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PubMed1983

Demonstration of Aujeszky's disease virus antigen in trypsin-digested paraffin-embedded tissue sections by immunofluorescence.

After trypsin digestion of buffered formalin-fixed, paraffin-embedded brain section, immunofluorescent indication of Aujeszky's disease virus antigen was successful in experimentally infected cow and goat. This result was comparable to those obtained with light and electron microscopic examinations. This method will be diagnostically useful where fresh-frozen tissue is not available.

Animals↗

PubMed1993

Immunohistochemical detection of CD1A antigen in formalin-fixed and paraffin-embedded tissue sections with monoclonal antibody 010.

The immunoreactivity of a CD1a monoclonal antibody (MAb), denoted 010, was investigated by means of the streptavidin-biotin-peroxidase method in formalin-fixed and paraffin-embedded tissues from 47 cases. The samples comprised reactive lymphoid proliferations of skin, tonsil, and lymph node including dermatopathic lymphadenopathy and Langerhans' cell histiocytosis, Hodgkin's and non-Hodgkin's lymphomas, and thymomas. Interdigitating and dermal dendritic cells, veiled cells, Langerhans' cells, and also cortical thymocytes and their neoplastic counterparts displayed immunostaining with MAb 010 in paraffin sections. These results are identical to previous ones reported for other CD1a MAbs in fresh or frozen specimens. The findings suggest that the binding site of 010 is a fixation-resistant epitope of CD1a antigen which has not been previously identified.

Antibodies, Monoclonal↗

PubMed2006

Real-time PCR assay for improved detection of Mycobacterium tuberculosis complex in paraffin-embedded tissues.

OBJECTIVE: To test the usefulness of a commercially available real-time polymerase chain reaction (PCR) kit for the detection of Mycobacterium tuberculosis complex (MTBC) in formalin-fixed, paraffin-embedded tissues. RESULTS: The examination of 24 specimens of patients with a final diagnosis of TB shows that the real-time PCR assay exhibits a higher sensitivity (66.7%) for the detection of MTBC DNA than an alternative in-house IS6110 PCR (33.3%), whereas staining detected acid-fast bacilli in only two cases (8.3%). CONCLUSION: The real-time PCR assay provides a highly sensitive and specific means for the detection of MTBC DNA in histopathological specimens.

Biopsy↗

PubMed1991

Detection of human papillomavirus type 16 E6/E7 transcripts in fixed paraffin-embedded cervical cancers by the polymerase chain reaction.

A simple, and sensitive method for the detection of human papillomavirus (HPV) 16 E6/E7 transcripts in RNA purified from formalin acetic alcohol (FAA)-fixed, paraffin embedded cervical cancer (CaCx) tissue is described. The entire procedure, including polymerase chain reaction (PCR) amplification of cDNA obtained from mRNA by reverse transcription, can be completed within a day. The results obtained compare favourably with those using total nucleic acids extracted from the same fixed material, and are consistent with results using RNA purified from corresponding fresh tumour tissue. Although developed for investigating the expression of HPV in tumours, this method should find general application in studies of gene expression and viral detection.

DNA, Single-Stranded↗

PubMed1997

Rapid polymerase chain reaction-based detection of the causative agent of cat scratch disease (Bartonella henselae) in formalin-fixed, paraffin-embedded samples.

Bartonella (formerly Rochalimaea) henselae (Bh) plays a central role in cat scratch disease. A polymerase chain reaction (PCR)-based assay that can detect Bh DNA in formalin-fixed, paraffin-embedded (FF-PE) samples would have utility in the evaluation of processed lymph nodes suggestive of this disorder. Fresh or FF-PE cultures of Bh and related species were analyzed. Thirteen lymph nodes (12 FF-PE and one fresh cell suspension) with necrotizing suppurative granulomatous inflammation and seven FF-PE negative control lymph nodes were also evaluated. PCR was performed using a novel, hemi-nested protocol. Amplified products were analyzed by gel electrophoresis. The fresh and FF-PE Bh cultures showed a specific PCR product with an analytical sensitivity of 0.5 pg bacterial DNA. Seven (54%) of 13 clinical lymph node samples with morphological features suggestive of cat scratch disease also had detectable Bh DNA, whereas none of the seven negative control lymph nodes yielded positive results. We have designed a rapid and sensitive PCR test that can reliably detect Bh DNA in fresh and FF-PE samples. Our findings indicate that this assay has clinical utility in the diagnosis of cat scratch disease.

Adult↗

PubMedDate not supplied

The accuracy of intraoperative diagnoses based on examination of frozen sections. A prospective comparison with paraffin-embedded sections.

The accuracy of diagnoses based on examination of frozen sections was determined by comparing the results to those obtained by examination of tissues prepared using conventional methods (formalin fixation, paraffin-embedded tissue). One hundred ninety-four specimens were examined using the frozen section technique; 37 were examined to confirm a tentative diagnosis or to document lymph node metastasis and the remainder were examined to diagnose an unknown pathologic process. Of the 194 specimens examined, an accurate, specific diagnosis was obtained in 161 (83%); in 19 (10%), the pathologic process was correctly identified, but a specific diagnosis was not obtained; and in 2 (1%) the diagnosis was deferred. The remaining 12 (6%) were incorrectly diagnosed by the frozen section technique. When the number of specimens in which a specific diagnosis was obtained was combined with the number of specimens in which the pathologic process was correctly identified, the overall accuracy rate of the frozen section technique was 93%. There was no difference in the accuracy of the frozen section technique based on the reason for submission of the sample, source of tissue submitted, or the type of pathologic process (i.e., inflammatory or neoplastic). Of the 12 incorrect diagnoses, 4 (33%) were because of sampling errors and 8 (67%) were caused by interpretation errors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

Identification of distinct regions of allelic loss on chromosome 13q in nasopharyngeal cancer from paraffin embedded tissues.

Our main purpose was to identify tumor suppressor gene loci on chromosome 13 responsible for nasopharyngeal cancer (NPC) development by analyzing loss of heterozygosity (LOH) and RB protein expression in paraffin embedded tissues. Normal and tumor DNA were extracted from microdissected samples, and their whole genomes were amplified using degenerate oligonucleotide primers. The polymerase chain reaction (PCR) products were analyzed by repeated amplification using primers derived from 16 microsatellite regions spanning the long arm of this chromosome. Among 50 informative cases, LOH was observed in 44 tumors. Thirty-one tumors displayed partial loss and provided an informative basis for detailed deletion mapping. Three minimal regions of loss were delineated; the first flanked by D13S120 and D13S219, the second by D13S126 and D13S119, and the third by D13S137 and 13qter. These 3 regions were linked to BRCA2 on 13q12, RB1 on 13q14, and 13q14.3-ter, respectively. Seven and 4 cases showed LOH either on 13q12 or 13q14, respectively. Nineteen cases showed LOH of both loci separately. One NPC displayed 13q12 and 13q14.3-ter LOH. RB protein expression was detectable in 76% of the cases. Ten out of 15 cases with the allelic losses limited to 13q14 showed RB protein expression. Contrasting that, 6 out of 7 cases devoid of RB protein expressions showed 13q14LOH. In conclusion, 13qLOH, involving 3 tumor suppressor gene loci, appears to be a frequent genetic event occurring during NPC development. However, other tumor suppressor genes besides RB1, may be responsible for the majority of 13q14LOH.

Adult↗

PubMed2004

Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

Artifacts↗

PubMed1994

Numerical abnormalities of chromosome 7 in human prostate cancer detected by fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections with centromere-specific DNA probes.

Fluorescence in situ hybridization (FISH) using chromosome-specific alpha-satellite DNA probes for chromosomes 7, 8, and 12 was performed on paraffin-embedded tissue sections and touch imprint preparations of 53 cases of human prostate cancer. Subsequent haematoxylin and eosin (H & E) staining of the hybridized tissue sections allowed unambiguous assignment of hybridization signals either to tumour or to non-tumorous parenchyma. Fifty-three cases of human prostate cancer were evaluated for numerical aberrations of chromosome 7. Scoring 200 cells of tumour and non-tumorous parenchyma in each case revealed abnormalities exclusively in tumour parenchyma in 41 cases (77 per cent). Ten of 41 cases (24 per cent) showed trisomy 7, and 15 cases (37 per cent) monosomy 7 or trisomy 7 in combination with monosomy 7, respectively. Sixteen cases (39 per cent) exhibited polysomy 7 in cells of the tumour parenchyma. In the tumour tissue in one case, different polyploid clones (triploid, tetraploid) and polysomy 7 could be identified by double hybridization with chromosome-specific DNA probes for chromosome 7, plus 8 or 12. The indicated numerical aberrations of chromosome 7 were correlated with 78 per cent of advanced pathological stages or poorly differentiated tumours (pT3/4 or G3) of prostate carcinomas. A statistical analysis of the data revealed significant relationships of particular numerical abnormalities of chromosome 7 to different pathological categories (pT, G, pN) of tumour classification. For the T-classification, the frequency of cells carrying polysomy 7 and polysomy 7/+7 increases significantly from pT1 to pT3/4 (P = 0.022).(ABSTRACT TRUNCATED AT 250 WORDS)

Centromere↗

PubMed2006

SARS-CoV virus-host interactions and comparative etiologies of acute respiratory distress syndrome as determined by transcriptional and cytokine profiling of formalin-fixed paraffin-embedded tissues.

These studies attempt to understand more fully the host response and pathogenesis associated with severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) by monitoring gene expression using formalin-fixed paraffin-embedded (FFPE) pulmonary autopsy tissues. These tissues were from patients in different hospitals in Singapore who were diagnosed with various microbial infections, including SARS-CoV, that caused acute respiratory distress syndrome (ARDS). Global expression patterns showed limited correlation between end-stage ARDS and the initiating pathogen, but when focusing on a subset of genes implicated in pulmonary pathogenesis, molecular signatures of pulmonary disease were obtained and appeared to be influenced by preexisting pulmonary complications and also bacterial components of infection. Many factors detected during pulmonary damage and repair, such as extracellular matrix (ECM) components, transforming growth factor (TGF) enhancers, acute-phase proteins, and antioxidants, were included in the molecular profiles of these ARDS lung tissues. In addition, differential expression of cytokines within these pulmonary tissues were observed, including notable genes involved in the interferon (IFN) pathway, such as Stat1, IFN regulatory factor-1 (IRF-1), interleukin-6 (IL-6), IL-8, and IL-18, that are often characterized as elevated in ARDS patients.

Aged↗

PubMed1998

Nested polymerase chain reaction for Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissues with granulomatous diseases for rapid diagnosis of tuberculosis.

We evaluated the sensitivity and specificity of a nested polymerase chain reaction (PCR) to the Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissue samples from patients with tubercular and other granulomatous lesions. Five groups of patients and samples were studied: (1) 28 samples from HIV-positive patients with tuberculosis, (2) 8 samples from HIV-negative patients with histologically suspected tuberculosis (confirmed by culture in 5 cases), (3) lymph nodes from 5 HIV-positive patients with Mycobacterium avium-intracellulare infection, (4) lymph nodes from 30 patients with sarcoidosis, and (5) specimens from 17 patients with other granulomatous diseases. The DNA was extracted from sections with a total thickness of 60 microm, and PCR amplified an internal fragment of 123 base pairs. All of the cases with M. tuberculosis infection were PCR-positive, although this sensitivity was partially related to the initial concentration of the DNA used for amplification. Two of the group 4 samples also were repeatedly positive, thus reducing the specificity of the method. All of the cases with granulomatous diseases other than sarcoidosis were negative. We propose a simplified and highly sensitive nested PCR for the diagnosis of M. tuberculosis infection on archived material in HIV-positive and HIV-negative patients.

AIDS-Related Opportunistic Infections↗

PubMed1991

Immuno- and enzymehistochemical characterization of 'plasmacytoid T-cells' in formalin-fixed paraffin-embedded tissue of reactive lymph nodes.

In eight (10.4%) of 77 randomly selected cases of lymphadenitis, foci of 'plasmacytoid T-cells' (PTCs) were found. We characterized the PTCs in formalin-fixed paraffin-embedded tissue by immuno- and enzymehistochemical techniques. In each case the PTCs formed nodular and patchy infiltration patterns around the high endothelial venules in the paracortex of the lymph nodes. The PTCs in half of the cases were present near the intermediary peritrabecular sinuses. The PTCs were found to be positive for CD 45 (LCA), CD 45 R (4 KB 5) and CD 43 (MT 1), but not for CD 45 RO (UCHL 1), L 26 (CD 20), myeloperoxidase, naphthol AS-D chloroacetate esterase, Mac 387, alpha-1-antichymotrypsin or S 100 protein and anti-dendritic reticulum cell antibody. Some lysozyme+ PTCs were mingled in and around the foci in each case. In five cases the PTCs showed weaker positive reactions for CD 68 (EBM 11) and HLA-DR than for T-zone macrophages and sinus histiocytes. Related to the degree of desintegration of the nodular PTC-foci increasing numbers of T- and B-lymphocytes, T-zone macrophages and interdigitating reticulum cells with HLA-DR were detected in the foci. In two cases, where there was no HLA-DR expression by PTCs, small amounts of HLA-DR in secondary lymph follicles and a decrease of HLA-DR+ T-zone macrophages and interdigitating reticulum cells were observed. Our results suggest that there is a high degree of interaction between PTCs and other cellular components in the lymph node. The PTC may represent a special type of resident macrophage with secretory capacities.

Adult↗

PubMed2004

Immunoperoxidase staining for C4d on paraffin-embedded tissue in cardiac allograft endomyocardial biopsies: comparison to frozen tissue immunofluorescence.

C4d deposition in microvasculature is a marker for humoral rejection. The authors compared a recently developed C4d immunoperoxidase (IP) method for paraffin-embedded tissue to immunofluorescence (IF) of frozen tissue. Of 315 frozen endomyocardial biopsies with IF staining for C4d, 280 were negative and 35 were positive. Negative controls were 17 negative biopsies and 11 biopsies with myocyte necrosis. The extent of IP and IF staining was graded as 0 to 3+. Staining intensity and the number and type of positive vessels were recorded. Staining patterns in Quilty lesions (QL) and foci of acute cellular rejection (ACR) were also evaluated. In 34 biopsies with sufficient tissue, IP criteria of 2+/3+, or more than 10 to 20 positive vessels per 10 high-power fields detected 25.0% (1/4), 18.2% (2/11), and 84.2% (16/19) of 1+, 2+, and 3+ IF-positive biopsies, respectively, without false positives. Considering C4d IF 3+ as positive resulted in 84.2% (16/19) sensitivity and 93.0% specificity (40/43). Intensely stained capillaries predominated in six of seven biopsies when more than 100 capillaries per 10 high-power fields were positive. Seventy percent (7/10) of IP 2+ and 3+ biopsies showed positive capillaries in QLs, while 36.4% (4/11) of IP 1+ and negative biopsies did. All eight IP 2+/3+ biopsies showed positive capillaries in ACR foci, while 25.0% (1/4) of IP-negative biopsies did. Capillary staining in QLs and areas of ACR reflects overall C4d deposition. In conclusion, IP staining of 2+/3+ is highly sensitive and specific for C4d positivity. The authors recommend considering 2+ and 3+ as positive staining when using the IP technique.

Adolescent↗

PubMed2004

Detection of Mycobacterium tuberculosis complex in formalin-fixed, paraffin-embedded tissue specimens with necrotizing granulomatous inflammation by strand displacement amplification.

Rapid, reliable diagnosis of tuberculosis is essential to initiate correct treatment, avoid severe complications, and prevent transmission. Conventional microbiological methods may not be an option if samples are formalin-fixed and paraffin-embedded (FFPE) for histopathological examination. With the demonstration of necrotizing granulomatous inflammation, tuberculosis becomes an important differential diagnosis, although it was not initially suspected. Following paraffin extraction, BDProbeTec ET strand displacement amplification for detection of Mycobacterium tuberculosis complex (MTC) was applied to 47 prospectively and 19 retrospectively collected FFPE samples from various sources with granulomatous inflammation and results were compared to tuberculosis notification. Of the prospective samples, 20 were from patients who were notified as having tuberculosis and the assay was positive in 18 (90%). Specificity was 100%. For 27 of the patients with prospectively collected FFPE specimens, culture was performed on a specimen collected at a later date from the same location. Culture revealed MTC in 14 and nontuberculous mycobacteria in four. BDProbeTec ET was positive in 13 (92.8%) of the patients with positive MTC culture and negative in the remaining. The sensitivity and specificity in 19 archival samples was 40% and 100%, respectively, compared to notification data. The assay provided rapid, correct diagnosis on different sources of FFPE samples collected prospectively and therefore offers an important supplementary method for patients where tuberculosis was not initially suspected.

Fixatives↗

PubMed1997

Nested polymerase chain reaction-based HLA class II typing for the unique identification of formalin-fixed and paraffin-embedded tissue.

Human leukocyte antigen (HLA) genotyping is routinely performed prior to organ transplantation using peripheral blood leukocyte-derived DNA. In addition, polymerase chain reaction (PCR)-based methods have permitted HLA genotyping using DNA extracted from formalin-fixed and paraffin-embedded tissue, with proven applications in HLA-disease association studies and surgical biopsy identification. The utility of current techniques may be limited by the poor yield of intact DNA from such paraffin biopsies. This paper describes a new nested PCR-based HLA class II genotyping method which reliably detects HLA DRB alleles within DNA extracted from even extremely small paraffin biopsies. This method comprises initial PCR amplification of exon II sequences of the HLA DRB1, 3, 4, and 5 genes using generic PCR primers. Identification of the HLA DRB1 alleles and detection of the DRB3, 4, and 5 genes is then performed using a series of separate individual second-round PCR reactions, each of which contains PCR primer pairs detecting a single HLA DRB allele or group of alleles (PCR-SSP). The ability of this method to detect 19 individual HLA DRB1 alleles or groups of alleles, covering all common DRB1 specificities, was confirmed via concordant results when compared with 'direct' (single amplification step) PCR-SSP analysis of one cell line-derived and nine peripheral blood-derived DNA samples, and with five DNA samples extracted from paraffin biopsies. The technique was then successfully applied to 11 further paraffin biopsy-derived DNA samples, of which ten were untypable by 'direct' PCR-SSP analysis, from five cases in which doubt existed as to the individual origin of the tissues.

Adult↗

PubMed2005

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction.

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.

Animals↗

PubMed1988

Effect of trypsinization on the immunostaining of formalin-fixed, paraffin-embedded tissues.

We have investigated the action of trypsin treatment on formalin-fixed, paraffin-embedded tissue sections prior to immunostaining with a variety of commercially available poly- and monoclonal antibodies commonly used in surgical pathology. Although enzymatic predigestion often markedly reduced the background and enhanced the immunoreaction for certain antibodies, false negative staining or no enhancement of the staining was observed with other antibodies. We conclude that the indiscriminate practice of trypsinization in immunohistochemical procedures could be a significant cause of false negative results and of discrepancies between laboratories. Therefore, each laboratory must develop its own quality control program for variables such as enzymatic predigestion.

Antigen-Antibody Reactions↗

PubMed2005

Determination of threshold values for determining the size of the fraction of steroid hormone receptor-positive tumor cells in paraffin-embedded breast carcinomas.

BACKGROUND: For 4 years we used a multiparameter DNA flow cytometric (MP-FCM) technique to assess steroid hormone receptor expression in the diagnostic workup of routinely processed formalin-fixed, paraffin-embedded breast carcinomas as an alternative to immunohistochemistry (IHC) for the quantification of hormone receptor-positive cells. In all cases a positive fraction of hormone receptor-expressing epithelial cells was detected. This observation raised the question of what the cutoff value might be to distinguish receptor-negative from receptor-positive tumors. METHODS: In our search for a possible threshold value of positivity for estrogen receptor (ER) and progesterone receptor (PR) in MP-FCM, we developed four steps. First, we compared IHC results in our own laboratory with the results obtained by MP-FCM on a small series of breast tumors (n = 42). Second, after collecting our first 843 tumors, we made a comparison with the literature of the distribution of receptor positivity according to age classes. Third, using the most likely threshold that resulted from this comparison, we compared a subset of 340 node-negative tumors for their combined ER/PR profiles with the data from a similar group of node-negative tumor cases from the National Cancer Institute's Surveillance, Epidemiology and End-Result (SEER) study. Fourth, with the results of these comparisons, we prospectively collected IHC data and MP-FCM results of the same tumor samples for a period of 1 year. In this way, we collected data for an additional 180 tumors. RESULTS: The first step in this process resulted in an previous publication where 20% of steroid hormone receptor-positive cells seemed to be an acceptable cutoff point for positivity. However, the second step provided the best correlation at approximately 35% of ER reactive cells in the cytokeratin-positive cell population. With this cutoff, the distribution of combined ER/PR profiles in our patient population of node-negative breast cancers also showed a distribution similar to the data from the SEER study. The fourth step, using the 35% threshold value, resulted in a good correlation (r = 0.85, P < 0.0001) for ER and PR between IHC and MP-FCM in the 180 tumors investigated. CONCLUSION: By comparing in-house data with those from large external data collections in the literature, a threshold percentage can be defined that distinguishes steroid hormone receptor-negative from hormone receptor-positive tumors. As a result, information about DNA content and cell cycle distribution can be obtained. This observational study provides additional support to our opinion that MP-FCM is an alternative for IHC determination of ER and PR positivity. It is more objective and quantification can be done more appropriately. The additional value of this approach is that we generate continuous variables of ER/PR content instead of categorical classes, which can be used at different threshold levels for evaluation of clinical relevance.

Adult↗