Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.     

Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM).

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.     

Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization

Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.     

Double-fluorescence image microscopy for quantitation of prostate-specific antigen in histologic sections of the prostate

BACKGROUND: Although the assessment of serum prostate-specific antigen (PSA) has become a powerful instrument in the diagnosis and for prognosis of prostate carcinoma, there are few quantitative studies of PSA in tissue sections. METHODS: We developed a technique using double-fluorescence image microscopy for quantifying immunohistochemical reactions in tissue sections. PSA was stained by Texas Red and the cellular DNA was counterstained with 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI). The fluorescence of Texas Red and DAPI was quantified separately after subtraction of background and shading correction. The amount of PSA related to the amount of DNA in identical tissue parts was studied in archival specimens from patients with hyperplasia and prostate carcinoma. RESULTS: The amount of tissue PSA decreased with the increase in tumor grade, Gleason score, and the change from diploid to aneuploid. CONCLUSION: Double-fluorescence image microscopy is a valuable technique for obtaining quantitative information of cellular constituents. For standardization of immunochemical reactions in tissue sections, cellular DNA seems to be most appropriate.     

Feulgen DNA stainability of bone tumors after demineralization

Microspectrophotometric DNA analysis of archival bone tumor tissue is often impeded by previous acid demineralization, which destroys Feulgen DNA stainability. To find an alternative to acid for prospective DNA studies of bone tumors in tissue sections, Feulgen stainability of fresh osteosarcoma specimens after demineralization in neutral EDTA was investigated. The reliability of DNA analysis of weakly Feulgen-stained sections from archival tissue was also studied. Demineralization of four fresh specimens in EDTA slightly reduced Feulgen DNA stainability compared to nondemineralized preparations but did not affect the determination of ploidy level. Hydrolysis tests of one diploid and one hyperploid osteosarcoma showed that the staining relationship between control and tumor cells was not altered by EDTA pretreatment. For DNA studies of bone tumors requiring demineralization, EDTA offers a means of retaining nuclear Feulgen stainability. In 22 archival osteosarcoma specimens of varying Feulgen stainability, three different upper limits of light transmission (75, 85, and 95%) were applied to test the significance of background disturbances in relation to nuclear stain intensity. The relationship between the median total extinction of the control and tumor cell populations was not significantly affected by altering the upper transmission limit except in four poorly stained lesions. The control cells of these four specimens exhibited a median total extinction less than one-third of the maximum encountered. The results suggest that weakly stained archival specimens can be tested for selecting those appropriate for ploidy determination.     

Discrete localization of various fatty-acid-binding proteins in various cell populations of mouse retina

The analysis of myenteric neurons is becoming increasingly important for the assessment of enteric nervous system injury and degeneration occurring in motor disorders of the gut. Limited information is presently available on the quantitative estimation of myenteric neurons and glial cells in paraffin-embedded colonic sections; additional data would be useful for diagnostic purposes. In this morphometric study, we performed immunohistochemistry to count myenteric neurons and glial cells in paraffin sections of human colon. Serial cross sections of formalin-fixed paraffin-embedded full-thickness normal human left colon (n = 10, age-range: 50–72 years) were examined. HuC/D and S100β antigens were found to be the best markers for the detection of neurons and glial cells, respectively. Significant correlations were noted between the numbers of neurons/glial cells and the respective myenteric ganglion areas. These findings suggest that HuC/D-S100β-immunostained paraffin cross sections of human colon can be regarded as valuable tools for the quantitative estimation of myenteric neurons and glial cells. Based on the present method, only a limited number of paraffin sections are needed for reliable quantitative assessments of myenteric ganglion cells, thus allowing fast and simple approaches in the settings of the histopathological diagnosis of colonic motility disorders and retrospective evaluations of pathological archival tissue specimens. R. De Giorgio is the recipient of grants from the Fondazione Del Monte di Bologna e Ravenna and from the Fondazione Cassa di Risparmio, Bologna, Italy. The authors declare no conflicting interests.     

Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization

Gene expression in diseased tissues can indicate the contribution to a disease process and potentially guide therapeutic decision-making. Archival tissues with associated clinical outcome may be useful to discover or validate the role of a candidate gene in a disease process or the response to therapy. Such archival tissues are commonly formalin-fixed and paraffin-embedded, restricting the methods available for gene expression analysis. Obviously, the detection of proteins in tissues requires adaptation for each protein and the detection of secreted proteins can prove difficult or of reduced value since the protein detected may not reflect the total amount produced. Thus, we describe here a reliable method for the detection of mRNA in archival tissues. The method for mRNA in situ hybridization (ISH) was adapted by us for >15 different genes and applied to several hundred tissue microarrays (TMAs) and full sections generating >10,000 expression data points. We also discuss the utility of TMAs to simultaneously analyze several hundred tissue samples on one slide to minimize variability and preserve valuable tissue samples. Experimental protocols are provided that can be implemented without major hurdles in a typical molecular pathology laboratory and we discuss quantitative analysis as well as advantages and limitations of ISH with a special focus on secreted proteins. We conclude that ISH is a reliable and cost effective approach to gene expression analysis in archival tissues that is amenable to screening of series of tissues or of genes of interest.     

The discrimination of high-risk HPV types by in situ hybridization and the polymerase chain reaction

Summary The parameter Tm^t has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tm^t) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tm^t is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tm^t is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy.Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material.     

Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.     

DNA extraction from archival formalin-fixed,paraffin-embedded tissues: heat-induced retrieval in alkaline solution

Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5–12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.     

Retrospective molecular detection of transhyretin Met 111 mutation in a Danish kindred with familial amyloid cardiomyopathy,using DNA from formalin-fixed and paraffin-embedded tissues

Severe familial amyloid cardiomyopathy (FAC) in a Danish kindred is associated with a specific mutation (Met for Leu 111) in the transthyretin (TTR) gene. The mutation causes the loss of a DdeI restriction site in the gene, allowing molecular diagnostic studies. We studied formalin-fixed, paraffin-embedded tissues, up to 39 years old, from 29 family members of this kindred. DNA was partially purified from deparaffinized tissue sections and a DNA sequence of the TTR gene flanking the mutation site was amplified by the polymerase chain reaction (PCR), followed by restriction enzyme analysis. Amplified DNA was obtained from tissues representing 23 of the 29 persons. Ten out of the 23 family members were found to carry the TTR Met 111 mutation, whereas 13 were not affected. The results were consistent with known clinical data and with corresponding serum TTR examinations. This retrospective study shows that archival tissues can be used to confirm the diagnosis and disease pattern in members of families affected by hereditary diseases.     

Effect of bone decalcification procedures on DNA in situ hybridization and comparative genomic hybridization. EDTA is highly preferable to a routinely used acid decalcifier.

Decalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on the use of these techniques on archival decalcified bony material. In this study we compared the effects of two commonly used decalcifiers, i.e. , one proprietary, acid-based agent (RDO) and one chelating agent (EDTA), in relation to subsequent DNA ISH and CGH to bony tissues (two normal vertebrae, six prostate tumor bone metastases with one sample decalcified by both EDTA and RDO). We found that RDO-decalcified tissue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal status of EDTA-decalcified material of both control and tumor material. Gel electrophoresis revealed that no DNA could be successfully retrieved from RDO-treated material. Moreover, in contrast to RDO-decalcified tumor material, we detected several chromosomal imbalances in the EDTA-decalcified tumor tissue by CGH analysis. Furthermore, it was possible to determine the DNA ploidy status of EDTA- but not of RDO-decalcified material by DNA flow cytometry. Decalcification of bony samples by EDTA is highly recommended for application in DNA ISH and CGH techniques.     

Postadhesive Technique for Archival Paraffin Sections

We present a postadhesive protocol for adhering paraffin sections of archival material to microscope slides. Appropriately posttreated sections, subsequently processed for immunohistochemistry, remained attached to the slides and were well preserved with no signs of artifacts, such as scratching and shrinkage. The immunohistochemical staining was intense and antigen-specific without nonspecific background. Specific staining intensity was equal to that produced in untreated control sections; however, the latter became partially or fully detached from the slides. The postadhesion protocol may be used with modern techniques and is recommended for reclaiming use of otherwise unsuitable paraffin sections of archival material.     

News in Brief

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.     

Cytophotometric DNA measurements of chondrosarcoma: methodologic aspects of measurements in tissue sections from old paraffin-embedded specimens

The methodologic prerequisites of cytophotometric DNA measurements of normal and tumor cells in tissue sections obtained from paraffin blocks and preserved as archival material were investigated. The optimal time of hydrolysis in 5-N HCl at room temperature was one hour for the different cell types analyzed. Paraffin-embedded tissues, stored for two decades, were still suitable for quantitative cytophotometric DNA determinations of Feulgen-stained nuclei. Different cell types in the Feulgen-stained sections could be identified with accuracy. The 90th percentile of fibroblast (internal control cells) DNA values was used as an upper limit of the diploid DNA content. By determining the number of tumor cells with DNA values exceeding this limit, nondiploid (hyperploid) tumor cell populations could be discriminated from diploid tumor cell populations. Cell population analysis of ploidy level, performed in this way, was found to be accurate in tissue sections of 4 micrometer. The accuracy of this analysis was not improved by increasing the section thickness. Since tissue sections obtained from old paraffin blocks could be used for the determination of hyperploidy, the prognostic significance of this parameter in different tumors can be assessed retrospectively.     

Characterization of diverse ploidy in the arctic‐alpine Arenaria ciliata species complex (Caryophyllaceae) using shoot meristem staining and flow cytometry analysis of archived frozen tissue

Ploidy levels were analyzed in 21 European populations of the Arenaria ciliata complex using baseline chromosome counts derived from Feulgen staining of HCl‐treated shoot meristems and calibrated flow‐cytometry analysis of fresh and archival frozen tissue. Calibration with two to three control samples of different ploidy facilitated rapid identification of ploidy states in unknown samples. Observed ploidy levels varied from 2N = 40–200, with the majority of populations showing 2N = 40–80. High‐altitude populations collectively showed the full range of ploidy states, but at low elevations only lower ploidy levels were observed. Populations with the highest observed ploidy contained the greatest observed phylogenetic diversity in the western and eastern Alps. Multiple polyploidization events are inferred in the continental European metapopulation, with lower, more stable ploidy characteristic of the west and north. The method deployed provides an effective approach to ploidy analysis for archival desiccated/frozen tissue samples from biogeographic collections.     

Detection of human papilloma virus DNA sequences by polymerase chain reaction

We have developed a polymerase chain reaction-based procedure for reproducible detection of the E6-E7 gene in human papilloma virus DNA sequences using formalin-fixed, paraffin-embedded tissue sections. This procedure is a simple one-step procedure which does not require any elaborate hybridization following polymerase chain reaction amplification. The protocol combines modified tissue treatment and proper primer selection for efficient amplification of target DNA in a highly specific manner allowing identification in ethidium bromide-stained gels. The procedure described here is useful for a variety of tissue preparations, particularly formalin-fixed, paraffin-embedded archival tissues.     

DNA content in fresh versus paraffin-embedded tissue. Flow cytometric analysis of 100 tumors.

DNA ploidy analysis was determined on 100 consecutive tumors from a wide variety of sites using both fresh and paraffin-embedded tissue on the same specimen. The correlation coefficient (r) value between the methods was 0.85. Aneuploidy was detected by both methods in 51/100 (51%) of the cases. Fresh tissue analysis yielded 10 additional cases (overall 61% aneuploidy) not detected on corresponding paraffin-embedded sections, whereas paraffin-embedded analysis detected 4 additional cases (overall 55% aneuploidy) not revealed by fresh tissue analysis. Fresh tissue analysis produced lower coefficients of variation and resulted in a cleaner preparation with less cellular debris. Fresh tissue analysis was also superior to paraffin for the detection of hypodiploid, near-diploid and multiple peaks. Analysis of paraffin-embedded material allows examination of archival tissue and provides a more rapid means of long-term follow-up and statistical correlations for prognostic studies. Although the overall correlation of both methodologies for DNA analysis showed a minimal variation in results, in our experience fresh tissue analysis has an advantage and is preferable, when available, for ploidy analysis.