Effects of short and long-term alcohol-based fixation on Sprague-Dawley rat tissue morphology,protein and nucleic acid preservation

Safety concerns on the toxic and carcinogenic effects of formalin exposure have drawn increasing attention to the search for alternative low risk fixatives for processing tissue specimens in laboratories worldwide. Alcohol-based fixatives are considered some of the most promising alternatives. We evaluated the performance of alcohol-fixed paraffin-embedded (AFPE) samples from Sprague-Dawley (SD) rats analyzing tissue morphology, protein and nucleic acid preservation after short and extremely long fixation times (up to 7 years), using formalin-fixed paraffin-embedded (FFPE) samples as a comparator fixative. Following short and long-term alcohol fixation, tissue morphology and cellular details in tissues, evaluated by scoring stained sections (Hematoxylin-Eosin and Mallory’s trichrome), were optimally preserved if compared to formalin fixation. Immunoreactivity of proteins (Ki67, CD3, PAX5, CD68), evaluated by immunohistochemistry, showed satisfactory results when the fixation period did not exceed 1 year. Finally, we confirm the superiority of alcohol fixation compared to formalin, in terms of quantity of nucleic acid extracted from paraffin blocks, even after an extremely long time of alcohol fixation.Our results confirm that alcohol fixation is a suitable and safe alternative to formalin for pathological evaluations. There is a need for standardization of formalin-free methods and harmonization of diagnosis in pathology department worldwide.     

Molecular fixative enables expression microarray analysis of microdissected clinical cervical specimens

Formalin-fixed tissue has been a mainstay of clinical pathology laboratories, but formalin alters many biomolecules, including nucleic acids and proteins. Meanwhile, frozen tissues contain better-preserved biomolecules, but tissue morphology is affected, limiting their diagnostic utility. Molecular fixatives promise to bridge this gap by simultaneously preserving morphology and biomolecules, enabling clinical diagnosis and molecular analyses on the same specimen. While previous reports have broadly evaluated the use of molecular fixative in various human tissues, we present here the first detailed assessment of the applicability of molecular fixative to both routine histopathological diagnosis and molecular analysis of cervical tissues. Ten specimens excised via the loop electrosurgical excision procedure, which removes conical tissue samples from the cervix, were cut into alternating pieces preserved in either formalin or molecular fixative. Cervical specimens preserved in molecular fixative were easily interpretable, despite featuring more eosinophilic cytoplasm and more recognizable chromatin texture than formalin-fixed specimens. Immunohistochemical staining patterns of p16 and Ki-67 were similar between fixatives, although Ki-67 staining was stronger in the molecular fixative specimens. The RNA of molecular fixative specimens from seven cases representing various dysplasia grades was assessed for utility in expression microarray analysis. Cluster analysis and scatter plots of duplicate samples suggest that data of sufficient quality can be obtained from as little as 50 ng of RNA from molecular fixative samples. Taken together, our results show that molecular fixative may be a more versatile substitute for formalin, simultaneously preserving tissue morphology for clinical diagnosis and biomolecules for immunohistochemistry and gene expression analysis.     

Ionic liquids as an alternative to formalin in histopathological diagnosis

Asymmetry of cations and the type of anions play a key role in the properties of ionic liquids (ILs) as fixatives for tissue preservation. 1-Methyl-3-octyloxymethylimidazolium tetrafluoroborate has proven to be a very good fixative, with similar effects as formalin. Our study shows that it is applicable for both histological and immunohistochemical purposes. After treatment with 1-methyl-3-octyloxymethylimidazolium tetrafluoroborate, tissue sections are more intensely stained. With respect to expression patterns and staining intensity, immunohistochemical staining is comparable in tissues fixed in formalin and the selected ILs. The present study demonstrates the properties of 1-methyl-3-octyloxymethylimidazolium tetrafluoroborate for tissue preservation in histopathological procedures, eliminating the requirement of formalin.     

Modified formalin and methanol fixation methods for molecular biological and morphological analyses

Several simplified fixation methods were examined to determine their suitability for both molecular biological analyses and morphological study. Fixation with 10% v/v formalin alone at 4°C and containing 5 mmol/L ethylenediamine-N, N, N', N'-tetraacetic acld (EDTA) at room temperature preserved significantly more high-molecular-weight DNA than 10% v/v formalin fixation at room temperature. The morphological differences between tissues fixed using these modified formalin fixation methods and conventional 10% v/v formalin fixation were negligible. Of the dehydration fixatives tested, 100% methanol did not cause regional differences due to artificial tissue shrinkage and the morphology of sections prepared by methanol fixation was preserved consistently better than that of acetone- or ethanol-fixed sections. All three dehydration fixatives preserved relatively higher-molecular-weight DNA and RNA, compared with formalin. Cold formalin, formalin containing EDTA at room temperature and 100% methanol are recommended as standard and additional fixatives routine clinic pathological laboratory use.     

Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity

Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson's II, 70% ethanol, UMFIX, modified Carnoy's, modified methacarn, Bouin's, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy's solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy's solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.     

Formaldehyde substitute fixatives. Analysis of macroscopy, morphologic analysis, and immunohistochemical analysis

Because formaldehyde is toxic and creates cross-links that may hinder immunohistochemical studies, we tested 3 new cross-linking (F-Solv [Adamas, Rhenen, the Netherlands]) and non-cross-linking (FineFIX [Milestone, Bergamo, Italy] and RCL2 [Alphelys, Plaisir, France]) alcohol-based fixatives for routine staining in comparison with neutral buffered formalin (NBF) as the "gold standard." Fresh tissue samples were divided into 4 equal pieces and fixed in all fixatives for varying times. After paraffin embedding, H&E staining, 7 common histochemical stains, and 9 common immunohistochemical stains were performed. RCL2 fixation resulted in soft and slippery tissue, causing sectioning difficulties. F-Solv and FineFIX led to partial tissue disintegration during fixation. F-Solv performed morphologically similar to NBF but needed considerable protocol adjustments before being applicable in daily histologic and immunohistochemical practice. FineFIX did not necessitate major protocol changes but caused shrinkage artifacts, degranulation, and lysis of RBCs. RCL2 generated morphologically overall good results without major protocol changes but caused pigment deposition, degranulation, and RBC lysis. The alcohol-based fixatives had positive and negative attributes and environmental drawbacks, and none was overall comparable to NBF with regard to macroscopy, morphologic evaluation, and immunohistochemical studies.     

A novel fixative improves opportunities of nucleic acids and proteomic analysis in human archive's tissues.

All tissues from biopsy or surgery origin are fixed and paraffin embedded as a routine procedure in the hospital departments of pathology. The traditional method of tissue preservation is the fixation in formalin, followed by paraffin embedding. In this way tissue's integrity is ensured also for future analyses, because there is no further chemical degradation of nucleic acids and proteins in tissues embedded in paraffin. After few sections for the histopathological examination the tissues are stored for decades in the hospital archives. Even if formalin fixation compromises the quality and integrity of nucleic acids, it has already been demonstrated that it is possible to recover and analyze DNA and RNA from these archive's tissues, even of autopsy origin. Protein analysis is on the contrary completely blocked, due to the fact that formalin fixation creates covalent links between proteins and the only way to study protein expression is immunohistochemistry. In this study we present our results concerning the use of a new formalin free fixative, the FineFIX. After extraction of nucleic acids, PCR and RT-PCR analyses were performed in DNA and RNA respectively. For DNA analysis it was possible to obtain amplicons of 2400 bps, while in formalin-fixed samples the maximum length achieved was less than 400 bps. RT-PCR analysis show that it was possible to study RNA fragments of 600 bps from FineFIX fixed tissues, against a maximum length of about 150 bps achieved by formalin-fixed tissues. These tissues were analyzed also by Western Blot analysis, showing that the proteins obtained from FineFIX treated samples are amenable and comparable in quality with those obtained from fresh frozen tissues. Protein extracts from FineFix treated tissues were also compared with fresh tissues'ones by two dimensional electrophoresis, demonstrating that the protein pattern were well comparable for number and distribution of the spots.     

Effect of delayed formalin fixation on estrogen and progesterone receptors in breast cancer: a study of three different clones

We previously reported that delayed formalin fixation (DFF) has a negative effect on immunohistochemical staining of estrogen receptor (ER), progesterone receptor (PR), and HER2. The primary aim of the study was to determine if DFF affected commonly used clones of the ER and PR differentially. The specific clones evaluated were ER clones 1D5, 6F11, and SP1 and PR clones 16, 1E2, and PgR636. Ten breast cancer cases were dissected and fixed at different times (0, 10, and 30 minutes; 1, 2, 4, and 8 hours; and overnight) and were then stained with anti-ER and anti-PR antibodies. The mean Q score for ER started to decline at 2 to 4 hours for clones 1D5 and 6F11 and at 1 hour for SP1. SP1 was superior to 1D5 at the 8-hour mark (P = .03). The Q score for PR started to decline at 1 hour for clones PgR636 and 16 and 4 to 8 hours for 1E2 (P = .03). Based on our findings, it appears that regardless of the antibody clones evaluated, DFF has a negative effect on hormone receptors.     

Estrogen,progesterone, and HER‐2 receptor immunostaining in cytology: The effect of varied fixation on human breast cancer cells

The ASCO/CAP Expert Panel recommends that all invasive breast carcinomas and breast cancer recurrences be tested for ER, PR and HER‐2 expression. The guidelines for testing of surgical specimens by immunohistochemistry (IHC) are well defined, whereas they are lacking for cytological samples. We evaluated various fixation protocols for optimal receptor testing by immunohistochemistry and immunocytochemistry (ICC) of human breast cancer cell lines MCF‐7 (ER/PR positive) and SKBR‐3 (overexpressing HER‐2). The cells were fixed in 10% neutral buffered formalin or Saccomanno Fixative (SF) for various time points, and either embedded in paraffin as cell blocks or prepared as cytospins. ER and PR slides were assigned a proportion score (PS; 0–5), an intensity score (IS; 0–3) and a total score (TS = PS + IS). Standard DAKO scoring system ranging from 0 to 3+ was used for the evaluation of HER‐2 staining. Human breast cancer cells stained successfully for ER, PR and HER‐2 when fixed in formalin and prepared as cell blocks. The optimal fixation time for formalin‐fixed cells ranged from 2 to 96 hours. Cells fixed in SF from 2 to 96 hours also stained well for ER and PR. However, SF produced variable results for HER‐2 staining; particularly, SF fixation beyond 24 hours caused false negative results. The interpretation of HER‐2 staining on cytospins was not feasible irrespective of the fixative and fixation time. In summary, formalin fixation from 2 to 96 hours and preparation of cells as cell blocks produces optimal results for ER, PR, and HER‐2 testing in human breast cancer cells. Diagn. Cytopathol. 2013;41:864–870. © 2013 Wiley Periodicals, Inc.     

Human intestinal mucosal mast cells: evaluation of fixation and staining techniques

The staining properties of tissue mast cells are influenced by the method of fixation. Differences in fixation and staining techniques may explain the contradictory results in the published reports on the number of human mucosal mast cells (MMC) in the gastrointestinal mucosa in health and disease. We have examined the influence of fixatives on the staining properties of human MMC in operative biopsy specimens of human jejunum. Specimens were divided into pieces, each of which was fixed in one of the following fixatives: Carnoy's, basic lead acetate (BLA), Baker's, Bouin's, isotonic formol-acetic-acid (IFAA), 10% neutral buffered formalin, formol sublimate, and formol saline. Thereafter, tissues were paraffin-embedded and 5 micron sections were cut and stained with either astra-blue/safranin pH 0.3, or toluidine blue pH 0.5. Counts of the number of MMC/mm2 were obtained for each fixation method. The results show a critical influence of the fixative on the number of mast cells identified after staining. For example with astra-blue/safranin the mean MMC/mm2 count was 40 in formol-saline-fixed specimens, and 268 in Carnoy's-fixed specimens. In biopsies fixed with formalin-based fixatives, mast cells were more readily stained with toluidine blue. It is recommended that Carnoy's or BLA be used as the fixative for any light microscopic study of human MMC.     

Efficiency of in situ hybridization as a function of probe size and fixation technique

In an attempt to improve fixation technique for viral RNA detection by in situ hybridization, we have quantitatively compared the hybridization signal obtained when measles virus or visna virus infected cell cultures were fixed with eight different fixatives and hybridized with 35S-labeled virus-complementary DNA probes of several size ranges. Small probes (mean length, 70 bases) gave higher signals than larger probes (mean lengths 140, 350, and 780 bases) with all fixatives. This increase in signal was minimal with acetic ethanol or formalin, but was dramatic with fixatives containing glutaraldehyde; with these fixatives the signals with small probes were 6.5- to 22-fold greater than with large probes. The highest signals were obtained with periodate-lysine-paraformaldehyde-glutaraldehyde (PLPG) fixed cells hybridized with small probes, and were 1.5- to 6.7-fold greater than those obtained with the commonly used fixative acetic ethanol. PLPG and other glutaraldehyde based fixatives also greatly improved the preservation of cellular morphology compared to acetic ethanol.     

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.     

Immunohistochemical demonstration of different latent membrane protein-1 epitopes of Epstein-Barr virus in lymphoproliferative diseases.

AIM--To compare the immunoreactivity of monoclonal antibodies S12 and CS1-4, which recognise different epitopes of the Epstein-Barr virus (EBV) latent membrane protein-1 (LMP-1), in EBV associated benign and malignant lymphoproliferative disorders and control tissues processed using different methods. RESULTS--Both monoclonal antibodies gave comparable results on frozen tissue sections and formalin fixed, paraffin wax embedded samples from cases with Hodgkin's disease and infectious mononucleosis. In all cases S12 stained more cells than CS1-4. For EBV associated B and T non-Hodgkin's lymphomas, frozen tissue sections yielded better LMP-1 staining results than formalin fixed material. Again, in all these cases S12 stained more cells and gave stronger results than CS1-4. For EBV negative tissues, both monoclonal antibodies showed cross-reactivity with melanocytic-like cells in the basal cell layer of the skin, synaptophysin-like staining in layers three and four of the cortex of the brain, and myelin-like staining in peripheral nerves and peripheral ganglion cells. Staining with S12 was always much stronger. Moreover, in contrast to CS1-4, S12 stained pancreatic islands in formalin fixed material but not in frozen tissue sections and sporadically stained solitary epithelial cells in the large bowel especially in formalin fixed tissue sections. CS1-4 also cross-reacted with myoepithelial cells around hair follicles and other adnexa of the skin. CONCLUSION--The results indicate that for optimal detection of LMP-1, S12 yields better results than CS1-4 and that tissue processing is very important especially when B and T non-Hodgkin's lymphomas are examined.     

The effect of fixation and trypsinization on the immunohistochemical demonstration of intracellular immunoglobulin in paraffin embedded material

Using an indirect labelled immunoperoxidase technique the influence of fixation time on the antigenicity of intracellular immunoglobulin in lymphoid tissue fixed in buffered formalin has been investigated. Within a fixation period of 96 hours a decrease of 15% of stainable immunoglobulin containing cells was found, for every 24 hours the fixation time was prolonged. By comparing sections from tissue fixed in buffered formalin and selected fixatives (Lillie's AAF, Bouin's fluid, Clarke's fluid and 96% ethanol 1% acetic acid (E--A) processed at 4 degrees C and at 25 degrees C) an increased number of stained immunoglobulin containing cells was found in tissue fixed in Lillie's AAF, Bouin's fluid, Clarke's fluid and E--A processed at 4 degrees C. No difference was found between tissue fixed in buffered formalin and E--A processed at 25 degrees C. In addition the effect of pretreatment of the sections with trypsin on the number of stainable immunoglobulin containing cells was investigated. Trypsinization of sections from formalin fixed material increased the number of stainable cells substantially. No essential effect was seen on tissue fixed in Lillie's AAF and Bouin's fluid. In contrast trypsin treatment of sections from tissue fixed in Clarke's fluid and E--A completely destroyed the tissue. No differences were observed between different immunoglobulin classes examined as regards the effect of fixation time, fixatives and trypsinization.     

Systematic comparison of tissue fixation with alternative fixatives to conventional tissue fixation with buffered formalin in a xenograft-based model

In our study we systematically compared the alternative fixatives acidified formal alcohol (AFA), PAXgene?, HOPE?, and combinations of AFA or formalin with ultrasound treatment to standard (buffered) formalin fixation. We examined general morphology and detectability of protein structures by immunohistochemistry of the membrane receptors epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), and phosphorylated human epidermal growth factor receptor 2 (phospho-HER2). In order to allow for stringent comparability of different fixation techniques, we used matched mouse xenograft tumor samples from three different human cancer cell lines (colon, ovarian, and non-small cell lung cancer), either fixed conventionally with formalin or an alternative fixative. Tissue morphology after fixation with AFA and PAXgene? was comparable to formalin-fixed paraffin-embedded tissue (FFPET) morphology. Ultrasound fixations resulted in slightly inferior morphology and HOPE? fixation preserved morphology only poorly compared to FFPET in this system. None of the tested alternative fixatives enabled immunohistochemical detectability of all three targets in the same manner as FFPET. Pronounced staining was possible for EGFR and IGF-1R with all alternative fixatives but HOPE?, and phospho-HER2 staining was only noteworthy with formalin-ultrasound-fixed tissue. Therefore, the use of alternative fixatives comes with the need for careful validation of obtained IHC results individually for each target.     

Impact of fixative on recovery of mRNA from paraffin-embedded tissue.

Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.     

Immunohistochemical assays in prostatic biopsies processed in Bouin's fixative

AIMS: To investigate the problems involved in undertaking immunohistochemistry (IHC) and nuclear morphometry using Bouin's fixed prostate biopsies. METHODS: Archival Bouin's fixed and formalin fixed, paraffin wax embedded prostatic biopsies were immunostained for three nuclear biomarkers (minichromosome maintenance protein 2 (MCM-2), p27, and Ki-67), one membrane localised biomarker (C-erb-B2), CD34, and alpha methylacyl-CoA racemase (AMACR). The quality of IHC staining was compared between tissues prepared separately in both fixatives. Feulgen staining was also performed on Bouin's fixed tissues to check its suitability for nuclear morphometry. RESULTS: MCM-2 staining was completely negative in Bouin's fixed tissues, whereas p27 showed more background and excess cytoplasmic staining in Bouin's fixed versus formalin fixed tissues. C-erb-B2 showed non-specific, strong luminal cell staining in the Bouin's fixed tissue. Feulgen staining was also very weak in Bouin's fixed tissue. However, Ki-67, AMACR, and CD34 worked equally well in Bouin's and formalin fixed tissues. CONCLUSIONS: Bouin's fixed tissues may be unsuitable when subsequent IHC and morphometry are contemplated. An awareness of which antibodies are suitable for use in Bouin's fixed biopsies is essential.     

Monoclonal antibody (UCHL1) that recognises normal and neoplastic T cells in routinely fixed tissues.

UCHL1 is a murine monoclonal antibody that recognises a 180-185 kD determinant on CD4 (72%) and CD8 (36%) positive T cells. This antibody is effective in formalin fixed and paraffin embedded tissues, using the immunoperoxidase method. One hundred and forty three cases of malignant lymphoma were examined. Neoplastic cells in 100% of cases of Mycosis fungoides (n = 10), 83% of cases of peripheral T cell lymphoma (n = 25), and 78% of cases of (T-ALL) T acute lymphoblastic lymphoma (n = 9) were stained by this antibody. In addition, staining was seen in 100% of cases of malignant histiocytosis of the intestine (n = 13), a condition now thought to be a T cell lymphoma. Two cases of true histiocytic lymphoma were also positive. This antibody stained neither the neoplastic cells in a wide range of B cell lymphomas (n = 62) nor Reed-Sternberg cells in 16 cases of Hodgkin's disease. UCHL1 also stained neoplastic cells in four cases of granulocytic sarcoma. A panel of normal tissues was similarly studied. Staining was seen in normal T cells and mucosal intraepithelial lymphocytes, macrophages, mature myeloid cells, and endometrial stromal granulocytes. UCHL1 is a monoclonal antibody that identifies T cells in formalin fixed paraffin embedded tissues, and should prove useful for diagnosing T cell lymphomas, especially when only formalin fixed tissue is available for diagnosis.     

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.     

Rabbit monoclonal antibodies show higher sensitivity than mouse monoclonals for estrogen and progesterone receptor evaluation in breast cancer by immunohistochemistry

A novel generation of rabbit monoclonal antibodies for estrogen (ER) and progesterone (PR) receptor evaluation in breast cancer by immunohistochemistry has been released recently. We compared the novel RabMab anti-ER and anti-PR antibodies with the mouse monoclonal antibodies using a tissue microarray of breast carcinomas. Two cylinders (2mm diameter) of formalin-fixed, paraffin-embedded tissue were obtained from 24 invasive breast cancers and were immunostained using anti-ER mouse (1D5 and 6F11) and rabbit antibodies (SP1 and B644), and anti-PR mouse (PgR312 and PgR636) and rabbit antibodies (SP2 and B645). The immunohistochemistry was evaluated by considering positive those tumors in which more than 10% of the tumor cell nuclei stained independently on the staining intensity. Our results demonstrated that rabbit antibodies against ER have a similar staining pattern compared to the 6F11, but better than 1D5 from three different suppliers. The rabbit antibodies against PR (SP2 and B645) provide a stronger and sharper immunohistochemical signal compared to mouse antibodies (PgR636 and PgR312). Both ER and PR rabbit antibodies allow a lower cost per test because of higher working dilutions compared to mouse antibodies using the same procedure. The novel rabbit antibodies against ER and PR are highly sensitive for immunohistochemical testing of breast carcinomas.