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Identification of Trichophyton rubrum by nested PCR analysis from paraffin embedded specimen in trichophytia profunda acuta of the glabrous skin.

Trichophytia profunda acuta of the glabrous skin (TPAGS) arose in a 67-year-old Japanese man. The patient presented indurated erythematous plaques and nodules on his left forearm. Direct microscopic examination of the scale in KOH preparation was negative for fungal elements, and culture for dermatophytes was also negative. Although fungal infection could not be proven in hematoxillin-eosin stained sections, deep-cut sections of the biopsied skin lesion with PAS stain revealed the ectothrix presence of fungal elements. Nested PCR was done with Trichophyton specific primers directed to internal transcribed spacer gene 1 (ITS1), using template DNA obtained from formalin fixed, paraffin embedded skin sections. A single band corresponding to T. rubrum was obtained, and the etiological agent was thus identified. KOH tests and cultures may often turn out unsuccessful, perhaps reflecting the hair follicle dominant fungus growth in TPAGS. Although these tests are most important for diagnosis of TPAGS, nested PCR using paraffin embedded skin sections may be an alternative method to identify the etiological agent.

Aged↗

Detection of HLA-DR antigens in paraffin-embedded thyroid epithelial cells with a monoclonal antibody.

The human Class II major histocompatibility (MHC) antigens, or Ia antigens, which are thought to regulate immune cell interaction, can be detected in paraffin-embedded tissues by immunoperoxidase staining with a recently developed monoclonal antibody (LK8D3). HLA-DR antigens were observed in lymphoid tissues, Langerhans cells of the skin, some epithelial cells, and pulmonary alveolar macrophages. The expression of HLA-DR antigens was analyzed in formalin-paraffin sections by immunoperoxidase in 86 normal and abnormal thyroid epithelial tissues. All patients with Hashimoto's disease (8/8) and most patients with Graves' disease (6/8) expressed HLA/DR antigens in the thyroid epithelial cells and in adjacent inflammatory cells. Most papillary carcinomas (12/18), including 3 of 5 follicular variant of papillary thyroid carcinomas, had HLA-DR antigens detected in epithelial cells; whereas medullary thyroid carcinomas (0/5), follicular carcinomas (0/5), and multinodular goiters (0/4) did not have detectable HLA-DR immunoreactivity. A few other thyroid lesions had HLA-DR antigens detected in epithelial cells, including anaplastic carcinomas (2/5), Hurthle-cell tumors (1/16), and thyroid lymphomas (2/2). Monoclonal antibody LK8D3 and two other commercially available monoclonal antibodies against HLA-DR-stained tissues equally well in cryostat sections, but only antibody LK8D3 was effective in formalin-fixed paraffin-embedded tissue sections. These results indicate that epithelial cells from thyroids of patients with autoimmune diseases commonly express HLA-DR antigens. The presence of HLA-DR antigens in most papillary thyroid carcinomas may be helpful diagnostically in cases of follicular variants of papillary carcinomas. The role of HLA-DR expression in autoimmune thyroid disease and in papillary thyroid carcinoma remains to be determined.

Antibodies, Monoclonal↗

Detection of the bcl-1 rearrangement at the major translocation cluster in frozen and paraffin-embedded tissues of mantle cell lymphomas by polymerase chain reaction.

The t(11;14)(q13;q32) translocation and its molecular counterpart bcl-1 rearrangement are highly characteristic of mantle cell lymphomas (MCLs). Most of these translocations occur at the major translocation cluster (MTC) in a tight area that makes this rearrangement identifiable by the polymerase chain reaction (PCR). In this study, the specificity and sensitivity of the PCR technique in the identification of bcl-1 rearrangement and its suitability to amplify the t(11;14) MTC in fixed, paraffin-embedded tissues were analyzed. Genomic DNA was obtained from 21 MCLs and 1 chronic lymphocytic leukemia (CLL) with the t(11;14) translocation. The bcl-1 rearrangement was studied by Southern blot with the MTC, p94PS, and PRAD-1 probes. Polymerase chain reaction was performed using a JH consensus primer and specific primers for chromosome II in the MTC region. bcl-1 rearrangement was identified by Southern blot in the MTC in nine (43%) MCLs and in the p94PS region in the CLL. Polymerase chain reaction analysis of genomic DNA showed that the nine MCLs with MTC rearrangement also had an amplifiable band of the expected size (100%). No amplifiable products were detected in the negative MCLs or in the CLL. The specificity of the PCR products was confirmed by hybridization with an internal MTC oligonucleotide probe. Amplifiable DNA was obtained from the paraffin blocks of 7 cases with MTC rearrangement and 11 negative tumors. bcl-1 rearrangement was detected in this DNA of 6 positive MCLs (86%) by PCR and in none of the negative cases. In conclusion, this study demonstrates that the PCR technique is highly sensitive and specific for the detection of the bcl-1 rearrangement at the MTC. It can be used with both high molecular weight DNA and DNA obtained from formalin-fixed, paraffin-embedded tissues.

Base Sequence↗

Keratin specificity analyses of eight anti-keratin monoclonal antibodies, and their immunostaining patterns in normal skin using formalin-fixed and paraffin-embedded tissue specimens.

Keratin specificity analyses of eight anti-keratin antibodies (34 beta B4 (K1), 35 beta H11 (K8), Ks 13.1 (K13), Ks 19.1 (K19), PKK1, LP34 (CK1), KL1 and AE1) using keratin protein derived from normal thigh epidermis, normal parotid gland and a human squamous cell carcinoma cell line (HSC-5) were performed, and compared with those described in the data sheets. The reactivities of LP34, KL1 and PKK1 were markedly different from those mentioned in the data sheets. The immunostaining pattern of these antibodies in normal skin using formalin-fixed and paraffin-embedded tissue specimens was also examined. The staining patterns of suprabasal keratinocytes (K1, K13, CK1 and KL1 positive), basal cells of the epidermis (PKK1 and AE1 positive), inner cells of the ducts (K8, K13, CK1, KL1 and AE1 positive) and secretory cells of sweat glands (K8, K19, PKK1, KL1 and AE1 positive), mature cells (K8 and KL1 positive) and peripheral cells (CK1, KL1 and AE1 positive) of sebaceous glands and outer root sheaths (PKK1, CK1, KL1 and AE1 positive) were specific. Thus, we conclude that the differentiation of epidermis and skin appendages is possible by immunostaining with these eight anti-keratin antibodies using formalin-fixed and paraffin-embedded tissue specimens with proper protease pretreatment.

Adolescent↗

Immunohistochemical detection of 8-hydroxy-2'-deoxyguanosine in paraffin-embedded sections of rat liver after carbon tetrachloride treatment.

To test the applicability of an anti-8-hydroxy-2'-deoxyguanosine (8-OH-dG) antibody for immunohistochemistry using paraffin-embedded sections, carbon tetrachloride (CCl4)-induced rat liver injury was evaluated. Male rats were given a single dose of CCl4 and killed at 6 hr, 12 hr, 1, 2, 3, and 7 days thereafter. Severe centrilobular necrosis was evident at 1 day. At 2 days, moderate mononuclear cell infiltration was present in centrilobular necrotic regions. Infiltrating mononuclear cells, surrounding sinusoidal endothelial cells and hepatocytes were stained with anti-8-OH-dG antibody at 2 and 3 days. Formation of 8-OH-dG in DNA and 8-oxo-dGTPase mRNA expression were also increased at these time points, the amounts of malondialdehyde and 4-hydroxy-2-nonenal showed 2 peaks at 6 hr and 3 days. The findings suggest that the main contributory factor in the massive hepatic necrosis was increased lipid peroxidation, rather than excessive formation of 8-OH-dG, and that the observed increase in the latter was largely due to infiltrating mononuclear cells. The agreement between biochemical data and the results for immunohistochemical analysis confirms that the anti-8-OH-dG antibody is applicable for detection of cells targeted by free radicals in paraffin-embedded sections and also for investigation of the mechanisms of oxidative damage-related disease, including carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗

Quantitation of intraepidermal T-cell subsets in formalin-fixed, paraffin-embedded tissue helps in the diagnosis of mycosis fungoides.

Differentiation between mycosis fungoides (MF) and cutaneous inflammatory processes can usually be made on clinical and histologic grounds. In difficult cases, immunohistochemical studies can be helpful since MF infiltrates usually contain a predominance of CD4+ lymphocytes, while most inflammatory lesions usually have a mixture of CD4+ and CD8+ lymphocytes. However, this determination has traditionally required the use of frozen tissue, thus severely limiting its usefulness. Recently, antibodies that differentially label CD4+ and CD8+ lymphocytes in formalin-fixed, paraffin-embedded tissue have become available (OPD4 and C8/144B respectively, DAKO (Carpinteria, CA, USA). This study tests the utility of these antibodies in the pathologic diagnosis of MF and inflammatory lesions with significant exocytosis. In 9 cases of MF for which both frozen and fixed tissues were available for comparison, the OPD4+ cell count in fixed tissue was significantly lower than the Leu-3a+ cell count in frozen tissue. Also, the C8/144B+ cell count in fixed tissue was higher than the Leu-2a+ cell count in frozen tissue, although this difference was not significant statistically. In a larger series for which only fixed tissue was available, epidermal CD4:CD8 ratios were significantly greater in 23 MF cases (mean 4.0+/-4.76) than in 35 inflammatory cases (mean 0.6+/-0.42; p = 0.001). Thus, although the studied antibodies appear to detect different epitopes in frozen versus paraffin-embedded tissue, demonstration of an elevated CD4:CD8 ratio in fixed tissue supports the diagnosis of MF, and is a helpful adjunct to routine histopathology.

Aged↗

Comparison of allelic ratios from paired blood and paraffin-embedded normal tissue for use in a polymerase chain reaction to assess loss of heterozygosity.

BACKGROUND: One method to assess loss of heterozygosity (LOH) of various genes is the amplification of DNA from neoplastic tissue by using microsatellite markers. LOH can best be considered on a quantitative basis as a comparison of allelic ratios of neoplastic tissue to that of the normal control. We will illustrate through quantitative methods the importance of using the appropriate controls when determining allelic loss. METHODS AND RESULTS: DNA extracted from 28 paired blood and formalin-fixed, paraffin-embedded normal mucosal tissue was amplified using the DP1 microsatellite marker, consisting of a variable number of CA repeats. This marker is located within the D5S346 (DP1) region on chromosome 5 and is linked to the adenomatous polyposis coli gene. Allelic ratios were calculated after scanning autoradiographs on a densitometer. Ratio values approaching 1 were observed when the two alleles were close in molecular weight, whereas ratios less than 1 were detected when the two alleles had very different molecular weights. This discrepancy was more pronounced in paraffin-embedded tissue than with blood samples. CONCLUSION: For LOH amplification assays, it is best to use normal control samples that are of the same tissue source as the neoplastic sample being analyzed. When assessing LOH in neoplastic tissue, a quantitative value rather than visual assessment of the alleles should be considered. The values may be normalized by dividing the ratio of the two tumor alleles by the ratio of the two normal alleles.

Alleles↗

Immunohistochemical detection of thrombospondin-1 in formalin-fixed, paraffin-embedded tissue.

Thrombospondin-1 (TSP) is a 450-KD glycoprotein that was initially discovered in the platelet alpha-granule. It now appears that TSP is intimately involved in the regulation of a variety of cellular functions and cell-to-cell interactions. Recently, it has been demonstrated that TSP functions as a p53-dependent inhibitor of angiogenesis in cultured fibroblasts from Li-Fraumeni patients and therefore may be an important factor involved with tumor invasion and metastasis. It has previously been demonstrated that TSP can be detected in frozen tissue sections by immunohistochemical methods. Our objective in this study was to determine the optimal antigen retrieval (AR) protocol for detection of TSP in formalin-fixed, paraffin-embedded tissue by using tissue sections from patients with invasive transitional cell carcinoma of the bladder. The optimal AR protocol was determined utilizing a variety of heating conditions and antigen retrieval buffers. Our results demonstrate that TSP can be reliably detected in paraffin-embedded tissue by immunohistochemical techniques that utilize AR with high-temperature microwave heating and a low-pH Tris-HCI buffer. The importance of this method is that it allows the reliable detection of TSP in archival tissue. This should facilitate further investigation into TSP's role in the regulation of cellular processes, including its influence on tumor angiogenesis and metastasis.

Carcinoma, Transitional Cell↗

Immunohistochemical demonstration of carcinogen-DNA adducts in tissues of rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): detection in paraffin-embedded sections and tissue distribution.

A polyclonal antibody against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts was raised for their immunohistochemical demonstration in paraffin-embedded sections. Specificity of this antibody was confirmed by competitive ELISA. Positive signals were immunohistochemically detected in acetone-fixed but not in formalin- or ethanol-fixed sections from F344 rats treated by gavage with a single dose of PhIP at 37.5-300 mg/kg and killed at 1, 2, and 7 days thereafter. Dose-dependent positive staining was observed in almost all organs of both sexes, including the colon, prostate, and mammary gland but largely independent of the tumor response. Repair activity, judged by disappearance of adducts with time, differed according to the organ or cell type. One exception was hepatocytes, the liver incidentally being a nontarget organ. The results suggest that the generated antibody is applicable for detection of cells targeted by PhIP in paraffin-embedded sections and also for the investigation of the mechanisms of PhIP-carcinogenesis.

Administration, Oral↗

Abundant expression of EBER1 small nuclear RNA in nasopharyngeal carcinoma. A morphologically distinctive target for detection of Epstein-Barr virus in formalin-fixed paraffin-embedded carcinoma specimens.

The highly restricted expression of the Epstein-Barr virus (EBV) genome in malignancy has limited the use of EBV detection methods applicable to formalin-fixed paraffin-embedded carcinoma specimens. In EBV-transformed lymphocytes very short nonprotein coding EBV transcripts (EBERs) are expressed in much higher abundance (10(7) copies per cell) than other EBV latency transcripts. Using a 3H riboprobe, the authors demonstrated EBER1 expression in NASOPHARYNGEAL CARCINOMAS (NPCs) as well as in parotid salivary gland. Recognition of EBER1 expression was facilitated by the intensity of hybridization and its characteristic morphology (nuclear with nucleolar sparing). EBER1 expression was not demonstrated in other epithelial malignancies arising from mucosal surfaces (oropharynx, uterine cervix) from which EBV shedding has been detected. Repeat study of the NPC specimens with digoxigenin-labeled probe yielded hybridization signal with subcellular morphologic detail and without background in a 12-hour procedure. Thus the EBER1 transcript is an appropriate target for in situ hybridization detection of EBV in formalin-fixed paraffin-embedded carcinoma specimens.

Carcinoma↗

Immunocytochemical characterization of a monoclonal antibody directed against mitochondria reactive in paraffin-embedded sections.

The monoclonal antibody mES 13 was previously produced against bacterially expressed BALB ras p21 and was reported to have both membrane and cytoplasmic reactivity in formalin-fixed, paraffin-embedded tissue sections. In the current study, the cytoplasmic reactivity of mES 13 is investigated and demonstrated to be mitochondrial. Immunoelectron microscopic studies showed specific labeling of mitochondria without labeling of other organelles. In normal tissues, the antibody strongly labeled tissues known to have large amounts of mitochondria such as renal tubules, hepatocytes, and myocardium. The pattern of reactivity of tumors generally mimicked that of normal tissues, with carcinomas and melanomas usually showing stronger staining than sarcomas and lymphomas. Two granular cell tumors were negative. Among renal neoplasms, mES 13 strongly labeled renal oncocytomas and granular cell renal cell carcinomas and showed weaker staining of clear cell and chromophobe cell tumors. The mES 13 antibody should be useful in the characterization and diagnosis of tumors in which oncocytoma is in the differential diagnosis, especially when only paraffin-embedded tissue is available for study.

Adenoma↗

[Expression of proliferating cell nuclear antigen in superficial bladder cancer--application to paraffin-embedded tissue sections].

It is important to know the proliferating ability and the malignant potential of each tumor. We studied 56 cases of pTa to pT1 superficial bladder tumors using immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and compared the results with the clinical course of each patient. We obtained the following results. 1) We detected the PCNA positive nuclei in all cases, and the PCNA positive rates varied within a range of 1.1-77.5% with a mean of 34.0%. 2) The PCNA positive rate showed no correlation with age, sex, duration of paraffin-embedded, or pathological stage, but showed a significant correlation with the number of tumors, pathological grade of malignancy, or non-recurrence rate. PCNA positive rate of Grade 1 cases (n = 19, 15.6%: mean) was significantly lower than those of Grade 2 cases (n = 27, 39.9%) or Grade 3 cases (n = 10, 53.1%) (P < 0.01). The recurrence rate of the cases with PCNA positive rates of more than 34% (n = 24) was significantly higher than that of the cases with a PCNA positive rate of less than 34% (n = 32) (P < 0.05). In conclusion, the method of counting the rate of PCNA positive nuclei is considered to be very useful because of its applicability to paraffin-embedded tissue sections and the simple and rapid techniques. Our results in bladder cancer tissues suggest that this method may also be useful for investigating the proliferating ability and the malignant potential of tumors in general.

Adult↗

Detection of mycobacterial DNA in formalin-fixed, paraffin-embedded tissue specimens by duplex polymerase chain reaction: application to histopathologic diagnosis.

Granuloma is a chronic inflammatory process associated with noninfectious agents or infectious diseases such as tuberculosis. Determination of the causative agent might be occasionally difficult in histopathologic sections. In this study, we examined 60 specimens of granuloma or inflammatory lesions that were originally diagnosed as 51 cases of granulomatous inflammation, 6 of leprosy, and 3 of atypical mycobacteriosis. The diagnoses in the last two categories were made both histologically and clinically. All of the sections and DNA were prepared from formalin-fixed, paraffin-embedded blocks. Histopathologic and immunohistochemical findings were compared with the results of duplex polymerase chain reaction (PCR) using two primers to amplify mycobacterial-common 383-base pair (bp) DNA and Mycobacterium tuberculosis-complex-specific 240-bp DNA. Six samples of leprosy and three of atypical mycobacteriosis showed the 383-bp but not the 240-bp band. Among the 51 specimens of granulomatous inflammations, nine showed no band of even the beta-globin, the cases being excluded from this analysis. The 42 specimens of granulomatous inflammation were subdivided into three categories by PCR: (1) 383- and 240-bp positive; (2) 383-bp positive and 240-bp negative; and (3) both negative. Category 1 included 32 specimens (76.2%), being considered as tuberculosis. One specimen was classified into Category 2, indicating possible atypical mycobacterium. Category 3 included nine specimens, composed of five of sarcoidosis and four other agent-induced granulomas, when compared with histologic and clinical findings. These findings indicate that the PCR assay using DNA extracted from paraffin-embedded materials provides useful information to differentiate tuberculosis from other type of granulomas.

DNA Primers↗

A novel monoclonal antibody, RM-4, specifically recognizes rat macrophages and dendritic cells in formalin-fixed, paraffin-embedded tissues.

An anti-rat macrophage/dendritic cell monoclonal antibody, RM-4, was produced using a homogenate of silica-induced lung granulomas of rat as immunogen. Immunohistochemistry demonstrated that RM-4 was specific for macrophage and dendritic cell populations residing in various organs and tissues. It did not react with any cells other than macrophage/dendritic cells. In the double staining of the spleen, RM-4-positive macrophages showed wider distribution than those of the four other anti-rat macrophage monoclonal antibodies compared. The immunoreactivity of RM-4 was well preserved not only in frozen sections but also in formalin-fixed, paraffin-embedded tissues. The isotype of the monoclonal antibody was IgG1 kappa and its antigen molecular weight was 46 kDa. Immunoelectron microscopy revealed positive reaction products for RM-4 on the membrane of endosomes and lysosomes in macrophages and epidermal Langerhans cells. Reaction intensity increased after thioglycolate elicitation or endocytosis regardless of ingested materials. From these data, it is concluded that RM-4 recognizes a membrane protein of endolysomes in macrophages and dendritic cells. The antigen may play a role in endolysosomal processing. RM-4 is considered to be a useful tool not only for identifying macrophage/dendritic cells both in frozen and paraffin-embedded tissues, but also for evaluating their endolysosomal processing.

Animals↗

Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes.

A method of in situ cytohybridization is described for the detection of specific viral genomes in infected cell cultures or paraffin-embedded tissue sections without the use of radioisotopes. Biotin-labeled analogs of TTP are incorporated into viral DNA in vitro by nick translation and the resultant DNA probes hybridized to cytologic samples. Cells containing viral genetic material are then revealed by standard immunofluorescence, immunoperoxidase, or affinity cytochemical techniques that are based on the specific interaction between biotin and antibiotin IgG or avidin. Hybridization probes containing nucleotides that have an 11- or 16-atom spacer arm between the biotin molecule and the pyrimidine ring interact with these detector proteins more efficiently than probes containing biotin-nucleotides with a 4-atom spacer arm. The total procedure can be performed fairly rapidly (24 hr or less) and numerous samples can be processed simultaneously. Although the detection methods employed to date are not as sensitive as autoradiographic procedures with high specific activity probes, more sensitive protein detector complexes are currently being constructed. The speed, specificity, and resolving power of this technique should be of general utility in screening for the presence of infectious agents in cell or tissue samples. Here we report the visualization of parvovirus, polyomavirus, herpes simplex virus, adenovirus, and retrovirus genetic material in infected cell cultures and herpes simplex and adenovirus DNA in paraffin-embedded autopsy tissues.

Adenoviruses, Human↗

High-resolution array-based comparative genomic hybridization for distinguishing paraffin-embedded Spitz nevi and melanomas.

Distinguishing between Spitz nevus and melanoma presents a challenging task for clinicians and pathologists. Most of these lesions are submitted entirely in formalin for histologic analysis by conventional hematoxylin and eosin-stained sections, and fresh-frozen material for ancillary studies is rarely collected. Molecular techniques, such as comparative genomic hybridization (CGH), can detect chromosomal alterations in tumor DNA that differ between these 2 lesions. This study investigated the ability of high-resolution array-based CGH to serve as a diagnostic test in distinguishing Spitz nevus and melanoma using DNA isolated from formalin-fixed and paraffin-embedded samples. Two of 3 Spitz nevi exhibited no significant chromosomal alterations, while the third showed gain of the short arm of chromosome 11p. The latter finding has previously been described as characteristic of a subset of Spitz nevi. The 2 melanomas showed multiple copy number alterations characteristic of melanoma such as 1q amplification and chromosome 9 deletion. This study has shown the utility of array-based CGH as a potential molecular test in distinguishing Spitz nevus from melanoma. The assay is capable of using archival paraffin-embedded, formalin-fixed material; is technically easier to perform as compared with conventional CGH; is more sensitive than conventional CGH in being able to detect focal alterations; and can detect copy number alterations even with relatively small amounts of lesional tissue as is typical of many skin tumors.

Adult↗

[Flow cytometric analysis of DNA ploidy using paraffin-embedded germ cell tumors].

The DNA content of paraffin-embedded materials was determined retrospectively using flow cytometry (FCM) in 36 germ cell tumors, and related to histological type, clinical staging and tumor marker. These histograms were classified from the basis of mode and variance into a diploid and an aneuploid pattern. We could evaluate the DNA histograms in 20 of 36 specimens (56%). Aneuploid patterns were found in 11 of 20 evaluated cases, but there was no correlation between ploidy patterns and histological types. Aneuploid patterns were demonstrated in 2 of 6 stage I cases (33%), and 4 of 5 stage II cases (80%) in seminomas. The difference between stage I and II cases was not statistically significant. There was no correlation between clinical staging and DNA content in non-seminomas. Of the seminomas with elevated human chorionic gonadotropin (HCG) titers, 3 of 4 cases showed aneuploid patterns. These findings indicate that the determination of DNA ploidy in seminomas may prove to be of prognostic value.

Adult↗

[Detection of enteroviral RNA in paraffin-embedded myocardial tissue from patients with Keshan by nested PCR].

Enteroviral RNA in paraffin-embedded myocardial specimens from 21 patients with Keshan disease and 21 patients with cerebral trauma was investigated by nested polymerase chain reaction. Of the 21 patients with Keshan Disease, 18 (85.7%) showed positive findings. Their myocardial tissues were infected by enterovirus previously. The results suggest that enterovirus infection is the major cause of Keshan disease in the Chuxiong district, Yunnan province.

Base Sequence↗