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PubMedDate not supplied

Immunostaining of cardiac biopsy specimens in the diagnosis of acute vascular (humoral) rejection: a control study.

The diagnosis of acute vascular (humoral) rejection in heart transplant biopsies is classically based on immunofluorescent studies of frozen tissue that show vascular staining for immunoglobulin and complement. We have noted that some pathologists have used immunostaining of formalin-fixed, paraffin-embedded tissue in testing for vascular rejection. To determine the specificity of immunostaining of heart biopsy specimens in the diagnosis of vascular rejection, we studied tissue from 68 consecutive endomyocardial biopsies from 16 patients without clinical or histologic evidence of vascular rejection. In each case, routinely processed formalin-fixed, paraffin-embedded tissue was stained for immunoglobulin G and immunoglobulin M with an avidin-biotin immunoperoxidase technique. Frozen tissue from each case was also stained for immunoglobulin G, immunoglobulin M, C3, and Clq by immunofluorescence. Immunoperoxidase stains on formalin-fixed tissue showed vascular staining for immunoglobulin in 67 of 68 (99%) of the cases. Staining was ablated if the antibodies were absorbed with their appropriate immunoglobulin. Immunofluorescent studies on frozen tissue showed no vascular staining for immunoglobulin or complement. We conclude that immunoperoxidase studies of routinely processed, formalin-fixed, paraffin-embedded tissues are nonspecific in the diagnosis of heart acute vascular rejection. The positive staining in fixed tissues may be due to labeling of passive immunoglobulins that are "fixed" in the vessels during routine processing but are washed away in techniques using frozen tissue.

Acute Disease↗
PubMed1993

Immunocytochemical localization of androgen receptor with polyclonal antibody in paraffin-embedded human tissues.

We investigated the immunohistochemical localization of androgen receptor (AR) using a polyclonal antibody for 55 KD recombinant human AR in human tissues fixed with 4% paraformaldehyde solution and embedded in paraffin. Immunoreactive AR was restricted to the nuclei of various tissues. Among the well-known androgen target organs, secretory cells and basal cells of the prostate, spermatogonia, spermatocytes, Sertoli cells and Leydig cells of the testis, epithelial cells of the rete testis, fibroblasts in the whole organ, squamous cells, sweat gland and hair follicle cells of the skin, and hepatocytes of the liver were positive for AR. In addition, smooth muscle cells of the prostate, uterus, urinary bladder, gastrointestinal tract, arteries, and arterioles were strongly immunoreactive for AR. Cardiac muscle and striated muscle of psoas were positive for AR. Acinar cells, ductal cells, and myoepithelial cells of the breast, endocervical and endometrial cells of the uterus, cyto- and syncytiotrophoblast of the chorionic villi, and tubules of the kidney were also positive for AR. Most FSH, LH, and some GH endocrine cells in the anterior and posterior lobes of the pituitary gland, follicular cells of the thyroid gland, and adrenocortical cells were positive for AR. Cells immunoreactive for AR were ubiquitously distributed throughout the entire body. The present study demonstrated the diversity of androgen effects on many target tissues.

Antibodies↗
PubMed1977

Combined Nomarski interference contrast and immunofluorescent study of neuropathological specimens: CSF sediments and paraffin embedded brain tissues.

The direct immunofluorescent technique may be easily improved by the use of the Nomarski optics. This contrast allows accurate identification of fluorescent CSF cells and structures in formalin fixed paraffin embedded brain tissues; in the latter, the combined optical procedure is fruitfull in order to avoid fluorescent artifacts misinterpretation. Furthermore, it is emphazised that the conditions in which routine neuropathological specimens are removed and stored usualy does permit the application of the immunofluorescent technique.

Brain↗
PubMedDate not supplied

In situ detection of reovirus in formalin-fixed, paraffin-embedded chicken tissues using a digoxigenin-labeled cDNA probe.

An in situ hybridization (ISH) technique using a digoxigenin (DIG)-labeled cDNA probe detected avian reovirus (ARV) RNA in formalin-fixed, paraffin-embedded chicken tissues. Tissues were collected 3 and 10 days following inoculation with the R-2 or the S1133 strain of ARV. The cDNA clone HJp1, located on the S1 gene segment of the ARV S1133 strain, was used to prepare a nonradioactive probe. The ISH assay localized ARV RNA in infected tissues including heart, liver, intestine, pancreas, and tendon. No positive-stained cells occurred in sections from uninfected chickens.

Animals↗
PubMed2001

[Interphase FISH analysis of frozen or fixed tissues for the detection of t(11;14) (q13;q32) in mantle cell lymphoma].

Cytogenetic analyses have revealed that mantle cell lymphomas (MCL) are closely associated with the t(11;14)(q13;q32). This translocation juxtaposes the immunoglobulin heavy chain gene (IGH) sequences with the BCL-1 locus, leading to up-regulation of the CCND1 gene and consequently to an overexpression of cyclin D1 protein. We studied 27 MCL with characteristic morphological and immunological (CD5+, CD10-, CD20+, CD23-) features and 2 controls (reactionnal lymphadenitis) to evaluate the feasibility and the interest of FISH analysis on interphase cells from frozen or paraffin-embedded tissues. Sections (CC) and touch preparations (EC) of frozen tissues and sections of paraffin-embedded tissues (CF) were successfully hybridized with the Vysis LSI IgH/CCND1 dual color dual fusion translocation probe. The touch preparations presented a lower cellularity than sections, therefore allowing an easier analysis. Hybridization spots intensities were found stronger in CC and EC than in CF. The percentages of t(11;14) positive cells were similar in CC, EC and CF from a same patient. The percentage of non hybridized cells, analogous in CC and EC, was higher in CF. However, the CF were directly analysed on microscope without the need of any numerical picture treatment. The t(11;14) was detected in all the cases (27/27) and positive cells percentages were always higher than the probe cut-off (5%). The FISH analysis on interphase cells appears a performing and rapid technique to detect t(11;14) in MCL on both frozen and paraffin-embedded tissue, thus extending its practical and diagnostic use.

Cyclin D1↗
PubMed1984

Dibasic staining of large epoxy tissue sections and applications to surgical pathology.

A variety of normal and pathologic aldehyde-fixed osmium postfixed human tissues were prepared as large sections embedded in Spurr epoxy. They were stained with a sequential basic fuchsin--methylene blue stain which gives "hematoxylin- and eosin-like" staining and additionally functions as several special stains. This technic also allows for electron microscopy directly on the embedded tissue. The histologic and cytologic preservation and overall staining was superior to tissue embedded in glycol methacrylate. The methods and technics presented in this article have important applications in diagnostic surgical pathology and histology in general.

Epoxy Resins↗
PubMed1990

[Comparison of flow cytometric, static cytometry and tumor cytogenetic investigation results human renal cell carcinomas].

Published data about the frequency of DNA-aneuploidy of renal cell tumours show a great variation. The own study was performed to clarify whether this fact is due to methodological discrepancies or to intratumoural heterogeneity of DNA ploidy. The comparison of DNA ploidy assessed by flow cytometry (fresh and paraffin-embedded tissue) and static cytometry (paraffin-embedded tissue) in equivalent tumour regions showed identical results. 71% of clear cell renal carcinomas showed intratumoural heterogeneity of DNA ploidy. In 55% there was either a combination of diploid and aneuploid stem lines and/or a combination of different aneuploid stem lines ("heterogenous aneuploidy"). The rate of aneuploidy was found to be higher with increasing cytoplasmic eosinophilia of the clear cell renal carcinoma cells. Studies which address the prognostic significance of DNA ploidy of renal carcinomas should specifically investigate on the significance of heterogenous aneuploidy for prognosis.

Aneuploidy↗
PubMed2002

Detection of LOH of the RB1 gene in bladder cancers by PCR-RFLP.

OBJECTIVES: Retinoblastoma (RB1) gene involves in retinoblastoma, osteosarcoma, bladder, prostate, lung, breast carcinomas, and soft tissue sarcomas. Loss of heterozygosity (LOH) is the most common mutation of the gene. METHODS: Xba I polymorphism in intron 17 of the gene was used to detect LOH in 20 bladder cancer patients. A cystitis and an osteosarcoma were used as control. LOH was investigated in three different kinds of samples (blood, paraffin-embedded tissue and fresh tissue) belonging to the same patients, and 20 blood samples, 20 paraffin-embedded tissue samples and 16 fresh tissue samples were obtained from 20 cancer patients. RESULTS: None of the 20 blood samples showed LOH. Eleven out of 20 paraffin-embedded bladder tissues were amplified, 3 of them homozygous and all 8 informative paraffin-embedded tissues showed LOH. Five out of 16 fresh tumor tissues obtained were amplified, in 1 the fresh tissue was normal, 1 fresh tissue showed LOH and 3 were not digested by Xba I. CONCLUSION: The results of the study have suggested that detection of LOH of the RB1 gene by PCR-RFLP can be a good adjunctive test for evaluation of the bladder cancer.

Carcinoma, Transitional Cell↗
PubMed1996

[Detection of hepatitis C virus RNA in paraffin embedded liver tissues by in situ PCR].

The new technique of in situ polymerase chain reaction (ISPCR) was used to study hepatitis C virus (HCV). ISPCR and in situ hybridization (ISH) were performed to detect HCV in 7 paraffin embedded liver tissues and 8 para-hepatocellular cancer tissues. The positive rates were 6/7 and 4/8 respectively, significantly higher than results obtained by ISH, which were 3/7 and 1/8 respectively. It also demonstrated that the specificity of ISPCR was better than ISH. HCV RNA was not only found in hepatocyte plasma but also in cholangiolar epithelia and monocytes. We conclude that ISPCR is a sensitive and specific method that can improve the accuracy in diagnosis of HCV.

Base Sequence↗
PubMed2004

Numeric aberrations of HER-2 and chromosome 17 detected by fluorescence in situ hybridization in urine-exfoliated cells from patients with urothelial carcinoma.

OBJECTIVES: To elucidate the clinical significance of the HER-2 gene alterations in urine-exfoliated cells detected by fluorescence in situ hybridization (FISH) in patients with urothelial transitional cell carcinoma. METHODS: The relative increase of HER-2 (RI-HER2) and gain of chromosome 17 (G-17) in urine-exfoliated cells were examined using DNA probes for HER-2 and the chromosome 17 centromere in 103 patients. In addition, FISH analysis was performed using corresponding paraffin-embedded tissue sections from 45 cases to compare the results obtained using urine-exfoliated cells and those obtained using paraffin-embedded tissue. RESULTS: RI-HER2 and G-17 was found in 23 (22.3%) and 46 (44.6%) of 103 patients, respectively. RI-HER2 was significantly more frequent in tumors with two or more recurrences (40.7% versus 15.8%, P = 0.010) and in those with carcinoma in situ (CIS) (35.4% versus 15.9%, P = 0.029). G-17 was more frequent in high-grade tumors (69.1% versus 16.7%, P = 0.032), invasive tumors (63.6% versus 14.3%, P < 0.001), and in patients with CIS (77.1% versus 29.0%, P < 0.001). The positive rate for FISH (presence of RI-HER2 and/or presence of G-17) tended to be more frequent in FISH than in cytology. A comparison of the analyses using urine-exfoliated cells and paraffin-embedded tissue showed identical results in 36 (80.0%) of 45 cases. CONCLUSIONS: Numeric alterations of the chromosome 17 centromere in urine-exfoliated cells detected by FISH may reflect the malignant potential of urothelial carcinoma. In addition, a relative increase in HER-2 was associated with the number of recurrences and the presence of CIS.

Aged↗
PubMed1997

Polymerase chain reaction for the detection of Helicobacter pylori in formaldehyde-sublimate fixed, paraffin-embedded gastric biopsies.

To improve morphologic detail and immunohistochemical staining, mercuric chloride-containing fixatives such as formaldehyde-sublimate (FS) has been widely used as an alternative for neutral buffered formalin. FS-fixed, paraffin-embedded tissue, however, is considered to be an unreliable source of DNA. We used an adapted DNA-extraction method for FS-fixed, paraffin-embedded tissue. In all cases tested we obtained amplifiable DNA with polymerase chain reaction (PCR), after FS-fixation and after fixation in neutral buffered formalin as well. A PCR assay for the 16S-rRNA region of Helicobacter pylori was developed amplifying a fragment of 145 bp. The specificity of this PCR assay was tested on a range of different microorganisms. PCR was performed on 46 archival FS-fixed paraffin-embedded gastric biopsies. The results were compared with histologic examination and with immunohistochemical detection using a polyclonal antibody against H. pylori. Both PCR and immunohistochemistry are very sensitive methods for the detection of H. pylori. A PCR offers the possibility of additional subtyping in archival FS-fixed, paraffin-embedded tissue.

Biopsy↗
PubMed1999

Molecular detection of EWS-FLI1 chimeric transcripts in Ewing family tumors by nested reverse transcription-polymerase chain reaction: application to archival paraffin-embedded tumor tissues.

Chromosomal translocations generating unique chimeric genes are highly characteristic of specific sarcomas, and their use as diagnostic markers has been suggested. From a diagnostic pathologic point of view, detection of such cytogenetic or molecular aberrations applicable to routinely processed archival tissue specimens is considered a powerful tool for tumor diagnosis. To assess the feasibility and reliability of the molecular detection of the transcript originating from the chimeric gene in paraffin-embedded tumor specimens, we performed a nested reverse transcription-polymerase chain reaction (RT-PCR)-based assay to detect the EWS-FLI1 chimeric message in a series of Ewing family tumors. Of 24 paraffin-embedded tumor specimens from 23 cases analyzed, the chimeric message was detectable in 20 (83%) specimens from 20 cases (87%) by this nested RT-PCR assay, whereas none of 7 small round cell tumors not from this family (3 alveolar rhabdomyosarcomas, 2 neuroblastomas, 2 malignant lymphomas) showed detectable chimeric messages. In the sequence analysis of the PCR products, the amplified chimeric messages contained the junctions between exon 7 of the EWS gene and any one of exons 5, 6 and 8 of the FLI1 gene. The detection process was usually completed within 3 days, except for the subseqent sequence analysis. Our results endorse the use of this molecular assay as an ancillary technique in the diagnosis of Ewing family tumors using paraffin-embedded material.

Adolescent↗
PubMed1991

Glycol methacrylate embedding in diagnostic pathology. A standardized method for processing and embedding human tissue biopsy specimens.

A standardized "low-cost" processing and embedding procedure is described for the preparation of semithin glycol methacrylate (GMA) sections of human tissue biopsy specimens for use in routine diagnostic pathology. The method is highly reliable and reproducible and is based on many years of experience in fundamental GMA research. The procedure combines the application of new, less toxic, plasticizers and the preparation of high-quality GMA mixtures. Microwave irradiation was used to improve staining qualities and to reduce staining times. An improved reticulin staining procedure is presented as well.

Biopsy↗
PubMed2003

Detection of Candida albicans mRNA from formalin-fixed, paraffin-embedded mouse tissues by nested reverse transcription-PCR.

Histopathology archives represent a vast source of infectious disease specimens that can be used to elucidate important disease processes. In this report, we describe a method to detect Candida albicans gene expression from infected, formalin-fixed, paraffin-embedded mouse tissue. By use of glass beads to break open the fungal cells and proteinase K treatment, RNA was extracted routinely from tissue sections that had been fixed for up to 72 h. Upon reverse transcription of the RNA and nested PCR, the procedure detected C. albicans "housekeeping" and putative virulence genes.

Animals↗
PubMed1997

Ultrastructural immunocytochemistry of collagenous and non-collagenous proteins in fast-frozen, freeze-substituted, and low-temperature-embedded renal tissue in Alport syndrome.

This paper describes the ultrastructural immunolocalization of the alpha 2 chain of collagen IV, laminin, and the amino terminal propeptide of collagen I (N-Pro I) in glomeruli of rapidly frozen, freeze-substituted, and low-temperature-embedded renal biopsy specimens from two cases of Alport disease and from normal kidneys. The alpha 2 chain of collagen IV is present in the whole thickness of the basement membrane in glomeruli of Alport patients, while it is limited to the subendothelial portion of the basement membrane of normal glomeruli. Laminin has the same distribution in both normal and Alport glomeruli, but is apparently more concentrated along the basement membrane of normal glomeruli. N-Pro I is localized in mesangial areas and in the basement membrane in Alport cases, while it is not detected in normal glomeruli. These data suggest complex rearrangements of major constituents of the glomerular basement membrane network and demonstrate early deposition of fibrillary collagen proteins in the matrix before the appearance of banded collagen fibres. This finding could be an indicator of early evolution towards glomerulosclerosis.

Basement Membrane↗
PubMed1988

Routine diagnostic immunohistochemical labeling of extracellular antigens in formol saline solution-fixed, paraffin-embedded cutaneous tissue.

A technique involving modification of the routine paraffin embedding procedure that allows immunohistochemical examination of extracellular antigens by light microscopy is described. The method is thus suitable for routine diagnostic immunofluorescence studies, permitting reliable, reproducible immunolabeling of immunoglobulins and complement components usually not preserved by routine processing procedures. Immunoreactivity as measured by direct immunofluorescence and immunoenzymatic (peroxidase-antiperoxidase) histochemistry is comparable to results with fresh-frozen cryostat sections. Morphologic preservation is superior, however, and the processed material is suitable for routine hematoxylin-eosin staining.

Antigens, Surface↗
PubMed1996

Detection of cytomegalovirus DNA in paraffin-embedded lung tissue specimens using in situ polymerase chain reaction.

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.

Cytomegalovirus↗
PubMed1991

Application of lectin and B-lymphocyte-specific monoclonal antibodies for the demonstration of human microglia in formalin-fixed, paraffin-embedded brain tissue.

To evaluate the usefulness of microglial markers for routine neuropathological material, we studied formalin-fixed, paraffin-embedded human brain tissue with the immunoperoxidase method using the lectin Ricinus communis agglutinin (RCA-1) and four monoclonal antibodies (LN-1, LN-2, LN-3, anti-HLA-DR/alpha). RCA-1 stained resting microglia, but the staining intensity was mostly weak. LN-1 also stained resting microglia in paraffin sections first treated with protease. In contrast to LN-1, RCA-1 stained blood vessels heavily. LN-1 stained resting microglia more markedly than RCA-1 in brains fixed for a prolonged period of time. However, LN-1 recognized a small number of astrocytes in routine paraffin sections. LN-3 reactivity was detected on a few resting microglia, but was intensely expressed on large numbers of reactive microglia in many neurological diseases. Both LN-2 and anti-HLA-DR/alpha labelled microglia, but the reactions were inconsistent. This study suggests that the monoclonal antibodies LN-1 and LN-3 are useful for the demonstration of microglia in paraffin sections, and a combination of these antibodies and the antibody to glial fibrillary acidic protein is recommended in attempting to identify microglia.

Antibodies, Monoclonal↗