Role of EMG in the evaluation of presumed myopathies in the era of DNA analysis.
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DNA Mutational Analysis↗
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Diagnostic uses of DNA analysis: powerful but problematic.
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DNA Mutational Analysis↗
Characterization of Streptococcus agalactiae isolates of bovine and human origin by randomly amplified polymorphic DNA analysis.
Streptococcus agalactiae is considered one of the major causes of bovine intramammary infections. It is also found in the vaginas of women without any apparent clinical symptoms, but reports of neonatal infections, causing significant morbidity, are relatively frequent. The aim of this study was to evaluate the genetic diversity of S. agalactiae strains isolated from bovine milk and from asymptomatic women in Québec, Canada, by randomly amplified polymorphic DNA (RAPD) analysis. A total of 185 bovine isolates and 38 human isolates were first serotyped for capsular polysaccharide by double diffusion in agarose gel (bovine isolates) and coagglutination (human isolates). Strains were then studied by RAPD using 3 primers, designated OPS11, OPB17, and OPB18, which were selected from 12 primers. Thirty-eight percent of bovine isolates and 82% of human isolates could be serotyped. Prevalent serotypes were type III (28%) for bovine isolates and types V (26%) and III (24%) for human isolates. RAPD results showed that, taken together, all isolates (of bovine and human origin) shared 58% similarity. Ninety-four percent of these isolates were clustered in four groups (I, II, III, and IV) with 70% similarity among them. Three clusters, A (48 isolates), B (14 isolates), and C (32 isolates), with 79 to 80% similarity were identified within group IV, whereas the three other groups did not present any clusters. Despite some clustering of human isolates, relatively high diversity was seen among them. Relatively high heterogeneity was observed with the RAPD profiles, not only for field strains belonging to different serotypes but also for those within a given serotype.
Animals↗
Dose-dependent effect of 17 beta-estradiol determined by growth curves and flow cytometric DNA analysis of a human breast carcinoma (T61) grown in nude mice.
An estrogen and progesterone receptor-positive human breast carcinoma (T61) grown in nude mice was exposed to 1.0, 0.1, 0.01, and 0.001 mg 17 beta-estradiol. These doses resulted in serum peak concentrations (day 1) of estradiol ranging from 3.5 X 10(-8) to 6.9 X 10(-10) M. The effect of the treatment was evaluated using growth curves and flow cytometric DNA analysis. The treatment induced a dose-dependent growth delay and dose-dependent changes in the cell cycle distribution. The cell cycle changes comprised a decrease in the G1 phase, an accumulation of cells in the S phase, and an increasing fraction of polyploid cells. The results suggest that estradiol induces a dose-dependent cell killing effect in the T61 human breast carcinoma. The correlation between the treatment-induced growth delay and the effect on the cell cycle distribution indicates that the changes in the cell cycle are a reflection of the estradiol-induced cell destruction. Since no tumor growth stimulation could be observed even at very low serum estradiol concentrations, the T61 human breast carcinoma may represent a new aspect in the study of human breast cancer.
Animals↗
Differentiation of distribution systems, source water, and clinical coliforms by DNA analysis.
During a 2-week period, Enterobacter cloacae was isolated from throughout the water distribution system in New Haven County, Connecticut. There was no forewarning of this event and no apparent reasons for it. Several epidemiologic and public health questions required rapid answers. Were these E. cloacae isolates the result of treatment failure and breakthrough or was regrowth occurring within the system? Did the E. cloacae isolates represent a health threat and were they causing infection? Pulsed-field gel electrophoresis utilizing whole-cell DNA digestion with restriction endonuclease SpeI permitted the rapid generation of specific information to answer these questions. Gel bands were stained with ethidium bromide and photographed with UV illumination. Homogeneity among isolates was confirmed by repeat digestion with XbaI. From each of the water distribution isolates, a single pattern of restriction endonuclease fragments was generated, indicating that only one clone of E. cloacae was in the distribution system. There was no homogeneity between source and distribution water E. cloacae isolates. Moreover, E. cloacae clinical isolates from patients from New Haven area hospitals showed no identity with E. cloacae isolated from the distribution system. Therefore, pulsed-field gel electrophoresis DNA analysis demonstrated that the E. cloacae from the distribution system was the result of a regrowth bloom within the system and not the result of treatment failure and that this clone was not causing a public health risk.
Bacterial Typing Techniques↗
Carrier detection in X-linked retinitis pigmentosa by multipoint DNA analysis. Problems due to genetic heterogeneity.
DNA diagnosis of X-linked retinitis pigmentosa (XLRP) is hampered by its genetic heterogeneity, while a clinical subdivision is almost impossible to make. So far, diagnostic services have been offered only to those families in which linkage to one RP locus (RP2 or RP3) has been clearly established. In most families, however, the nature of the XLRP type cannot be distinguished on the basis of linkage analysis. Here the authors describe that in some families DNA diagnosis is nonetheless feasible, when polymorphic DNA markers are used which span the entire Xp21.1-Xcen region and when no recombination between these markers disturbs the phase.
Blotting, Southern↗
DNA analysis in the case of Kaspar Hauser.
In 1828 a mysterious young man appeared in Nürnberg, Germany, who was barely able to speak or walk but could write down his name, Kaspar Hauser. He quickly became the centre of social interest but also the victim of intrigue. His appearance, his origin and assassination in 1833 were, and still are, the source of much debate. The most widely accepted theory postulates that Kaspar Hauser was the son of Grand Duke Carl von Baden and his wife Stephanie de Beauharnais, an adopted daughter of Napoleon Bonaparte. To check this theory, DNA analysis was performed on the clothes most likely worn by Kaspar Hauser when he was stabbed on December 14th, 1833. A suitable bloodstain from the underpants was divided and analysed independently by the Institute of Legal Medicine, University of Munich (ILM) and the Forensic Science Service Laboratory, Birmingham (FSS). Mitochondrial DNA (mtDNA) was sequenced from the bloodstain and from blood samples obtained from two living maternal relatives of Stephanie de Beauharnais. The sequence from the bloodstained clothing differed from the sequence found in both reference blood samples at seven confirmed positions. This proves that the bloodstain does not originate from a son of Stephanie de Beauharnais. Thus, it is becoming clear that Kaspar Hauser was not the Prince of Baden.
Base Sequence↗
Methodological aspects of flow cytometry DNA analysis in endometrial carcinoma, with special reference to sampling and reproducibility.
Two adjacent paraffin-embedded sections manifested concordance in ploidy status in 96% of cases (45/47), and the standard deviation (SD) for SPF was 2.7%. Analysis of 'micro-heterogeneity', within a distance of < or = 700 microm, yielded results for concordant ploidy status in 94% of cases, and the SD for SPF was 1.9% (n = 17). Frozen and paraffin-embedded material yielded concordant results for ploidy status in 87% (39/45) of cases, and SPF values were significantly lower (mean difference 1.5%) in the frozen samples. Diagnostic and repeat curettage material yielded concordant results for DNA ploidy status in 85% (40/47) of cases, and no significant difference in mean SPF (12% vs. 11%) was found. Discordant DNA ploidy results were attributable to small differences in the DNA histograms influencing the interpretation of near-diploid, near-tetraploid and small non-diploid cell populations, and the influence of debris on SPF estimation. On the basis of our findings and the practical advantage we recommend paraffin-embedded material from diagnostic curettage for FCM DNA analysis; the results are available sooner and the handling and transportation of tumor samples is more convenient.
Cryopreservation↗
The application of flow cytometric DNA analysis in detecting the presence of malignant cells in ovarian carcinoma peritoneal fluids.
OBJECTIVE: To compare flow cytometric detection of malignant cells with standard cytological evaluation in patients with ovarian carcinoma. SETTING: The City Hospital Trust, The Women's Hospital and CRC Trials Unit, Birmingham. SUBJECTS: Forty-three patients with histologically proven ovarian carcinoma and positive cytology, and a control population of 20 patients undergoing surgery for benign gynaecological conditions. METHODS: Prospective, blinded study examining ascitic fluid or peritoneal washings obtained at primary surgery by flow cytometric DNA analysis and cytological examination. RESULTS: Flow cytometry detected aneuploid cells in 27/43 (63%) of malignant and 7/20 (35%) of benign fluid specimens. In malignant samples the mean aneuploid count was 38.5% (range 1-98%) with a mean S-phase fraction of 5.2% (range 0-33.9%). In benign specimens the mean aneuploid count was 30.4% (range 14.5-66.4%). Based on these results, the overall sensitivity of cytometric detection of malignant cells was 71.4%, specificity 65%, with a positive predictive value of 85.1%. False positivity was found mainly in patients with benign ovarian cysts. Further examination revealed four false negative and four false positive results, where the peritoneal fluid and ovarian tissue DNA ploidy status concurred. Assuming such results to be correct increased the sensitivity of the test to 88.5% and specificity to 85%. CONCLUSIONS: Although flow cytometry can glean information beyond the capabilities of cytological assessment, using the premise that aneuploid cells alone indicate malignancy, it remains secondary to cytology in the detection of malignant cells in peritoneal fluids.
Adult↗
Multicystic mesothelial proliferation. Immunohistochemical, ultrastructural and DNA analysis of five cases.
We investigated the clinicopathological findings in five cases of multicystic mesothelial proliferation (MMP). All masses consisted of multiloculated cysts attached to pelvic organs and sometimes growing into the upper abdominal cavity. The cystic spaces were lined by flattened or cuboidal cells. The stroma showed fibrosis, oedema and chronic inflammation. Immunohistochemistry revealed strong positive staining for cytokeratin and epithelial membrane antigen, and focal positivity for vimentin and carcinoembryonic antigen. The endothelial markers were negative. Electron microscopy showed abundant surface microvilli and well-developed basal lamina. DNA analysis identified euploid cell populations in all cases. All but one case had a previous history of abdominal surgery. Despite the worrying appearance the clinical outcome was favourable in all cases; there was one recurrence. Clinical and pathological data support the hypothesis that MMP represent a reactive mesothelial proliferation and not a neoplastic process.
Adult↗
Comparative flow DNA analysis of different cell suspensions in breast carcinoma.
This study compared three methods of dissociation of breast lesions for DNA flow cytometry. Eleven benign lesions and 66 cancers were dissociated using mechanical, Ficoll, or enzymatic methods. DNA flow analysis showed that the DNA index did not vary from one method of dissociation to another. All benign lesions were diploid and 67% of all cancers were aneuploid. Enzymatic dissociation gave a lower percentage of aneuploid cells with a diminution of the proportion of cells in the G2 + M phase (13.2% enzymatic against 17.6% Fi-coll); on the other hand, it provided cell populations of greater viability than the other methods (32.6% enzymatic, 17.2% Fi-coll; P less than 0.01). The mechanical and Ficoll suspensions did not differ significantly when they were analyzed on the basis of their DNA content and their cellular viability. When compared with mechanical preparation, Ficoll suspension showed a lower recovery of tumor cells, but this inconvenience was compensated for by a more homogeneous aspect where the contribution of aggregates and debris was clearly lessened. Therefore, this study led us to choose Ficoll suspension for subsequent flow analysis of breast tumors.
Adult↗
Aspiration of musculoskeletal tumors for cytodiagnosis and DNA analysis.
Preoperative fine-needle aspirates of 25 soft tissue tumors and four bone tumors were used for cytodiagnosis and flow cytometric DNA ploidy analysis. The results were compared with the histopathologic diagnosis and flow cytometric DNA ploidy analysis on tissue samples after surgery. There was good agreement between the cytodiagnoses and histopathologic diagnoses with regard to benign tumor or sarcoma. There was good agreement between the ploidy determinations in aspirates and tissue samples in 20 out of the 26 tumors that could be analyzed. Aneuploid cell populations were found in 13 tumors cytologically classified as sarcoma and in one tumor classified as unspecified malignant tumor. These 14 tumors also showed aneuploid cell populations in the tissue samples and were histologically diagnosed as high-grade malignant sarcomas. The combined evaluation of preoperative cytodiagnosis and DNA ploidy on aspirates may give valuable prognostic information.
Adult↗
Two-color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte-common antigen.
Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.
Antibodies, Monoclonal↗
Assays for microbial contamination and DNA analysis based on electrorotation.
A new assay is described for determining the concentration and viability of waterborne micro-organisms. The method exploits the fact that when a latex bead coated with a specific binding agent is complexed to the target analyte, the analyte complex formed assumes new dielectric properties that can be monitored by its electrorotation response. This generic technology is applicable to a wide range of organism and toxicological diagnostic tests, as well as to other areas of biotechnology. An example is given of its possible application to DNA sequence analysis.
Animals↗
Restorative proctocolectomy: histological assessment and cytometric DNA analysis of ileal pouch biopsies.
BACKGROUND/AIMS: The pathological changes and the risk of developing cancer in the ileal pouch mucosa of patients who received restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) were studied. The presence or absence of remaining rectal mucosa below the IPAA in both patients with stapled and handsewn IPAA was also examined. MATERIALS AND METHODS: Endoscopy of the ileal pouch was performed on 38 patients at 4, 12, 18 and 36 months after restorative proctocolectomy with ileal pouch. Mucosal biopsy specimens were taken from the ileal reservoir in order to assess the histological incidence of inflammation. In 23 patients, biopsies were taken to perform cytometric DNA analysis. Clinical symptoms of pouchitis (over six evacuations in 24 hours, night-time evacuations, leakage of feces, bloody diarrhea, abdominal pain and fever) were recorded and correlated with the histological findings. Biopsies were also sampled below the ileo-anal anastomosis (IPAA) in order to identify residual rectal mucosa. RESULTS: Results of histological assessment showed various degrees of chronic inflammation increasing over time (from 42 to 60%) while the presence of both acute and chronic inflammation of the reservoir was less frequent (from 18 to 30%). Villous atrophy was present in 39-68% of patients and the grade of villous atrophy was correlated to the grade of inflammation. Clinical pouchitis was present in 3 to 8% of cases at the different controls and it was always associated with the highest grade of histological inflammation and severe villous atrophy. No significant alteration of the DNA cellular content was observed. Very low incidence of aneuploidy (0.7-1% Ex.R.) has been reported in three cases. However, we found dysplasia in only one patient who underwent surgical treatment for familial polyposis coli. IPAA evaluation showed no residual rectal mucosa in 40% of cases with stapled IPAA; in the remaining 60%, we found a small amount of rectal mucosa (maximum 1 cm). We did not find rectal mucosa after handsewn IPAA with mucosectomy. CONCLUSIONS: Patients treated with restorative proctocolectomy with IPAA showed a higher and increased incidence of inflammation during follow-up. No significant alteration of DNA cellular content nor dysplasia of the pouch mucosa were observed. In this study the chance of leaving rectal mucosa after stapled IPAA was about 60%.
Adolescent↗
Prenatal detection of 46,XY,rec(5),dup q, inv(5)(p13q33) using DNA analysis, flow cytometry, and in situ hybridization to supplement classical cytogenetic analysis.
Distinguishing between balanced and unbalanced chromosome complements segregating from parental rearrangements may be difficult using only classical cytogenetic techniques if banding morphology is similar under both expectations. In these situations, supplementing cytogenetic analysis with molecular genetic techniques and flow cytometry may provide increased diagnostic accuracy. To illustrate this, we present a case in which similar band pattern morphology would be expected for both the balanced carrier (heterozygote) and the recombinant dup q chromosome complements segregating from a mother with a balanced inversion [46,XX,inv(5)(p13q33)]. The parents came to Northwestern for consultation after receiving conflicting interpretations of their first amniotic fluid cultures. An ultrasound examination was said to be normal. They inquired whether there were ways to increase their confidence that the complement was unbalanced. Their reluctance to terminate the pregnancy was due to a 6-year history of infertility. After extensive counselling, the couple elected repeat amniocentesis. Further cytogenetic analysis of repeat amniotic fluid cultures by G-banding and R-banding, molecular genetic analysis with highly polymorphic DNA probes, and quantitative flow cytometry were performed. Results agreed that an unbalanced fetal complement was present. Southern blot analysis with a 5p marker definitively demonstrated a lack of maternal 5p material in the fetus, and in situ hybridization showed a 5q marker at either end of the recombinant chromosome. Flow cytometry was consistent with this interpretation. Because of the advanced gestational age, the parents elected to terminate based on cytogenic results of the second amniocentesis, rather than to wait another 1-2 weeks for results of other methods.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Reciprocal translocation between the proximal regions of the long arms of chromosomes 13 and 15 resulting in unbalanced offspring: characterization by fluorescence in situ hybridization and DNA analysis.
We describe two female siblings with similar clinical features consisting of hydrocephalus, scaphocephaly, hypotonia, mongoloid eye slant, blepharophimosis, micrognathia, supernumerary mouth frenula and mental retardation. Routine cytogenetic studies in the elder patient did not reveal any abnormality, and initially it was assumed that the syndrome had an autosomal recessive inheritance. However, a slightly larger chromosome 13 was seen in routine G-banded metaphases of the mother and the youngest of the two siblings. A shorter chromosome 15 was detected in the mother only. High resolution banding showed that the abnormal chromosome 13 contained an extra G-positive band at 13q12. The short chromosome 15 in the mother appeared to have a deletion of band q12. Fluorescence in situ hybridization using DNA markers specific to chromosomes 13 and 15 unequivocally showed that the mother was a carrier of a balanced reciprocal translocation t(13;15)(q12;q13), whereas the youngest sibling's karyotype was 46,XX,-13,+der(15)t(13;15)(q12;q13)mat, resulting in partial monosomy 13pter----q12 and partial trisomy 15pter----q13. The proband is thus trisomic for the critical region responsible for Prader-Willi syndrome and Angelman syndrome; this was confirmed by DNA analysis demonstrating one paternal and two maternal alleles from multiallelic marker loci mapping to 15q11-q13. This report illustrates the sensitivity and specificity offered by fluorescence in situ hybridization and its usefulness in the diagnosis and delineation of subtle chromosomal rearrangements.
Abnormalities, Multiple↗
Concentrations and tracking of listeria monocytogenes strains in a seafood-processing environment using a most-probable-number enrichment procedure and randomly amplified polymorphic DNA analysis.
Concentrations of environmental microflora and Listeria monocytogenes were monitored at multiple environmental locations within a seafood-processing facility over the course of 6 months. Concentrations of L. monocytogenes were determined using a most-probable-number (MPN) enrichment procedure. Two floor drains had persistent low concentrations of L. monocytogenes (0.03 to >1,100 MPN/cm2). In comparison, concentrations of the other organisms in the drain were much higher (heterotrophic plate count range of 10(5) to 10(8) CFU/cm2). Concentrations of environmental organisms (heterotrophic aerobic plate counts and counts of pseudomonads, Shewanella spp., Aeromonas hydrophila, and coliforms) were not correlated with concentrations of L. monocytogenes. The 178 confirmed L. monocytogenes isolates from the MPN procedure were further characterized by randomly amplified polymorphic DNA analysis. Sixteen different banding patterns were identified, and nine of the patterns were identified from samples collected on two or more collection dates. From all locations, banding type A was observed in 98 confirmed isolates (55%). Although present, L. monocytogenes was a relatively minor component in the ecosystem of the floor drains in this seafood-processing facility.
Animals↗