The Contribution of Bifunctional SkipDewax Pretreatment Solution, Rabbit Monoclonal Antibodies, and Polymer Detection Systems in Immunohistochemistry

2007 ◽  
Vol 131 (7) ◽  
pp. 1047-1055
Author(s):  
Sze Chuen Cesar Wong ◽  
John K. C. Chan ◽  
Elena S. F. Lo ◽  
Amanda K. C. Chan ◽  
Manson C. K. Wong ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Colorectal Carcinoma ◽  
Diagnostic Accuracy ◽  
Poor Quality ◽  
Antigen Retrieval ◽  
Ki 67 ◽  
Detection Systems ◽  
Edta Solution ◽  
Endogenous Biotin ◽  
Pretreatment Solution

Abstract Context.—In immunohistochemistry, nonstandardized antigen retrieval protocols and fluids, poor-quality antibodies, and the presence of endogenous biotin frequently lead to incorrect results. Recently, advanced reagents including bifunctional SkipDewax pretreatment solution (BSPS), rabbit monoclonal (RM) antibodies, and biotin-free polymer detection systems (PDSs) have been developed, which, it is claimed, resolve these problems. Objectives.—To determine whether BSPS, RM antibodies, and biotin-free PDSs improve the accuracy of immunohistochemistry; to optimize a new protocol consisting of a combination of BSPS, RM antibodies, and PDSs; and to compare it with a conventional protocol. Design.—The efficacies of BSPS, RM antibodies, and PDSs were compared with those of their respective conventional reagents using multitissue spring-roll sections. The new protocol was compared with a conventional protocol using Ki-67 immunostaining of 49 colorectal carcinoma specimens. Results.—For antigen retrieval, BSPS resulted in similar or better tissue staining than an EDTA solution, but the efficacy of BSPS decreased when it was reused. Most RM antibodies resulted in a greater proportion of positive cells than the corresponding non-RM antibodies, which did not produce satisfactory results in the absence of antigen retrieval. The PDSs Bond, ChemMate, and SuperPicture resulted in a high percentage of positive cells, good staining intensities, and low backgrounds. Other PDSs, except that from Ventana, resulted in high backgrounds and false positivity. The new combined protocol resulted in better Ki-67 staining than the conventional assay. Conclusions.—Bifunctional SkipDewax pretreatment solution, RM antibodies, and PDSs improve staining quality and diagnostic accuracy of immunohistochemistry assays and provide a foundation for standardization.

Expression of C-kit in Ewing Family of Tumors: A Comparison of Different Immunohistochemical Protocols

2004 ◽  
Vol 7 (4) ◽  
pp. 342-347 ◽  
Author(s):  
Atif Ahmed ◽  
Enid Gilbert-Barness ◽  
Atilano Lacson
Keyword(s):  
Monoclonal Antibodies ◽  
Ewing Sarcoma ◽  
Polyclonal Antibodies ◽  
Growth Factor Receptor ◽  
Myelogenous Leukemia ◽  
Antigen Retrieval ◽  
Tyrosine Kinase Receptor ◽  
Ki 67 ◽  
Blue Cell ◽  
Round Blue Cell Tumor

Ewing sarcoma is a small round blue cell tumor with a high incidence of metastasis and poor survival. The tyrosine kinase receptor, c-kit, is a growth factor receptor that is expressed in a variety of tumors including Ewing sarcoma. Blockade of c-kit by imatinib mesylate (Gleevec; Novartis Pharmaceuticals Corp, East Hanover, NJ) has been successfully used in the treatment of chronic myelogenous leukemia and gastrointestinal tumors. Detection of c-kit expression in Ewing sarcoma indicates a possible role of c-kit in tumor progression and a potential use of anti-c-kit therapy in Ewing sarcoma. Ki-67 is a proliferation marker found at all stages of the cell cycle. Expression of c-kit and Ki-67 was studied in 17 patients with Ewing sarcoma. Sections from paraffin-embedded tumor samples were immunostained, using standard immunohistochemical protocols, with c-kit and Ki-67 monoclonal antibodies, polyclonal c-kit antibody without antigen retrieval, and c-kit polyclonal antibody with antigen retrieval. Eleven out of 17 cases (65%) stained with c-kit monoclonal antibody; the staining was diffuse in 6/17 (35%) cases. C-kit expression did not correlate with Ki-67 proliferation rates. Using the polyclonal c-kit-antibody without antigen retrieval methods, c-kit expression was demonstrated in 1/11 (9%) cases. Incorporating antigen retrieval methods, c-kit expression increased to 53%. Concordance between monoclonal antibodies in detecting c-kit expression was observed in 12/17 cases (71%). We conclude that c-kit is variably expressed in Ewing sarcoma, using either monoclonal or polyclonal antibodies. Detection of c-kit expression in Ewing sarcoma improves with the use of antigen retrieval methods.

Technical pitfalls potentially affecting diagnoses in immunohistochemistry

2008 ◽  
Vol 61 (11) ◽  
pp. 1184-1192 ◽  
Author(s):  
G Bussolati ◽  
E Leonardo
Keyword(s):  
High Frequency ◽  
Cross Validation ◽  
Antigen Retrieval ◽  
Diagnostic Process ◽  
Extensive Review ◽  
Related Literature ◽  
Detection Systems ◽  
Incorrect Diagnosis ◽  
Proper Interpretation ◽  
Endogenous Biotin

Result of the immunohistochemical reactions routinely used in diagnostic surgical pathology should be properly interpreted, since false results, related to technical and interpretative pitfalls may lead to incorrect diagnosis. The main sources of such pitfalls are reviewed, analytically described and related to different steps (fixation, tissue processing and embedding, decalcification, antigen retrieval) which may affect the accuracy of immunohistochemistry. In addition, the presence of endogenous enzyme activity, improper binding of avidin to endogenous biotin, incorrect use of antibodies, chromogen and detection systems, as well as incorrect interpretation may produce unreliable data. The high frequency and extension of such pitfalls make mandatory the use of internal and external controls and adoption of cross-validation programmes. The present study, supported by an extensive review of the related literature, is intended as a guideline leading to proper interpretation of immunohistochemical data, an essential component of the diagnostic process. Experience on the antigen retrieval procedures for different antigens is also presented.

Strategic Aspects of NPY-Based Monoclonal Antibodies for Diagnosis and Treatment of Breast Cancer

2020 ◽  
Vol 21 (11) ◽  
pp. 1097-1102
Author(s):  
Drashti Desai ◽  
Pravin Shende
Keyword(s):  
Breast Cancer ◽  
Monoclonal Antibodies ◽  
Diagnosis And Treatment ◽  
Detection Methods ◽  
Active Immunotherapy ◽  
Immune Checkpoints ◽  
Proliferation Index ◽  
Protein Fusion ◽  
Ki 67 ◽  
Cost Efficient

: Immunotherapy emerges as a treatment strategy for breast cancer marker, diagnosis and treatment. In this review, monoclonal antibodies (mAbs)-based passive and peptide vaccines as active immunotherapy approaches like activation of B-cells and T-cells are studied. Passive immunotherapy is mAbs-based therapy effective against tumor cells, which acts by targeting HER2, IGF 1R, VEGF, BCSC and immune checkpoints. Neuropeptide Y (NPY) and GPCR are the areas of interest to target BC metastases for on-targeting therapeutic action. Neuropeptide S (NPS) or NPS receptor 1, acts as a biomarker for Neuroendocrine tumors (NET), mostly characterized by synaptophysin and chromogranin-A expression or Ki-67 proliferation index. The protein fusion technologies arise as a promising avenue in plant expression systems for increased recombinant Ab accumulation and cost-efficient purification. Recently, mAbs-based immunotherapy effectiveness is appreciated as a novel therapeutic combination of chemotherapy and immunotherapy to reduce the side effects and improve therapeutic responsiveness. Synthetic drug resistance will be overcome by mAbs-based therapy through several clinical trials and detection methods need to be optimized for accuracy and precision. Pharmacokinetic attributes need to be accessed for preferred receptor-agonist activity without ligand accumulation.

scholarly journals Role of p16/INK4a and Ki-67 as specific biomarkers for cervical intraepithelial neoplasia: An institutional study

2017 ◽  
Vol 9 (02) ◽  
pp. 104-110 ◽  
Author(s):  
Ankitha Hebbar ◽  
Venkataramappa Srinivasa Murthy
Keyword(s):  
Diagnostic Accuracy ◽  
Cervical Intraepithelial Neoplasia ◽  
Squamous Metaplasia ◽  
Intraepithelial Neoplasia ◽  
P Value ◽  
Kappa Statistics ◽  
Histopathological Diagnosis ◽  
Ki 67 ◽  
Cervical Biopsy ◽  
P16 Ink4a

Abstract BACKGROUND: P16/INK4a and Ki-67 have emerged as important biomarkers for the detection of high-risk human papilloma virus (HR-HPV) associated dysplastic changes in the cervical biopsy samples. The increasing inter- and intra-observer variability in the diagnosis of dysplastic lesions and immature squamous metaplasia on histopathology has led to the advent of these biomarkers. This study was taken up with an aim to study their role in increasing the diagnostic accuracy in equivocal cases on histopathology. MATERIALS AND METHODS: Fifty cervical biopsy specimens were stained with p16/INK4a and Ki-67 consisting of 10 cases each of cervical intraepithelial neoplasia (CIN I/II/III) along with five cases of squamous metaplasia. Histopathological diagnosis was considered as the gold standard. Statistical analysis was done by kappa statistics, and P value was calculated. RESULTS: The sensitivity and specificity of p16/INK4a and Ki-67 were 76.2%, 87.5%, 90.5%, and 87.5%, respectively. The overall agreement of both the immunostains with histopathological diagnosis was statistically significant (P < 0.05) and the diagnostic accuracy improved when both the stains were used in conjunction. CONCLUSION: Ki-67 and p16/INK4a can be used as complimentary tests in differentiating dysplastic and nondysplastic lesions and help in confirming the histopathological diagnosis. They aid in recognition of dysplasias caused by HR-HPV, which have higher tendency to progress to neoplasia. However, further research is advocated before the widespread use of these markers for screening of dysplasias.

Clinical significance of Ki-67 proliferation index in disease progression and prognosis of patients with resected colorectal carcinoma

2005 ◽  
Vol 92 (8) ◽  
pp. 1002-1007 ◽  
Author(s):  
V. Valera ◽  
N. Yokoyama ◽  
B. Walter ◽  
H. Okamoto ◽  
T. Suda ◽  
...  
Keyword(s):  
Colorectal Carcinoma ◽  
Disease Progression ◽  
Clinical Significance ◽  
Proliferation Index ◽  
Ki 67

scholarly journals Antigen Masking During Fixation and Embedding, Dissected

2016 ◽  
Vol 65 (1) ◽  
pp. 5-20 ◽  
Author(s):  
Carla Rossana Scalia ◽  
Giovanna Boi ◽  
Maddalena Maria Bolognesi ◽  
Lorella Riva ◽  
Marco Manzoni ◽  
...  
Keyword(s):  
Room Temperature ◽  
Detection System ◽  
Antigen Retrieval ◽  
Fixation Time ◽  
Water Removal ◽  
Immunohistochemical Detection ◽  
Frozen Sections ◽  
Ki 67 ◽  
Paraffin Embedding ◽  
Multiple Factors

Antigen masking in routinely processed tissue is a poorly understood process caused by multiple factors. We sought to dissect the effect on antigenicity of each step of processing by using frozen sections as proxies of the whole tissue. An equivalent extent of antigen masking occurs across variable fixation times at room temperature. Most antigens benefit from longer fixation times (>24 hr) for optimal detection after antigen retrieval (AR; for example, Ki-67, bcl-2, ER). The transfer to a graded alcohol series results in an enhanced staining effect, reproduced by treating the sections with detergents, possibly because of a better access of the polymeric immunohistochemical detection system to tissue structures. A second round of masking occurs upon entering the clearing agent, mostly at the paraffin embedding step. This may depend on the non-freezable water removal. AR fully reverses the masking due both to the fixation time and the paraffin embedding. AR itself destroys some epitopes which do not survive routine processing. Processed frozen sections are a tool to investigate fixation and processing requirements for antigens in routine specimens.

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