Wet Autoclave Pretreatment Research Articles

Cytokeratin 5/6 immunohistochemistry assists the differential diagnosis of atypical proliferations of the breast.

This study was performed to determine the diagnostic value of keratin 5/6 (CK 5/6) immunophenotyping on routinely processed breast tissues. Six hundred and ninety-nine breast lesions, including normal tissues as well as benign and malignant lesions in 321 formalin-fixed, paraffin-embedded samples from 158 different patients were investigated immunohistochemically, following wet autoclave pre-treatment for antigen retrieval. In normal breast tissues, both myoepithelial and luminal epithelial cells expressed CK 5/6 in varying amounts. While myoepithelial immunoreactivity was most pronounced in the duct system, luminal epithelial immunoreactivity was strongest in the terminal duct lobular units. In ductal hyperplasias (DH), luminal epithelial cells predominantly revealed CK 5/6 immunoreaction. In contrast, neoplastic epithelial cells in atypical ductal and lobular hyperplasias (ADH and ALH) lacked such an expression, whereas in ductal in-situ carcinomas (DCIS) and in infiltrating ductal carcinomas 3.7% and 7.7%, of the cases respectively, showed positive immunostaining for CK 5/6. Immunophenotyping of keratin 5/6 expression can be helpful in the diagnosis of atypical hyperplasias and in-situ carcinomas of the breast. It is particularly valuable in the differential diagnosis between benign and atypical proliferative lesions.

Gains and losses of CD44 expression during breast carcinogenesis and tumour progression.

This study was performed to investigate whether the CD44 immunophenotype of breast lesions correlates with the clinical evolution and prognosis of breast cancer. One-hundred and fifty-two routinely processed normal, benign and malignant breast tissue samples were investigated by the following monoclonal antibodies: CD44s (F10-44-2), CD44v3 (3G5), CD44v4 (11.10), CD44v5 (VFF-8), CD44v6 (VFF-18), CD44v7 (VFF-9), CD44v9 (11-24) after wet autoclave pretreatment for antigen retrieval. We found that: (1) in normal breast tissues luminal epithelial cells lacked detectable CD44 in contrast to basal cells, which constitutionally expressed CD44s, v3, v5 v6 and v9 isoforms; (2) in the intraductal compartment of benign hyperplastic lesions, there was scattered or focal staining for CD44s, v5, v6, v7 and v9 isoforms; (3) in neoplastic lesions restricted neo-expression of CD44v3 and v4 was detected; and (4) the CD44 immunophenotype of invasive breast carcinomas was influenced largely by differentiation grade, steroid receptor status of the tumours and significantly correlated with metastatic involvement of the axillary lymph nodes. Qualitative and quantitative changes of CD44 expression are implicated in early stages of breast carcinogenesis. The restricted neo-expression of certain CD44 isoforms in breast neoplasias suggests that CD44 might be a potential target for future antibody-based tumour therapy.

Demonstration of silver-stained nucleolar organizer region associated proteins (AgNORs) after wet autoclave pretreatment in breast carcinoma: correlation to tumor stage and long-term survival.

Argyrophilic nucleolar organizer region associated proteins (AgNORs) are known to reflect cellular and nucleolar activity. Due to a novel staining procedure, which substantially improves visualisation of AgNORs on formalin-fixed and paraffin-embedded material, AgNORs can be reliably demonstrated as true substructures of the nucleoli. The aim of the present study was to apply a standardized morphometric AgNOR quantification on a large series of breast carcinomas with regard to its prognostic relevance. AgNOR quantity was evaluated on archival tumor tissues of 115 adenocarcinomas of the breast treated with the wet autoclave method prior to standardized silver-staining and morphometric analysis. AgNOR parameters were correlated to prognostic features (steroid hormonal receptor status, tumor type, tumor size, histological grading, pTNM, and UICC stage) carrying out both univariate and multivariate survival analyses. AgNOR number and area were proven to be statistically significantly related (Pearson correlation coefficient: 0.67, Bonferroni adjusted P = 0.0001). Almost all AgNOR parameters, in particular CV (coefficient of variation) of corrected area (delta-area) and CV of number, were statistically significantly correlated to estrogen and progesterone receptor status as well as histological grading of tumors. Increased AgNOR parameters were statistically significantly associated with early tumor relapse and cancer related death. Univariate and multivariate analysis by means of Cox regression revealed independent prognostic significance for CV of delta-area and number of AgNORs. Various AgNOR parameters (CV of number, CV of delta-area, CV of area, mean delta-area, and mean area of AgNORs per nucleus) determined on wet autoclave pre-treated formalin-fixed and paraffin-embedded breast cancer tissues are statistically highly significantly associated with the prognostic outcome, independently predicting tumor-free and overall survival.

Significance of Wet Autoclave Pretreatment in Immunohistochemistry.

Until recently the only way to rescue masked epitopes in routinely processed surgical pathological material was enzymatic digestion. The use of heat for antigen retrieval, first by microwave irradiation, represents an important breakthrough in immunohistochemistry. With the acceptance of microwave oven pretreatment, various modified techniques and alternative heating methods have also been proposed. Wet autoclave pretreatment for tissue proteolysis is a highly reliable alternative to the microwave antigen retrieval technique. It provides uniform heating of the slides, hence an even enhancement of staining intensity in a variety of formalin-sensitive antigens, and it also offers consistent interlaboratory results. The method has been introduced in routine diagnostic immunohistochemistry for the detection of estrogen- and progesterone receptors, L26-, Ki-67- and bcl-2 antigens and variable types of cytokeratins (1/5/10/11, 8, 13, 19). Experimentally, wet autoclaving can be used very successfully for the immunophenotyping of p53 and mdm2 expression, for the detection of adhesion molecules (CD44, integrins) and some anti-inflammatory molecules (annexins), among others. It has produced a substantial improvement in the visualisation of silver-stained nucleolar organizer regions- associated proteins (AgNORs) in routine paraffin sections and along with modified silver staining and standardized AgNOR parameters assessed by image analysis. Wet autoclaving-based AgNOR staining has been proposed by a European multicentric study group as the standardized method for AgNOR analysis in archival material.

Expression of p53 protein in oral squamous cell carcinomas and adjacent non-tumorous mucosa of the floor of the mouth: an archival immunohistochemical study using wet autoclave pretreatment for antigen retrieval.

Routinely formalin-fixed and paraffin-embedded material of 22 squamous cell carcinomas of the floor of the mouth (T2NoMo, Ro), together with adjacent dysplastic or normal mucosa, were immunohistochemically investigated using a panel of four anti-p53 antibodies (CM1, PAb1801, DO7, PAb240) subsequent to wet autoclave pretreatment for antigen retrieval. p53 immunoreactivity was detected in 9/22 (40%) carcinomas with PAb1801 and DO7 antibodies, and in 8/22 cases using CM1 and PAb 240. p53-positive tumour cells accumulated predominantly at the invasive front of the carcinomas. A focal or scattered p53 immunoreactivity was observed in the adjacent normal and/or dysplastic mucosa in 17/22 (77%) cases using both CM1 and PAb1801 antibodies, in 10/22 with DO7, and in 8/22 with PAb240. This study has demonstrated examples of different p53 immunophenotypes in the non-tumorous and neoplastic oral mucosa of the same patient without significant correlation to the clinicopathological parameters studied.

Immunohistochemical detection of p53 protein in archival tissues from squamous cell carcinomas of the oral cavity using wet autoclave antigen retrieval

Using a panel of four anti-p53 antibodies (CM-1, PAb 1801, DO7, and PAb 240), the phenotypic characteristics of p53 protein overexpression were investigated in 22 routinely formalin-fixed and paraffin-embedded squamous cell carcinomas of the oral cavity. Conventional immunohistochemical staining without pretreatment for antigen retrieval was compared with wet autoclaving (AC) and microwave irradiation (MW). Applying AC or MW, p53 immunoreactivity was demonstrated in 9/22 cases with PAb 1801 and DO7 antibodies and in 8/22 cases with the CM-1 antibody. Immunostaining with the PAb 240 antibody after AC pretreatment showed p53 positivity in 8/22 tumours, but in only 4/22 following MW pretreatment. Immunostaining without pretreatment revealed only 2/22 p53-positive cases using CM-1, 4/22 with PAb 1801, and 3/22 with DO7. Without pretreatment, the PAb 240 antibody was consistently negative. Wet autoclave pretreatment was significantly superior for all the antibodies used in this study when compared with conventional immunohistochemistry without pretreatment (P < 0.005). It yielded better results than microwave irradiation for antigen unmasking in relation to the PAB 240 antibody (P < 0.05). Wet autoclave pretreatment is recommended as a reliable and highly reproducible method for p53 antigen retrieval in routinely processed archival material. The advantages over microwave pretreatment include simple handling and good preservation of morphology.

Standardized staining and analysis of argyrophilic nucleolar organizer region associated proteins (AgNORs) in radically resected colorectal adenocarcinoma--correlation with tumour stage and long-term survival.

Quantification of silver-stained nucleolar organizer region associated proteins (AgNORs) was introduced in histopathology as a marker of cellular and nucleolar activity. However, due to the poor staining quality obtained on routinely processed archival material, the method yielded controversial and sometimes non-reproducible results. The recent introduction of wet autoclave pretreatment has reliably improved AgNOR staining quality on routinely formalin-fixed and paraffin-embedded tissues. In the present study, 92 routinely processed colorectal carcinomas were investigated, applying this novel staining technique. Subsequent standardized morphometric analysis revealed, irrespective of common tumour staging or grading classifications, a statistically highly significant correlation between AgNOR parameters and clinical course. The usefulness of standardized AgNOR parameters for the independent prediction of patient survival was proven by uni- and multivariate analysis.

Wet autoclave pretreatment for immunohistochemical demonstration of oestrogen receptors in routinely processed breast carcinoma tissue.

The immunohistochemical demonstration of oestrogen receptor (OR) was performed on 32 randomly selected and routinely processed breast carcinomas after wet autoclave pretreatment of sections. The autoclave method was compared to the OR status found on frozen sections as well as to alternative pretreatment methods such as enzymatic predigestion and microwave irradiation. Using four different monoclonal antibody clones (H222, LH1, CC4-5, 1D5.26), the OR status was evaluated for each of the various pretreatment methods applied. All cases with a high OR content on frozen sections (n = 11) also showed a high OR status on wet autoclave-pretreated paraffin tissues using antibody clones 1D5.26 and CC4-5; in cases with low OR content on frozen sections, no false-negative cases were recorded using only the antibody 1D5.26 neither after wet autoclave nor microwave pretreatment. In addition, with this antibody, OR was detectable after autoclave pretreatment in two cases which were considered to be OR-negative even on frozen sections. When the primary antibody was omitted, no false-positive cases were observed after wet autoclave pretreatment. Thus, in our hands, wet autoclave pretreatment, in combination with the antibody 1D5.26, offers a highly sensitive method for the immunohistochemical demonstration of OR in routinely formalin-fixed, paraffin-embedded sections of breast carcinomas.

The effect of wet autoclave pretreatment on silver staining of nucleolar-organizer-region-associated proteins (AgNORs) in archival oral squamous cell carcinomas

The effect of wet autoclave pretreatment on silver staining of nucleolar-organizer-region-associated proteins (AgNORs) in archival oral squamous cell carcinomas

A standardized AgNOR stain method for formalin fixed and paraffin embedded material

Visualization of proteins associated with nucleolar organizer regions proteins (AgNORs) on formalin-fixed and paraffin-embedded archival tissues is substantially improved after application of wet autoclave pretreatment. Silver staining results are comparable to those obtained on tissues processed in alcohol based fixatives, illustrating AgNORs as substructures of the nucleoli without any staining artefacts. A highly reproducible staining quality was achieved irrespective of tissue origin or duration of formalin fixation. As a result of this novel and simple method, the grounds have been prepared for standardized AgNOR quantification on archival material.