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PubMed1987

Norrie's disease in a French-Canadian kindred: attempt to detect carriers by DNA analysis.

In a French-Canadian kindred four male cousins are affected with Norrie's disease, a rare X-linked recessive disorder. Three have university education, and the fourth has some developmental delay. Only one is microcephalic. All have mild to severe hearing deficit, although only three were aware of their hearing loss. Linkage analysis of DNA from family members with the probe L1.28 failed to detect female carriers.

Adult↗

PubMed1989

Flow cytometric DNA analysis of bone tumors.

Flow cytometric DNA analysis was performed in a total of 203 bone tumors, benign and malignant. In more than 80% of cases the material studied was paraffin-embedded tumor tissue, mainly from the archives of the Bone Tumor Registry of Westphalia in Münster. Compared with ethanol-fixed fresh tumor samples, the variation coefficient in DNA histograms of the stored material was increased by a factor of 1.2-1.5, which means that resolution was decreased and that, in many cases, accurate cell cycle analysis was not feasible. However, the results of cell cycle analysis in bone tumors, even if performed on optimally fixed specimens, have to be evaluated with caution and full reference to the corresponding histological slides, since these histograms are apt to show various superpositions from the inflammatory infiltrate. The assessment of DNA ploidy is unimpaired if, in agreement with most researchers today, deviations smaller than +/- 10% from the diploid standard are still defined as DNA diploid, peridiploid, or pseudodiploid. The coefficient of variation should be kept as low as possible. If it is between 10% and 15%, the near-diploid stemlines with DNA indices of 0.9 or 1.1 may be hard to delineate. On account of the particularly marked regressive changes, the resolution of DNA histograms was most strongly impaired in chondromatous tumors, whereas it was mostly excellent in highly cellular viable tumor tissue, such as that from Ewing's sarcoma or osteoblastoma. On the whole, there was a distinct correlation between DNA ploidy and the biological behavior of bone tumors (Table 8). The highest rates of DNA aneuploidy were found in highly malignant OSs (18/21) and FSs (14/16), thus reflecting their poor prognosis. Of six juxtacortical OSs, three well-differentiated parosteal OSs and two periosteal OSs were DNA diploid, whereas one highly malignant surface OS and five highly malignant extraskeletal OSs, all DNA aneuploid, corresponded fully to the medullary OSs. Judging by preliminary results, adjuvant preoperative chemotherapy (COSS 80/82: Bösing et al. 1987) may reduce the rate of DNA aneuploidy and, consequently, of stem cell heterogeneity in general. A selective destruction of those stemlines that respond particularly to chemotherapy appears probable. In contrast to their high malignancy, Ewing's sarcomas showed an unexpectedly low proportion of DNA aneuploid stemlines (14/24). The comparatively favorable prognosis of MFH of bone is reflected in a lower rate of aneuploidies (2/10), which is also rather low (probably too low) when compared to our own data from soft tissue MFH (9/19).(ABSTRACT TRUNCATED AT 400 WORDS)

Bone Neoplasms↗

PubMed1996

Peripheral blood mononuclear cells are a reliable internal standard for the flow cytometric DNA analysis of frozen malignant breast biopsies.

DNA ploidy and S-phase estimations are widely used as prognostic indicators in treatment decisions for breast cancer patients. For accurate calculation of the DNA index there is a need to identify the G0G1 diploid peak position. This study tested the reliability of peripheral blood mononuclear cells (PBMCs) as a diploid internal standard in the flow cytometric DNA analysis of 60 frozen malignant breast tumors. For each tumor, PBMCs were added to a duplicate sample prior to staining. There were 55 tumors with interpretable histograms; 18 were DNA diploid, 37 were DNA aneuploid, and all tumors had coefficients of variation < 5.0%. For each of the 55 tumors, whether diploid or aneuploid, the addition of PBMCs gave a unimodal peak at the diploid peak position. The results show that PBMCs are a reliable standard to identify the G0G1 diploid peak position.

Aneuploidy↗

PubMed2004

[Flow cytometric DNA analysis and neoadjuvant chemotherapy of cervix cancer (stage IB2-IIIB)].

The purpose of the present study is to determine the predictive role of the data of flowcytometric DNA-analysis--aneuploidity DNA-index, proliferative index in patients with squamous cervical cancer who was given chemotherapy as a initial treatment. The data of 12 patients in stage IB2-IIIB, who were divided into two groups according to the second-line treatment--surgery of definitive radiotherapy, were analysed. These data were correlated with the respond of the tumor to neoadjuvant chemotherapy (clinical and histopathological) as well as other factors such as stage, size of tumor lesion, grading disease free survival. The method of flowcytometric DNA-analysis was presented briefly. The results we have obtained, although in quiete limited number of patients, are interesting and justify prospective studies of the problem.

Carcinoma, Squamous Cell↗

PubMed1993

Plasmid DNA analysis, biotyping, and antimicrobic susceptibility as subtyping tests for Klebsiella pneumoniae and Klebsiella oxytoca.

We compared plasmid DNA analysis, biotyping by Vitek, and disk diffusion antimicrobic susceptibility as subtyping tests of Klebsiella pneumoniae and Klebsiella oxytoca. The 92 tested isolates were from alternate, culture-positive patients over 6 months. No outbreak or cluster of infections was recognized during this interval. Plasmid DNA was detected in 85% of the isolates. Each isolate except one had a reproducible absence of plasmid DNA or a reproducible plasmid DNA profile on repetitive testing. Restriction endonuclease enzyme analysis of plasmid DNA was necessary to distinguish differences among some isolates that had only large plasmids. Isolates with only large plasmids represented 18% of the collection. Of the 78 isolates with plasmid DNA, all but two were considered different from one another by plasmid DNA analysis. Biotyping and antimicrobic susceptibility testing were not highly reproducible. In addition, biotyping did not demonstrate a sufficient variety of patterns among the isolates for subtyping purposes. We conclude that plasmid DNA analysis is very useful as a subtyping test for isolates of K. pneumoniae and K. oxytoca. Neither biotyping nor antimicrobial susceptibility as performed in our laboratory had sufficient discriminatory power and reproducibility for subtyping these organisms.

Anti-Bacterial Agents↗

PubMed1989

[Flow cytometric DNA analysis in colorectal cancer].

Flow cytometric DNA analysis for assessing malignant potential of colorectal carcinoma was investigated by paraffin-embedded materials. Preservation time of paraffin blocks and formalin fixation time of surgical specimens within 14 days do not influence the nuclear DNA content. There was seen a good correlation between the DNA contents of paraffin-embedded and fresh materials obtained from the same surgical specimens. Using deparaffinized tumor specimens, the nuclear DNA content was measured by flow cytometry in 144 patients with primary colorectal cancer, who had been treated from 1971 to 1985. Forty-four percent of colorectal cancer were diploid and 56% were aneuploid. There was no significant correlation between ploidy pattern and clinicopathological factors. However, the patients with aneuploid tumor had a significantly worse survival than those with diploid tumor (Generalized Wilcoxon test, p less than 0.001). The patients with aneuploid seemed to have an unfavorable survival than those with diploid in the same stage, and had a significantly worse survival in each group of negative nodes, P0 and H0. It is concluded, therefore, that the nuclear DNA content of colorectal cancer may be an important prognostic factor, being independent of pathological stage.

Cell Nucleus↗

PubMed1997

Molecular DNA analysis for differentiation of persistence or relapse from recurrence in treatment failure of Streptococcus pyogenes pharyngitis.

In the evaluation of treatment failure in group A streptococcal pharyngitis, it is essential to distinguish persistence or relapse with homologous streptococcal strains from the acquisition of new, unrelated strains. Randomly amplified polymorphic DNA analysis and restriction fragment length polymorphism analysis of total DNA were used as epidemiological tools to compare 122 pre- and post-treatment Streptococcus pyogenes isolates obtained from 61 patients. The results obtained by molecular typing showed that bacteriological failures were due to the original strains in 43 cases (70%) and to new strains in 18 cases (30%). In the present study, restriction fragment length polymorphism analysis of total DNA appeared to be more discriminative than randomly amplified polymorphic DNA analysis. Thus, molecular analysis of DNA is an effective way to distinguish recurrence from persistence or relapse and will be useful in assessing the efficacy of new antibiotic treatments.

Adolescent↗

PubMed2001

Implementation of forensic DNA analysis on casework evidence at the Palm Beach County Sheriff's Office Crime Laboratory: historical perspective.

Palm Beach County is the largest of the 64 counties in the state of Florida, USA, with most of the area uninhabited and the population concentrated near the coastal region. The Serology/DNA Section of the Palm Beach County Sheriff's Office (PBSO) Crime Laboratory serves a community of approximately one million residents, and an additional million tourists visit Palm Beach County every year. In addition to the unincorporated county regions, there are thirty-four city police agencies, the Florida State Highway Patrol, several university security agencies, the local Federal Bureau of Investigation, and the county Medical Examiners Office that all use the PBSO Serology/DNA Laboratory for the analysis of casework evidence. The purpose of this manuscript is to provide laboratories that are in the process of initiating DNA analysis on casework with practical information regarding the decision-making processes that occurred during the development of the DNA testing program at PBSO. Many of the concerns addressed in the early 1990's are still a guide to the development of a quality forensic DNA analysis program in the year 2001. Issues, such as personnel, laboratory space, internal standard operating procedures, implementation of DNA analysis on casework evidence, and building a relationship with law enforcement personnel are discussed.

Clinical Laboratory Techniques↗

PubMed1990

DNA analysis of periampullary cancers.

The nuclear DNA content in 71 cases of periampullary cancer (27, cancer of the head of the pancreas (Ph); 24, cancer of the ampulla of Vater (A); and 20, cancer of the inferior common bile duct (Bi] was measured cytofluorometrically using the archival paraffin-embedded specimens of the primary lesions. They were analyzed in relation to prognosis, tumor size, histological differentiation, lymph node metastasis, lymphatic invasion, venous invasion, perineural invasion, and growth pattern. The results show that "Ph" has more unfavorable prognosis compared with the other two and it has more DNA content under the same conditions such as tumor from 2.1 to 4 cm in its greatest dimension, well differentiated adenocarcinoma, with or without lymph node metastasis, with or without venous invasion, with lymphatic invasion, with perineural invasion, and in the intermediate growth pattern between expansive and infiltrative. In conclusion, this study demonstrates a close correlation between the DNA content and prognosis and the significant clinical value of DNA analysis for predicting the prognosis in patients of periampullary cancer.

Adenocarcinoma↗

PubMed2005

Probing the kinetics of SYTOX Orange stain binding to double-stranded DNA with implications for DNA analysis.

Rapid binding kinetics of SYTOX Orange stain with double-stranded DNA (dsDNA) was revealed on the DNA fragment sizing flow cytometer. We demonstrated for the first time that the dye molecules could be adsorbed onto the capillary surface and native DNA fragments can be dynamically stained while passing through the capillary. High-quality burst size distribution histograms were obtained for DNA samples analyzed immediately after staining, dilution, or mixing. These observations indicated that rapid interactions exist between SYTOX Orange dye molecules and dsDNA. A stopped-flow fluorescence apparatus was set up to capture the fast association traces of intercalating dyes binding to dsDNA. Kinetic equations were derived to fit the association curves for determination of association rates and to model the dynamic staining, dilution, and mixing processes of DNA samples stained with intercalating dyes. The measured association rates for both SYTOX Orange and PicoGreen stains intercalating into dsDNA were on the order of 10(8) M-1 s-1, suggesting a diffusion-controlled process. Simulations indicate that reequilibration can be reached in seconds upon staining, dilution, or mixing. Insight into the kinetics of DNA binding dyes will help implement efficient sample-handling practices in DNA analysis, including DNA fragment sizing flow cytometry.

Binding Sites↗

PubMed1988

Impulse cytophotometric DNA analysis in pituitary adenomas.

Flow cytometric DNA analysis was carried out on 32 microsurgically removed pituitary adenomas. Additionally, the histograms of tumor cell nuclei of 7 patients were compared with those of the cultured cells from the same tumor samples. The tumors were classified into 3 groups according to the proliferation index (PI) of the flow cytometric results: 1) tumors with DNA patterns of slow proliferation (PI under 10), to which the majority of the examined pituitary adenomas belonged; 2) pituitary adenomas with diploid karyograms and PI values from 10 to 15; 3) diploid or aneuploid karyograms with PI values above 15. The third group were characterized histologically by increased chromatin content, nuclear polymorphism, mitoses, and extrapituitary infiltration of the tumor cells, and were, therefore, no longer benign. However, there was no direct relationship between the intensity of hormone secretory activity of the tumors and DNA ploidy. Cultured adenoma cells examined by flow cytometry remained stable in all cases but one.

Adenoma↗

PubMed1993

Prognostic value of DNA analysis in colorectal carcinoma.

BACKGROUND: Reported experiences regarding the prognostic significance of DNA content in colorectal carcinoma have been a matter of controversy. METHODS: DNA analysis with image cytometry was performed in 137 patients with colorectal cancer. Only patients who had resection without tumor residual and who did not die postoperatively as a consequence of the operation were entered in the study. At the time of DNA analysis, neither the histomorphologic data nor the relapse-free survival time of the patients were known. RESULTS: In this investigation the DNA content of tumor cells had no univariate or multivariate influence on the relapse-free survival time. The prognosis was dependent on the tumor localization, depth of tumor infiltration, lymph node metastasis, and grade. CONCLUSIONS: DNA content provides no additional prognostic information in colorectal carcinoma.

Adult↗

PubMed1987

Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis.

We have used a new method of DNA analysis for the rapid prenatal diagnosis of sickle cell anemia in two fetuses at risk for this disease. This method of detecting the sickle gene is a modification of standard restriction-enzyme techniques and requires only a small amount of DNA. The first step involves a 200,000-fold enzymatic amplification of the specific beta-globin DNA sequences that may carry the sickle mutation. This provides a sufficient quantity of DNA for the analysis. Next, a short radiolabeled synthetic DNA sequence homologous to normal beta A-globin gene sequences is hybridized to the amplified target sequences. The hybrid "duplexes" are then digested sequentially with two restriction endonucleases. The presence of beta A- or beta S-globin gene sequences in the amplified target DNA from the patient determines whether the beta A-hybridization probe anneals perfectly or with a single nucleotide mismatch. This difference affects the restriction-enzyme digestion of the DNA and the size of the resulting radiolabeled digestion products, which can be distinguished by electrophoresis followed by autoradiography. This method is sufficiently sensitive and rapid that the prenatal diagnosis of sickle cell anemia can be made on the same day that the fetal DNA is made available. It can also be applied to the diagnosis of hemoglobin C disease.

Anemia, Sickle Cell↗

PubMed1999

Adenosquamous carcinoma of the prostate: case report with DNA analysis, immunohistochemistry, and literature review.

We diagnosed prostatic adenosquamous carcinoma by prostate core needle biopsy in a 55-year-old man with no history of prostate cancer. The prognosis, DNA analysis, and histogenesis of this extremely rare tumor are controversial. To our knowledge, this is the first case of adenosquamous carcinoma diagnosed by core needle biopsy in a patient with no history of prostate cancer or hormonal therapy. We performed immunohistologic and DNA analysis to further characterize this cancer. The clinical presentation and abnormal DNA analysis portend an aggressive course.

Carcinoma, Adenosquamous↗

PubMed1989

Prenatal diagnosis of homozygous alpha 0-thalassaemia by direct DNA analysis of chorionic villi in Singapore.

First-trimester prenatal diagnosis by DNA analysis was carried out for seven pregnancies at risk for homozygous alpha 0-thalassaemia. Transabdominal placental biopsy was carried out at 10-12 weeks' gestation. The presence of alpha-globin genes in the fetal DNA was determined by restriction endonuclease mapping and hybridization with cloned alpha-globin probe. Homozygous alpha 0-thalassaemia was detected in two fetuses and the pregnancies were interrupted. Alpha 0-thalassaemia in both cases was confirmed by electrophoresis of the umbilical cord blood where only haemoglobin Bart's was detected. The remaining five fetuses were diagnosed as normal or as possessing alpha-thalassaemia-1 trait and the pregnancies are being carried to term. The use of DNA analysis in prenatal diagnosis of fetuses at risk for homozygous alpha 0-thalassaemia enables detection of the haemoglobinopathy at 10 weeks' gestation.

Chorionic Villi↗

PubMed1994

Effects of thermocyclers and primers on the reproducibility of banding patterns in randomly amplified polymorphic DNA analysis.

The effects of thermocyclers and primers on the reproducibility of banding patterns in randomly amplified polymorphic DNA analysis were tested. Purified Bordetella pertussis DNA was analysed with four primers (12-mer), which did not differ from each other in their GC-content. Three different thermocycler models from two manufacturers were tested. Three of the primers produced consistent banding patterns in separate wells of the reaction plates of individual thermocyclers, and in different thermocycler models. In contrast, the fourth primer showed extensive between-instrument variation, and the within-instrument variation with this primer was acceptable only when the most advanced thermocycler model was used. We conclude that only primers that provide consistent banding patterns in different environments should be chosen for interlaboratory use in randomly amplified polymorphic DNA analysis. The primers showing variability in banding patterns can only be used if they produce consistent results in the thermocycler used.

Base Sequence↗

PubMedDate not supplied

Flow cytometric DNA analysis of oral squamous cell carcinoma in Iraqi patients.

We studied the DNA content, DNA index and cell cycle parameters that are reliable markers for assessing the proliferative activity and aggressiveness of malignancies. Cytometric DNA analysis was performed on formalin-fixed paraffin embedded sections from 36 Iraqi patients with oral squamous cell carcinoma. The results showed that 20 of 36 cases (55.5%) were diploid, while 15 cases (41.7%) were aneuploid. Significantly higher S-phase fractions and higher DNA indices characterized aneuploid tumours. Nuclear DNA analysis as part of the evaluation of oral squamous cell carcinoma will influence the selection of appropriate treatment strategies.

Adolescent↗

PubMed1986

Flow DNA analysis in human lung cancer: potentiality and limitations due to sampling methods.

Flow DNA analysis was performed on samples from 71 surgically removed lung cancers and from 145 patients undergoing bronchoscopy. Abnormal DNA stem lines, characterized by their DNA index (DI), were frequently observed in operated lung carcinomas (87%). Two or three abnormal DNA stem lines were discovered simultaneously in 10% of the samples. The mean DI of all abnormal tumor stem lines was lowest for rare tumor cell types and highest for adenocarcinomas. Intermediate mean DI values were found for epidermoid and small cell carcinomas, which were among the most proliferative tumors. The high rate of false negative results suggests poor diagnostic reliability of flow DNA analysis on bronchoscopic samples. However, the method appears to provide a promising objective tool capable of evaluating tumor behavior and prognosis.

Adenocarcinoma↗