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PubMed1984

[Cytophotometric DNA analysis of the early and superficial esophageal cancer].

Although it is widely known that early esophageal cancer (n-) patients have a favorable prognosis after surgery and the superficial esophageal cancer (n+) patients have a poor one, the differences between them have not been found out under the microscopic examination of the primary lesion. Cytophotometric DNA analysis of the primary lesion was done to search for the differences between early and superficial esophageal cancer. Significant differences were obtained in the mean DNA value (p less than 0.01), the percentage of nuclei beyond tetraploid (p less than 0.001) and the product of 2 indices above (p less than 0.001). The result suggests that cytophotometric DNA analysis is a useful means for differentiating the early esophageal cancer from the superficial esophageal cancer and that the most suitable perioperative combined therapy can be chosen through the Feulgen-stained biopsy specimen.

Aged↗
PubMed1995

Significance of Flow Cytometric DNA Analysis from Freshly Aspirated Samples.

The procedure of aspiration biopsy cytology by fine needle aspiration (ABC) is as option in establishing definitive diagnoses for breast cancers. In this series, a needle size of 21G was considered most suitable for ABC as well as flow cytometric DNA analysis. Histograms from fresh samples aspirated by fine needle clearly delineated a sharp peak in G&sup0;G(1) phases and also a better CV was obtained than with paraffin-embedded preparations. In addition, fresh samples gave more reliable DI and suggested the value of measuring nuclear DNA contents. It is believed that the prognoses of breast cancers are closely associated with DNA ploidy patterns. In this sense, flow cytometric DNA analysis of fresh samples of ABC is regarded as important in clinical use.

Journal Article↗
PubMed1994

DNA analysis in forensics, disease and animal/plant identification.

During 1993, significant advances have been achieved in applications of DNA analysis to forensic science, disease assessment, and animal/plant identification. These advances include the development of simple and high sample-throughput techniques for highly informative personal identification, rapid screening for pathogens and the development of polymorphic genetic markers in plants and animals.

Animals↗
PubMed1993

Transitional cell carcinoma of the renal pelvis and ureter in Taiwan. DNA analysis by flow cytometry.

BACKGROUND: The incidence of upper urinary tract tumors is relatively high in southern Taiwan. DNA analysis by means of flow cytometry is not well investigated with regard to tumors of the upper urinary tract and the differences in DNA ploidy between transitional cell carcinomas in endemic and nonendemic areas. METHODS: A retrospective nuclear DNA ploidy analysis by flow cytometry comprised 41 formaldehyde solution-fixed, paraffin-embedded tissue specimens of transitional cell carcinoma of the renal pelvis and ureter. The preparation of nuclear suspensions from paraffin-embedded tissue blocks and staining were modified by means of the techniques of Hedley and Vindeløv. RESULTS: There was no statistical correlation between DNA ploidy, histologic grade, and pathologic stage, however, 82% of the DNA nondiploid tumors showed tumor progression. In contrast, only 46% of the DNA diploid tumors revealed tumor progression. Among Grade 2 tumors, 85% of the DNA nondiploid tumors showed postoperative tumor progression, whereas only 31% of the DNA diploid tumors showed tumor progression. Seventy-nine percent of the nondiploid patterns were present in patients native to the Pa Chang Valley, where the so-called "blackfoot disease" and urothelium tumor are endemic, whereas only 22% appeared in patients living in other areas. CONCLUSIONS: DNA flow cytometry can identify a group of patients with poor outcome unpredictable by pathologic examination, and is an important tool in research into the pathogenesis of cancer.

Carcinoma, Transitional Cell↗
PubMed1987

Approach to the diagnosis of beta-thalassaemia by DNA analysis.

Over the last few years, the new techniques of DNA analysis have enabled efficient strategies for the detection and characterisation of the different mutations causing beta-thalassaemia of which a total of 41 have now been characterised. Since the majority of the beta-thalassaemia molecular defects are of the non-deletion type and not directly detectable by Southern blotting, a comprehensive approach to the identification of these mutations entails the use of several different techniques, including direct identification by restriction enzyme analysis and synthetic oligonucleotide probes, indirect identification by linkage analysis to restriction fragment length polymorphisms and globin chain synthesis analysis. As the majority of beta-thalassaemia in each population is accounted for by a few mutants, it should be possible to work out a combination of techniques to directly detect the majority of beta-thalassaemia in a defined population.

DNA↗
PubMed2001

Seizures in myoclonic epilepsy with ragged-red fibers detected by DNA analysis: a case report.

A 19-year-old Thai woman presented with progressive ataxia and generalized tonic-clonic seizures. Later on, she developed status epilepticus. Blood was tested by molecular DNA analysis which showed A8344G mitochondrial DNA mutation associated with myoclonic epilepsy with ragged-red fibers (MERRF). We confirmed this finding in other members of this family. This is an interesting case report in Thailand of MERRF identified to have A-->G transition mutation at nucleotide 8344 of mitochondrial tRNA(lys) gene without ragged-red fibers from histopathologic studies of muscle. Molecular genetic analysis in suspicious cases of mitochondrial disorders is necessary for proper management and genetic counseling.

Adult↗
PubMed1990

Flow cytometric DNA analysis of corneal epithelium.

We have modified an existing technique in order to perform DNA analysis by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statistically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken 1) from three individuals who had epithelial dysplasia and 2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amount of DNA or had higher than normal percentages of cells in the S and G2 + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G2 + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.

Animals↗
PubMed2000

Fingerprint enhancement revisited and the effects of blood enhancement chemicals on subsequent profiler Plus fluorescent short tandem repeat DNA analysis of fresh and aged bloody fingerprints.

This study was aimed at determining the effect of seven blood enhancement reagents on the subsequent Profiler Plus fluorescent STR DNA analysis of fresh or aged bloody fingerprints deposited on various porous and nonporous surfaces. Amido Black, Crowle's Double Stain. 1,8-diazafluoren-9-one (DFO), Hungarian Red, leucomalachite green, luminol and ninhydrin were tested on linoleum, glass, metal, wood (pine, painted white), clothing (85% polyester/15% cotton, 65% polyester/35% cotton, and blue denim) and paper (Scott 2-ply and Xerox-grade). Preliminary experiments were designed to determine the optimal blood dilutions to use to ensure a DNA typing result following chemical enhancement. A 1:200 blood dilution deposited on linoleum and enhanced with Crowle's Double Stain generated enough DNA for one to two rounds of Profiler Plus PCR amplification. A comparative study of the DNA yields before and after treatment indicated that the quantity of DNA recovered from bloody fingerprints following enhancement was reduced by a factor of 2 to 12. Such a reduction in the DNA yields could potentially compromise DNA typing analysis in the case of small stains. The blood enhancement chemicals selected were also evaluated for their capability to reveal bloodmarks on the various porous and nonporous surfaces chosen in this study. Luminol. Amido Black and Crowle's Double Stain showed the highest sensitivity of all seven chemicals tested and revealed highly diluted (1:200) bloody fingerprints. Both luminol and Amido Black produced excellent results on both porous and nonporous surfaces, but Crowle's Double Stain failed to produce any results on porous substrates. Hungarian Red, DFO, leucomalachite green and ninhydrin showed lower sensitivities. Enhancement of bloodmarks using any of the chemicals selected, and short-term exposure to these same chemicals (i.e., less than 54 days), had no adverse effects on the PCR amplification of the nine STR systems surveyed (D3S 1358, HumvWA, HumFGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) or of the gender determination marker Amelogenin. The intensity of the fluorescent signals was very similar and the allele size measurements remained constant and identical to those of untreated bloody fingerprints. No additional background fluorescence was noted. Continuous exposure (for 54 days) to two of the seven enhancement chemicals selected (i.e., Crowle's Double Stain and Hungarian Red) slightly reduced the amplification efficiency of the longer STR loci in profiles of fresh and 7 to 14-day-old bloodprints. This suggests that long-term exposure to these chemicals possibly affects the integrity of the DNA molecules. This study indicates that significant evidence can be obtained from fresh or aged bloody fingerprints applied to a variety of absorbent and nonabsorbent surfaces which are exposed to different enhancement chemicals for short or long periods of time. It also reaffirms that PCR STR DNA typing procedures are robust and provide excellent results when used in concert with fluorescence-based detection assays after fingerprint identification has taken place.

Blood Chemical Analysis↗
PubMedDate not supplied

Prognostic significance of DNA-analysis by flow cytometry in non-Hodgkin's lymphoma.

DNA analysis by flow cytometry was performed on lymph node cells obtained from 65 untreated patients with non-Hodgkin's lymphoma. According to the Kiel classification 41 cases were of low grade malignancy and 24 cases belonged to the high grade malignancy group. 47 out of 61 evaluable cases were diploid/near-diploid, 12 were aneuploid and 2 cases showed polyploidy. No difference in survival was found between diploid/near-diploid and aneuploid cases. The percentage of S-phase cells was found to be a valuable prognostic parameter. Using a cut point at 4 per cent S-phase cells between low and high S-phase lymphomas a highly significant difference in survival was demonstrated (p = 0.0004). The actuarial survival up to 3 years was about 77 per cent for lymphomas with less than 4 per cent S-phase cells to be compared with 20 per cent for patients with high S-phase (greater than or equal to 4 per cent) lymphomas. Subdivision according to the percentage of S-phase cells seemed to be a better prognostic predictor than morphologic subclassification.

DNA, Neoplasm↗
PubMed2002

Combined ribotyping and random multiprimer DNA analysis to probe the population structure of Listeria monocytogenes.

To improve our understanding of the genetic links between strains originating from food and strains responsible for human diseases, we studied the genetic diversity and population structure of 130 epidemiologically unrelated Listeria monocytogenes strains. Strains were isolated from different sources and ecosystems in which the bacterium is commonly found. We used rRNA gene restriction fragment length polymorphism analysis with two endonucleases and random multiprimer DNA analysis with seven oligonucleotide primers to study multiple genetic features of each strain. We used three clustering methods to identify genetic links between individual strains and to determine the precise genetic structure of the population. The combined results confirmed that L. monocytogenes strains can be divided into two major phylogenetic divisions. The method used allowed us to demonstrate that the genetic structure and diversity of the two phylogenetic divisions differ. Division I is the most homogeneous and can easily be divided into subgroups with dissimilarity distances of less than 0.30. Each of these subgroups mainly, or exclusively, contains a single serotype (1/2b, 4b, 3b, or 4a). The serotype 4a lineage appears to form a branch that is highly divergent from the phylogenetic group containing serotypes 1/2b, 4b, and 3b. Division II contains strains of serotypes 1/2a, 1/2c, and 3a. It exhibits more genetic diversity with no peculiar clustering. The fact that division II is more heterogeneous than division I suggests that division II evolved from a common ancestor earlier than division I. A significant association was found between division I and human strains, suggesting that strains from division I are better adapted to human hosts.

DNA Primers↗
PubMed1989

[Flow cytometric DNA analysis of normal adrenal tissues and adrenal tumors].

Flow cytometric DNA analysis (FCM) of adrenal tumors was studied to evaluate whether FCM will be a useful examination for differentiating between benign and malignant adrenal tumors. 10 specimens of surgically resected (for renal cell carcinoma confined within the middle or lower pole) normal adrenal glands and 20 specimens of surgically resected adrenal tumors were submitted for the study. Hyperplastic adrenal cortex as well as normal adrenal gland showed normal diploid pattern. On the other hand, some of the cortical adenomas showed tetraploid patterns, which has been known to be an index of malignancy in most of the flow cytometric intervention to other solid tumors. Conn adenomas were especially apt to show this tendency, in which as much as 86% showed tetraploid pattern. Proliferation Index (PI) (ratio of S + G2 +M cells for the whole population of analyzed cells) were as much as 9.45 +/- 6.97% in normal adrenal cells, whereas it was much higher in cortical adenomas (17.75 +/- 8.53%). As a matter of fact, PI of hyperplastic adrenal cortex was within the same range as that of the normal adrenal glands. In pheochromocytomas, aneuploid pattern, which has been believed to be a definite index of malignancy, was shown in 60% of the cases, tetraploid pattern in 20%, and normal diploid pattern in only 20%. As a matter of fact, a case of non functioning cortical adenocarcinoma and a case of malignant pheochromocytoma were judged to show typical aneuploid pattern. Thus, the application of the flow cytometric diagnosis for adrenal tumors was supposed to require some refinement in understanding the significance of aneuploidy or tetraploidy.

Adrenal Gland Neoplasms↗
PubMed1996

[DNA analysis of a case of so-called blood group chimerism].

A 46-year-old woman who has three children, whose red blood cells exhibit a dual population of groups A and B, was investigated. Her lymphocytes had an XX/XY karyotype and Y chromosome markers were detected from buccal cells and hair root cells in DNA analysis. Three bands were detected in D1S80 VNTR locus (MCT118), two bands from father and one from mother. Her ABO genotype should be genetically AO, however, the PCR-RFLP method revealed a pattern of BO genotype. From these findings, it is concluded that she is a so-called dispermic chimera which is the result of the fertilization by two sperms and that DNA analysis is useful for distinguishing between twin and dispermic chimerism.

ABO Blood-Group System↗
PubMed1994

[DNA analysis of cytochrome b positive chronic granulomatous disease (a case report)].

A patient was diagnosed as having chronic granulomatous disease (CGD). This case seems to have been transmitted in an X-linked from judging from the family history. We had previously suggested that the patient's cytochrome b was normal both qualitatively and quantitatively. Thus, we thought that there might be mutation in the gp91-phox (one of the two components of cytochrome b) gene affecting electron transport but leaving other functions intact. To confirm this speculation, we performed DNA analysis. Complementary DNA (cDNA) was obtained from messenger RNA (mRNA) derived from peripheral blood lymphocytes. By using primers specific for the gp91-phox cDNA, the cDNA was amplified by polymerase chain reaction (PCR). The amplified cDNA was then ligated into Blue Script vector and transfected into E. coli (JM109) in order to clone the cDNA of gp91-phox. Then, the cloned cDNA was sequenced. Sequence analysis showed that the nucleotides 1521-1525 were deleted and a new sequence of 8 nucleotides was substituted. This mutation converted Glu-Lys-Thr into His-Ile-Trp-Ala. To confirm that the mutated allele came from the patient's mother; we performed mismatched PCR. PCR using a mutated allele could produce approximately 250 base pair products only when the patient's cDNA was used. PCR using a wild type primer could produce 250 base pair products only when cDNA from a healthy donor was used.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Multiple-channel microchips for high-throughput DNA analysis by capillary electrophoresis.

Capillary electrophoresis on microfabricated multiple-channel chips has great potential for high-throughput analysis. This review focuses on multiple-channel chips used for high-throughput DNA analysis. It covers progress in the design and fabrication of multiple-channel chips and detection schemes used on these chips. Applications are concentrated on DNA fragment sizing, genotyping, and sequencing.

Animals↗
PubMed1996

Slit-scan flow cytometry for consistent high resolution DNA analysis of X- and Y-chromosome bearing sperm.

This paper describes the application of slit-scan flow cytometry for accurate DNA analysis of X- and Y-chromosome bearing sperm. The introduction of the slit-scanning technique was initiated to improve the consistency in resolution of the X and Y population from donor to donor. An optimal resolution is essential for high purity sorting of X and Y sperm, as the difference in DNA content is small (3-4%) in most mammals. This difference is the discriminatory parameter for the flow cytometric sorting of the two populations. Our approach was to focus on the role of the sperm tail in the detection process. Slit-scan flow cytometric analysis allows the whole sperm to be spatially analyzed along the direction of flow. Sperm were stained with Dansyl Lysine, a UV excitable fluorescent membrane dye, which stained the head, midpiece, and principal piece. Analysis of these stained sperm showed that there was no difference between the relative number of sperm that travel headfirst or tailfirst through the detection zone of the flow cytometer. The influence of sperm with coiled tails on DNA analysis was also investigated. The proportion of sperm with coiled tails influences semen quality. The standard X-Y separation procedure uses Hoechst 33342, which stains all intact sperm, both living and dead. Propidium iodide was added to discriminate the dead sperm population. Slit-scan analysis showed that measurement of a sample containing a high proportion of living sperm with coiled tails results in an inferior DNA histogram and reduced X-Y resolution. Sperm with coiled tails can result in a lower detected fluorescence intensity, but the reason for this is unclear. Slit-scan flow cytometry allows exclusion of sperm with coiled tails from the analysis, resulting in a restoration of high resolution of X- and Y-chromosome bearing sperm populations.

Animals↗
PubMed2006

Application of Gleason analogous grading system and flow cytometry DNA analysis in a novel knock-in mouse prostate cancer model.

OBJECTIVE: A new knock-in mouse adenocarcinoma prostate model (KIMAP) was established, which showed a close to human kinetics of tumour development. This study used a new mouse histological grading system similar to the human Gleason grading system and flow cytometry DNA analysis to measure and compare the new KIMAP model with human CaP and transgenic mouse adenocarcinoma prostate (TGMAP) model. METHODS: According to heterogeneity of the clinical standard for prostate cancer diagnosis, a close to human mouse standard for histological grading and scoring system, Gleason analogous grading system, was established in this study. Sixty KIMAP and 48 TGMAP prostate cancer samples were measured and compared with human CaP. Flow cytometry DNA analysis was performed on malignant prostate tissues obtained from both TGMAP and KIMAP models. RESULTS: Mice with CaP from KIMAP (n = 60) and TGMAP (n = 48) models showed a different distribution of histological scores (p = 0.000). KIMAP mice showed higher percentage (53.3%) of compound histological score rate than TGMAP (25%), but closer to the human clinical average (50%), which showed significant correlation with age (p = 0.001), while TGMAP mice showed unbalanced and random score distribution in all age groups. Flow cytometry analyses showed that most tumour tissues in KIMAP were diploid, analogous to the human condition, while all the TGMAP mice showed aneuploid tumours. CONCLUSIONS: Results of this study further show that KIMAP, a new generation of murine prostate cancer model, could be used as a supplementary model in addition to the currently widely used transgenic models.

Adenocarcinoma↗
PubMedDate not supplied

[Microsatellite DNA analysis as a tool for forensic paternity testing (DNA paternity testing)].

UNLABELLED: MICROSATELLITE ANALYSIS: By using serological or HLA-testing, the alleged father can be excluded as the biological father, but, regardless of the degree of probability, positive paternity results cannot be obtained without DNA testing. According to the results of the National Human Genome Project, human genome consists of approximately 30.000 genes. The vast majority of human DNA is not organized in genes and has no genetic expression or visible function. Non-coding DNA contains genetic markers important for human identification. Short tandem repeats, or STRs, are a class of microsatellites consisting of tandemly repeated sequences of 2 to 6 base pair length monomers. Most of the microsatellites show a high degree of polymorphism, which can be evaluated by PCR technique, and used in criminalistics, forensic identification and parentage testing. A source of DNA in parentage testing are blood samples or buccal swabs which are routinelly used. Amplification of isolated DNA can be performed in 25-30 cycles by PCR, and fragments are separated by capillary electrophoresis. CONCLUSION: The probability of paternity of 99.99% or higher corresponds to the paternity "practically proven", indicating that the alleged father is the biological father. Such results can be obtained only by DNA testing. DNA-testing laboratories are required to conduct validation of laboratory facilities, equipment and staff and are subject to permanent control by the society.

DNA↗
PubMedDate not supplied

Preneoplastic oral lesions: the clinical value of image cytometry DNA analysis, p53 and p21/WAF1 expression.

BACKGROUND: Various mucosal lesions are frequently encountered in the oral cavity. Neither macroscopic nor microscopic evaluation of these lesions gives any reliable information concerning the risk of cancer development. MATERIAL AND METHODS: From 21 patients, 29 mucosal lesions were found to precede development of invasive squamous cell carcinoma or carcinoma in situ at the same location. The lesions were matched to 29 control lesions, with the same grade of dysplasia and from exactly the same locations but without subsequent cancer during a mean follow up of 112 months (46-194). The specimens were evaluated using Image Cytometry DNA analysis and immunohistochemical analysis of p53 and p21/WAF1 expression. RESULTS: Lesions prior to carcinomatous development displayed a higher degree of DNA aberration as compared with the control lesions. p53 and p21/WAF1 evaluation did not reveal any differences between cases and controls. CONCLUSION: Image Cytometry DNA analysis is an useful adjunct to histopathological evaluation of oral mucosal lesions for prediction of risk of malignant transformation.

Adult↗