Interphase cytogenetics in paraffin sections of routinely processed hydatidiform moles and hydropic abortions.

The differential diagnosis of complete (CM) and partial (PM) hydatidiform moles and hydropic abortions (HA) can be difficult when based on histology alone. Therefore, a more objective approach of chromosome ploidy analysis as detected by in situ hybridization (ISH) was performed on 6 microns paraffin sections of seven cases, originally classified as three CM, two PM, and two HA with a histologic pattern suggestive of triploidy. Probes for repetitive DNA targets in the (peri)centromeric region of chromosomes 1 and X and in the q arm of chromosome Y were used to determine chromosome ploidy and sex chromosome composition. The findings in the three CM were consistent with diploidy: two copies of chromosomes 1 and X and none of chromosome Y. In the two HA with a histologic pattern suggestive of triploidy, three copies of chromosomes 1 and X and none of chromosome Y confirmed triploidy. Two cases originally classified as PM both appeared to have two copies of chromosome 1 with an XX pattern in one case and an XY pattern in the other case, which is consistent with diploidy instead of triploidy. After reviewing, both cases most likely represented CM. We conclude that interphase cytogenetics by ISH on paraffin sections of hydatidiform moles and hydropic abortions enables chromosome ploidy analysis with preservation of histological context.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers

An improved protocol for in situ hybridization (ISH) to routinely processed, paraffin-imbedded tissue sections from transitional bladder carcinoma (TCC) is presented. The protocol to detect numerical chromosome aberrations involved treatment of sections with thiocyanate prior to proteolytic digestion, resulting in reproducible ISH reactions. It was used to explore the influence of nuclear truncation in the detection of numerical chromosome aberrations and the detection of tumor cells among stromal and inflammatory cells, to compare the flow cytometric DNA index with chromosome copy number, and to study chromosome heterogeneity within tumors. For this study, a DNA probe for the chromosome region 1q12 was used. Hybridization of model systems with known chromosome numbers, such as sections of paraffin-embedded lymph nodes, paraffin-embedded human peripheral lymphocytes, T24 and Molt-4 cells with two, three, and four chromosomes 1, respectively, showed in at least 50% of the cells the proper number of chromosome hybridization signals in standard 6-microns-thick sections. Depending on the size of the nucleus, a certain percentage of the cells showed lower copy numbers as a result of truncation. In four cases of normal urothelium in paraffin sections, the percentage of nuclei with more than two chromosome spots did not exceed 5%. Comparison of the number of ISH signals, as detected in ethanol-fixed single cell suspensions of 11 TCCs [five flow cytometric (FCM) diploid, three FCM aneuploid, and three FCM tetraploid], with ISH results obtained in paraffin sections of the same tumors showed that typical numerical chromosome aberrations, such as trisomy and tetrasomy up to nonasomy, could be detected. However, the real chromosome copy number is underestimated, especially in tumors with high copy numbers, as detected in the single cell suspensions of the same tumors. Hybridization of a TCC with extremely large nuclei (DNA index = 3.2) containing six to nine ISH signals as detected in the isolated tumor cells, showed that an indication of these real chromosome copy numbers could be obtained in 6-microns paraffin sections. The accuracy for the detection of the chromosome copy number was even higher in cases where hybridization signals were counted in the mitotic cells. Furthermore, chromosome heterogeneity was detected by ISH using centromeric probes for chromosomes 7, 9, and 18, even though nuclei are truncated in the section. The surplus value of ISH on paraffin sections, as compared with ISH on isolated tumor cells, can be summarized as follows. (a) The focal tumor cell areas with chromosome aberrations can be recognized in the sections and be correlated with the histologic appearance.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of messenger RNA in routinely processed tissue sections with biotinylated oligonucleotide probes

In situ hybridization (ISH) with a radioactively 35S-labeled probe and a biotinylated oligonucleotide probe for human calcitonin was used to analyze eight cases of medullary thyroid carcinoma (MTC) in paraffin sections. Three of these cases were also studied with frozen tissue sections. The biotinylated probe readily detected calcitonin messenger RNA (mRNA) in routinely processed formalin-fixed paraffin-embedded tissue section within 24 hours. Northern hybridization and other control studies demonstrated the specificity of the calcitonin probe. Biotinylated oligonucleotide probes for other mRNAs present in high abundance such as adrenocorticotroipic hormone (ACTH), prolactin, and growth hormone also detected the respective mRNAs in pituitary tissues. These result show that biotinylated oligonucleotide cDNA probes can be used to detect specific mRNAs present in large amounts in some endocrine cells and tumors by ISH. This approach offers an alternative that does not require the use of molecular cloning or radioactive probes for this investigative and diagnostic technique.     

Immunoperoxidase staining for identification of Aspergillus species in routinely processed tissue sections.

AIMS: To evaluate the performance of an immunoperoxidase stain using the monoclonal antibody EB-A1 to detect Aspergillus species in formalin fixed, paraffin wax embedded tissue. METHODS: The monoclonal antibody EB-A1 directed against galactomannan was used to detect Aspergillus species in 23 patients with suspected or confirmed invasive aspergillosis. Immunostaining was performed on formalin fixed, paraffin wax embedded tissue using the streptavidin-biotin method and compared with conventional haematoxylin and eosin, periodic acid-Schiff, and Gomori-Grocott stains. Results of immunostaining were semiquantitatively analysed with regard to both intensity of staining and number of positively staining micro-organisms. Tissue sections from 16 patients with confirmed invasive mycoses due to Candida species, Apophysomyces elegans, Rhizopus oryzae, Pseudallescheria boydii and Histoplasma capsulatum were used as controls. RESULTS: In 19 (83%) of 23 cases invasive aspergillosis was confirmed by both histological examination and culture (18 Aspergillus fumigatus and one A flavus). Immunoperoxidase stains were positive in 17 (89%) of 19 cases including one case of disseminated infection due to A flavus. Furthermore, the immunoperoxidase stain was positive in a culture negative tissue section with histological evidence of mycelial development, indicating the presence of Aspergillus species. Some cross-reactivity was observed with the highly related fungus P boydii, although the number of mycelial elements that stained was low. CONCLUSIONS: Immunoperoxidase staining using the monoclonal antibody EB-A1 performs well on routinely processed tissue sections and permits detection and generic identification of Aspergillus species, although it was no better than conventional histopathology in identifying the presence of an infection. An additional advantage is that the immunostain may help to provide an aetiological diagnosis when cultures remain negative.     

Ki-M6 immunostaining in routinely processed sections of reactive and neoplastic human lymphoid tissue

A monoclonal antibody, termed Ki-M6 (CD68), which shows a restricted reactivity to cells of the monocyte/macrophage system, has been evaluated primarily with the use of cryostat sections. In this study the authors could assess that the Ki-M6 antibody recognizes a fixation-resistant epitope in most human macrophages. The Ki-M6 immunoreactivity with monocyte/macrophage-related cells was established by testing on routinely processed samples of reactive and neoplastic lymphoid tissues; it was compared with the staining for vimentin (V9) and S-100 protein antibodies, with visualization of the stationary elements of lymphoid tissues, with the aim of establishing its value in the study of the nonlymphoid microenvironment. The Ki-M6 antibody reactivity could be achieved with Bouin-fixed, paraffin-embedded tissue sections, without any proteolytic treatment, with the use of the avidin-biotin complex (ABC) method, especially after overnight incubation time at 4 degrees C. Some reduction in antigenic reactivity was observed in B5- or formaldehyde-fixed samples. The antibody reacted with macrophages of all different lymph node compartments; a broad reactivity against cells of macrophage lineage, including multinucleated giant cells, was observed in epithelioid granulomas. Ki-M6-positive cells other than classic macrophages were the so-called "plasmacytoid T-cells" and cells displaying elongated cytoplasms with fibroblastic-like features. Granulocytes, follicular dendritic reticulum cells, and interdigitating reticulum cells did not reveal any reactivity with Ki-M6 antibody. In malignant lesions, neoplastic cells of follicular and diffuse B- and T-cell lymphomas, including large cell non-Hodgkin's lymphomas, and Reed-Sternberg cells of Hodgkin's disease were negative in all cases studied. This study shows that Ki-M6 seems to be another anti-macrophage-specific antibody that reacts, in routinely processed tissue sections, with tissue macrophages but not with accessory cells. Thus, it may be a valuable addition to vimentin and S-100 protein antibodies for investigation of the microenvironmental organization of lymphoid tissues both in normal and neoplastic conditions.     

PCR based detection of mycobacteria in paraffin wax embedded material routinely processed for morphological examination.

BACKGROUND: The incidence of mycobacterial infections has increased during the past five years. A prompt diagnosis is indispensable for initiating appropriate treatment. Because culturing of mycobacteria takes three to six weeks and sensitivity of microscopic detection of acid fast bacilli is low, amplification methods provide promising possibilities. Recently, the polymerase chain reaction (PCR) has been shown to be useful for confirming a mycobacterial infection, especially in cases with unexpected histological findings or lack of suitable material for culturing. AIMS: To evaluate the impact of PCR based techniques in the detection of mycobacterial infections in uncultured routine histological specimens as an alternative to surgical pathology. METHODS: Two hundred and twenty nine formalin fixed and paraffin wax embedded samples from 141 patients with clinical or histological suspicion of a mycobacterial infection were investigated using three different PCR assays and Southern blotting. PCR results were compared with histology and culture and the patients' clinical findings. RESULTS: When using culture as the reference method, the sensitivity for the detection of mycobacteria of the tuberculosis complex was 90%, specificity was 92%, the positive predictive value was 81%, and the negative predictive value was 96%. The sensitivity for the detection of nontuberculous mycobacteria was 100% and specificity was 78%, the positive predictive value was 26%, and the negative predictive value was 100%. The patients' clinical findings supported the PCR positive results, indicating a mycobacterial infection in 11 of 18 initially culture negative cases and in 21 of 35 PCR positive cases without culture results. CONCLUSIONS: These results indicate that PCR based techniques are sensitive, specific, and rapid methods for the detection of mycobacteria in routinely processed paraffin wax embedded and formalin fixed histological samples.     

PCR based detection of mycobacteria in paraffin wax embedded material routinely processed for morphological examination.

BACKGROUND: The incidence of mycobacterial infections has increased during the past five years. A prompt diagnosis is indispensable for initiating appropriate treatment. Because culturing of mycobacteria takes three to six weeks and sensitivity of microscopic detection of acid fast bacilli is low, amplification methods provide promising possibilities. Recently, the polymerase chain reaction (PCR) has been shown to be useful for confirming a mycobacterial infection, especially in cases with unexpected histological findings or lack of suitable material for culturing. AIMS: To evaluate the impact of PCR based techniques in the detection of mycobacterial infections in uncultured routine histological specimens as an alternative to surgical pathology. METHODS: Two hundred and twenty nine formalin fixed and paraffin wax embedded samples from 141 patients with clinical or histological suspicion of a mycobacterial infection were investigated using three different PCR assays and Southern blotting. PCR results were compared with histology and culture and the patients' clinical findings. RESULTS: When using culture as the reference method, the sensitivity for the detection of mycobacteria of the tuberculosis complex was 90%, specificity was 92%, the positive predictive value was 81%, and the negative predictive value was 96%. The sensitivity for the detection of nontuberculous mycobacteria was 100% and specificity was 78%, the positive predictive value was 26%, and the negative predictive value was 100%. The patients' clinical findings supported the PCR positive results, indicating a mycobacterial infection in 11 of 18 initially culture negative cases and in 21 of 35 PCR positive cases without culture results. CONCLUSIONS: These results indicate that PCR based techniques are sensitive, specific, and rapid methods for the detection of mycobacteria in routinely processed paraffin wax embedded and formalin fixed histological samples.     

Simplified procedures for applying the polymerase chain reaction to routinely fixed paraffin wax sections.

The polymerase chain reaction was applied to the analysis of DNA contained in archival paraffin wax embedded material. DNA suitable for the reaction was obtained from these tissues by simple extraction methods, without previous dewaxing of tissue sections. When compared with unfixed material, the reaction efficiency was compromised, so that an increased number of amplification cycles were required to produce equivalent amounts of amplified product. This in turn led to an increase in amplification artefacts, which can be minimised by a simple modification of the standard reaction. Amplification of relatively large DNA fragments was not always successful, and it seems prudent to bear this in mind when designing oligonucleotide primers which are to be used for the amplification of archival material. The efficiency of the procedure can be improved by dividing the amplification cycles into two parts: this reduces the amount of reagent needed, is relatively simple and inexpensive, and can be performed in one working day.     

Preparation of paraffin sections for immunofluorescence analysis of antigens differing in their chemical nature

A technique is suggested for preparing paraffin sections from tissues fixed with acetone which can be used for the immunohistochemical detection of antigens which differ in their chemical nature, including -fetoprotein, antigens of mouse leukemia viruses, alcohol-soluble antigens of hepatocyte membranes, and certain phospholipids.Laboratory of Immunochemistry and Diagnosis of Tumors, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR. Laboratory of Pathological Anatomy, N. N. Burdenko Institute of Neurosurgery, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR G. V. Vygodchikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 1018–1020, August, 1976.     

Detection of estrogen receptors with monoclonal antibodies in routinely processed formalin-fixed paraffin sections of breast carcinoma. Use of DNase pretreatment to enhance sensitivity of the reaction

This study describes an improved immunohistochemical method for the sensitive and specific identification of estrogen receptors (ERs) in paraffin sections from formalin-fixed and routinely processed breast carcinoma tissues, using DNase pretreatment to expose nuclear antigenic sites and commercially available immunoreagents (including monoclonal antibody) in kit form. Results were compared with dextran-coated charcoal cytosolic assay (DCC) and with conventional immunohistochemistry on frozen sections. Sensitivity and specificity for determinations on paraffin sections were 88% and 86%, respectively, and statistical analysis showed very good agreement between DCC and paraffin sections (kappa = 0.805). The DNase technic on paraffin sections allows excellent correlation between histologic characteristics and ER status and reduces DCC sampling error resulting from stromal dilution and tumor variability. This method offers a reliable and reproducible alternative when tissue is not suitable or unavailable for DCC or frozen tissue analysis and can be used for retrospective studies on stored tissue blocks.     

Fluorescent in situ hybridization in routinely processed bone marrow aspirate clot and core biopsy sections.

Fluorescent in situ hybridization (FISH) is a technique which complements conventional cytogenetic banding analysis by allowing the evaluation of cells in interphase as well as metaphase. This technique has been used to study air-dried peripheral blood and bone marrow aspirate smears. We have applied the FISH technique to study routinely processed sections of bone marrow aspirate clot and decalcified core biopsy specimens, fixed in either formalin or B5 and embedded in paraffin. We evaluated 28 specimens (8 aspirate clot and 20 core biopsy sections) for chromosome 8 copy number, studied previously by conventional cytogenetics, and found the following distribution: 15 with disomy, 11 with trisomy, and 2 with tetrasomy. Using a chromosome 8 alpha-satellite probe, we detected fluorescent hybridization signals in 18 of 28 specimens (64%); 6 of 8 (75%) aspirate clot sections, and 12 of 20 (60%) core biopsy sections. Ten of 13 (77%) B5-fixed and 8 of 15 (53%) formalin-fixed specimens had hybridizing signals. Specimen age was a significant factor; 10 of 11 (91%) specimens processed within the last 6 months showed signals, in contrast with 8 of 17 (47%) specimens older than 6 months. In the positive specimens, 200 cells were analyzed in areas where individual cells could be identified. In the disomic specimens, two signals per cell were seen in 34 to 66% of the cells. Rare cells (0-2%) with three signals were detected. In the trisomic specimens, three signals per cell were seen in 19 to 46% of the cells. In the tetrasomic specimens, four signals per cell were seen in 15 to 25% of the cells. We conclude that the FISH technique may be useful in the detection of numerical chromosomal abnormalities such as trisomy and tetrasomy 8 in routinely processed bone marrow aspirate clot and decalcified core biopsy sections.     

Monoclonal antibodies reactive in routinely processed tissue sections of malignant lymphoma, with emphasis on T-cell lymphomas

The immunoreactivity of eight monoclonal antibodies was evaluated on 45 routinely processed lymphomas (22 T-cell lymphomas, 11 B-cell lymphomas, and 12 cases of Hodgkin's disease). Two antibodies reactive with leukocyte common (T200) antigens (PD7/26 and 2B11) stained most of the B- and T-cell lymphomas but did not stain the Reed-Sternberg cells and variants in Hodgkin's disease. Two antibodies known to stain B cells (LN-1 and LN-2) reacted with some of the B-cell lymphomas, but LN-2 also reacted with the neoplastic cells in six of 22 T-cell lymphomas and with the Reed-Sternberg variants in eight of 12 cases of Hodgkin's disease. The granulocyte antibody anti-Leu M1 reacted with most cases of Hodgkin's disease but also reacted with two of 11 B-cell non-Hodgkin's lymphomas. An antibody to epithelial membrane antigen (anti-EMA) stained some cases of T-cell lymphoma, B-cell lymphoma, and Hodgkin's disease. Leu 7 was expressed in one T-cell lymphoma and in one case of Hodgkin's disease. A novel antibody reactive with T cells (L60) stained all cases of T-cell lymphoma but also stained some cases of B-cell lymphoma and one case of Hodgkin's disease. We conclude that none of these antibodies, when used alone on routinely fixed paraffin-embedded material, is completely sensitive and specific for T-cell lymphoma, B-cell lymphoma, or Hodgkin's disease. However, a panel of antibodies is useful in distinguishing Hodgkin's disease from non-Hodgkin's lymphoma and in suggesting the B- or T-cell phenotype of non-Hodgkin's lymphomas.     

Immunohistochemical staining for terminal deoxynucleotidyl transferase (TDT). An enhanced method in routinely processed formalin-fixed tissue sections

This report describes an improved technique for sensitive and specific localization of terminal deoxynucleotidyl transferase (TdT) in routinely processed paraffin-embedded, formalin-fixed tissue sections using DNAse pretreatment and the avidin-biotin complex (ABC) technique. This method is useful in identifying lymphoblastic lymphomas (14/15 cases positive), with all other B- and T-cell lymphomas tested negative for the reaction. Used in conjunction with monoclonal antibodies immunoreactive for T- and B-cells in paraffin sections this technique should prove helpful in immunophenotyping malignant lymphomas where fresh tissue is unavailable for study.