppressor gene; putative suppressor genes - BRCA-1 gene, DNA repair genes (microsatellite instability) are all examples of genogenic IHC.. The genetic mutations such as loss of E-cadherin protein in lobular carcinoma of the breast, ALK over-expression to recognize the t(2;5) translocation in anaplastic large cell lymphoma, FLI-1 over-expression for the t(11;22) translocation of PNET/ES; WT-1 over-expression for the t(11;22) translocation of DSRCT are the newer examples of genogenic IHC markers. 
For targeted therapy, trastuzumab, a monoclonal antibody to HER-2 NEU gene (Cerb2) (breast cancer); rituximab, an anti-CD20 monoclonal antibody (B-cell NHL); and imatinib against C-kit positive tumors (GIST) have demonstrated success ,, [Figure - 12]. Research and trials are in vogue for monoclonal antibodies against several growth factor-related receptors such as vascular endothelial growth factor (VEGF), platelet-derived growth factors (PDGFR-B), and epidermal growth factors (EGFR) for the treatment of cancers of breast, colon, lung; and renal cancers.
Newer technology for the development of more specific antibodies from recombinant antibody fragments has paved the way for molecules with ultra-high affinity, high stability, and increased potency, which is almost unattainable by the traditional immunization methods.  Automation in IHC has been advocated for carrying out the procedures for consistency in performance.  Methods using automated computerized image capture and analysis systems as opposed to the traditional subjective observations of IHC stains are being introduced.  The emergence of tissue microarrays (TMA) as a high-throughput technique for examining hundreds of marker molecules in histological microarray sections comprising between 100 and 1,000 core tissues on a single glass slide enables economical evaluation in terms of sample utilization and reagent costs. ,, In future, TMA will be an increasingly sought-after tool for evaluating the expression of proteins by IHC and thus validating the findings of DNA microarrays. This technique holds the promise of better understanding of the genetically heterogeneous groups of diseases, such as lymphomas, which have shown different response to treatment despite identical international prognostic index (IPI). , The results obtained by these high-throughput methods can be analyzed by automated image analyzers. Finally it is of interest that IHC has a newfound role to detect agents of bioterrorism, and it is also of value in the field of veterinary pathology. ,
| Quality Assurance in Immunohistochemistry|| |
Standardization in IHC is the need of the day.  The steps towards quality assurance include ensuring technical reproducibility, uniformity in interpretation, quantification of extent of immunoreactions by the use of a scoring system, and participation in an external quality assessment program.
Standardization of monoclonal antibody dilutions and validation of the control paraffin blocks are basic requirements of the IHC laboratory. Multi-tissue blocks and tissue microarray blocks have been advocated for use as controls.  It is necessary to put up positive and negative controls with each run for every case on a daily basis. Negative control usually involves replacing the primary antibody with the normal serum or unreactive antibody and not merely subtracting the specific antibody. 
All validation records elaborating the protocol, the clone of the antibody tested, the antigen retrieval method employed, the identity of the control blocks tested, the optimal dilution, and also record of details of repeat requests need to be maintained. A regular audit of all repeat IHC requests helps identify the problem areas. Such records form part of the guidelines for quality control and also for accreditation.
Monoclonal antibodies are expensive, and most laboratories retain the infrequently requested antibodies beyond their expiry dates. It is being debated whether or not there is enough evidence to prove that such expired antibodies are unsuitable for use. Such antibodies have been found to be useful beyond their shelf life.  According to Chan, there is no need to discard these antibodies if the immunoreactivity on the controls is good. 
Providing quality IHC services is often handicapped by several pre-analytical and analytical procedures. Repeat performance of IHC tests at different laboratories or within the same laboratory at different times may yield results at variance with the original results. Such discordance may have a major impact on the diagnosis (as in lymphoma versus carcinoma) or therapeutic management (Cerb2 or C-kit positive versus negative tumors). A recent audit at the Fox Chase Cancer Center aimed at assessing the utility of re-performing and/or performing IHC on referred cases which had a previous diagnosis of cancer, revealed that IHC on these referrals had led to a change in diagnosis in 18.3% of cases, with an overall nonconcordance to the extent of 21.2%.  The antibody which has been subjected to maximum scrutiny is HER-2/neu over-expression. The reliability of the HER-2 assay could be greatly improved by stringent quality control, and an ongoing quality assurance program utilizes a standard reference obtained from cell lines. ,, A large study, wherein 172 laboratories participated, evaluating inter-laboratory and inter-observer agreement for semi-quantitative assessment of estrogen receptor (ER) using tissue array technology suggested that neither pH of the formalin buffer nor the duration in the fixative greatly influenced the detection of ER. Variability in subsequent IHC practices (such as antigen retrieval) and interpretation of results were a greater source of diagnostic error. 
The UK National External Quality Assessment Service organizes an EQA program specifically for IHC-based work (http://www.ukneqas.org.uk). The scheme offers different modules for general pathology, breast pathology, neuropathology, lymphoma, and nongynec cytology. Participants demonstrate appropriate diagnostic antigens on the distributed slides, as well as on "in-house" sections, and receive result sheets and yearly "performance record."
The US-based Association of Directors of Anatomic and Surgical Pathology (www.adasp.org) recommends that a quality assurance and improvement plan (QA and I) should have a departmental committee to oversee its implementation, utilizing inbuilt QA and I monitors, to audit the processes involved in generation of the final result.  Certification and proficiency-testing programs and a checklist for laboratories going in for accreditation can be read at the website of College of American Pathologists ( http://www.cap.org/apps/docs/.....anatomic_pathology_december2006_changes.doc ). Participants receive unstained paraffin sections with methodological instructions and a detailed critique of the results at the end. 
| IHC Information Sources|| |
Referral to updated and comprehensive resources which give information of the possible range of reactivity is necessary, and some of the helpful links are listed below:
http://immunoquery.com, http://immunohypermart.net, http://www.ncbi.nlm.nih.gov/prow, http://www.e-immunohistochemistry.info/'vade mecum'. The vade mecum website provides a regularly updated comprehensive knowledge bank on IHC.  Two other useful sites are WWW.DAKO.COM for the newer antibodies and http://www.mh-hannover.de/aktuelles/projekte/hlda7/hldabase/select.htm for the leukocyte antibodies. The journals focused on IHC techniques include Immunocytochemistry, Journal of Histochemistry, Cytochemistry, Applied Immunohistochemistry , in addition to the standard journals in diagnostic histopathology. The Society for Applied Immunohistochemistry ( www.appliedimmuno.org ), the Histochemical Society ( http://www.histochem.org/ ) and the internet group's sci.bio.immunocytochem and http://www.mailman.srv.ualberta.ca/mailman/listinfo/patho are further sources of information. The websites for IHC External Quality Assurance include http://www.ukneqas.org.uk and http://www.cap.org/apps/....tomic_pathology_december2006_changes.doc
| Conclusions|| |
Immunohistochemistry is an essential extension of surgical pathology practice. Even in our resource-crunched setting, IHC has become necessary for the work-up of lymphomas, for the detection of ER-PR and HER-2/neu status, for diagnosing MRCT, and in case of metastasis from an unknown site. Immunohistochemistry, however, is not a surrogate for routine histopathology diagnostic skill; a good tentative diagnosis is a prerequisite for the success of IHC. There will always be a limitation to the number of IHC antibodies one can possibly do. Health care administrators trained in evidence-based medicine are likely to question the utility and the cost-benefit ratio of expensive tests.  Developing diagnostic algorithms based on available "best evidence" will help decide the issue of how many immunostains and/or molecular methods should be performed in a given case.  As opposed to the past decade, many laboratories in the country are now offering IHC services. Nonconcordance of IHC results from different laboratories, from the same laboratory at different times or by different reporting pathologists causes confusion. A system to obtain reproducible results, no matter where and when the tests are done, is a pressing need. In future the practice of IHC will witness more automation, shorter turn-around time, better visualization methods, and hopefully better concordance of IHC results.
| Acknowledgments|| |
The authors are sincerely grateful to all departmental colleagues, comprising consultant staff members, scientific and technical personnel, present and past, whose individual contributions over the past two decades have added immense value to the functioning of the diagnostic IHC services of the Institute. The authors regret their inability to thank everyone individually. We also acknowledge accessing information from the several internet sites as alluded in the text.
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Nirmala Ajit Jambhekar
Department of Pathology, Tata Memorial Hospital, E. Borges Marg, Parel, Mumbai - 400 012, Maharashtra
Source of Support: None, Conflict of Interest: None
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7], [Figure - 8], [Figure - 9], [Figure - 10], [Figure - 11], [Figure - 12]
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