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PubMed2003

Detection of hepatitis C virus RNA in formalin-fixed, paraffin-embedded thin-needle liver biopsy specimens.

We examined the feasibility of detecting hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) specimens obtained by thin-needle biopsy (TNB). Specimens obtained by large-needle biopsy (LNB) and unfixed frozen tissue served as controls. A total of 23 biopsy specimens, 13 obtained by TNB and 10 by LNB, from 20 patients with chronic hepatitis C were included in the study. HCV RNA was detected by nested reverse transcription-polymerase chain reaction (RT-PCR). HCV RNA was found in FFPE archival specimens obtained by TNB (11 [85%]) or LNB (7 [70%]). The sensitivity was similar in unfixed tissue in which HCV RNA was found in 7 (88%) of 8 TNB specimens and 6 (86%) of 7 LNB specimens. The detection of HCV RNA did not seem to be affected by storage of the paraffin blocks, the presence of advanced fibrosis and cirrhosis, or fragmentation of the core cylinder. TNB yields enough qualitative suitable material to detect HCV RNA by RT-PCR.

Adult↗

PubMed1988

Detection of early myocardial infarction in formalin-fixed, paraffin-embedded tissue.

Whether the early infarct area in formalin-fixed, paraffin-embedded tissue could be delineated by the immunohistochemical method using myoglobin-antibody was studied in 23 pig hearts without collateral circulation. Five hearts were examined at 20 minutes, 2 hours, 4 hours, and 6 hours after occlusion of the distal one third of the left anterior descending coronary artery, respectively. Three pigs were killed 24 hours after occlusion. Heart rate and aortic pressure before and after occlusion did not change in any groups. The subepicardial and subendocardial regional blood flows were reduced to almost zero in all hearts after occlusion (0.88 +/- 0.10 to 0.02 +/- 0.02 mL/g/min). Slight myoglobin defects in the ischemic tissue were noted in the five pigs examined 2 hours after occlusion and definite myoglobin defects were detected in all pigs examined at 4, 6, and 24 hours after occlusion. Nitrotetrazorium blue stain of myocardial tissue before formalin fixation showed slight demarcation of the ischemic area at 4 hours after occlusion and definite demarcation at 6 and 24 hours after occlusion. Slight demarcation was noted at 2 hours after occlusion in Masson trichrome stain and 4 hours after occlusion in the hematoxylin-eosin stain. However, definite demarcation of the ischemic area was seen in Masson trichrome stain only at 24 hours after occlusion and was not noted in hematoxylin-eosin stain even at 24 hours after occlusion. Our previous electron microscopic study revealed that, in the pig heart, irreversible cellular damage was transmurally seen at two hours after occlusion of the coronary artery. Therefore, a definite myoglobin defect reflects irreversible cellular damage such as infarction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Enhancement of hybridization efficiency in interphase cytogenetics on paraffin-embedded tissue sections by microwave treatment.

To enhance the efficiency of in situ hybridization in interphase cytogenetics on formalin-fixed and paraffin-embedded tissue sections we studied the effect of microwave heating as an addition to tissue pretreatment. The modified protocol resulted in a reduction of proteolytic digestion time and of effective enzyme concentration. Apart from that, reproducibility was raised and in large sections successful hybridization was more homogenous. We conclude, that the given technical recommendations should be useful for the study of chromosomal aberrations in routinely processed tissues.

Chromosome Aberrations↗

PubMed1980

Glial fibrillary acidic protein in the retina of the developing albino rat: an immunoperoxidase study of paraffin-embedded tissue.

The peroxidase-anti-peroxidase method was used on paraffin-embedded material to demonstrate the distribution of glial fibrillary acidic (GFA) protein, an astrocyte-specific protein, in the developing retina of the albino rat. At birth activity was scant and was confined to scattered, poorly differentiated cells in the inner retinal layers near the optic disc. At 3 days primitive astrocytes which displayed GFA protein activity were confined to the stratum opticum near the optic disc. With increasing age these cells were found at greater distances from the optic disc and began to assume the appearance of typical fibrous astrocytes. By 30 days the perikarya of these cells were confined almost exclusively to the region between the nerve fiber layer and the inner limiting membrane. The processes of these cells terminated either in suckerlike end-feet upon blood vessels or, to a lesser extent, ended in relation to axon fascicles of the nerve fiber layer. A second population of GFA protein-active cells existed as perivascular glia which were found upon vessels in the inner portion of the stratum opticum in young animals. In the mature retina perivascular glia were found on vessels throughout the stratum opticum and in the inner portion of the inner plexiform layer. Unequivocal staining of Müller cells or their processes was not obtained. The best staining was obtained with fixatives containing minimal concentrations of aldehydes, especially in tissue from younger animals. The fixative which gave the best preservation of cellular structure along with preservation of GFA protein antigenicity was Perfix (Fischer Scientific Company).

Animals↗

PubMed1983

Plasma cell lesions of the head and neck: immunofluorescent determination of clonality from formalin-fixed, paraffin-embedded tissue.

An immunofluorescent technique was used on formalin-fixed, paraffin-embedded tissue to characterize and diagnose plasma cell lesions of the head and neck. Infiltrates were classified as either monoclonal or polyclonal with rhodamine and fluorescein staining of light chains and heavy chains. In the cases in which histopathologic diagnoses were relatively certain, immunofluorescence provided good correlation. In those cases in which histopathologic diagnoses were equivocal, immunofluorescence distinguished between reactive and neoplastic infiltrates through the determination of clonality of the infiltrates.

Clone Cells↗

PubMed1990

Acute myeloid leukemia: immunohistologic findings in paraffin-embedded bone marrow biopsy specimens.

Immunohistochemical investigations were performed on decalcified, paraffin-embedded iliac crest trephine biopsy specimens from 30 cases of acute myeloid leukemia (AML, as defined by the FAB classification) with antibodies against B cells (L26, 4KB5, MB1, Ki-B3), T cells (UCHL1, MT1), myeloid/histiocytic cells (anti-neutrophil elastase, MAC387, anti-S-100 protein, anti-alpha 1-antichymotrypsin, DAKO-M1), natural killer/killer cells (anti-Leu-7), and megakaryocytes (anti-factor VIII-related antigen). (1) The blast cells of all the cases reacted with from at least two to at most eight different antibodies. Each antibody reacted with blast cells in a minimum of two (maximum 30) cases. (2) MT1, Ki-B3, anti-alpha 1-antichymotrypsin anti-neutrophil elastase, anti-S-100 protein, and MAC387 stained blast cells in more than 50% of the cases; MB1, L26, UCHL1, 4KB5, and DAKO-M1 in 20% to 50% of the cases; and anti-Leu-7 and anti-factor VIII-related antigen in less than 20% of the cases. (3) In the majority of cases many T lymphocytes, a small-to-moderate number of B lymphocytes, and a few Leu-7-positive lymphoid cells were intermingled with the blast cells. In some cases, especially where only a minor proportion of the blast cells was immunostained, it was nearly impossible to distinguish the lymphocytes of the tumor's stromal reaction from small blast cells. Thus, AML exhibits a heterogeneous immunophenotype in trephine biopsy specimens. Immunohistologic diagnosis of this disease in such specimens may be extremely difficult. Since staining of the blast cells with one or more of the antibodies generally used to define B cells, T cells, or their neoplastic derivatives is not uncommon, misinterpretation as non-Hodgkin's lymphoma of high-grade malignancy could easily occur. These findings also suggest that mixed-type (hybrid) acute leukemias with coexpression of myeloid and lymphoid cell markers could be more common than generally realized.

Adolescent↗

PubMed1999

Use of nested polymerase chain reaction (PCR) for detection of retroviruses from formalin-fixed, paraffin-embedded uveal melanomas in cats.

Thirty-six formalin-fixed, paraffin-embedded enucleated globes from cats with a diagnosis of diffuse anterior uveal melanoma were obtained. Sections of tumor were excised, deparaffinized, and subjected to nested polymerase chain reaction (PCR) to identify proviral DNA sequences from the feline leukemia virus (FeLV)-feline sarcoma virus (FeSV; 36 eyes), and the feline immunodeficiency virus (FIV; 18 eyes). All samples tested were negative for FIV DNA. Three samples were positive for FeLV-FeSV DNA. This is the first reported evidence of a possible link between naturally occurring feline anterior uveal melanoma and the presence of FeLV-FeSV DNA.

Journal Article↗

PubMed2006

Mass spectrometric analysis of formalin-fixed paraffin-embedded tissue: unlocking the proteome within.

The predominance of tissues stored worldwide in hospitals and clinical laboratories exist in formalin-fixed paraffin-embedded (FFPE) blocks that are generated by simple and well-established protocols. Although generation of FFPE tissues has facilitated their characterization by such techniques as histopathology, they have proven refractory to biomarker discovery investigations using state-of-the-art MS-based proteomic methodologies. Very recently new methods have been developed that enable proteins extracted from FFPE tissues to be analyzed by MS. This review will highlight and discuss those efforts that have led to this exciting recent progress. Although these developments are quite new, the ability to conduct MS-based proteomic analyses of FFPE tissues opens heretofore intractable clinical samples for discovery-based biomarker research.

Cryopreservation↗

PubMed2000

Quantitative molecular analysis of laser-microdissected paraffin-embedded human tissues.

Laser microdissection enables the contamination-free isolation of morphologically defined pure cell populations from archival formalin-fixed paraffin-embedded tissue specimens. Cells isolated by this method have been characterized by a wide variety of qualitative molecular assays, e.g. loss of heterozygosity, point mutations, clonality and lineage origin. The recently introduced real-time PCR technology renders the reliable quantification of very small amounts of nucleic acids possible. Several groups including our own showed that this technique can be successfully applied for the quantification of DNA and RNA isolated from microdissected archival tissue sections, even after immunohistochemical staining. The exact analysis of quantitative changes of nucleic acids during the course of pathological alterations has thus become possible. In many situations these quantitative changes can be expected to be more important than qualitative changes. The new technology for the quantification of structural genomic alterations and changes in the gene expression pattern in conjunction with microdissection have equipped morphologists with a powerful tool to study reactive and neoplastic changes of tissues.

Cell Separation↗

PubMedDate not supplied

Immunohistochemical detection of infectious bursal disease virus in formalin-fixed, paraffin-embedded chicken tissues using monoclonal antibody.

Immunoperoxidase and immunofluorescence techniques detected and localized infectious bursal disease virus (IBDV) in formalin-fixed paraffin-embedded sections of the bursa of Fabricius of experimentally and naturally infected chickens. The primary antibody was a monoclonal antibody that bound all IBDV serologic subtypes, including the GLS isolate. Both techniques were valuable in detecting IBDV. The presence and severity of microscopic lesions in the bursa correlated with the location and number of positive IBDV-infected cells as measured by either test. Mild vaccine strains induced minimal microscopic lesions and resulted in a few cells positive by either test. In contrast, virulent IBDV produced widespread lymphoid necrosis and numerous cells positive by both assays. The immunoperoxidase test was more useful than the immunofluorescence test, since the same section could be stained and examined by immunoperoxidase and then restained and examined for microscopic pathology. The presence of immunoperoxidase-positive stained cells associated with areas of microscopic pathology suggested that microscopic lesions were induced by IBDV.

Animals↗

PubMed1993

Molecular detection of Borrelia burgdorferi in formalin-fixed, paraffin-embedded lesions of Lyme disease.

A system for the detection of a Borrelia burgdorferi (Bb)-specific gene segment in formalin-fixed, paraffin-embedded skin lesions is described. A nested polymerase chain reaction technique is used to selectively amplify in vitro a short segment of a Bb-specific gene recently described by Rosa et al. (J Infect Dis 1989: 160: 1018). The design of oligonucleotide primers for the amplification of a relatively short gene segment allows the successful analysis of DNA which has been altered by fixation in formalin. Using this technique, Bb-specific DNA was clearly identified in 8 of 12 specimens of erythema chronicum migrans and in 1 case of lymphadenosis benigna cutis. These skin lesions are known to represent cutaneous manifestations of Lyme disease. Negative control reactions, using DNA from borrelial strains not related to Lyme disease, were negative. The system enables the dermatopathologist to identify Bb in routinely fixed clinical specimens and allows the rapid analysis of various skin diseases for which an association with Bb so far has only been hypothesized.

Base Sequence↗

PubMed1994

The prognostic role of the DNA ploidy pattern in colorectal cancer analysis using paraffin-embedded tissue by an improved method.

To assess the prognostic value of DNA ploidy in colorectal cancer, compared with the histopathological findings, paraffin-embedded surgical specimens from 330 patients who underwent resection for primary adenocarcinoma were studied using a new modified method of flow cytometry. Of these specimens, 141 were DNA diploid and 189, DNA aneuploid, among which there were 3 DNA hypodiploid lesions. Of the ten variables studied in curative resection, DNA ploidy ranked fourth in prognostic significance according to the linear trend by the chi 2 test, after nodal status, grade of cellular differentiation, and degree of invasive growth, if the DNA ploidy pattern was classified into three categories. Conversely, DNA ploidy was the sixth most significant factor if DNA hypodiploidy was included in the DNA aneuploidy. The Cox multivariate analysis showed that DNA ploidy was one of the five significant factors independently determining prognosis; however, if adjustment for the modified Dukes' stage was made by the Mantel-Haenszel test, the survival difference between the diploid and aneuploid groups did not reach a statistically significant level. Thus, we conclude that from a practical point of view, DNA ploidy is not an essential factor which must be combined with histopathological variables for a better prediction of patient outcome.

Colorectal Neoplasms↗

PubMed1992

DNA quantitation by image analysis of paraffin-embedded colorectal adenocarcinomas and its prognostic value.

DNA content was measured by image analysis in a retrospective study of formalin-fixed paraffin-embedded colorectal carcinomas from 213 patients who were followed up for at least 5 yr. DNA histograms were classified as diploid, aneuploid, or tetraploid. Diploid tumors comprised 29% of all cases, aneuploid 50%, and tetraploid 21%. Aneuploid tumors were found more often in patients with advanced disease and in carcinomas arising in the rectum. Pathologic stage, histologic grade, and ploidy were individually related to survival and recurrence. However, after stage stratification, histologic grade was no longer a significant prognostic factor. In patients without regional or distant metastases (Dukes' Stage A and Stage B), patients with aneuploid tumors had a statistically worse prognosis than patients with diploid or tetraploid tumors (P less than 0.01). The prognostic value of ploidy in this group of patients was maintained only in tumors arising in the distal colon and rectum (P less than 0.04). In patients with regional or distant metastases, DNA content did not provide additional prognostic information. In conclusion, DNA quantitation can be evaluated reliably by image analysis of archival material and can provide valuable prognostic information, especially in patients with Dukes' Stage A and Stage B disease. It may prove useful in guiding adjuvant therapy in these patients.

Adenocarcinoma↗

PubMed1996

Immunohistochemical identification of B and T lymphocytes in formalin-fixed, paraffin-embedded feline lymphosarcomas: relation to feline leukemia virus status, tumor site, and patient age.

The lymphocyte phenotype of 70 formalin-fixed, paraffin-embedded feline lymphosarcomas (LSAs) was determined immunohistochemically using a T cell polyclonal antibody, and a B cell monoclonal antibody. Forty-seven of 70 (67%) tumors were T cell, 19/70 (27%) were B cell, and 4/70 (6%) did not stain with either marker. Thirty-eight of 70 (54%) tumors were positive for feline leukemia virus (FeLV) antigen by immunohistochemistry (IHC), and 52/70 (74%) tumors were positive for FeLV DNA using the polymerase chain reaction (PCR). B cell tumors were as frequently FeLV-positive as T cell tumors using either IHC or PCR. Intestinal tumors were more likely to be B cell than T. The incidence of B and T cell tumors was not different among young (< or = 3 y), middle-aged (> 3 y to < or = 8 y), and old (> 8 y) cats. Both B and T cell tumors from old cats were FeLV-positive more often by PCR than by IHC. Feline leukemia virus DNA but not antigen, was detected in B cell tumors and intestinal tumors from cats > 8 y as often as it was detected in B cell tumors and intestinal tumors from cats < or = 8 y. Previously, most B cell and intestinal tumors from old cats were considered to be negative for FeLV. Here, the results suggest involvement of latent or replication-defective forms of the virus in such tumors from old cats. This study supports a role for FeLV in feline B cell as well as T cell tumorigenesis.

Aging↗

PubMed1991

Detection of P-glycoprotein with JSB-1 monoclonal antibody in B-5 fixed and paraffin-embedded cell lines and tissues.

We analyzed the expression of P-glycoprotein (Pgp) by immunohistochemistry using JSB-1 monoclonal antibody (MAb) on paraffin-embedded sections of the multi-drug resistant (MDR) (CHrC5 and CEM-VLB), and sensitive (AuxB1 and CEM) cell lines, and also in normal kidney, colon, adrenal and in kidney and colon carcinomas. After comparing the sensitivity of three different immunohistochemical techniques the peroxidase-antiperoxidase method was found to be the best. We then tested six different fixation methods. The MDR cell lines and human tissues demonstrated the strongest staining with B-5 fixative. Both MDR cell lines, but not the tissues fixed in 1% paraformaldehyde and Zamboni's fixative demonstrated weak staining. No immuno- reactivity could be detected in MDR cell lines and tissues fixed in 10% buffered or nonbuffered formalin or by the AMeX method of tissue processing. The present study clearly shows that the type of fixative is critical for the preservation of Pgp epitope recognized by JSB-1 MAb, and that B-5 fixative is expected to be equally applicable for the detection of Pgp in normal and neoplastic tissues.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Methacarn fixation for genomic DNA analysis in microdissected, paraffin-embedded tissue specimens.

We recently found methacarn to be a versatile fixative for analysis of RNA and protein applicable for microdissected specimens from paraffin-embedded tissue (PET). In this study we investigated the performance of methacarn for genomic DNA analysis using microdissected rat tissues. We found that extensive portions of DNA up to 2.8 kb could be amplified by nested PCR using DNA templates extracted by a simple and rapid extraction procedure from a 1 x 1-mm area of cerebral cortex of a 10-microm-thick section. By nested PCR, a 522-bp fragment from a single cell could be amplified in 20% of cresyl violet-stained Purkinje cells, and the minimal number of cells required, as estimated using hippocampal neurons, was on the order of 10-20. Although tissue staining with hematoxylin and eosin affected the PCR, amplification of a 522-bp fragment was successful, with 150-270 cells by 35 cycles of single-step PCR. Immunostaining resulted in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, a 184-bp DNA fragment could be amplified with 150-270 cells by 35 cycles of PCR. The results thus demonstrate the superior performance of methacarn to that reported with formalin in genomic DNA analysis using microdissected PET specimens.

Acetic Acid↗

PubMed2003

Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCRgamma PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.

Clone Cells↗

PubMed2000

Immunoglobulin heavy-chain gene rearrangement studies by Southern blot using DNA extracted from formalin-fixed, paraffin-embedded tissue.

BACKGROUND: In most clinical molecular diagnostics laboratories, Southern blots for gene rearrangement studies are not routinely performed on formalin-fixed, paraffin-embedded (FFPE) tissue samples. In this study, immunoglobulin heavy-chain gene rearrangements by Southern blot using DNA extracted from FFPE tissue samples were studied. METHODS AND RESULTS: Eleven paired freshly frozen and FFPE tissue samples were evaluated for immunoglobulin gene rearrangements by PCR and Southern blot analyses. An additional 14 selected samples sent to our laboratory to rule out lymphoma, for which only FFPE tissue (no frozen tissue) was available and for which PCR was interpreted as negative, were evaluated by the same techniques. Southern blots generated from DNA extracted from FFPE tissues were qualitatively identical to those generated from DNA extracted from fresh or freshly frozen tissue and correlated well with the final diagnoses. Ten interpretable Southern blots were generated in the 14 cases in which no frozen tissue was available. Four of these ten blots were interpreted as positive for an immunoglobulin gene rearrangement. Although the number of samples analyzed is small, success with Southern blotting correlated with increased sample size and sample width (1.17 vs 0.49 cm(2); P <.024; 0.71 vs 0.43 cm; P <. 049, respectively). CONCLUSION: DNA extracted from FFPE tissue samples using the simple, efficient, and nontoxic techniques described in this report can be used in many cases for Southern blotting for the detection of clonality by gene rearrangement studies.

Blotting, Southern↗