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PubMed2006

Detection of the activating JAK2 V617F mutation in paraffin-embedded trephine bone marrow biopsies of patients with chronic myeloproliferative diseases.

The discovery of the activating V617F mutation in the JAK2 tyrosine kinase in a high proportion of patients with Ph- chronic myeloproliferative diseases (CMPD) represents a diagnostic breakthrough for these disorders. Trephine bone marrow biopsy is an essential part of the diagnostic workup of CMPD and represents a valuable archival source of DNA. Therefore, we studied 152 paraffin-embedded trephines with CMPD and related disorders for the presence of the V617F mutation, using both allele-specific polymerase chain reaction (PCR) and nested PCR with subsequent digestion with BsaXI. Only 6 of 152 (4%) samples were not evaluable because of poor DNA quality. The V617F mutation was detected in 27 of 28 (96%) cases of polycythemia vera, 17 of 23 (74%) cases of essential thrombocythemia, 28 of 45 (62%) cases of chronic idiopathic myelofibrosis, six of eight (75%) cases of CMPD unclassified, and two of four (50%) cases of myelodysplastic/myeloproliferative syndrome. Ph+ chronic myelogenous leukemia (four cases), reactive (secondary) erythrocytosis (14 cases), and thrombocytosis (one case) as well as normal controls (19 cases) all lacked the V617F mutation. Based on results of BsaXI digestion and sequencing, 24 of 54 (44%) evaluable V617F+ cases were considered homozygously mutated. Thus, detection of the V617F JAK2 mutation is feasible in paraffin-embedded trephine biopsies and represents a major advance in the diagnostic evaluation of CMPD.

Adult↗

PubMed1990

DNA flow cytometry of myocardial cell nuclei in paraffin-embedded, human autopsy, cardiac tissue.

Changes in nuclear DNA occur in myocytes as they grow and hypertrophy. We used DNA flow cytometry to study myocardial nuclei from paraffin-embedded human autopsy tissue. Forty hearts ranging from 310-1040 g were studied. Histograms generated for each heart showed peaks at the 2N and 4N positions. Two of 9 hearts weighing 310-430 g had 8N peaks as well. Fourteen of 17 hearts weighing 450-690 g also had 8N peaks but no 16N peaks. Twelve of 14 hearts weighing 710 to 1040 g had 8N peaks and 4 had 16N peaks as well. Contingency analysis showed statistically significant differences in the ploidy of nuclei from the three different groups of hearts. This study corroborates previous work indicating that DNA ploidy in myocardium increases with hypertrophy. Thus, flow cytometry of paraffin-embedded myocardium should be a useful technique to study DNA from archival material, obviating the need for fresh or frozen tissue.

Cardiomegaly↗

PubMed2001

The application of a PCR technique for the detection of immunoglobulin heavy chain gene rearrangements in fresh or paraffin-embedded skin tissue.

Although detection of a clonal sequence of the heavy chain gene of immunoglobulin by the polymerase chain reaction (PCR) is frequently used to assess lymphoid infiltrates in skin biopsy specimens, there are no data on the sensitivity and specificity of this test in detecting clonal B cell populations. Having refined a PCR technique for the detection of immunoglobulin heavy chain (IgH) gene rearrangement in both fresh and formalin-fixed, paraffin-embedded skin samples, we undertook to define the role of this assay in the diagnostic setting. Thirty-one cases of cutaneous B cell lymphoma (CBCL), 19 cases of B cell pseudolymphoma (lymphocytoma cutis), 34 cases of benign lymphocytic infiltrates of the skin and one case of cutaneous T cell lymphoma (CTCL) were studied using the polymerase chain reaction assay. All biopsies were formalin-fixed, paraffin-embedded skin sections apart from 13 of the 31 CBCL specimens which were fresh skin specimens. DNA from the framework region 3 (FR3) sequence of the IgH genes was amplified to ascertain the presence of a clonal IgH gene rearrangement. The findings were correlated with histological and immunophenotyping results on all samples. The assay performed with 73% sensitivity and 100% specificity, comparable to results obtained examining fresh lymphoid tissue specimens from patients with B cell tumours. The results indicate that this technique is a useful tool in the work up of suspected CBCL and in differentiating between CBCL and mixed lymphocytic infiltrates, a clearly important distinction with regards to prognosis and treatment.

Clone Cells↗

PubMed1995

Identification of abnormal chromosomal complement in formalin-fixed, paraffin-embedded placental tissue.

The objective of this project was to assess the efficacy of fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes to identify chromosome number in formalin-fixed, paraffin-embedded placental specimens. Using this approach, 75 per cent of the karyotypes in 20 formalin-fixed placental samples (comprising aneuploids, triploids, and normals) were correctly identified. As this technology improves, the ability to obtain information regarding chromosomal abnormalities in formalin-fixed, paraffin-embedded placental tissue should improve as well. This technology can potentially provide important cytogenetic information even when fresh tissue is not available for standard karyotypic analysis.

Chromosome Aberrations↗

PubMed1997

MIC2, TdT, bcl-2, and CD34 expression in paraffin-embedded high-grade lymphoma/acute lymphoblastic leukemia distinguishes between distinct clinicopathologic entities.

We propose that 12E7 (CD99) expression, along with TdT, bcl-2, and CD34 reactivity in lymphoblastic lymphoma (LyL)/acute lymphoblastic leukemia (ALL), distinguishes this group of neoplasms from small noncleaved cell lymphomas (SNCLs) in both pediatric and adult patients, thereby narrowing the differential diagnosis of high-grade non-Hodgkin's lymphomas and acute lymphoblastic leukemias in paraffin sections. 12E7 (CD99) is one of a group of available antibodies that recognizes the product of the mic-2 gene, which was originally identified in ALL. Despite this, most clinicopathological research has focused on the reactivity of 12E7 in a subset of the small round cell tumors of childhood. Although TdT is widely used in the subtyping of blastic leukemias, its use in the distinction of high-grade lymphomas in paraffin sections has been limited. We collected 24 cases of LyL/ALL (13 B-cell and 11 T-cell) and 15 cases of SNCL from 1984 through 1993. We confirmed the diagnoses using morphology and analysis of immunologic data. We performed immunohistochemistry with the 12E7 antibody, TdT, bcl-2, and CD34 on formalin-fixed, paraffin-embedded material. The patients' ages ranged from 4 to 81 years; nine of the study patients were children. Sixteen of the 24 LyL/ALLs stained with 12E7. In contrast, none of the 15 cases of SNCL reacted with this antibody (chi-square P < .0001). A larger percentage of T-cell LyL/ALLs reacted with 12E7 than did B-cell LyL/ALLs (82% v 54%). Sixteen of 20 LyL/ALLs reacted with the anti-TdT antibody, as compared with none of 11 SNCLs (chi-square P < .0001). Six LyL/ALLs were CD34 positive (of 23), and none of the SNCLs were CD34 positive (0 of 12) (chi-square P = .0519). Bcl-2-positive cases were found among both LyL/ ALLs and SNCLs, although they were more prevalent among LyL/ ALLs (92% v 25%; chi-square P < .0001). When one considers the differential diagnosis of a high-grade lymphoma/acute lymphoblastic leukemia, positive reactions with 12E7, TdT, bcl-2, and CD34 support the diagnosis of LyL/ALL over SNCL. Moreover, we present data that suggests that evaluating for TdT in formalin-fixed paraffin-embedded tissue is a more sensitive test than using either 12E7, bcl-2 or CD34 alone.

Adult↗

PubMed1999

Evaluation of rapid microsatellite analysis of paraffin-embedded specimens in screening for hereditary nonpolyposis colorectal cancer.

Length alterations in short repetitive DNA sequences, termed microsatellite instability (MSI), are used as a diagnostic criterion of replication errors caused by various mutations in at least five mismatch repair genes. Therefore, MSI analysis is useful in clinical practice to identify patients with hereditary nonpolyposis colorectal cancer (HNPCC). MSI can be detected by amplification of microsatellite loci in DNA extracted from paraffin-embedded tumor and corresponding peritumoral specimens after numerous time consuming steps limiting the clinical utilities. Rapid microsatellite analysis, a efficient and rapid DNA extraction technique based on Triton X-100 preincubation, was compared with the conventional DNA extraction for HNPCC screening in colorectal tumor specimens from 12 patients. Five complex and two noncomplex (CA)n microsatellite loci were tested, with use of multicolor fluorescent analysis. MSI and loss of heterozygosity in colorectal tumor samples could equally be assessed with the two DNA preparation methods, whereas the number of initially unsuccessful DNA extractions from paraffin-embedded tissue specimens and overall duration for MSI analysis were significantly reduced when rapid microsatellite analysis was used. A replication error-positive phenotype was detected in 2 of 10 patients with a positive family history for colorectal cancer, and diagnosis of HNPCC was finally confirmed by detection of a specific germline mutation. The described rapid microsatellite analysis is less time consuming and more efficient, and, in general, it reduces the risk of contamination by limiting the number of steps required. Therefore, it might replace current DNA extraction procedures. Furthermore, techniques using fluorescent polymerase chain reaction and semiautomated DNA sequencer allow for precise, observer-independent, and rapid scoring in MSI and loss of heterozygosity assessment. A combination of our rapid DNA extraction method and the use of a highly specific microsatellite marker might improve replication error analysis in HNPCC screening.

Adult↗

PubMed2004

Reactivity of anti-Nipah virus monoclonal antibodies to formalin-fixed, paraffin-embedded lung tissues from experimental Nipah and Hendra virus infections.

The immunohistochemical reactivity of seven clones of mouse monoclonal antibodies raised to Nipah virus antigens were investigated using formalin-fixed, paraffin embedded porcine and equine lung tissues from experimental Nipah and Hendra virus infection, respectively. Either microwave irradiation or enzymatic digestion effectively unmasked the viral antigens in formalin-fixed, paraffin-embedded tissue sections. Four clones showed positive reaction to both Nipah virus-infected porcine lung tissue and Hendra virus-infected equine lung tissue. Two clones (11F6 and 13A5) reacted with Nipah virus-infected porcine lung tissue, but not with Hendra virus-infected equine lung tissue. These Nipah virus-specific monoclonal antibodies may therefore be useful for immunohistological diagnosis of Nipah virus infection and for further research on Nipah virus pathogenesis.

Animals↗

PubMed1994

Comparison of fluorescence in situ hybridization with flow cytometry and static image analysis in ploidy analysis of paraffin-embedded prostate adenocarcinoma.

Nuclear DNA ploidy has been shown to have an important prognostic association for patients with adenocarcinoma of the prostate. Flow cytometry and static image analysis are ploidy methods that have been used in prostate carcinoma. Fluorescence in situ hybridization (FISH) using chromosome-specific probes can be used to evaluate the ploidy of interphase nuclei. In this study FISH was compared with flow cytometry and static image analysis in determining ploidy in paraffin-embedded tissue from 34 prostatic adenocarcinomas. Ploidy status using FISH was determined by enumerating centromeres of two chromosomes (8 and 12) by use of directly-labeled alpha-satellite DNA probes in isolated whole nuclei obtained by the Hedley technique. All three methods identified 11 of 34 cases as diploid and 17 of 34 cases as nondiploid (82% concordance). Six cases were discordant; two cases had discrepant results by each method. Ploidy classification as determined by FISH had an 88% concordance with ploidy classification by either flow cytometry or static image analysis. In conclusion, FISH was found to be a sensitive method of ploidy analysis in isolated paraffin-embedded nuclei from prostate adenocarcinomas. When the chromosomes commonly involved in aneuploidy have been identified in prostate adenocarcinoma, FISH has the potential to provide greater sensitivity for aneuploidy detection compared with currently available methods.

Adenocarcinoma↗

PubMed2005

Diagnosis of Helicobacter pylori infection and determination of clarithromycin resistance by fluorescence in situ hybridization from formalin-fixed, paraffin-embedded gastric biopsy specimens.

A reliable diagnostic test for Helicobacter pylori is important in clinical practice and research. The ideal diagnostic test for H. pylori should be sensitive, specific, and cost-effective. Helicobacter pylori resistance to clarithromycin is a common reason for failure of eradication therapy. The aim of this study was to evaluate the fluorescent in situ hybridization (FISH) method to detect H. pylori and determine clarithromycin resistance in formalin-fixed, paraffin-embedded gastric biopsy specimens. One hundred seventeen gastric biopsy specimens from patients with dyspepsia were examined for the presence of H. pylori by conventional culture, FISH, and histopathological methods. A set of fluorescent-labeled oligonucleotide probes binding to either H. pylori 16S rRNA or 23S rRNA sequences were used for FISH analysis. Phenotypic antibiotic susceptibilities of the isolates were tested using the Epsilometer test method (E test). Helicobacter pylori was detected in 70 of 117 biopsy specimens by histopathological examination and FISH, whereas it was detected in 47 specimens by culturing. Histopathology and FISH techniques failed to identify H. pylori in 1 biopsy sample isolated by culture. Clarithromycin resistance was found in 11 of 46 H. pylori isolates using the E test method. All of the phenotypic resistance measurements of isolates were correlated with genotypic clarithromycin resistance. Eleven clarithromycin-resistant strains were identified by FISH. The diagnosis of H. pylori infection and the determination of clarithromycin resistance in formalin-fixed, paraffin-embedded specimens using FISH is promising because it provides a rapid, reliable, and culture-independent diagnosis.

Anti-Bacterial Agents↗

PubMed2001

Determination of loss of heterozygosity in frozen and paraffin embedded tumors by denaturating high-performance liquid chromatography (DHPLC).

Spontaneous renal cell carcinoma (RCC) occurs with a high frequency in Eker rats carrying a germline alteration of the tuberous sclerosis-2 (Tsc-2) tumor suppressor gene. To determine the frequency with which the wild-type allele of the Tsc-2 gene is lost in RCC and the ability of DHPLC to detect loss of heterozygosity (LOH) at this gene locus, fresh-frozen and paraffin-embedded formalin-fixed tumors from heterozygous Eker rats (Tsc-2(Ek/+)) were examined for LOH at the Tsc-2 locus. LOH was determined by quantitation of peak areas of PCR products specific for the mutant and wild-type Tsc-2 alleles. For normal DNA isolated from heterozygous animals, the allele ratio (AR) of mutant to wild-type PCR products was empirically determined to be 1.5+/-0.3 (n=30) and LOH was defined as >2 standard deviations away from this mean, i.e. any AR >2.1. Analysis of 15 spontaneous frozen RCC samples showed LOH in 10/15 samples (66%). Carcinogen-induced tumors exhibited an even higher frequency of LOH, with 6/6 paraffin-embedded, formalin-fixed tumors exhibiting LOH. 100% concordance was observed between the results obtained by DHPLC and traditional methodologies. Therefore, LOH appears to occur with a high frequency in both spontaneous and carcinogen-induced RCC in this animal model and DHPLC is a sensitive and high throughput methodology for detecting this type of genetic alteration.

Alleles↗

PubMed1991

PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies.

The development of polymerase chain reaction (PCR) DNA amplification methods has afforded molecular studies of fixed paraffin-embedded tissue samples and other archival material. Some fixation methods damage DNA, and thus deleteriously affect subsequent PCR analysis. This study addressed the effect of short- and long-term storage (2 hr to 30 days) in a variety of fixatives (10% buffered-neutral formalin [BNF], 95% ethanol, acetone, and OmniFix) before paraffin embedding. We tested the ability of prepared tissue sections to yield DNA amplification products ranging from 268 to 1327 bp. Results indicated that tissues fixed for 8 days in BNF were able to amplify 536-bp but very little 989-bp DNA fragments; after 30 days of BNF fixation only a 268-bp fragment was amplifiable. Samples fixed in OmniFix and acetone yielded products of 989 and 1327 bp, respectively, after 8 days of fixation; both fixatives yielded 989-bp amplification products after 30 days of fixation. Tissues fixed in 95% ethanol for up to 30 days efficiently produced DNA amplification fragments of up to 1327 bp in length. The results provide important information for prospective studies that involve PCR analysis from archival material. Furthermore, fixation and long-term storage in ethanol should prove particularly useful in remote areas where refrigeration or immediate sample-processing is unavailable.

DNA↗

PubMed1990

Oestrogen receptor staining of paraffin-embedded breast carcinomas following short fixation in formalin: a comparison with cytosolic and frozen section receptor analyses.

This paper describes an improved immunohistochemical method for demonstrating oestrogen receptor (OR) protein in paraffin-embedded sections of tissue fixed for 1.5 h in formalin. Thirty-two cases of infiltrating ductal breast carcinoma were stained with a monoclonal anti-OR antibody (H222), using a standard streptavidin-biotin method, following pretreatment with pronase. OR counts in paraffin sections were compared with those of frozen sections and with cytosolic values determined by a dextran-coated charcoal method. Twenty-seven of the carcinomas were OR-positive in paraffin sections. There was concordance between the paraffin section and the frozen section-determined receptor status in 30 cases (94 per cent) and a strong correlation was observed (r = 0.76; P less than 0.0001). Similarly, OR counts in paraffin sections correlated with cytosolic OR values (r = 0.60; P less than 0.001) and there was concordance in 97 per cent of cases. The percentage of positively-stained tumour cells in paraffin sections ranged from 0 to 94 per cent with staining intensities comparable to those seen in frozen sections. Staining of paraffin sections identified more OR-positive tumours than either frozen section staining or cytosolic assay. This study validates immunohistochemical OR analysis in formalin-fixed, paraffin-embedded breast carcinomas using a commercial anti-OR antibody.

Adult↗

PubMed1996

Barrett esophagus with dysplasia. Flow cytometric DNA analysis of routine, paraffin-embedded mucosal biopsies.

Flow cytometric DNA ploidy analysis has been reported to be more objective and sensitive than morphologic evaluation as a surveillance method in patients with Barrett esophagus (BE) for the development and progression of precancerous lesions. Such analyses are typically performed using fresh samples that require a separate or "jumbo" biopsy, are prone to false DNA aneuploidy if not promptly processed, and do not allow for retrospective studies. The feasibility of performing flow cytometric DNA analysis on paraffin-embedded biopsies was studied to circumvent some of these problems using 12 squamous esophageal mucosa with inflammation and 58 BE cases showing varying degrees of dysplasia. Among the BE cases, 12 had no dysplasia, 20 were indefinite for dysplasia, 14 had low grade dysplasia, and 12 had high grade dysplasia. Satisfactory histograms were obtained in 86% of the analyzed samples. Among cases with adequate histograms, DNA aneuploidy was identified in 77% with high grade dysplasia, 16% with low grade dysplasia, 23% of indefinite for dysplasia, and 0% without dysplasia. One of the esophagitis samples was also DNA aneuploid. Correlation of DNA aneuploidy and degree of dysplasia is highly significant (P = .001). The authors have demonstrated that routinely processed paraffin-embedded biopsies can be used for flow cytometric ploidy analysis. DNA aneuploidy was highly correlated with degree of dysplasia and serves as a quantitative prognostic indicator for prospective as well as retrospective studies of the evolution of BE to carcinoma.

Barrett Esophagus↗

PubMed1997

Detection of thymidylate synthase gene expression levels in formalin-fixed paraffin embedded tissue by semiquantitative, nonradioactive reverse transcriptase polymerase chain reaction.

We describe a new, simple and reliable semiautomated strategy for quantifying mRNA from archival specimens by using oligo(dT)25 paramagnetic beads and the reverse-transcriptase polymerase chain reaction (PCR) coupled with quantitative digital image analysis (Q-DIA). To evaluate the experimental conditions, we examined thymidylate synthase (TS) gene expression in mRNA isolated from both flash-frozen and formalin-fixed paraffin-embedded human biopsy samples using biopsy material obtained from 2 patients prior to chemotherapy with 5-fluorouracil. Following the electrophoretic separation of the PCR products through a 20% polyacrylamide gel, quantitation of the perimeters of the silver-nitrate-stained PCR products will be done by Q-DIA using a video frame-grabber board attached to a CCD camera using Image-Pro+ software. Validation of this approach will involve a comparison of the observed gene expression levels to TS protein levels obtained by tissue homogenization assays of TS, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 2'-deoxyuridine-5'-monophosphate and 5-fluoro-2'-deoxyuridylate (FdUMP), by established [3H]FdUMP ligand-binding assays. The novelty of this method is that it offers a low-cost means whereby Q-DIA is performed directly from the gel to rapidly and accurately determine the level of TS gene expression, which is standardized against the beta-actin housekeeping gene. In the protocol described herein, gene expression studies can be done quickly and without the use of radioactive substances in both normal clinical samples shock frozen at the time of surgical excision and in formalin-fixed paraffin-embedded archival samples, which are commonly available in all hospital pathology departments. To demonstrate the utility of this method, mRNA was extracted from both nonpathological and tumor biopsies originating from both types of material from the same patients. TS gene expression in the flash-frozen and archival materials was compared to the level of TS intracellular enzyme activity in the same samples and a correlation of 89 and 80% between the shock-frozen and archival material relative to TS intracellular enzyme activity levels was observed. These findings suggest that routine semiautomated quantitative analysis of rare mRNA transcripts, e.g. TS, from archival material can be applied for retrospective studies.

DNA, Complementary↗

PubMed2001

Detection of gp43 and ITS1-5.8S-ITS2 ribosomal RNA genes of Paracoccidioides brasiliensis in paraffin-embedded tissue.

Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. An increase in PCM has been reported in recent years and the disease is now recognized as one of the imported fungal infections in Japan. To date, more than 15 cases of PCM have been reported in our country, and five of them were diagnosed by clinical and histopathological findings without mycological study. We applied 2 nested polymerase chain reaction (PCR) amplification methods for detecting P. brasiliensis genes from paraffin-embedded tissue specimens. Successfully amplified were: a 473 base pairs fragment of gp43 gene of P. brasiliensis (located from 741st to 1,213rd base), and a 418 base pairs fragment of 5.8S ribosomal RNA gene of P. brasilienisis which included internal transcribed spacers (ITS) 1 and 2 (located from 131st at ITS1 to 195th at ITS2) in paraffin-embedded murine tissues infected with P. brasiliensis yeast cells. The authenticity of the PCR products was confirmed by nucleotide sequence analysis. These results indicate that the two nested PCR methods may be useful for diagnosis of PCM.

Animals↗

PubMed2004

Efficient treatment of paraffin-embedded cervical tissue for HPV DNA testing by HC-II and PCR assays.

BACKGROUND AND OBJECTIVES: High-risk human papillomaviruses (HR-HPVs) are the primary cause of cervical cancer. In order to meet with clinical requirements, a direct capture test with signal amplification (HC-II), able to detect the 13 prevalent HR-HPVs, has been developed and validated for cytological specimens. STUDY DESIGN: the use of HC-II assay with formaldehyde-fixed paraffin-embedded cervical biopsies, for retrospective studies or to support histological findings, was investigated by analysing three different sample treatments. The efficacy of this test was compared with a reference PCR-ELISA, using MY09/11 and GP5+/6+ consensus primers and the use of a single extraction method for both HC-II and PCR-ELISA assays was validated. RESULTS: protease treatment of dewaxed biopsy sections allowed an optimal performance of HC-II and has also been validated for PCR-ELISA. Overall, on the analysis of 50 cervical samples HC-II and PCR-ELISA assays showed a high concordance (K=0.80). Compared with PCR-ELISA, the HC-II had a sensitivity of 86.4% and a specificity of 92.9%. The results showed that short amplimers are necessary for a sensitive PCR-ELISA from formaldehyde-fixed paraffin-embedded biopsies, while HC-II showed a relatively low sensitivity for HPV18 within the HR probe pool. CONCLUSIONS: HC-II can be a valid tool for the diagnosis of HPV infection in biopsy material. The possibility to use the same specimen preparation material for both HC-II and PCR-ELISA allows HC-II positive specimens to be further processed by PCR-ELISA if specific genotyping is needed.

Biopsy↗

PubMed2001

Detection of novel gene expression in paraffin-embedded tissues by isotopic in situ hybridization in tissue microarrays.

Correlating altered gene expression patterns with particular disease states is a critical step in understanding disease processes and developing treatment strategies. Many thousands of novel gene sequences have recently been annotated in public and private databases and are now available for analysis. Tissue-specific expression patterns of these sequences can be evaluated physically on DNA arrays and other high throughput assays, or virtually by bioinformatics mining of expressed sequence tag (EST) databases. As a secondary screening tool, in situ hybridisation (ISH) not only confirms tissue specificity, but also reveals what is often valuable information about cell-type expression patterns of nov16l sequences. Due to their availability and long-term stability at room temperature, formalin-fixed paraffin-embedded clinical specimens provide an invaluable resource for evaluating expression patterns of novel human genes. We describe a high-throughput approach for identifying and quantifying the expression of novel genes in paraffin-embedded human tissues using isotopic in situ hybridisation and tissue microarrays (TMA).

Blotting, Northern↗

PubMedDate not supplied

Detection of cutaneous Leishmania infection in paraffin-embedded skin biopsies using the polymerase chain reaction.

In this study the polymerase chain reaction (PCR) with previously developed oligonucleotide primers was used to detect Leishmania aethiopica in paraffin-embedded skin biopsy specimens. The Leishmania-specific 120 base pair fragment of the kinetoplast deoxyribonucleic acid (kDNA) minicircles has been amplified from all parasitologically or histologically confirmed cases of cutaneous leishmaniasis (CL), as demonstrated by gel electrophoresis and hybridization with L. aethiopica kDNA. Control specimens from patients with skin diseases other than CL were all negative. Using PCR, Leishmania were demonstrated in the skin lesions of 7 cases in a group of 40 patients in whom the parasites could not be demonstrated by histopathology or culture in vitro although lesions were clinically suggestive of CL. These data indicate that PCR, carried out on DNA extracted from formalin-fixed and paraffin-embedded tissue specimens, is a valuable method for the diagnosis of CL, especially in chronic cases where the parasite load in the lesion is low.

Animals↗