Use of freeze-drying technology for the study of renal biopsies

Adequate handling of renal biopsies requires processing for light and electron microscopy, as well as for immunohistochemical study. Problems of adequate sampling are frequently encountered, and tissue size can be insufficient, since morphologic examination requires chemical fixation, while immunofluorescence study is currently performed on snap-frozen, cryostat-cut tissue. The application of freeze-drying technology on 20 renal needle biopsies has been investigated to assess the feasibility and reliability of the technique as a routine diagnostic tool in renal pathology. Morphologic as well as immunohistochemical studies were performed on freeze-dried, paraffin-embedded specimens, including immunofluorescence and PAP techniques to detect immunoglobulins, complement fractions, fibrinogen, collagen types, and laminin. Our results showed good preservation of tissue morphology, similar to standard formalin fixation. Moreover, absence of diffusion artifacts and intensity of immunohistochemical reactions were comparable to what is obtained with cryostat sections. We therefore suggest that freeze-drying before paraffin embedding is, at least for diagnostic purposes, preferable to formalin fixation; unfixed, cryostat-cut sections can still be informative and should be used whenever tissue is available in selected cases, and/or in experimental work.     

New monoclonal antibodies to oestrogen and progesterone receptors effective for paraffin section immunohistochemistry

Assessment of oestrogen and progesterone receptors (ER and PgR) in breast cancer is widely used for the prediction of response to endocrine therapy and as a prognostic marker. Cytosolic assays have been replaced in many centres by immunochemical techniques, which have many advantages including applicability to small samples, simplicity, and cost-effectiveness. This study describes the generation and characterisation of two novel murine monoclonal antibodies recognizing ER and PgR, designated NCL-ER-6F11 and NCL-PGR respectively, which are effective in heat-treated formalin-fixed, paraffin-embedded tissue. The antibodies have been characterized by Western blotting and by immunohistochemistry on normal and pathological breast and other tissues. NCL-ER-6F11 has been shown to compare favourably with a currently available ER antibody. These antibodies may prove of value in the assessment of hormone receptor status in human breast cancer. © 1997 John Wiley & Sons, Ltd.     

Effects of formalin fixation, paraffin embedding, and time of storage on DNA preservation in brain tissue: a BrainNet Europe study

There is a large amount of tissue stored in brain collections and brain banks, but little is known about whether formalin-fixed tissues and paraffin blocks stored for years in brain banks are suitable for the retrospective genetic studies. The study was carried out in order to: (i) compare DNA preservation in frozen, formalin-fixed and paraffin-embedded tissues stored for different periods; (ii) study point mutations and triplet expansions in frozen, formalin-fixed and paraffin-embedded material stored for variable periods, and using different fixative solutions; (iii) compare different methods to optimize DNA extraction and DNA amplification from suboptimally preserved brain tissue. DNA preservation is suitable for genetic studies in samples stored at -80 degrees C for several years. Formalin-fixed, paraffin-embedded tissue was inferior to frozen tissue, but did yield adequate results in many cases depending on the type of fixative solution and time of fixation before embedding. Prolonged fixation in formalin rarely yielded useful DNA. Similar results were obtained in samples from prion diseases. The best results were obtained by using the Qiagen kits (QIAmp DNA Micro) in frozen material, paraffin blocks and formalin-fixed tissue. Genomiphi and TaKaRa Ex Taq methods were also assayed in paraffin blocks and in formalin-fixed samples with limited success.     

Oestrogen receptor, progesterone receptor, pS2, ERD5, HSP27 and cathepsin D in invasive ductal breast carcinomas

Hormonal receptors and markers for prognostic evaluation were detected immunohistochemically in 196 infiltrating ductal breast carcinomas. Immunohistochemical detection of progesterone and oestrogen receptor is a method giving results generally concordant with those of the binding assay. However, immunohistochemical detection seems better. It allows the detection of hormonal receptors on small carcinomas, it is not modified by the endogenous hormones, and it has a slightly better correlation with prognosis and with the response to hormone therapy. Immunohistochemical detection of progesterone receptor has a prognostic value, sorting a negative subgroup with a poor prognosis from the oestrogen receptor positive tumours. These results can be obtained without quantitative immunohistological methods. ERD5, pS2, HSP27 and cathepsin D are associated with oestrogen receptor positivity. pS2 and HSP27 are interesting markers. They characterize a subgroup of oestrogen receptor negative tumours with a good prognosis. Moreover, pS2 is a marker of response to hormone therapy. ERD5 and cathepsin D do not appear to be of value as markers of prognosis.     

Immunohistochemical quantitation of oestrogen receptors and proliferative activity in oestrogen receptor positive breast cancer.

AIM--To evaluate the effect of the duration of formalin fixation and of tumour heterogeneity on quantitative estimates of oestrogen receptor content (oestrogen receptor index) and proliferative activity (MIB-1 index) in breast cancer. METHODS--Two monoclonal antibodies, MIB-1 and oestrogen receptor, were applied to formalin fixed, paraffin wax embedded tissue from 25 prospectively collected oestrogen receptor positive breast carcinomas, using a microwave antigen retrieval method. Tumour tissue was allocated systematically to different periods of fixation to ensure minimal intraspecimen variation. The percentages of MIB-1 positive and oestrogen receptor positive nuclei were estimated in fields of vision sampled systematically from the entire specimen and from the whole tumour area of one "representative" cross-section. RESULTS--No correlation was found between the oestrogen receptor and MIB-1 indices and the duration of formalin fixation. The estimated MIB-1 and oestrogen receptor indices in tissue sampled systematically from the entire tumour were closely correlated with estimates obtained in a "representative" section. The intra- and interobserver correlation of the MIB-1 index was good, although a slight systematical error at the second assessment of the intraobserver study was noted. CONCLUSION--Quantitative estimates of oestrogen receptor content and proliferative activity are not significantly influenced by the period of fixation in formalin, varying from less than four hours to more than 48 hours. The MIB-1 and the oestrogen receptor indices obtained in a "representative" section do not deviate significantly from average indices determined in tissue samples from the entire tumour. Finally, the estimation of MIB-1 index is reproducible, justifying its routine use.     

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.     

Immunohistochemical demonstration of androgen receptor in breast cancer and its relationship to other prognostic factors

Androgen actions and androgen receptors (ARs) have been described in human breast cancer cells both in vivo and in vitro. With the use of a new monoclonal anti-AR antibody, AR was immunohistochemically demonstrated in 76 primary breast cancers. Positive immunostaining was found in 79 per cent of tumours. Benign ductal epithelium was often AR-positive whereas the tumour stroma lacked AR immunoreactivity. At the subcellular level, nuclear localization was evident using either cross-linking (Zamboni's fluid) or precipitating (acetone) fixatives on frozen sections. The use of archival paraffin-embedded tissue yielded negative results. A significant association was found between expression of AR and oestrogen receptor (ER) (P=0.0006) determined immunohistochemically on adjacent sections. Most progesterone receptor (PR)-negative cases were also AR-negative (P=0.02), but significant proportion (38 per cent) of AR-positive tumours did not contain PR. Unlike ER, AR was not associated with aneuploidy or erb-B2 oncogene overexpression, and was only marginally associated with tumour proliferation rate (S-phase fraction by DNA flow cytometry). In conclusion, the close association of AR with ER and PR suggests that immunohistochemical determination of androgen receptors may have value as a prognostic factor and/or predictor of response to endocrine therapy.     

Oestrogen and progesterone receptors in screen-detected breast carcinoma: an immunohistological study using paraffin sections

The presence of oestrogen and progesterone receptors was studied in paraffin sections of 81 screen-detected breast carcinomas using the monoclonal antibodies ER-ICA and PgR-ICA (Abbott) and the immunoperoxidase technique. The immunohistological results were compared with the results of the standard dextran-coated charcoal biochemical assay in 28 tumours which were big enough to provide tumour tissue for this assay. Sixty-three cases (78%) were oestrogen receptor positive and 62 (77%) were progesterone receptor positive. There was no statistical difference between receptor positivity in palpable or impalpable, in situ or invasive tumours. In the 28 cases where the biochemical assay was carried out, the two methods gave similar results in 23 (82%) and 21 (75%) tumours for oestrogen and progesterone receptors respectively. The majority of the remaining tumours, with one exception, were positive with immunohistology and negative with biochemistry. A good correlation was also present between the mean numerical biochemical values and the semiquantitative histological scores for both receptors. It is concluded that assessment of receptor status of small screen-detected carcinomas is feasible using routinely processed paraffin sections. There is reasonably good correlation with the results obtained by the standard dextran-coated charcoal biochemical assay, but more genuine receptor positive cases are detected by immunohistology.     

Progesterone receptors in human oestrogen target tissues

The levels of specific oestrogen and progesterone receptors have been measured in myometrial, endometrial and vaginal tissues obtained from 18 women between the ages of 28–73 yr.High speed cytosols of the three tissues were incubated at four different concentrations of specific ligands: E₂ for the oestrogen receptor (ERc) and Org-2058 for the progesterone receptor (PgRc). Separation of bound and free hormones was done by dextran-coated charcoal; data were analysed according to Scatchard.In the myometrium and endometrium both PgRc and ERc were found. In the vaginal tissues obtained from the same patients only ERc was present. There was no evidence of specific progesterone receptors in the vagina. Both clinical and histological findings indicate that the vagina is an oestrogen-sensitive organ. Therefore, it is surprising that progesterone receptors, which are considered to be a specific response of oestrogen target tissue, are absent in the human vagina.This finding suggests that the induction of the progesterone receptors is not a specific response to oestrogen stimulation in the human vagina.     

Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.

In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.     

Immunocytochemical staining of progesterone receptor in paraffin sections of human breast cancers.

Monoclonal antibodies (mAB) against progesterone receptor (PR) and the peroxidase-antiperoxidase (PAP) method to visualize PR in paraffin sections from 68 human breast cancers were used. Ten mAB, which recognize human PR on frozen sections, were tested. Six could detect PR in paraffin sections, with Li 417, LET 456, and LET 126 giving the best results. LET 126 antibody was used for most further studies. The effects of fixation with picric acid-formaldehyde (PAF), buffered-formalin, or with Bouin solution were investigated; all fixation methods allowed PR immunolabeling, although PAF or buffered formalin usually gave the best results. Positive staining was seen in the nucleus of carcinoma cells. Variations in intensity and extent of immunoreactivity were observed in all sections and among different regions of the same specimen. These were probably related to the heterogeneity of the tumor cell population. Results were compared with the PR content in the respective tumor tissues, determined by steroid-binding assay, and with immunocytochemistry on frozen sections. It was shown that there were correlations between the immunocytochemical staining (positive or negative) and the steroid binding assay (80%) and between the immunocytochemical staining on paraffin sections and on frozen sections (78%). Weaker intensity and fewer number of PR-positive cells were found for paraffin-embedded tumors. Estrogen receptors were also detected on adjacent sections from the same paraffin-embedded tissues by use of monoclonal anti-ER antibodies (ERICA-kit[Abbott Laboratories, Chicago, IL]) and DNase pretreatment. In conclusion, this immunocytochemical method for detection of PR and ER on paraffin sections offers an alternative to frozen tissue. It allows histologic and immunocytochemical studies on the same sample and retrospective studies on stored tissue blocks.     

The effects of glyoxal fixation on the histological evaluation of breast specimens

Glyoxal (GL), a non-formalin-containing aldehyde tissue fixative, is advocated as a superior fixative that is environmentally safe and lacks the purported carcinogenic health hazards associated with formalin use. In addition, it is advertised as requiring no antigen retrieval before immunohistochemical staining. We compared GL fixation to standard formalin fixation of breast specimens removed for microcalcifications or breast tumors. Although the hematoxylin and eosin morphology of GL-fixed and formalin-fixed tissues was equivalent, detection of microcalcifications in GL-fixed breast specimens was hampered by loss of basophilia, likely due to increased calcium solubility in glyoxal. Moreover, estrogen receptor detection in GL-fixed specimens was diminished compared to formalin and did require antigen retrieval.     

Steroid hormone receptors in human salivary gland tumours.

Major salivary gland tumours were studied for the presence of hormone receptors for oestrogen and progesterone. Of the eight salivary gland tumours exhibiting varied histology, none showed high affinity receptors for oestrogen or progesterone. Salivary tissue from four patients with non-neoplastic salivary gland disease was also studied and found not to contain high affinity receptor sites. The absence of hormone receptors in these glands suggests that such tumours are not dependent on endocrine function.     

Differential expression of glucocorticoid receptor in human breast tissues and related neoplasms

Glucocorticoid receptor (GR) is a steroid hormone receptor that has been shown to play important roles in mammary development and differentiation, and has been implicated in breast tumourigenesis, but its precise biological significance in mammary pathophysiology remains unclear. In order to generate a comprehensive expression profile for GR in normal versus neoplastic breast tissues, GR expression was investigated in situ in 400 human breast tissue samples, comprising normal tissue and a range of benign, pre-invasive, and invasive lesions, using immunohistochemical assays. The novel expression of GR in myoepithelium, not observed in luminal epithelium, not only demonstrates expression patterns exclusive to the alpha form of oestrogen receptor and progesterone receptor and suggests distinctive functions between GR and these two important steroid hormone receptors in the breast, but may also indicate unique physiological and perhaps pathological roles for the myoepithelium in mediating the effects of glucocorticoid hormones in the breast. The strong expression of GR in metaplastic carcinomas (94.4%) and malignant phyllodes tumours (92.3%) suggests a pathogenetic role for GR, and implies that targeting GR in these tumours may have potential therapeutic application. However, studies on the roles of GR in mammary carcinogenesis should be interpreted with great caution, based on the lack of GR expression in cancer cells in the great majority (98.2%) of non-metaplastic carcinomas, which has gone unnoticed in previous studies. This marked discrepancy warrants a re-examination of the biological roles of GR in the pathophysiology of breast malignancy. The lack of methylation in the promoter region of the GR gene in all 118 non-metaplastic carcinomas, as demonstrated by methylation-specific PCR and bisulphite DNA sequencing analysis, indicates that methylation is less likely to play a role in the reduction of GR expression in non-metaplastic carcinoma of the breast.     

Immunohistochemical localisation of androgen receptor in apocrine metaplasia and apocrine adenosis of the breast: relation to oestrogen and progesterone receptors

AIM: To investigate the receptor status of the sex steroid hormones in apocrine metaplasia of the breast. METHODS: 82 cases of apocrine metaplasia, including 18 of the rare lesion apocrine adenosis, were studied immunohistochemically for the expression of androgen receptor, oestrogen receptor, and progesterone receptor proteins on formalin fixed, paraffin embedded tissue sections. The standard avidin biotin complex (ABC) technique was followed and appropriate positive and negative controls were used. RESULTS: All the studied cases (82/82) were positive for androgen receptor, but were negative for oestrogen receptor and progesterone receptor. CONCLUSIONS: Apocrine metaplastic epithelium, unlike the normal breast epithelium, is responsive to androgens, through androgen receptors, rather than to the female sex hormones. This may have clinical implications.     

Ultrasound-accelerated formalin fixation of tissue improves morphology, antigen and mRNA preservation.

Formalin fixation and paraffin embedding are conventional tissue preservation and processing methods used for histologic diagnosis in over 90% of cases. However, formalin fixation has three disadvantages: (1) slow fixation (16-24 h) hinders intraoperative decision making, (2) slow quenching of enzymatic activity causes RNA degradation, and (3) extensive molecule modification affects protein antigenicity. Applying high-frequency, high-intensity ultrasound to the formalin fixative cuts fixation time to 5-15 min. Fixation of various tissues such as lymph node, brain, breast, and prostate suggests that, compared to the conventional method, implementation of ultrasound retains superior and more uniform tissue morphology preservation. Less protein antigenicity is altered so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. Better RNA preservation results in stronger signals in in situ hybridization and longer RNA fragments extracted from fixed tissues, probably due to rapid inhibition of endogenous RNase activity. Molecules extracted from ultrasound-fixed tissues are of greater integrity and quantity compared to conventionally fixed tissues, and thus better support downstream molecular analyses. Overall, ultrasound-facilitated tissue preservation can provide rapid and improved morphological and molecular preservation to better accommodate both traditional and molecular diagnoses.     

Triple negative breast carcinoma: the good,the bad and the ugly

Triple-negative breast cancers (TNBC) are a heterogeneous group of breast cancers defined by their lack of expression of oestrogen and progesterone receptors as well as human epidermal growth factor receptor 2 amplification, and therefore, are resistant to hormonal and Trastuzumab therapy. TNBC accounts for 15% of all breast cancers, and are more common in African-American women than in Whites. Also, BRCA-1 associated tumours are usually TNBC. Since the majority of TNBC fall into the basal-like breast cancer category by molecular studies, they are generally regarded as tumours of poor prognosis. However, some TNBC, such as adenoid cystic carcinoma and medullary carcinomas have excellent prognosis. Others, like metaplastic carcinoma have a prognosis that is comparable to infiltrating ductal carcinoma, not otherwise specified (NOS). Many immunohistochemical markers have been studied as an adjunct tool in classifying TNBC. However, microscopic evaluation remains an important tool in classifying these tumours and therefore predicting their prognosis.     

Expression of the pS2 peptide in primary breast carcinomas: Comparison of membrane and cytoplasmic staining patterns

The pS2 protein is oestrogen-regulated in breast cancer cell lines. Previous studies have shown a relationship to oestrogen receptor in primary breast carcinomas. This study examined 178 breast carcinomas for pS2 using immunohistochemistry. A high frequency (77 per cent) of positive tumours was found, using a 10 per cent cut-off point to define a positive tumour. There was no relationship with menopausal status or node status, a significant association with differentiation, a weak association with oestrogen receptor, and no association with progesterone receptor or overall survival. Two patterns of cellular localization were observed: cytoplasmic and membrane. The former showed a stronger relationship with oestrogen receptor status, although there were oestrogen receptornegative tumours with marked pS2 staining. Membrane staining showed a stronger relationship with differentiation, with a staining pattern similar to that observed for milk fat globule membrane. The staining patterns observed may support a role for pS2 in a secretory mechanism. However, the expression and function of pS2 in breast carcinomas emain complex, and are not simply related to oestrogen regulation.     

The cytochemical demonstration of oestrogen receptors in human breast carcinomas

A cytochemical method has been used to demonstrate oestrogen receptor in histological sections of breast carcinomas, and has given similar qualitative results to the biochemical dextran-coated charcoal method. Two conjugation techniques were employed to couple horseradish peroxidase to 17β-oestradiol-6-O-carboxymethyloxime—bovine serum albumin. The periodate conjugation method led to a greater degree of coupling but a higher concentration was required for demonstration of oestrogen receptor than that needed when the two-stage glutaraldehyde conjugation method was used, even though this resulted in a lesser degree of coupling. Care in the preparation of tissue has been found to be essential. Formalin-fixed, paraffin-embedded tissue was unsuitable; rapid freezing of fresh tissue, only brief air-drying or acetone fixation of tissue sections and short-term storage were important. A correlation has been shown between the presence of oestrogen receptor in breast carcinomas and good histological differentiation. The poorer differentiated tumours were also found to contain fewer reactive cells.