Potential value of estrogen receptor immunocytochemical assay in formalin-fixed breast tumors.

Sixty-two primary breast carcinomas were analyzed for estrogen receptor (ER) by both the dextran-coated charcoal (DCC) technique and estrogen receptor immunocytochemical assay (ER-ICA) on cryostat and permanent sections. Paraffin sections of formalin-fixed breast tissue underwent DNase pretreatment to expose the nuclear antigenic site as described by P. Shintaku and J. H. Said (Am J Clin Pathol 87:161, 1987). The results of immunocytochemical staining agreed with those of the DCC biochemical assay in 89% of paraffin-sectioned tissue and in 94% of the cryostat sections. Comparison of the results of ER-ICA on permanent and frozen sections showed 85% agreement (kappa statistic = 0.704). This study suggests that ER can be demonstrated immunocytochemically on paraffin-sectioned breast tissue. However, although highly specific, immunoperoxidase determination on paraffin-embedded tissue is less sensitive than that on frozen tissue. The commercial source of DNase, length of incubation, and tissue fixation are important factors in the demonstration of ER immunoreactivity. The assay may offer an alternative for assessment of ER when tissue is not suitable or available for biochemical assay or conventional cytochemical analysis.     

Image analysis for quantitation of estrogen receptor in formalin-fixed paraffin-embedded sections of breast carcinoma.

Monoclonal antibody to human estrogen receptor (ER) provides a useful immunohistochemical tool for the evaluation of ER content in breast carcinoma, but visual interpretation is subjective. Computer-assisted image analysis has proved effective in immunohistochemical quantitation of ER in fresh tumor imprints and cryostat sections. We examined the usefulness of this technique in 5-microns-thick formalin-fixed paraffin-embedded tissue sections of 66 cases of primary breast carcinoma previously assayed by dextran-coated charcoal (DCC) analysis. Immunohistochemistry was automated and performed on a Code-on slide stainer (Instrumentation Laboratories, Lexington, MA) using Pronase predigestion, a monoclonal antibody (ER-ICA; Abbott, Chicago, IL), and a biotin-labeled secondary antibody. Detection was achieved with an avidin-alkaline phosphatase conjugate and nitroblue tetrazolium (NBT) bromochloroindoyl phosphate (BCIP) substrate. The immunohistochemical ER staining was analyzed visually and with the CAS/200 image analyzer (Elmhurst, IL). The visual semiquantitative histologic scores (HSCORE), the automated quantitative assays including the percentage of positive nuclear areas (PNA), and the quantitative immunocytochemical scores (QIC SCORE = PNA x % of positive stain/10) were compared with the corresponding DCC results. Linear correlations were demonstrated between all immunohistochemical assays and the logarithm of DCC, the strongest correlation seen with PNA (r = 0.91). Threshold points for positive HSCORE, QIC SCORE, and PNA assays were extrapolated using DCC as the reference. ER immunodetection by PNA as compared with visual examination alone was enhanced by 18% (up to 88%) in sensitivity and 34% (up to 94%) in specificity, and the DCC concordance rate increased by 26% (up to 91%). A comparative chart extrapolating DCC from PNA was thus established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utility of the paraffin-embedded section method on the detection of estrogen receptor from breast cancer tissues--comparison of the paraffin-embedded section method (6F11 and 1D5) with frozen section (H222) and dextran-coated charcoal (DCC) ones]

From March 1988 to December 1990, we detected estrogen receptor (ER) from breast cancer tissues with the dextran-coated charcoal (DCC) method. From September 1990 to March 1998, we repeated the above with the frozen section method (cloneH222; DAINABOT). With the paraffin-embedded section method using two antibodies of anti-ER (clone6F11; NOVOCASTRA and clone 1D5; MBL), we examined the ER of the same 185 primary breast cancer tissues. We had already detected these tissues with the frozen section method, and we also applied the same procedure for the 43 primary breast cancer tissues which had already been detected with the DCC method. We compared these data. The positive rates of ER with DCC, H222, 6F11 and 1D5 were 49%, 53%, 53% and 52% respectively, which were within the reported range. The accuracy between H222 and 6F11 which was calculated as the percentage that were in positive and negative concordance by the two methods, was 88%. The accuracies between H222 and 1D5, between 6F11 and 1D5, between DCC and 6F11, between DCC and 1D5 were respectively 89%, 96%, 79%, and 77%. Although the accuracy between DCC and the paraffin-embedded section was not necessarily high, we obtained a higher concordance between the frozen and paraffin-embedded sections. The highest concordance existed between 6F11 and 1D5. The 30% of negative cases with DCC were positive with paraffin-embedded section. Among these methods of ER detection from breast cancer tissues, the paraffin-embedded section method seemed to be the most useful.     

Automated immunohistochemical estrogen receptor in fixed embedded breast carcinomas. Comparison with manual immunohistochemistry on frozen tissues

Estrogen receptor (ER) status of breast carcinomas determines prognosis and treatment. Biochemical ER assays are expensive and time-consuming and require fresh tumor. Immunohistochemical ER was assessed in 68 breast carcinomas, by an automated method using routinely processed formalin-fixed paraffin-embedded tissues, and manually with the use of snap-frozen tissues with a monoclonal anti-ER and peroxidase-antiperoxidase technique. The paraffin sections were digested with DNase to enhance development of signal. Positive nuclear ER was obtained in 9 (13%) fixed tissues and 36 (53%) frozen tissues. The sensitivity, specificity, and predictive value of a positive test result, as compared with the biochemical assay, were 25%, 100%, and 100% for the paraffin section technique, and 89%, 88%, and 89% for the frozen sections. Although it is specific, lack of sensitivity, resulting from loss of ER with fixation and room temperature handling, renders this immunohistochemical technique unacceptable on fixed tissues. However, ER immunostain on frozen tissue is an acceptable alternative to biochemical assay.     

Immunohistochemical estrogen receptor assay: quantitation by image analysis

The immunohistochemical estrogen receptor (ER) assay quantitated by image analysis was compared with the biochemical dextran-coated charcoal (DCC) assay in 97 primary breast carcinomas. Frozen sections immunostained for ER with a monoclonal antibody (ER-ICA; Abbott Laboratories, Chicago, IL) and the peroxidase-antiperoxidase technique were scored semiquantitatively by microscopy (HSCORE) and quantitatively [percentage of nuclear area immunopositivity (PNA)] using the CAS 200 image analyzer (Elmhurst, IL). There was a significant and identical correlation between DCC and both HSCORE and PNA. According to sensitivity/specificity calculations, the cut-off points of 10 for HSCORE and 1% for PNA were chosen with positive and negative predictive values of 97.7% and 74.5%, respectively. Low DCC-positive cases between 10 and 50 fmol of ER/mg of protein were immunohistochemically ER positive only in 61.5%. Immunohistochemical results of greater than 150 HSCORE and greater than 15% PNA corresponded to high DCC results of greater than 150 fmol/mg of protein. Immunohistochemical assay quantitated by image analyzer (PNA) is a comparable alternative to biochemical ER, especially when insufficient fresh tissue is available.     

Ward Burdick Award Lecture: Blood, sweat, and cheers!

This study compares the estrogen receptor (ER) content of 26 breast neoplasms as determined by dextran-coated charcoal (DCC), fluorescein-labeled estradiol conjugate (FITC) on frozen sections, and immunoperoxidase (IP) on sections of formalin-fixed, paraffin-embedded tissues. Concordance between results of the three technics was found in 16 (62%) cases. DCC and IP agreed in 18 (69%) cases, DCC and FITC agreed in 22 (85%) cases, and IP agreed with FITC in 18 (69%) cases. Most of the concordant cases (15/16) were in patients with ER-positive neoplasms. The relative merits and applications of the three technics are discussed.     

Comparative analysis of various biochemical and immunohistochemical methods for evaluating hormone receptors in breast carcinoma

The aim of this study was to compare the grade of concordance between three methods to evaluate and to quantify the expression of estrogen and progesterone receptors in 180 cases of breast cancer. The methods compared were dextran-coated charcoal (DCC) method, immunochemical analysis (ICA), and immunohistochemistry (IHC). The grades of concordance are so summarized: (1) 77.8% for estrogen receptors (ER) and progesterone receptors (PgR) with DCC and ICA methods (r = 0.53 and r = 0.64, respectively); (2) 83.3% for ER (r = 0.53) and 75.6% for PgR (r = 0.64) with DCC and IHC methods; (3) 87.8% for ER (r = 0.98) and 86.7% for PgR (r = 0.99) with ICA and IHC methods. Nowadays, the technique carried out on tissue sections provides the most reliable information. The IHC method performed on formalin-fixed and paraffin-embedded tissue is preferred in laboratories with a high work load because it is easy and inexpensive and can be automated.     

Detection of estrogen receptors with monoclonal antibodies in routinely processed formalin-fixed paraffin sections of breast carcinoma. Use of DNase pretreatment to enhance sensitivity of the reaction

This study describes an improved immunohistochemical method for the sensitive and specific identification of estrogen receptors (ERs) in paraffin sections from formalin-fixed and routinely processed breast carcinoma tissues, using DNase pretreatment to expose nuclear antigenic sites and commercially available immunoreagents (including monoclonal antibody) in kit form. Results were compared with dextran-coated charcoal cytosolic assay (DCC) and with conventional immunohistochemistry on frozen sections. Sensitivity and specificity for determinations on paraffin sections were 88% and 86%, respectively, and statistical analysis showed very good agreement between DCC and paraffin sections (kappa = 0.805). The DNase technic on paraffin sections allows excellent correlation between histologic characteristics and ER status and reduces DCC sampling error resulting from stromal dilution and tumor variability. This method offers a reliable and reproducible alternative when tissue is not suitable or unavailable for DCC or frozen tissue analysis and can be used for retrospective studies on stored tissue blocks.     

Demonstration of oestrogen receptors in paraffin wax sections of breast carcinoma using the monoclonal antibody 1D5 and microwave oven processing.

This study aimed at assessing the usefulness of a new monoclonal antibody (1D5) for the demonstration of oestrogen receptors (ER) in paraffin wax sections, using brief microwave processing rather than proteolytic predigestion. Routinely processed paraffin wax sections of 50 cases of breast carcinoma with known ER concentrations, estimated by the standard dextran-coated charcoal (DCC) biochemical assay, were examined using the avidin-biotin complex-immunoperoxidase technique. The results were assessed semiquantitatively, using a five grade scoring system. Of the 50 cases examined, 37 were positive and six were negative by both DCC and immunohistology. Of the remaining seven cases, three (6%) were negative by DCC but positive with immunohistology, and four (8%) were positive with DCC and negative with immunohistology. The DCC results of the latter four cases were 10, 14, 14 and 16 fmol/mg protein which is at the lowest level of positivity, our cutoff point being less than 10 fmol. The monoclonal antibody 1D5, as used in this study, can provide easily assessed reliable information about the ER status of breast carcinoma using routinely processed paraffin wax sections.     

Estrogen and progesterone receptor detection in archival formalin-fixed, paraffin-embedded tissue from breast carcinoma: a comparison of immunohistochemistry with the dextran-coated charcoal assay.

The steroid receptor (estrogen (ER) and progesterone receptor (PR] status was studied in 94 cases of invasive breast carcinoma from three separate institutions. All cases had fresh tissue examined for ER and PR by the dextran-coated charcoal cytosolic assay (DCC), and each case was examined immunohistochemically for ER and PR from archival formalin-fixed, paraffin-embedded tissue. Immunohistochemical assays (IH) were reviewed blinded to the DCC results and scored in a semiquantitative fashion prior to comparison to the DCC results. Overall, there was agreement between DCC and IH in 89% of ER and in 87% of PR assays. Some 50% of the ER discorrelations were of the IH-positive DCC-negative type, while 27% of the PR discorrelations were of this type. In four cases, both ER and PR did not correlate between IH and DCC determinations, with two being IH (ER and PR) positive and DCC negative, and two of the opposite type. The results of the study show that steroid receptor assays performed on routinely processed formalin-fixed archival material are reliable and closely recapitulate the results of traditional biochemical assays. Results suggest that, in the cases where IH is positive while DCC is negative, the IH result may actually provide a more reliable receptor status of the tumor than does the DCC result. Semiquantitation of fixed tissue IH assays shows a trend toward quantitative correlation with DCC results, but this correlation is weak, and factors concerning fixation and processing are most likely to be responsible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical analysis of progesterone receptors in breast cancer.

Breast cancer specimens from 116 patients were assayed for the presence of progesterone receptor (PR), with the use of a highly specific monoclonal antibody and the peroxidase-antiperoxidase technique on cryostat and permanent sections. Results were compared with those obtained by the conventional PR determination by dextran-coated charcoal (DCC) assay; they were in concordance in 90% of cryostat sections and 85% of paraffin-embedded tissue. The sensitivity and specificity of the PR immunocytochemical assay (PR-ICA) were 91% and 89% for frozen sections and 83% and 89% for permanent sections, respectively. The immunostained slides also were evaluated for several semiquantitative features, including staining intensity, heterogeneity of staining, and the proportion of positive tumor cells. A statistically significant correlation was found between the percentage of tumor cells stained with the PR immunocytochemical technique and the PR-cytosol levels (P less than 0.05). These results suggest that the PR-ICA is an effective tool in the evaluation of PR content in breast cancer and can be applied in paraffin as well as frozen sections. This technique provides excellent morphologic detail, as well as tissue localization for PR. It also offers an alternative for assessment of PR when fresh tissue is not available for conventional hormone receptor analysis. The immunocytochemical assay can be performed easily at community hospitals. Because it requires only a small amount of tissue, PR-ICA is an ideal method for analyzing specimens of insufficient size for the DCC assay. This technique also is suited to the evaluation of fine-needle aspiration biopsy specimens.     

Quantification of estrogen receptors on paraffin-embedded tumors by image analysis

Emphasis on early detection of breast carcinomas has increased the number of instances in which an insufficient amount of tissue is available for biochemical estrogen receptor (ER) assay. Image analysis, used together with immunohistochemistry, introduces the possibility of quantifying molecules, such as ER, in routinely processed tissues. To explore that possibility, sections from 40 formalin-fixed, paraffin-embedded breast carcinomas with biochemically determined ER values were reacted with H222 anti-ER antibody (ER-ICA) and quantified on a CAS 200 image analysis system. A minimum of ten fields comprising at least 15,000 microns 2 of nuclear area were analyzed in each case. If the antigen distribution were not homogeneous, proportional sampling of the different tumor areas was carried out. Of the 31 ER-positive (10 to 344 fmol/mg) tumors, 27 (87%) were immunoreactive by the ER-ICA assay (greater than 5% positive nuclear area), which represents an improvement over simple microscopic evaluation (68%). Bivariate analyses showed statistically significant, albeit only modest, correlation between ER values and the percentages of positive area (r = 0.556) and positive stain (r = 0.518). The following obstacles were found to interfere with a proper correlation: (a) antigen loss during fixation and processing; (b) intratumoral antigenic heterogeneity; and directly associated with it, (c) interobserver variability. Uniform tissue handling or, alternatively, the use of internal controls to compensate for fixation-induced differences, together with thorough assessment of the tissue, should reduce these obstacles and facilitate accurate and reproducible quantification.     

Estimation of estrogen receptor content in fine-needle aspirates from breast cancer using the monoclonal antibody 1d5 and microwave oven processing: Correlation with paraffin embedded and frozen sections determinations

We describe a method of immunocytochemically assessing estrogen receptor (ER) status on alcohol-fixed smears obtained by fine-needle aspiration (FNA) from breast cancer patients, using a commercially available monoclonal antibody (1D5) with microwave oven processing. A series of 31 cases of aspirates from breast cancer were analysed and the results were compared with assessment by ER immunocytochemical assay using the same procedure on formalin-fixed tissue and with assessment by ER-ICA assay on frozen sections. The results were scored semiquantitatively using a five grade scoring system. Of the 31 cases examined, 21 were positive at least by two methods and 10 were negative for all three determinations. The results obtained in the ER immunocytochemical assay on aspirates and paraffin-sections using the antibody 1D5 and those obtained on frozen sections using the antibody H222 were closely similar. In only one case was it not possible to interpret the reaction in the cytological specimen because there was a strong background in the smear. In general, we obtained more intense positivity with the antibody 1D5 in aspirates and formalin-fixed material than with the antibody H222 in frozen sections. The scoring results of the three methods were almost identical. We conclude that the application of ER method on alcohol-fixed smears will eliminate the need for using a special fixation procedure and will provide several advantages, such as: improvement in morphological concomitant analysis, utilization whenever malignancy is found without necessity to re-aspirate the patient, and adequacy of archival material. © 1995 Wiley-Liss, Inc.     

Methodology of immunohistological detection of oestrogen receptor in human breast carcinoma in formalin-fixed, paraffin-embedded tissue: a comparison with frozen section methodology

We describe a method of immunohistochemically assessing oestrogen receptor status on routinely processed formalinfixed tissue, using a commercially available monoclonal antibody (Abbott H222). with pronase predigestion of tissue sections and overnight antibody incubation. The staining was assessed using the H score system. A series of 94 cases of breast cancer were analysed and the results were compared with assessment by oestrogen receptor immunocytochemical assay performed on frozen section. Direct comparison of the paired sets of H scores obtained with frozen tissue and formalin-fixed tissue showed a highly significant correlation of 0.8 ( P < 0.001) between the two methods of oestrogen receptor assessment. Chi-squared analysis using H score cut off points of 50 and 100 also showed a similar significant association ( P < 0.001). We conclude that this oestrogen receptor method, applicable to formalin-fixed, paraffin-embedded tissue, gives accurate results on routinely fixed tissue and could be used as an alternative to other methods.     

IMMUNOHISTOCHEMICAL ASSAY FOR OESTROGEN RECEPTORS IN PARAFFIN WAX SECTIONS OF BREAST CARCINOMA USING A NEW MONOCLONAL ANTIBODY

The aim of this study was to evaluate the utility of a new monoclonal antibody (AER311) that targets the oestrogen receptor (ER) in an immunohistochemical assay (IHA) applied to breast cancers. Ninety-seven cases of invasive ductal carcinoma were studied by AER311-IHA using a pressure-cooking antigen retrieval technique applied to formaldehyde-fixed, paraffin-embedded tissue sections; immunostaining was assessed by semi-quantitative scoring ( H score). There was 80 per cent concordance between the ER status measured by dextran-coated charcoal (DCC) assay and AER311-IHA, with 63/97 (65 per cent) tumours positive and 15/97 (15 per cent) tumours negative by both assays. Of the 12 DCC-positive cases that were negative by AER311-IHA, 11 were borderline positive (3–8 fmol/mg). Similarly, six of seven DCC-negative cases that scored positive by AER311-IHA had only borderline positive H scores (<50). When AER311-IHA was compared with 1D5-IHA, there was good concordance in ER status (77 per cent) and a significant correlation ( r =0·7, P <0·001) between H scores. Nevertheless, the correlation between ER level determined by AER311-IHA and that measured by DCC ( r =0·53, P <0·001) was higher than that for 1D5-IHA ( r =0·32, P =0·002). AER311-IHA can therefore provide reliable information about the ER status of breast carcinoma on paraffin sections and is an acceptable alternative to other commercially available monoclonal antibodies.     

Comparative evaluation of ER-ICA and enzyme immunoassay for the quantitation of estrogen receptors in breast cancer

Evaluation of estrogen receptors (ERs) is essential for the treatment and prognosis of human breast cancer. The authors have undertaken a study of 225 primary breast cancers to compare the receptor values using immunohistochemistry and enzyme immunoassay (EIA) techniques. The results are compared with the biochemical assay with the use of the dextran-coated charcoal (DCC) method done on the same specimens. The overall concordance between ER-ICA and DCC was 77%. Ninety-seven percent concordance was observed for specimens with a positive ER-ICA score (greater than 70 and the DCC more than 10 fmol/mg protein). Ninety-two percent of tumors had negative results by ER-ICA when the DCC was less than 10 fmol/mg protein. When the ER-ICA score was plotted against the DCC or EIA value, a significant correlation was obtained with a P value of less than 0.001. The correlation between the ER-ICA score and the total ER content (cytosol and nuclear) assayed by EIA was similar to comparison with cytosol alone. The authors conclude that the ER-ICA can be useful in assessing ERs in breast especially in hypocellular or small tumors and in assessing the degree of heterogeneity in a given tumor. The immunohistochemical and biochemical assay methods are complementary and provide greatly needed information for the management of breast cancer.     

Oestrogen receptor staining of paraffin-embedded breast carcinomas following short fixation in formalin: a comparison with cytosolic and frozen section receptor analyses

This paper describes an improved immunohistochemical method for demonstrating oestrogen receptor (OR) protein in paraffin-embedded sections of tissue fixed for 1.5 h in formalin. Thirty-two cases of infiltrating ductal breast carcinoma were stained with a monoclonal anti-OR antibody (H222), using a standard streptavidin-biotin method, following pretreatment with pronase. OR counts in paraffin sections were compared with those of frozen sections and with cytosolic values determined by a dextran-coated charcoal method. Twenty-seven of the carcinomas were OR-positive in paraffin sections. There was concordance between the paraffin section and the frozen section-determined receptor status in 30 cases (94 per cent) and a strong correlation was observed (r = 0.76; P less than 0.0001). Similarly, OR counts in paraffin sections correlated with cytosolic OR values (r = 0.60; P less than 0.001) and there was concordance in 97 per cent of cases. The percentage of positively-stained tumour cells in paraffin sections ranged from 0 to 94 per cent with staining intensities comparable to those seen in frozen sections. Staining of paraffin sections identified more OR-positive tumours than either frozen section staining or cytosolic assay. This study validates immunohistochemical OR analysis in formalin-fixed, paraffin-embedded breast carcinomas using a commercial anti-OR antibody.     

Estrogen receptor protein in bone and soft tissue tumors

Thirty-three histologically diverse bone and soft tissue tumors were analyzed biochemically for the presence of estrogen receptor protein (ERP) and progesterone receptor by means of a conventional, commercially available, steroid-binding assay (dextran-coated charcoal method) on fresh frozen tissue. These results were compared with analysis of ERP by using a specific monoclonal antibody both in an enzyme immunoassay and on frozen tissue sections by using immunohistochemical procedures. Frozen tissue sections were also examined for the presence of estrogen and progesterone receptors using fluorescein-labeled steroids. Six of the 33 tumors (18%) contained low levels of ERP ranging from 19 to 73 fmol/mg as determined by the dextran-coated charcoal method. The remaining 27 cases contained no (less than 10 fmol/mg) ERP. The ERP-positive group included a fibromatosis, leiomyosarcoma, liposarcoma (2 cases), neural sarcoma, and a synovial sarcoma. Four were high grades sarcomas, and two were low grade sarcomas. There was excellent agreement between the ERP levels determined by the dextran coated charcoal method and those determined by enzyme immunoassay. ERP could not be demonstrated immunohistochemically on frozen tissue sections of the tumors even though it could be demonstrated in breast carcinomas serving as positive controls. The failure of the immunohistochemical technique may be related to the low levels of ERP in these tumors and the difficulty of detecting antigen at threshold levels. Cytochemical localization of receptor protein employing fluoresceinated steroids did not correlate with cytosolic ERP as determined by enzyme immunoassay or the dextran coated charcoal method. Moreover, the high level of background fluorescence gave rise to a significant amount of intraobserver and interobserver variation. Although the clinical significance of ERP protein in mesenchymal tumors is still uncertain, the present findings, coupled with various clinical observations suggesting hormonal dependency of some mesenchymal tumors, indicate that investigation of a larger group of patients amenable to statistical analysis is warranted.     

Enzyme-labeled Antigen Method: Factors Influencing the Deterioration of Antigen-binding Activity of Specific Antibodies during Formalin Fixation and Paraffin Embedding

The enzyme-labeled antigen method is an immunohistochemical technique detecting plasma cells producing specific antibodies in tissue sections. The probe is an antigen labeled with an enzyme or biotin. This immunohistochemical technique is appliable to frozen sections of paraformaldehyde (PFA)-fixed tissues, but it has been difficult to apply it to formalin-fixed, paraffin-embedded (FFPE) sections. In the current study, factors inactivating the antibody reactivity during the process of preparing FFPE sections were investigated. Lymph nodes of rats immunized with horseradish peroxidase (HRP) or a mixture of keyhole limpet hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) or biotinylated proteins in 4% PFA-fixed frozen sections, significantly decreased in unbuffered 10% formalin-fixed frozen sections. The positive cells were further decreased by paraffin embedding following formalin fixation. In paraffin-embedded sections fixed in precipitating fixatives such as ethanol and acetone and those prepared with the AMeX method, the antigen-binding reactivity of antibodies was preserved. Fixation in periodate-lysine-paraformaldehyde and Zamboni solution also kept the antigen-binding reactivity in paraffin to some extent. In conclusion, formalin fixation and paraffin embedding were major causes inactivating antibodies. Precipitating fixatives could retain the antigen-binding reactivity of antibodies in paraffin-embedded sections.     

Expression of estrogen and progesterone receptors in cytologic specimens using various fixatives

Estrogen and progesterone receptor reactivity may be useful in identifying possible primary sites of metastatic disease or directing therapy in tumors of the female genital tract, including breast, ovary, and endometrium. Various methods have been described for the immunocytochemical evaluation of estrogen receptor (ER) and progesterone receptor (PR) status of cytologic specimens but our results have been variable. We evaluated the effectiveness of various fixatives [cytospin collection fluid, Shandon, Pittsburgh, PA (SH); ethanol (ETH); and formalin (FOR)] for fixation of smears (SM) and cell block (CB) material. The percentage and intensity of tumor nuclei of SM, CB, and tissue sections (TS) stained for ER and PR by the avidin-biotin-peroxidase complex technique were compared. Samples were considered ER or PR positive when ≥20% of tumor nuclei were stained. The sensitivity of ER analysis of SMs and CBs in each fixative compared to formalin-fixed paraffin-embedded tissue sections were as follows, SM (SH) 88%, SM (ETH) 14%, CB (SH) 58%, CB (ETH) 43%, and CB (FOR) 70%. The sensitivity of PR determination on SMs and CBs was SM (SH) 71%, SM (ETH) 6.0%, CB (SH) 25%, CB (ETH) 33%, CB (FOR) 80%. These findings indicate that of the fixatives evaluated for ER analysis SMs fixed in SH provided the best results. For PR evaluation, CBs fixed in FOR gave the best results. Diagn Cytopathol 1996;15:78–83. © 1996 Wiley-Liss, Inc.