Demonstration of estrogen receptor by immunohistochemical staining in paraffin sections of breast carcinoma

Paraffin embedded sections of 64 breast carcinomas were stained immunohistochemically using a commercially available monoclonal antibody to estrogen receptor. To improve the sensitivity of the staining, the authors used a Pronase enzyme pretreatment, biotinylated antibody to rat IgG as secondary antibody, streptavidin-alkaline phosphatase as tertiary reagent and fast red as chromogen. When compared to the results of estrogen receptor enzyme immunoassay, this method yielded an 85.9% concordance rate, 86.2% specificity and 85.7% sensitivity. When compared to estrogen receptor immunocytochemistry(ER-ICA) in frozen section and considering the inherent advantages of immunohistochemical staining over biochemical assay, the major advantages of this method are good morphology, suitability for retrospective study and reduced cost of staining due to dilution of expensive primary antibody. Thus, this method offers an alternative to ER assay using fresh tissue and should provide additional valuable information about estrogen receptor.     

Improved method for immunoperoxidase detection of membrane antigens in frozen sections.

The visualisation of membrane antigens in frozen sections using metal-enhanced 3,3'diaminobenzidine (DAB) was investigated. Particular attention was paid to the degree of reaction enhancement, the disruptive effect on morphological detail, and the ease of the techniques. The best result was obtained using nickel-modified DAB at pH 6.0 to develop the peroxidase enzyme, with further enhancement in cobalt chloride at neutral pH. Silver methenamine enhancement is also possible but can give rise to non-specific or background staining.     

Immunohistochemical detection of estrogen receptors in paraffin sections from primary and metastatic breast cancer

With the availability of monoclonal antibodies against the estrogen receptor (ER) it is possible to demonstrate the presence of ER immunohistochemically. Some of the antibodies are claimed to be reactive in formalin fixed, paraffin embedded tissue. We have evaluated the reactivity of one of these antibodies, D75 and found an acceptable reaction in routinely formalin fixed, paraffin embedded tissue. The antibody was applied to both primary and secondary tumors from a group of patients with recurrent breast cancer. The metastatic lesions consisted of lymph node metastases, bone marrow metastases, and liver metastases. While 41% of the primary tumors were ER-positive, this was only the case with 35%, 20%, and 17% of the lymph node, bone marrow, and liver metastases, respectively. The discordance between the ER-status of the primary tumor and the distant metastasis was 41% in cases of bone marrow metastases, and 44% in liver metastases. In most cases the shift was from an ER-positive primary tumor to an ER-negative metastasis. The results support the hypothesis that ER-negative tumor cells are probably more aggressive with a larger metastatic potential than the higher differentiated, ER-positive tumor cells.     

Rapid immunoperoxidase staining of axons for frozen section diagnosis of nerve biopsy specimens.

A new method of freezing and embedding a nerve biopsy specimen and staining it with the immunoperoxidase technique for neurofilaments was developed to overcome the difficulties normally encountered in the assessment of tiny portions of nerve. The method clearly shows the architecture of the nerve, the exact number and size of all axons present, and the degree of fibrosis present. The entire procedure may be accomplished in 20 minutes.     

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.     

Peroperative fat staining of frozen sections in primary hyperparathyroidism.

Roth and Gallaher recently described a fat staining method for rapid peroperative differentiation between parathyroid adenoma and chief cell hyperplasia. They used Sudan IV in a solution of ethanol and acetone. This solution, however, was found to cause a considerable dissolution of small lipid droplets from the tissue; in our hands sections stained with this technique were diffcult to interpret. To diminish the loss of fat from the tissue, we have used a modification of Lillie's supersaturated ispropanol method with oil red O. This method gave a deeper staining and increased the difference between hyperfunctioning and unnivolved parathyroid tissue with respect to the amount of stainable lipid in the chief cells. It was found to be a valuable supplement, adding a functional dimension to the structural interpretation of the tissue, and it facilitated the peroperative distinction between ademona and hyperplasia. The pattern of lipid distribution within the glands from patients with nodular hyperplasia suggests that the compact nodules of such glands are autonomously hyperfunctioning, whereas the intervening parts of the parenchyma are more or less responsive to the serum calcium level.     

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)     

Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma

To identify the minimum time necessary for consistent immunohistochemical estrogen receptor (ER) results in our laboratory, we evaluated results in timed fixation blocks and cases with disparate and similar needle core biopsy and partial mastectomy specimens. Tissue sections of 24 ER-positive, invasive breast carcinomas were fixed for 3, 6, 8, and 12 hours and 1, 2, and 7 days. ER values were quantified using the Q score (0-7). In timed fixation blocks, the mean Q score per block was 2.46 for blocks fixed for 3 hours, 5.75 for blocks fixed for 6 hours, and 6.70 for blocks fixed for 8 hours (P < .001). The difference between the case maximum and mean block Q scores was a plateau of almost 0 at 6 to 8 hours of formalin fixation. For needle core biopsy specimen fixation times, the means for specimens with ER-disparate and ER-similar results were 1.2 and 6.3 hours, respectively (P = .01). The minimum formalin fixation time for reliable immunohistochemical ER results is 6 to 8 hours in our laboratory, regardless of the type or size of specimen.     

Critical evaluation of monoclonal antibody staining in breast carcinoma.

The immunoperoxidase staining of 84 primary invasive breast carcinomas with four monoclonal antibodies (BRST-1, HMFG1, EMA, B72.3) was evaluated by semiquantitative light microscopical examination and quantitative image analysis. Major differences in the staining of the tumours for each of the monoclonal antibodies was observed. Correlation between monoclonal antibody staining and patient age, survival, histological grade, tumour diameter and cellularity was also carried out. This showed a significant association between histological grade and staining with BRST-1 and EMA.     

Proliferative activity and steroid receptors determined by immunohistochemistry in adjacent frozen sections of 102 breast carcinomas

Adjacent frozen sections of 102 consecutive female breast carcinomas were examined for the expression of the Ki-67 antibody-reactive proliferation-associated nuclear antigen and of estrogen and progesterone receptors with the use of monoclonal antibodies and peroxidase histochemistry. The results of steroid receptor stainings were semiquantitatively assessed (histoscore) on the basis of nuclear staining intensity and the percentage of positively stained carcinoma cell nuclei. Carcinomas negative for either receptor had significantly higher percentages of Ki-67-positive cells. The highest percentages of Ki-67-positive cells were observed in carcinomas negative for both estrogen and progesterone receptors. There was a highly significant decrease in receptor histoscores with increasing proliferative cell fractions as determined by Ki-67 positivity. No significant (progesterone receptor) or poor negative correlation (estrogen receptor) was observed when proliferative cell fractions were related to receptor concentrations from conventional steroid-binding assays. Immunoperoxidase staining for the Ki-67 antibody-defined proliferation antigen and steroid receptors in tissue sections provides a simple means to gain information of therapeutic and prognostic importance.     

Demonstration of progesterone receptors in paraffin wax sections of breast carcinoma.

Several immunohistological methods for the demonstration of progesterone receptors were tried on routinely processed paraffin wax sections of breast carcinoma, using Abbott's PgR-ICA monoclonal antibody. The best results were obtained with the avidin-biotin-immunoperoxidase complex method with no prior trypsinisation or DNAse digestion, and with imidazole added to the final diaminobenzidine developing solution. A simple semiquantitative scoring system was used to assess the staining results which were then compared with the results obtained by a standard dextran-coated charcoal biochemical assay. Of 31 cases examined, the results of the two methods were concordant in 25 (81%) of cases. This is near the higher end of the concordance range obtained by several other authors using frozen sections. The discordance encountered in a few cases was possibly the result of sampling errors which are more likely to occur with the chemical rather than the histological method. It is concluded that the method described here is fairly reliable and would greatly simplify the process of assessment of progesterone receptors in breast, and possibly other tumours.     

Immunocytochemical evaluation of estrogen receptors in histological specimens of primary breast cancer

Immunocytochemical methods (ER-ICA) by using monoclonal antibodies were applied to determine the presence of estrogen receptors (ER) in 44 primary breast cancers in women. Of this 48% of the tumours were classified as receptor positive. In these tumours the nuclei had a clearly positive heterogenous colouration. In three cases a positive reaction was also found in benign epithelial cells of the breast. ER determinations by ER-ICA method were compared with quantitative analysis carried out by using radioligand and immunoenzymatic methods. There was a strong correlation between immunocytochemical ER evaluations and quantitative methods. We also found a correlation of the menopause state and patients age and ER content.     

Demonstration of immunoglobulin in malignant lymphomas. Use of an immunoperoxidase technic on frozen sections

We used an immunoperoxidase technic to detect surface and cytoplasmic immunoglobulin in frozen sections of 46 malignant lymphomas. With the immunoperoxidase technic, differential staining with antisera to the various heavy and light chain classes permitted detection and characterization of monotypic immunoglobulin in frozen sections of 15 of 15 nodular lymphomas and 24 of 31 diffuse lymphomas. The immunoperoxidase technic applied to frozen sections is a convenient and reliable method for the detection of immunoglobulin in lymphoid tissues, which can be performed in a pathology laboratory without the need for special equipment.     

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.     

Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections

Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections was done on 30 haematological problems. Core needle biopsy of the bone marrow is a safe and useful procedure. It is a valuable diagnostic aid for measurement of marrow cellularity, metastatic tumours and fibrosis. It should not be taken as a substitute for examination of the marrow by aspiration smear but is a complementary procedure which affords several advantages. Bone marrow biopsy was of maximum utility in myelofibrosis which was diagnosed on biopsy alone. There were three additional cases with normal bone marrow aspiration in which specific diagnosis could only be made from bone marrow biopsy sections. New methodologies i.e. plastic embedding and semi thin sections of undecalcified bone marrow, can be expected to improve the cytological details of tissue obtained by biopsy. Imprint preparations obtained from biopsy can be useful in patients of malignancy but we have found them to be of limited value except in cases of dry tap.