ABC of clinical genetics. Techniques of DNA analysis.
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Blotting, Southern↗
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DNA analysis of atrial myxomas.
The atrial myxoma is a primary tumor of the heart which may have an uncertain clinical course. In this study, we performed flow cytometric DNA analysis of 15 paraffin-embedded atrial myxomas and correlated DNA ploidy status and proliferative fraction with clinical findings. Twelve of 15 cases (80 percent) were diploid and the remaining three cases (20 percent) were aneuploid. Two patients with aneuploid histograms were free of tumor at the time follow-up; the third patient experienced local tumor recurrence and metastases. Five patients with diploid myxomas demonstrated an elevated (greater than or equal to 17 percent) proliferative cell cycle fraction; four of these patients experienced embolic phenomenon or tumor recurrence. This pilot study suggests that an atrial myxoma with either aneuploid DNA content or elevated proliferative fraction may be associated with aggressive biologic behavior.
Aneuploidy↗
Breast stereotactic core biopsy washings: abundant cell samples from clinically occult lesions for flow cytometric DNA analysis.
RATIONALE AND OBJECTIVES: The object of this study was to determine by flow cytometry the cellular and DNA content of the washings of core biopsy samples and needles obtained during stereotactic core biopsies of clinically occult suspicious lesions. METHODS: Fourteen-gauge core multipass biopsy inner-needle saline washings obtained after dislodgment of the tissue core samples (n = 30) and 14-ga mammotomy core biopsy specimen washings (n = 30) were analyzed by flow cytometry. RESULTS: Multipass core biopsy needle washings of 7 malignant and 23 benign lesions yielded a median of 10,584 cells per lesion (range 562-29,019 cells). Mammotomy core biopsy specimen washings of 10 malignant and 20 benign lesions yielded a median of 12,164 cells per lesion (range 1,295-115,150). Forty-four (73%) washings in each technique had more than 5000 cells. Flow cytometric DNA ploidy analysis was possible in 60 (100%) and the S phase percentage determination was possible in 59 (98%) of the washings. Aneuploidy was detected in 53% of malignant lesions and 2% of benign lesions. CONCLUSIONS: Core biopsy specimen or needle washings provide abundant fresh cellular material from clinically occult lesions for DNA analysis by flow cytometry.
Adult↗
Comparative assessment of DNA analysis in effusions by image analysis and flow cytometry.
Cytologic evaluation of body cavity fluids is useful to detect malignancy within the pleural and peritoneal spaces. A definitive diagnosis cannot always be made on cytologic evaluation alone. As malignant processes may show abnormal DNA content, DNA analysis of effusions may be useful. Therefore, we determined the DNA content of 37 effusions by flow cytometry (FC) and image analysis (IA) using the CAS 200. Of the 37 fluids evaluated, 18 were cytologically malignant, 15 benign, and four atypical. Overall, 22 fluids (60%) showed concordance between FC and IA. None of the benign fluids were aneuploid. All showed diploid histograms or diploidy with increased proliferating cells. Three of four atypical fluids had increased proliferating cells by either FC or IA, whereas one was diploid by both methods. Aneuploidy was detected in 13 malignant fluids: five were aneuploid by both methods and eight by only one method. IA identified aneuploidy in five of those eight cases, while three were identified by FC. Three of the cytologically malignant fluids were diploid by both methods, and two showed increased proliferating cells by IA and diploidy by FC. The specificity of both methods was 100%. However, the sensitivity of identifying a malignant fluid by aneuploidy is low, 44% for FC and 55% for IA. IA appears to identify small aneuploid populations more frequently than FC. The detection of aneuploidy in effusions is highly suggestive of malignancy, and the combination of both techniques gives the highest detection rate (72%). However, neither are as sensitive as traditional cytologic evaluation with the occasional use of additional histochemical stains.
DNA, Neoplasm↗
Dysplasia and HPV infection initially detected by DNA analysis in cytomorphologically normal cervical smears.
OBJECTIVE: We examined the relationship between the risk of dysplasia and human papillomavirus (HPV) infection initially detected by DNA analysis in Japanese women with cytomorphologically normal cervical smears. METHOD: We applied the polymerase chain reaction method to detect HPV DNA in 800 women and subsequently followed up 49 women positive for HPV DNA. RESULTS: HPV 16 or HPV 18 DNA was detected in 53 (6.6%) of 800 women with normal smears (negative). During the follow-up period (23 months), the presence of HPV DNA could not be detected in 41 (83.7%) of 49 women after its initial presence. The remaining eight women had repeated HPV DNA, and four of these eight women had lesions that progressed to mild dysplasia. CONCLUSION: The results indicated that some women with cytomorphologically normal smears may have HPV DNA which can be transient, and persistence of HPV types 16 or 18 DNA plays an important role in the pathogenesis of cervical dysplasia.
Adolescent↗
Photographic densitometry for quantitative DNA analysis of cytologic and histologic specimens.
We found that photographic densitometry (PD) is a useful technique for quantitative determinations of nuclear DNA content in clinical tumor material. Optimum conditions for the use of PD in clinical cytology and histopathology were worked out. A quantitative evaluation of the method was performed, particularly with respect to errors that may appear when measuring clinical tumor material. Our study showed that PD offers accurate DNA measurements in cytologic and histologic specimens. Ploidy level determinations in tumor cell populations in clinical material could be as accurately performed with PD as with scanning microspectrophotometry (SMP). Nuclear DNA content of individual cells as determined by PD correlated highly with nuclear DNA content determined by SMP (correlation coefficient, 0.96). Since the PD method is less influenced by background variation than are other image techniques (due to measurement of a photographic image), it is particularly useful in measurement of histopathologic sections, in which the background variation can introduce considerable errors. The method is also valuable with clinical cytologic smears, in which the presence of blood and other material disturbs the background. PD represents a valid complement to scanning microspectrophotometry and TV imaging systems, particularly for DNA analysis of tissue sections. Moreover, it can be applied easily in the clinical routine. Relevant tissue areas are selected and photographed by the pathologist or cytopathologist, and the measurement is performed by a laboratory technician.
Cell Nucleus↗
Intracommunity relationships, dispersal pattern and paternity success in a wild living community of Bonobos (Pan paniscus) determined from DNA analysis of faecal samples.
Differences in social relationships among community members are often explained by differences in genetic relationships. The current techniques of DNA analysis allow explicit testing of such a hypothesis. Here, we have analysed the genetic relationships for a community of wild bonobos (Pan paniscus) using nuclear and mitochondrial DNA markers extracted from faecal samples. Bonobos show an opportunistic and promiscuous mating behaviour, even with mates from outside the community. Nonetheless, we find that most infants were sired by resident males and that two dominant males together attained the highest paternity success. Intriguingly, the latter males are the sons of high-ranking females, suggesting an important influence of mothers on the paternity success of their sons. The molecular data support previous inferences on female dispersal and male philopatry. We find a total of five different mitochondrial haplotypes among 15 adult females, suggesting a frequent migration of females. Moreover, for most adult and subadult males in the group we find a matching mother, while this is not the case for most females, indicating that these leave the community during adolescence. Our study demonstrates that faecal samples can be a useful source for the determination of kinship in a whole community.
Animal Communication↗
Personal identification from human remains using morphological characteristics and DNA analysis.
This report describes personal identification of four parts of a dismembered human body on the basis of physical characteristics and DNA analysis. A 69-year-old woman suffering from depression went missing from her home in May 1992. Five days after her disappearance, a female torso was found in a river. In a two-week period thereafter, a left forearm, a female head and neck and a right forearm were found consecutively in the same river. Although the end of the cervical vertebra was anatomically identical to that of the torso, the two forearms did not match. However, DNA analyses with a single-locus DNA probe pYNH24, DO alpha typing and a multi-locus DNA probe "Myo", indicated that these four parts came from the same person. The missing woman had undergone lobectomy of the right pulmonary upper lobe, and had suffered from slight scoliosis of the thoracic vertebrae and a second lumbar vertebral compression fracture. These characteristics were also observed in the torso. In order to obtain more conclusive evidence, DNA fingerprinting using DNA extracted from the torso, the missing woman's brother and her daughter was performed using the DNA probes described above. The data obtained indicated that the remains were those of the missing woman.
Aged↗
Rapid discrimination of Mycobacterium tuberculosis strains by random amplified polymorphic DNA analysis.
Investigations of the epidemiology of tuberculosis have been hampered by the lack of strain-specific markers that can be used to differentiate isolates of Mycobacterium tuberculosis. We report the development of a rapid protocol for random amplified polymorphic DNA analysis which included the use of a commercially available DNA extraction kit (GeneReleaser). This was applied to 14 strains of M. tuberculosis, including strains associated with temporal and geographical clusters of tuberculosis in the United Kingdom and those from India, Africa, and Saudi Arabia. Strains of M. tuberculosis could be discriminated in about 8 h by this method, which is therefore a rapid and simple alternative to restriction fragment length polymorphism analysis.
Africa↗
Biological characterization of human bone tumors. VII. Detection of malignancy in a giant cell tumor of bone by flow cytometric DNA-analysis.
The biological behavior of giant cell tumors of bone often cannot be definitively evaluated by light and electron microscopical criteria. As DNA aneuploidy was found to be a highly sensitive marker of malignant cells, we have analyzed this descriptive parameter in 4 giant cell tumors of bone by flow cytometry. Three of the four cases were non-metastasizing giant cell tumors, the fourth was a clinically malignant neoplasm presenting extensive visceral metastases four years after first diagnosis. Neither light- nor electron microscopical findings provided unequivocal criteria for defining the biological dignity of the four tumors. DNA aneuploidies were identified in the metastasizing case only, whereas the three clinically benign tumors showed a unimodal euploid DNA distribution. Thus flow cytometric DNA analysis is an additional diagnostic tool for the detection of malignancy in giant cell tumors of bone.
Adult↗
Bcr/abl recombinant DNA analysis versus karyotype in the diagnosis and therapeutic monitoring of chronic myeloid leukemia.
Karyotype and bcr/abl recombinant DNA analyses are two means of detecting the chromosomal aberration in chronic myeloid leukemia. The authors compared these two methods in a retrospective study of 36 patients with CML in which they found the bcr/abl DNA recombinant event in 100% (29 of 29) of those patients who had the Philadelphia chromosome. To achieve this sensitivity, a battery of two bcr probes and three restriction enzymes is necessary. The authors propose a sequential algorithm for efficient use of these probes and enzymes. In 76% of the patients, bcr/abl rearrangement can be detected with a Bgl II digest and a 3' commercial probe. An additional 21% of patients can be detected by a second assay in which the same membrane is rehybridized to a 3' and 5' combination bcr probe. One patient (3%) required an additional restriction enzyme digest with BamH I to detect the recombinant event by the same 3' probe. Karyotype analysis is used to determine cytogenetic remission in patients with CML under therapy. The authors studied the use of DNA analysis by the Southern blot technique to detect a decrease in the relative number of leukemic cells. By dilution studies and densitometric scanning of autoradiographs, the authors were able to detect a 15% decrease in the relative number of cells having the bcr/abl recombinant event. The authors report the preliminary results of three patients in whom they compared the karyotype and recombinant DNA analysis at multiple time points in their clinical course. In conclusion, the bcr/abl recombinant DNA analysis is superior to karyotype for the diagnosis of CML and can be used for monitoring treated patients.
Adolescent↗
Ploidy and proliferation in human bladder tumors as measured by flow-cytofluorometric DNA-analysis and its relations to histopathology and cytology.
Biopsies from bladder tumors of 41 patients were investigated by flow-cytofluorometric DNA analysis and compared with exfoliated cells. The degrees of ploidy and proliferation were determined. Good agreement was found between the degrees of ploidy and proliferation in the biopsies and the exfoliated cell material. Tumors Grade I-II were either euploid or aneuploid. All Grade III tumors were aneuploid. The S-phase fractions were about 6% in the diploid tumors and 17% with large variations in the aneuploid tumors. The histological grading was well correlated to the number of S-phase cells and the occurrence of aneuploidy. When the Grade II tumors were divided into two groups having lesser and more pronounced atypia, the two groups differed significantly with regard to their degrees of proliferation. In addition to aneuploidy as an important criterium for malignancy, the degree of proliferation appears to be of major biological significance.
Aneuploidy↗
Application of a DNA analysis method for the cultivar identification of grape musts and experimental and commercial wines of Vitis vinifera L. using microsatellite markers.
A DNA-based method has been applied to the identification of several musts and wines using microsatellite markers. DNA was extracted from the solid phases of sixteen monovarietal and five multivarietal musts (mixtures of two musts down to a 4:1 proportion) and they were genotyped at seven microsatellites through a multiplex PCR reaction and automated fluorescent detection. PCR multiplexing was successful in monovarietal musts, but should be used with caution with at least some markers and in multivarietal musts. The same extraction and detection methods were unsuccessfully applied to the solid and liquid phases of five monovarietal commercial wines, even after using different concentration procedures. Nucleic acids presence was then studied in a recent must, during the fermentation process, and during the subsequent steps of winemaking. Genotyping was possible in the resulting experimental wine until decanting, when the particles in suspension were removed. These results suggest that wine authentication through DNA analysis is not possible in commercial wines, in the tested conditions.
DNA, Plant↗
Image analysis DNA cytometry of bladder cancer.
In a retrospective analysis, the DNA histograms of 65 paraffin-embedded bladder carcinomas from radical cystectomy specimens (stage pT1-pT4a, pN0, pN1, pN2) were analyzed using an automated image analysis system (Leytas 2). Automated image analysis was able to characterize invasive bladder carcinoma as being either diploid, polyploid, or aneuploid. Within the group of aneuploid tumors, the DNA content of the stem-cell line allowed further subtyping of the tumors; hypotriploid, hypertriploid, hypertetraploid, and even hyperpentaploid tumors could be distinguished. Comparing different sites of identical tumors, the DNA histogram was found to be a stable and reproducible tumor characteristic. The various tumor types differed significantly in prognosis. This technique can also be applied to smears of urine sediment or transurethrally resected tumor chips. In the case of superficial tumors, DNA cytometry defines those tumors which are potentially invasive, requiring careful follow-up and/or early aggressive treatment.
DNA, Neoplasm↗
[Use of DNA analysis in the diagnosis and prevention of hemophilia A].
Hemophilia A belongs to the monogenically determined diseases. The methods of molecular genetics (recombinant DNA) have greatly contributed both to the elucidation of the genetic basis of these diseases and to the elaboration of effective preventive approaches either by prenatal genetic diagnosis or by detection of heterozygous transmitters. The authors present a short survey of the results in the field of genetic research of hemophilia A at molecular level achieved over the last years and they report on their own results of DNA analysis in 15 families with the occurrence of the disease. Of 17 potential subjects transmission was excluded in 10 and confirmed in 7 cases. The importance of early and complete detection of families with the occurrence of hemophilia A is emphasized particularly in the light of effective prevention.
DNA↗
Flow cytometric DNA analysis in fine needle aspiration biopsies from patients with prostatic lesions. Diagnostic value and relation to clinical stages.
Transrectal fine needle aspiration biopsy was carried out in 59 patients with cancer of the prostate, benign prostatic hypertrophy and prostatis for cytomorphological examination and flow cytometric DNA analysis. One major single peak in the DNA histogram was observed in patients with benign hyperplasia, prostatitis and in some patients with carcinoma. A DNA histogram with a second or third peak was always compatible with carcinoma and was found both in patients with carcinoma in stage I--II and stage III--IV. It is suggested that patients with hyperploid cancers in stage I--II could benefit from early treatment.
Biopsy, Needle↗
Perinatal hepatitis B virus detection by hepatitis B virus-DNA analysis.
Maternal transmission of hepatitis B virus infection in relation to the hepatitis B e antigen/antibody system and serum hepatitis B virus-DNA were evaluated. Results indicate that hepatitis B virus-DNA analysis can identify hepatitis B serum antigen positive mothers who may transmit infection to their offspring.
Autoradiography↗
Two-color multiparametric method for flow cytometric DNA analysis. Standardization of spectral compensation.
Spectral overlap of green fluorescence signals into the red detector (red-minus-green compensation) is one potential source of variation in two-color flow cytometric DNA analysis. Suboptimal compensation in a two-color propidium iodide (PI)/fluorescein isothiocyanate (FITC) system may be observed if compensation is adjusted using an inappropriate standard, or if changes to fluorescence detector high-voltage settings are made without corresponding readjustment of fluorescence compensation. To quantitate the influence of red-minus-green compensation on the quality of DNA histograms, data from 60 dual PI/cytokeratin (CK)-FITC stained carcinomas were acquired in parallel using two compensation standards: a PI/CK-FITC-stained T24 cell line calibrator overstained to achieve a high-intensity green fluorescence standard (HIGFS) with manually set compensation and automated compensation settings derived from commercial phycoerythrin/low intensity FITC beads (LIGF). Both compensation standards gave similar DNA hyperdiploidy results (DNA index, 1.1-2.8). However, LIGF standard yielded two falsely hypodiploid peaks (DNA index, .7 and .9). Eight left-skewed peaks became DNA diploid and symmetric, respectively, with the HIGFS. Use of HIGFS lowered the coefficient of variation percentage in 95% of cases, the greatest differences (maximum, 3.4%; mean, 1.81%) in tumors of highest intensity CK-FITC. The authors concluded that use of cell-based compensation standards (HIGFS) with intense green signals that mimic clinical tumor samples will avoid spurious aneuploidy and maximize resolution of near-diploid abnormalities.
Analysis of Variance↗