SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
A reliable method for the determination of total liver iron in formalin-fixed, paraffin-embedded tissue is presented. The correlation with total liver iron in fresh tissue is good (r = 0.92). Material, which is processed routinely in the pathological anatomical department and stored in the archives, can be used for a quantitative iron determination for clinical or research purposes.
Unlike other B cells, plasma cells (PC) react with only a few antibodies against haemopoietic antigens. We investigated 36 specimens exhibiting a reactive increase in PC numbers (i.e. plasmacytosis, PC hyperplasia) with a broad panel of antibodies suitable for use on formalin-fixed, paraffin-embedded tissue, and compared the findings with those obtained in 51 cases of multiple myeloma (plasmacytoma). Regardless of the immunostaining pattern for immunoglobulin light and heavy chains, reactive PC reacted with at least two and at most six of seventeen antibodies detecting haemopoietic antigens [Ber-H2/CD30 (91%), anti-leucocyte common antigen (LCA)/CD45 (86%), KP1/CD68 (64%), MB2 (57%), 4KB5/CD45RA (37%), DF-T1/CD43 (28%), UCHL1/CD45RO (20%), L26/CD20 (17%), MT2 (14%) and Mac387 (8%)], and with at least one and at most four of six antibodies against non-haemopoietic antigens [anti-epithelial membrane antigen (EMA) (94%), anti-vimentin (77%), anti-pan-cytokeratin/KL1 (74%), BMA120 (51%) and HMB45 (14%)]. Five antibodies stained reactive PC significantly more often than neoplastic PC: Ber-H2/CD30 (p < or = 0.0000), KP1/CD68 (p < or = 0.0000), anti-LCA/CD45 (p < or = 0.0000), anti-EMA (p < or = 0.0339) and anti-pancytokeratin/KL1 (p < or = 0.0000). The more frequent and more heterogeneous expression of antigens by reactive PC suggests that the aberrant immunoreactivity of neoplastic PC in plasmacytoma is not due to the process of malignant transformation in an early step of B-cell differentiation, but could reflect the heterogeneity of antigen expression by normal PC.
The gene encoding the specific glycosyltransferases which catalyze the conversion of the H antigen to A or B antigens shows a slight but distinct variation in its allelic nucleotide sequence and can be divided into 6 genotypes when digested with specific restriction enzymes. We extracted DNA from formalin-fixed, paraffin-embedded tissues using SDS/proteinase K treatment followed by phenol/chloroform extraction. The sequence of nucleotides for the A, B and O genes was amplified by the polymerase chain reaction (PCR). DNA fragments of 128 bp and 200 bp could be amplified in the second round of PCR, using an aliquot of the first round PCR product as template. Degraded DNA from paraffin blocks stored for up to 10.7 years could be successfully typed. The ABO genotype was deduced from the digestion patterns with an appropriate combination of restriction enzymes and was compatible with the phenotype obtained from the blood sample.
We investigated whether the determination of clonality by polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain (IgH) gene rearrangements could be helpful in the evaluation of B-cell lymphoma (BCL) involvement of bone marrow (BM) biopsy specimens. We evaluated 83 paraffin-embedded BM biopsy specimens from 26 patients with BCL. When BM biopsy specimens considered positive, "suspicious," or negative by morphologic and immunohistochemical examination were evaluated by PCR, a monoclonal B-cell population was detected in 81% (39/48), 64% (9/14), and 11% (2/18), respectively. In most cases, a reproducible monoclonal IgH gene rearrangement was observed from BM and extramedullary sites. Nevertheless, in 4 cases, a different and independent monoclonal IgH rearrangement was observed during the disease course. PCR is efficient and complementary to morphologic and immunohistochemical examination for the evaluation of BCL involvement of BM biopsy specimens, especially when a reproducible rearrangement is found in 2 different samples.
The histopathology archive represents a vast, well-characterized source of specimens covering virtually every disease and is available for molecular biological investigation. The archive has in recent years become widely used for molecular genetic analysis and DNA can be routinely extracted from formalin-fixed, paraffin-embedded tissue. More recently, archival specimens have become a source of material for extensive analysis of mRNA expression utilizing DNA microarrays, real-time quantitative reverse transcriptase polymerase chain reaction (PCR), and in situ hybridization and amplification techniques. These techniques will enable a greater understanding of the changes that occur in gene function during every stage of the development of disease and will lead to better diagnosis, better evaluation of prognosis, and better treatment through targeted therapeutic regimes.
We report the development of a reverse transcriptase polymerase chain reaction assay that reliably detects the ETV6-NTRK3 chimeric RNA characteristic of infantile fibrosarcoma and the cellular variant of congenital mesoblastic nephroma (CMN) in formalin-fixed, paraffin-embedded tissue blocks. The 188 base pair polymerase chain reaction fusion product was detected in 11 of 12 cases of cellular CMN from which a larger sized control RNA band could be amplified, and even in 7 of 8 cases in which the control band was not detectable. A variety of other tumors that are in the histologic differential diagnosis of cellular CMN yielded negative results, including four classic CMNs, four rhabdoid tumors of the kidney, and four clear cell sarcomas of the kidney, confirming the assay's specificity. We further demonstrate the assay's utility by illustrating two cases of molecularly confirmed cellular CMN that mimicked rhabdoid tumor and clear cell sarcoma of the kidney. In contrast to previous reports, five mixed CMNs that had both classic and cellular areas all lacked the ETV6-NTRK3 fusion transcript. These results suggest that cases morphologically defined as mixed CMN may represent a mixed group of genetically distinct entities.
OBJECTIVE: To evaluate a group of vaccine site-associated sarcomas (VSS) for the presence of feline foamy virus (FeFV) DNA, using polymerase chain reaction (PCR) methods. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded (FFPE) tissue blocks from VSS of cats. PROCEDURE: DNA was extracted from FFPE sections of each tumor, and regions of the gag and pol genes of FeFV were amplified by use of PCR methods, using 1 primer set for each region. Sensitivity of the method was compared between fresh and FFPE cells, using mouse kidney tissue that was injected with FeFV-infected cultured cells and using agarose-cell pellets. Results-Feline foamy virus DNA was not detected in VSS tissues. Sensitivity of the method was 10 times greater in fresh versus FFPE mouse tissues. Sensitivity of the method in fresh FeFV-infected cultured cells versus FFPE agarose-cell pellets was equal when fixation was 24 or 48 hours and 10 times greater when fixation was 72 hours or 1 week. CONCLUSIONS AND CLINICAL RELEVANCE: A PCR-based method can be successfully applied to FFPE tissues for FeFV DNA detection. Results suggest there is no direct FeFV involvement in the pathogenesis of VSS in cats.
A novel assay based on a nested PCR and restriction enzyme analysis of the PCR products was developed for the rapid detection and identification of Mycobacterium bovis and M. avium-M. intracellulare species in formalin-fixed, paraffin-embedded tissue (PET) specimens. On the basis of the nucleotide sequence data obtained in the present study, general nested primers were constructed to amplify a 424-bp segment of the gene encoding the 65-kDa surface antigen of mycobacteria. The nested PCR assay proved to be highly sensitive, since as little as 5 to 10 fg of extracted mycobacterial DNA was detected. The safety of the assay as a routine method for the diagnosis of M. bovis and M. avium-M. intracellulare in PET specimens was provided by taking various precautions. In order to prevent false positivity, specific tools and procedures were applied. To detect false-negative results and assess the efficiency of the PCR, an internal standard molecule of amplification was constructed. The digestion of the amplicons with the restriction endonuclease Sau96-I allowed the identification of M. bovis and M. avium-M. intracellulare in a large number of clinical specimens. The present results indicate that PCR combined with an internal control of amplification and restriction enzyme analysis of the amplicons provides a rapid, sensitive, and reliable method for routine diagnostic laboratories to detect and identify M. bovis and M. avium-M. intracellulare in PET specimens.
A rapid non-isotopic PCR-SSCP (polymerase chain reaction-single-stranded conformation polymorphism) method was developed in this study to detect polymorphism and loss of heterozygosity (LOH) of p53 in formalin-fixed and paraffin-embedded samples of normal breast tissue and of breast cancer. p53 expression was also examined by immunohistochemistry. In 35 paired samples, heterozygosity in exon 4 of p53 was detected in 17 cases (49 per cent) and LOH of the p53 gene in breast cancer tissues was observed in 7 out of 15 informative cases (47 per cent). The correlation of LOH of p53 with positive p53 immunostaining did not reach statistical significance, but all immunostaining-positive tumours among informative cases had LOH of p53. The results support the hypothesis that in most cases the allelic deletion of p53 may uncover the 'recessive mutation' in the remaining allele. However, LOH of p53 was more frequent than positive immunostaining and was significantly associated with poor differentiation of breast cancer (P < 0.05). The results suggest that the allelic deletion of p53 may also contribute to the development and progression of breast cancer by reducing the amount of normal p53 protein. These results show that non-isotopic PCR-SSCP is a simple, fast, and effective method for detecting polymorphism and LOH of the p53 gene, which is especially useful for retrospective studies.
PCNA is a 36-KD proliferating cell nuclear antigen associated with the cell cycle. The immunocytochemical detection of PCNA represents a useful tool for the study of tumor proliferation activity. This study documents the detection of PCNA, using antibody PC 10 in formalin-fixed, paraffin-embedded tissue, and correlates the proliferative activity of the non-Hodgkin's lymphomas (NHL) with histological grading assessed by the International Working Formulation (WF) and Kiel classification. In 92 cases of NHLs we found a strong correlation between the PCNA index and lymphoma grading. Statistically significant differences were also found between the proliferative index (PI) in low and high grade lymphomas according to the Kiel classification (t = 9.519; p < 0.001) and between low, intermediate and high grade lymphomas according to the WF classification (F = 79.01; p < 0.001). In the Kiel classification the mean of low grade lymphomas was 39.5% and of high grade 75.7%. In the WF the average of low grade lymphomas was 29.7%, intermediate 53.1% and high 75.1%. Although the differences among the groups had been significant, we found variations inside each histological subgroup in both classifications. The intermediate lymphomas were the most heterogeneous group, with PI inside the same histologic subtypes coincident with low and high grade lymphomas. Since PCNA may be used as a marker of cell proliferation in clinical studies to estimate the biological aggressiveness of lymphomas, its determination in intermediate grade NHL could be very useful to evaluate individual cases in this group and determine prognosis and probably the appropriate therapy.
A panel of monoclonal antibodies (Leucocyte Common Antigen [CD45], LN-1 [CDw75], LN-2 [CD74], LN-3, L-26, UCHL-1 [CD45RO] and MT-1) which identify B and T cell surface markers were applied to benign lymphocytoma cutis and malignant lymphoma of the skin in paraffin-embedded sections. In five cases of lymphocytoma cutis, both B and T cell markers were positive, consistent with the polyclonality of the proliferative lymphocytes. In contrast, in two cases of B cell lymphoma, only the B cell markers were positive, consistent with the monoclonality. Furthermore, the immunohistochemistry clarified lymph follicle structures of lymphocytoma cutis. These were occasionally not evident in routine hematoxylin and eosin stained sections. Thus an immunologic phenotyping study combined with other appropriate criteria for histologic evaluation and clonal gene rearrangement analysis appears to be useful in distinguishing reactive from neoplastic lymphoid lesions of the skin.
Although recent studies have analyzed Hepatitis C (HCV) infections in liver tissue by in situ hybridization (ISH), many of these studies have been of limited diagnostic utility because of the low copy numbers of HCV in formalin-fixed paraffin-embedded (FFPE) tissue and failure to correlate the ISH analysis with other methods of detecting HCV. Thirty six cases of liver biopsies from patients with known HCV antibody status including 20 cases of serum HCV positive and 16 cases of serum HCV negative were analyzed. All cases showed histologic features suggestion of HCV infection. Analyses of all 36 cases were done by RT-PCR combined with Southern hybridization (RT-PCR-SH) and in situ hybridization (ISH). A prolactin riboprobe was used as a negative control. Immunohistochemistry (IHC) with an antibody against HCV (Rb 246) was also used to analyze HCV viral protein in the tissues. Of the 20 serum antibody-positive cases, RT-PCR-SH detected 18 positive cases, while ISH and IHC detected 19 and 16 positive cases, respectively. Of the 16 serum antibody-negative cases, RT-PCR-SH detected 8 positive cases while ISH and IHC detected 8 and 11 positive cases, respectively. A positive ISH signal for HCV was also detected in some lymphocytes and bile ducts in the liver. These results show that ISH with a highly specific riboprobe is comparable to RT-PCR-SH for detection of HCV infection in liver tissue.
OBJECTIVE: To develop a possible alternative laboratory diagnostic method, an in situ polymerase chain reaction (ISPCR) for definitive diagnosis of human cytomegalovirus (HCMV) pneumonia in infants. METHODS: The material involved in this study was formalin-fixed paraffin-embedded lung tissue specimens from 6 infants died of histopathologically diagnosed cytomegalic inclusion body pneumonia (CMIBP) and from 4 infants died of other diseases. When DNA extracts from the specimens of these 10 infants were tested by using a previously applied conventional PCR, the specimens from the 6 infants with CMIBP were positive for HCMV DNA, while those from the other 4 infants were negative. In the ISPCR, biotin-11-dUTP was used for labeling the amplified products and streptavidin, biotin-alkaline phosphatase for in situ color development. RESULTS: The results of the study showed specific staining for HCMV DNA in the lung tissue slices from the 6 cases with CMIBP, while no staining was found in the specimens from the 4 infants who died of other diseases. The control ISPCR tests on the HCMV DNA positive specimens without adding either biotin-11-dUTP or Taq DNA polymerase or the primers for HCMV DNA showed negative results. The specific staining for HCMV DNA was seen in bronchial and alveolar epithelial cells, small vascular endothelial cells and leukocytes in the lumens of small blood vessels. CONCLUSIONS: The results suggested that the ISPCR was able to demonstrate both presence of HCMV DNA in the lung tissues and active HCMV infection; therefore the ISPCR may be applicable in definitive diagnosis of HCMV pneumonia when proper specimens such as lung biopsy material or bronchoalveolar lavage is available for the test.
We developed a real-time PCR which allowed the highly sensitive detection of Naegleria fowleri in histological brain tissue sections from experimentally infected mice. This genus-specific small-subunit (18S) rRNA gene-based PCR can complement conventional (immuno-) histology for the diagnosis of primary amoebic meningoencephalitis in paraffin-embedded brain necropsy specimens that had been fixed in formalin buffered with phosphate-buffered saline.
To overcome the degradation problem encountered in DNA extracted from formalin-fixed, paraffin-embedded tissue blocks, several methods of tissue fixation were examined in order to improve the quality of the DNA recovered for use in nucleic acid analysis. The fixation methods included formalin fixation alone, alcohol fixation alone, and microwave fixation with tissues immersed in phosphate-buffered saline (PBS), alcohol, or formalin. Unfixed fresh frozen tissue served as the control. Using hepatitis B virus (HBV) DNA sequences and the type I human procollagen gene as markers and liver tissue as a target, microwave fixation, with formalin omitted, not only preserved the DNA very well, but also the labile viral antigen. Both high molecular weight-integrated and free-form HBV DNAs were well preserved, and suitable for polymerase chain reaction and Southern blot analysis. The restriction enzyme fragment pattern of DNA recovered from these paraffin blocks was identical to that of unfixed fresh frozen tissue. Microwave fixation also preserved the labile preS2 epitope of the hepatitis B surface antigen (HBsAg) considerably better than formalin. These results suggest that microwave fixation is superior to routine formalin fixation for the preservation of excellent quality of genomic and viral DNAs for nucleic acid hybridization analysis. Alcohol, often used for nucleic acid purification, was also a good fixative for preserving DNA and the antigenicity of the labile antigen, especially when carried out in combination with microwave fixation.
This paper establishes a method of clonality analysis using the reverse transcription-polymerase chain reaction (RT-PCR) to amplify X-linked G6PD transcripts on defined cell populations microdissected from archival, paraffin-embedded tissue sections. Four known monoclonal low-grade B-cell lymphomas from females who were heterozygous (informative) at the 1131 exonic polymorphic locus of the G6PD gene were used to validate the method. Lymphoma and reactive lesions in each case were separated by microdissection. In order to preserve the intact RNA species in the lesion, sections were digested on the slides before microdissection. A one-step RT-PCR was performed with a single pair of primers, one of which contained a mismatched base adjacent to the polymorphic site, to generate a PvuI cutting site. Successful amplification and allele identification by PvuI digestion were achieved from all RNA samples studied. Three of four samples from non-neoplastic reactive lesions showed two bands with equal intensity, representing transcription of the two alleles of the G6PD gene, while the corresponding tumour samples demonstrated a biased intensity in one allele, indicating monoclonality. To assess the method further, the clonal nature of in situ and invasive breast cancers was examined, along with adjacent normal breast tissue and hyperplastic lesions from three informative females from our archives. Apart from the clusters of normal terminal duct-lobular units, all lesions were monoclonal. This result is in agreement with data derived from other X-linked gene studies and loss of heterozygosity (LOH) analyses of pre-invasive breast disease. The results suggest that the clonality analysis method presented here is simple and reliable, and is therefore potentially applicable in a wide range of pathological conditions.
A murine monoclonal antibody prepared against an ovine abortion isolate of Chlamydia psittaci (A22/Teramo) revealed specific binding to a 57 kDa chlamydial antigen in immunoblotting studies. The monoclonal antibody was able to detect intracytoplasmic chlamydial inclusions and scattered elementary bodies in infected McCoy cell culture, and on formalin-fixed paraffin-embedded tissue sections both from experimentally infected mice and from fetal membranes of cases of ovine enzootic abortion.
We present a strategy for the evaluation of numerical copy number changes of DNA segments within a solid tumor genome that allows the correlation of microscopic phenotype with genotype in formalin-fixed, paraffin-embedded tumor material. Cells from a human testicular germ cell tumor and adjacent tissue areas with normal seminiferous tubules were selected separately from microscopically analyzed histological tissue sections, and DNA was extracted from the selected areas. After universal DNA amplification, the amplification products were subjected to comparative genomic hybridization. The results confirmed balanced chromosome copy numbers for the normal tissue area, although the analysis of the tumor tissue area revealed numerous gains and losses of chromosome segments. The comparative genomic hybridization results were used to select DNA probes for interphase cytogenetics on serial sections. We conclude that this technique allows the screening of selected tissue areas for numerical DNA alterations, thus enabling a direct phenotype-genotype comparison.