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PubMed1992

Detection of c-erbB-2 activation in paraffin-embedded tissue by immunohistochemistry.

Commercially available monoclonal antibodies were tested for their ability to detect increased levels of c-erbB-2 protein in formalin-fixed, paraffin-embedded breast carcinomas. Of five antibodies studied, four (TAB-250, CB11, 3B5, and N3/D10) showed strong cytoplasmic membrane reactivity in 23% (11 of 47) of routinely processed tumors, although interpretation of the immunoreactivity with 3B5 and N3/D10 occasionally was difficult due to cytoplasmic granular staining. Since the c-erbB-2 oncogene is activated by DNA amplification and overexpression of mRNA and protein, the same tumors were analyzed for c-erbB-2 activation by other techniques. c-erbB-2 activation in these 11 tumors was confirmed by immunohistochemistry of frozen tissue (nine of nine tumors), in situ hybridization (nine of 11 tumors), and Southern blot analysis (five of eight tumors). In some of these tumors the failure to demonstrate c-erbB-2 DNA amplification may be due to the small percentage of malignant cells. One additional tumor showed probable c-erbB-2 protein overproduction based on strong immunoreactivity with two antibodies (TAB-250 and CB11), although no definite activation could be demonstrated by additional techniques. Three other tumors (6%) showed equivocal c-erbB-2 protein overproduction based on weak immunoreactivity only with TAB-250, although unequivocal activation could not be demonstrated by additional techniques. The 32 carcinomas (68%) that showed no significant immunoreactivity with any antibodies in routinely processed tissue also showed no detectable c-erbB-2 activation by additional techniques. We conclude that TAB-250 and CB11 are reliable antibodies for detecting c-erbB-2 protein overproduction in routinely processed tissue. TAB-250 also weakly stains a few tumors showing no definite c-erbB-2 activation by other techniques. Two additional antibodies (3B5 and N3/D10) detect c-erbB-2 protein overproduction in paraffin-embedded tissue, but are more difficult to interpret. A fifth antibody, TA-1, is an excellent reagent for use on frozen tissue, but prolonged formalin fixation may impair recognition of its antigenic epitope.

Adenocarcinoma↗

PubMed1994

New immunohistochemical "sandwich" staining method for mdr1 P-glycoprotein detection with JSB-1 monoclonal antibody in formalin-fixed, paraffin-embedded human tissues.

We have developed a new immunoperoxidase "sandwich" staining method for amplified detection of P-glycoprotein (Pgp) that is suitable for use on formalin-fixed, paraffin-embedded (conventional) tissue sections. This was accomplished by substantially changing the procedure described by Chan (1988) so as to increase specific staining intensity and to decrease nonspecific background staining. To determine the most appropriate primary antibody for the assay, we compared the immunoreactivity of JSB-1, C494, and C219 monoclonal antibodies recognizing internal epitopes of Pgp, and MRK16 and 4E3 monoclonal antibodies recognizing external epitopes of Pgp. Paraffin sections of Pgp-positive normal human tissues (adrenal, liver, kidney, and brain), of renal tumors, and of cell pellets of sensitive and multidrug resistant human tumor cell lines (MCF-7, KB) were used for comparisons. Immunostaining was excellent with JSB-1, moderate with C494, and very weak with C219. MRK16 and 4E3 showed no reaction. Nonspecific background staining was reduced by 1) omitting immunoglobulin G from secondary antibodies; 2) decreasing the concentration of peroxidase-antiperoxidase complex; and 3) utilizing casein solution for blocking and washing. Pretreatment of sections before immunostaining was also simplified. Using JSB-1, the threshold for detection of elevated Pgp corresponded to less than two-fold relative resistance to doxorubicin. Applying this method, we found two of 26 non-small cell lung cancers were positive for Pgp, consistent with previous results of others using frozen sections. This new immunoperoxidase sandwich staining method using JSB-1 now allows reliable Pgp detection in sections of formalin-fixed, paraffin-embedded (archived) surgical specimens and small biopsy materials commonly used for diagnostic purposes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Detection of bovine viral diarrhea virus RNA in formalin-fixed, paraffin-embedded brain tissue by nested polymerase chain reaction.

Isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissues is delicate due to its fragility and ubiquitous ribonucleases. For retrospective studies, however, a convenient procedure for the detection of RNA in archived material is of great value. Bovine viral diarrhea (BVD) virus is a member of the pestivirus genus in the family Flaviviridae. Different protocols for the isolation of BVD virus RNA from fresh and autolytic as well as from routinely formalin-fixed and paraffin-embedded brain tissue of BVDV-infected calves were compared. The polymerase chain reaction (PCR) after reverse transcription (RT-PCR) was carried out subsequently for the detection of viral RNA. Using proteinase K digestion of deparaffinized tissue sections without additional ribonuclease inhibitors and subsequent nested PCR, a 803 bp fragment of the gene coding for the nonstructural protein p125 of BVD virus could be consistently detected. In addition, BVD virus RNA was detected by RT-PCR from non-fixed brain tissue after 10 days of autolysis.

Animals↗

PubMed1992

Estimation of tumor growth fractions in archival formalin-fixed, paraffin-embedded tissues using two anti-PCNA/Cyclin monoclonal antibodies. Factors affecting reactivity.

Immunohistochemical detection of cell cycle-related markers for estimation of tumor growth fractions using paraffin-embedded tissue sections would have applications in experimental and clinical pathology as an in situ histologic alternative to flow cytometry. The monoclonal antibodies 19A2 and PC10 detect the proliferating cell nuclear antigen (PCNA/Cyclin), an auxiliary protein to DNA polymerase-delta. In a prospective group of uniformly handled, formalin-fixed malignant lymphomas we previously demonstrated 19A2 to be a reliable marker of proliferative activity similar to Ki-67 in frozen tissue. The present study examines the applicability of this technique in archival formalin-fixed material. Studies on tonsilar tissue revealed that formalin fixation beyond 30 hours adversely affected reactivity of 19A2, possibly explaining the variable results in nonuniformly fixed archival material. We found that only 27 (56%) of 48 archival cases of infiltrating ductal carcinoma showed sufficient reactivity with 19A2 to permit reliable quantification of the tumor growth fraction. Acid pretreatment with 2N HCl had no apparent effect on 19A2 reactivity. Using both antibodies on a group of 32 archival lymphomas, carcinomas, and sarcomas, significantly more biopsies stained reliably for PC10 (84%) than for 19A2 (72%; P < 0.036). Further, none of the cases that did not react with PC10 reacted with 19A2. PC10 may recognize a different epitope of PCNA/Cyclin which may be more resistant to alterations by fixation. In the 23 cases that reliably stained for both markers, largely carcinomas, there was excellent correlation between estimated growth fractions (r = 0.96). Although immunostaining provides a useful way to estimate tumor growth fractions in paraffin-embedded tissues, modifications of technique and cautious interpretation of results are advisable when using archival material.

Antibodies, Monoclonal↗

PubMed1990

Southern and dot blot analysis of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas.

Two extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods gave similar results. Formalin-fixation and paraffin-embedding irreversibly denatured DNA and consequently decreased the extraction yield and interfered with the quantitative measurement of DNA. Southern blot and dot blot analysis of processed and native DNA was performed using a c-myc and an actin probe. The results show that for Southern analysis processed DNA can be used but, due to the generation of random breaks, the restriction fragments have to be small. Furthermore, the fixation-induced crosslinking of DNA appears to hamper hybridization. For these reasons processed DNA can be analyzed better by dot blot rather than Southern blot hybridization.

Blotting, Southern↗

PubMed1986

DNA-measurements on formalin-fixed, paraffin-embedded squamous cell carcinomas from different ENT-regions.

DNA measurements were performed on formalin-fixed, paraffin-embedded squamous cell carcinomas, using the method of Hedley et al. Ultrasonication was sometimes necessary after enzymatic disintegration to dispose of residual cytoplasm. The isolated nuclei were stained with Hoechst 33258. DNA measurements were performed using a rapid system for static fluorometry (FLUORA-programme). The method proved to be highly accurate. The mean CV of the stem cell peaks from different tumours was 5.3. There was no difference between material less or more than 5 years old (mean CVs 5.2 and 5.5 respectively). RNase treatment had no effect and was therefore not considered necessary. The proliferative activity was calculated by computer assuming a rectilinear distribution of S-phase cells. Because routinely paraffin-embedded material can be used, DNA analysis can be performed on tumour material where the clinical outcome is known, in order to evaluate certain DNA variables.

Carcinoma, Squamous Cell↗

PubMed2007

Immunohistochemical detection of 3 viral infections in paraffin-embedded tissue from mink (Mustela vison): a tissue-microarray-based study.

Immunohistochemical (IHC) assays were developed and tested for the detection of 3 viral infections in archived paraffin-embedded mink tissue. Specimens had been obtained from mink with diagnoses of acute Aleutian disease (AD), mink parvoviral enteritis (MVE), or canine distemper (CD) made by means of routine diagnostic procedures. To improve the efficiency and reduce the costs of IHC analyses, tissue microarray (TMA) technology was used. Representative cores 2 mm in diameter from each tissue specimen and from positive- and negative-control specimens were collected in a TMA block. Immunohistochemical reactions to viral antigens were assessed and graded. Positive reactions were found in 91% of the 32 specimens from mink with AD, 53% to 80% of the 60 specimens from mink with MVE, and all 66 of the specimens from mink with CD. To validate the use of TMAs, the IHC methods were applied to whole-mount paraffin-embedded sections of 10 of the positive specimens for each disease, together with whole-mount sections of small intestine and lung tissue from 2 healthy mink. The IHC grading of the TMA cores and the whole-mount sections from the same animal corresponded completely. These results suggest that IHC demonstration of viral antigen allows rapid and reliable diagnosis of the 3 viral infections in mink and is a potential supplement to histologic diagnostic procedures. The TMA technique proved useful for screening large numbers of samples for expression of specific viral antigens, while reducing overall costs.

Aleutian Mink Disease↗

PubMed1998

Evaluation of PCR in detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues: comparison of four amplification assays.

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of the mtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 microg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.

Evaluation Studies as Topic↗

PubMed1989

Phenotypic analysis of infiltrating cells in human myocarditis. An immunohistochemical study in paraffin-embedded tissue.

To define the cellular subpopulations that infiltrate the heart in human myocarditis, formaldehyde-fixed, paraffin-embedded sections from 18 hearts with histologically proved myocarditis were examined immunohistochemically. Disease was classified on routine stains as follows: mixed mononuclear cell (7 cases), granulomatous (3), giant cell (1), rheumatic (2), and fungal (5) myocarditis, respectively. On immunohistochemical examination, T-lymphocyte and macrophage predominance was found in nearly every case, except in fungal myocarditis, in which polymorphonuclear leukocytes and macrophages prevailed. In contrast, B lymphocytes and natural killer cells were conspicuously absent, regardless of histologic classification. Giant cells in giant cell myocarditis and in the Aschoff lesions of rheumatic carditis expressed macrophage, but not myocyte, antigens, suggesting derivation along macrophage lineage. Immunohistochemical data obtainable in paraffin-embedded tissues supplement the study of myocarditis, providing information potentially relevant to immunopathogenesis, natural history, and therapy.

Cell Line↗

PubMed2004

Detection of t(14;18) in follicular lymphoma by dual-color fluorescence in situ hybridization on paraffin-embedded tissue sections.

We studied the incidence of t(14;18)(q32;q21) in 54 patients with follicular lymphoma (FL) by dual-color fluorescence in situ hybridization on paraffin-embedded tissue sections (tissue-FISH) using probes for BCL2 and immunoglobulin heavy chain (IGH) genes. The t(14;18) was detected in 28 (56%) of 50 patients, who were successfully analyzed. On the other hand, BCL2 protein expression was detected in 45 (83%) of 54 patients evaluated by immunohistochemical staining. There was a discrepancy between t(14;18) and BCL2 expression. The absence of t(14;18) was associated with disease-free survival (P=0.02). There was no statistical difference, however, in overall survival and failure-free survival between the t(14;18)-positive and -negative groups. Tissue-FISH should be of great value to detect t(14;18) in FL because this method enabled us to demonstrate specifically t(14;18)-positive individual cells in neoplastic follicles on paraffin-embedded tissue sections. Furthermore, tissue-FISH can be applied to small specimens obtained from endoscopic biopsy or specimens obtained more than 10 years ago. We validated the usefulness of tissue-FISH as a diagnostic procedure and retrospective meta-analysis for malignant lymphoma.

Adult↗

PubMed1999

Detection of COL1A1-PDGFB fusion transcripts in dermatofibrosarcoma protuberans by reverse transcription-polymerase chain reaction using archival formalin-fixed, paraffin-embedded tissues.

The reciprocal translocation t(17;22)(q22;q13) and a supernumerary ring chromosome, r(17;22), derived from the translocation, have been shown to be highly characteristic of dermatofibrosarcoma protuberans (DFSP). Its consequence is a fusion of two genes, a collagen type I alpha 1 gene (COL1A1) and platelet-derived growth factor B-chain gene (PDGFB). The COL1A1-PDGFB fusion gene, is expected to be a diagnostic molecular assay. However, previous studies on this subject were mostly based on frozen tissue specimens or cultured tumor cells. In this present study, the investigators conducted a reverse transcription (RT)-polymerase chain reaction (PCR) assay to detect the COL1A1-PDGFB fusion transcripts using archival formalin-fixed, paraffin-embedded tumor specimens from 12 patients with DFSP. To amplify the fusion transcripts, a specific COL1A1 forward and PDGFB reverse primers were designed for single step PCR. The COL1A1-PDGFB fusion transcripts could be detected in 10 of 12 paraffin-embedded DFSP tumor specimens (83%). Subsequent sequence analysis using the PCR products confirmed that the detected messages were derived from gene fusions composed of PDGFB exon 2 and different regions of the COL1A1 gene (exon 8, 10, 22, 24, 32, 38, 45 or 46). Two samples of Bednar tumor included in this series also contained the fusion transcripts. In sample of DFSP with fibrosarcomatous transformation, the COL1A1-PDGFB could not be detected in the fibrosarcoma areas of the third recurrence, though the chimeric transcripts were identified in the ordinary DFSP areas of the first recurrence. No fusion transcripts could be amplified in non-DFSP lesions, including 10 dermatofibromas and 9 malignant fibrous histiocytomas. These results indicate that this molecular assay could be applied to archival formalin-fixed, paraffin-embedded tumor tissues as a diagnostic aid for DFSP.

Adolescent↗

PubMed1999

PCR detection of Clostridium perfringens type D in formalin-fixed, paraffin-embedded tissues of goats and sheep.

A polymerase chain reaction (PCR) was used to identify the genes encoding the alpha, epsilon and beta toxins of Clostridium perfringens in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep. When pure cultures of Cl. perfringens types B and D were used as control templates in the PCR, products of the following sizes were observed on the agarose gel: 247 bp (alpha primers), 1025 bp (beta primers) and 403 bp (epsilon primers). When used to identify Cl. perfringens type D in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep, the PCR technique resulted in the detection of this micro-organism in 11 out of 13 samples known to be infected with Cl. perfringens. No false positive results were obtained when 13 culturally negative samples were analysed by the PCR technique.

Animals↗

PubMed1994

Detection of human papillomavirus by in situ polymerase chain reaction in paraffin-embedded cervical biopsies.

The efficiency of the in situ polymerase chain reaction (PCR) for the detection of human papillomavirus (HPV) DNA sequences in 20 cervical biopsies fixed with buffered formalin, paraffin-embedded and revealed negatively by conventional in situ hybridization (ISH) has been investigated. The biopsies were classified histologically into condylomata acuminata without dysplasia, cervical intraepithelial neoplasia and carcinoma in situ. Amplified HPV DNA was performed after an optimal proteolytic digestion using one pair of consensus oligonucleotide primers located in the L1 ORF of HPV 6 and ISH was carried out after the PCR assay with a cocktail of biotinylated HPV probes. Viral DNA was detected in 100% of high grade squamous intraepithelial lesions (SIL) and in 50 to 60% of low grade SIL. The high sensitivity of the in situ PCR applicable to paraffin-embedded archival biopsies facilitated the detection of cells poorly reactive by conventional ISH. In situ PCR appeared clearly an efficient tool to investigate HPV infection in tissue sections.

Base Sequence↗

PubMed1996

Multiple drug resistance-related messenger RNA expression in archival formalin-fixed paraffin-embedded human breast tumour tissue.

A method is described by which RNA, suitable for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, can be extracted from formalin-fixed paraffin-embedded (FFPE) tissues and subsequently used for detecting the expression of several genes. Using this technique, RNA can be extracted from specimens, quantified, reverse transcribed and regions of interest amplified and analysed within 36 h. The tissue specimens included in this study were from human breast carcinoma, investigating a range of genes associated with the development and/or maintenance of multiple drug resistance (MDR). This technique, applied to archival tissues, offers great potential for increasing our understanding of alterations in expression levels of genes associated with MDR. The method developed is also applicable to studies on expression of other genes in paraffin-embedded tissues.

Adult↗

PubMed2001

Inter-laboratory comparison of DNA preservation in archival paraffin-embedded human brain tissue from participating centres on four continents.

DNA extracted from formalin-fixed and paraffin-embedded brain tissue is known to contain as yet ill-characterized inhibitors of the PCR process. As part of a project that aims to clarify the role of mitochondrial DNA sequence variation in human neurodegenerative diseases using DNA from various ethnic backgrounds, we have investigated factors that influence the preservation of archival DNA and its suitability for PCR. In this study, neuropathological tissue samples were analysed that had been routinely processed in 18 international centres on four continents. Following DNA extraction, PCR amplification of mitochondrial and nuclear DNA sequences was performed with and without additional purification of the template DNA. In addition, the DNA used for PCR was analysed by HPLC. Phosphate-buffered formalin proved to be a superior fixative compared with unbuffered aldehyde: DNA extraction resulted in greater yields, the molecular weight of the isolated DNA was higher and PCR was more successful. PCR inhibitors were identified as (1) high concentrations of small (<300 bp) DNA fragments that competitively compete with template DNA and (2) contaminants of the DNA template solution including denatured protein that cannot be completely removed by phenolic extraction. HPLC analysis did not reveal significant qualitative differences between DNA isolated from fresh-frozen tissue samples and DNA recovered from formalin-fixed, paraffin-embedded brain tissue. The fact that DNA could be amplified from the majority of tissue specimens in this study suggests that rare diseases and diseases where ethnic background plays an important role can be sampled for genetic polymorphism analysis on a global scale using archival neuropathological collections.

Alzheimer Disease↗

PubMed2005

Detection and identification of Mycobacterium species by polymerase chain reaction (PCR) from paraffin-embedded tissue compare to AFB staining in pathological sections.

BACKGROUND: Polymerase chain reaction (PCR) is a recent, rapid and reliable method in the detection of causative organism. The authors tried to determine the possibility of using PCR technique as an alternative way to detect mycobacterial DNA from paraffin-embedded tissue to avoid repeated biopsy from the patient. MATERIAL AND METHOD: Paraffin-embedded tissue blocks, the corresponding histopathologic slides, and cultural results were retrospectively searched for according to the patient's records, the granuloma clinic, Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand from 1994-2000. One hundred and thirty-one tissue blocks and slides were found but only 120 cultural results were retrieved Histologic sections were reviewed for AFB findings and PCR was done using 16S rRNA sequences to detect M. tuberculosis by one-tube nested technique and multiplex PCR for M. marinum and M. fortuitum complex. RESULTS: The causative organisms were identified by AFB staining in pathologic sections 31.29%, by PCR 35.87%, and by culture 30.00% of tested samples. The sensitivity of PCR when compared to AFB result was 29.26%, specificity 61.11% but when compared to cultural results, the sensitivity of PCR was 66.67% and AFB sensitivity was 41.66% with specificity 76.19% and 72.61% respectively. CONCLUSION: The low sensitivity of the PCR method may be due to formalin fixation, deparaffinization process, DNA extraction method, the use of 16S rRNA-based primers and the length of the expected product, and the tissue type that may have Taq polymerase inhibitor. Therefore, PCR should be used to augment the information of the conventional method in the diagnosis of mycobacterial infection.

Adolescent↗

PubMed1992

Proliferating cell nuclear antigen counts in formalin-fixed paraffin-embedded tissue correlate with Ki-67 in fresh tissue.

Cell proliferation can be studied by a variety of techniques. However, most require fresh tissue. To evaluate cell proliferation in formalin-fixed paraffin-embedded sections, the authors immunohistochemically studied 35 tumors and 11 samples of normal/hyperplastic tissue with PC10, a monoclonal antibody directed against proliferating cell nuclear antigen. Results were compared with those obtained with Ki-67 on fresh tissues. There was no significant difference between proliferating cell nuclear antigen and Ki-67 counts, which were strongly correlated (r = 0.8). Proliferating cell nuclear antigen positivity was easier to evaluate because morphology was better preserved in formalin-fixed tissue. The authors conclude that PC10 is an alternative to Ki-67 in evaluating cell proliferation and has the advantage of reacting with formalin-fixed paraffin-embedded tissue.

Antibodies, Monoclonal↗

PubMed2005

[Detection of EWS-WT1 fusion transcripts in paraffin-embedded tissues for desmoplastic small round cell tumor].

OBJECTIVE: To detect the EWS-WT1 chimeric mRNA in desmoplastic small round cell tumor (DSRCT) and its clinicopathological significance. METHODS: Formalin fixed, paraffin-embedded tumor specimens from 4 examples of this entity were studied by reverse transcriptase polymerase chain reaction to detect the EWS-WT1 fusion transcripts resulting from the chromosomal translocation t(11;22)(p13;q12). The following tumor specimens were included as controls: 2 Ewing's sarcomas/primitive neuroectodermal tumors (PNET/ES), 2 alveolar rhabdomyosarcomas, and 2 lymphomas. RESULTS: EWS-WT1 fusion transcripts were detected in 3 of 4 desmoplastic small round cell tumors but not in any other tumor types studied as controls. CONCLUSION: The analysis of the EWS-WT1 fusion transcript which was performed on formalin-fixed, paraffin-embedded tissues is a sensitive and specific method in the diagnosis and differential diagnosis of DSRCT.

Abdominal Neoplasms↗