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This article discusses the specific use of DNA analysis as one scientific method used in solving crime. Case studies presented here provide examples of crime investigations where the use of DNA analysis can prove beneficial.
A survey of over 225 recent studies examining the relationship between the flow cytometric DNA analysis of solid tumors and clinical prognosis indicates that criteria used to classify DNA histograms are variable and often inconsistent with the recommendations proposed by the Convention on Nomenclature for DNA Cytometry. Numerous reports not only lack unambiguous descriptions of the histogram features used to differentiate diploid from aneuploid DNA distributions, but also inadequately describe the technical aspects of data acquisition, standardization, and inclusion or exclusion of subpopulations by gating. In many cases, the coefficient of variation of the diploid and aneuploid G0/1 peaks, which would allow an assessment of histogram quality, is not reported. Because of the differences in DNA histogram interpretation, extrapolation of the results among laboratories may be difficult and is probably not reliable. This review summarizes the criteria that have been used to classify the DNA histograms and illustrates the effects of these different classifiers on DNA ploidy analysis and clinical conclusions.
Now that flow cytometric DNA analysis is becoming an established procedure, many samples that are being received from various hospitals cannot all be analyzed within a day. Therefore, samples must be stored for subsequent analysis in a flow system. To determine a proper temperature for storage by freezing, samples of the same material were stored for 2 weeks at 5 degrees C, -80 degrees C, and -195 degrees C. Measurement and analysis at the end of this period revealed no major differences among the subsequent histograms of these samples. Thus, it seems that a flow cytometric DNA analysis can be made of frozen samples for diagnostic and prognostic evaluations.
Random amplified polymorphic DNA (RAPD) analysis was evaluated in comparison with restriction endonuclease analysis (REA) of genomic DNA and serotyping in the typing of 160 epidemiologically unrelated group A streptococci (GAS). Amplification of genomic DNA of GAS was performed with a single primer with an arbitrarily selected nucleotide sequence of 12 nucleotides. In total, 31 RAPD patterns and 15 REA patterns were observed among the isolates studied. The results of RAPD analysis were in accordance with the results of REA for 86% of the isolates, as both methods identified 15 different strains among 138 isolates. However, RAPD analysis differentiated 16 additional strains among 22 isolates. RAPD analysis was somewhat better than REA for differentiation of isolates of the same and different serotypes. However, not all of the serotypes were differentiated by RAPD analysis either. In conclusion, RAPD analysis provides a practical alternative for genomic typing of GAS. It can be recommended for the typing of GAS, especially if used in parallel with serotyping.
DNA flow cytometry is frequently used to determine the aggressiveness of breast cancer at the time of primary treatment. Because its role is not as clear in advanced disease, a retrospective investigation was conducted on 132 breast cancer patients with metastases or a subsequent recurrence of the disease. DNA analysis of 96 primary tumors and 53 metastases was performed. The follow-up time was up to 11 years. The mean DNA index of the primary tumors was 1.4 and the mean S-phase fraction (SPF) was 9.4%; 36.5% of the tumors was diploid. The DNA index did not differ significantly among the different stages, but the SPF was significantly higher in stage III than in stage IV. There was no significant difference in ploidy between the stages. There were no significant differences in DNA index and SPF between the primary tumors of the patients with local or distant metastases. The disease-free interval was independent of DNA index, ploidy, and SPF alone. There was no difference in DNA index, ploidy, or SPF between patients with an early or late relapse. Breast cancer-related mortality after metastasis was independent of ploidy alone. In stage I-II, patients with a low SPF had a significantly higher survival rate than other patients. The same was true in patients with a diploid tumor and a low SPF. When the DNA index, SPF, and ploidy of primary tumors and metastases/recurrences were analyzed, no combination of variables yielding an improved prognosis was found. Therefore, it seems that DNA flow cytometry has limited value in determining the prognosis in a patient with metastases or recurrence.
A highly polymorphic DNA probe (3'HVR) with genetic linkage to the locus of autosomal dominant polycystic kidney disease was used for screening. Families with subjects at risk were from the Po river delta region (Northern Italy), where the disease accounts for 24% of the demands for dialysis. 3'HVR alleles were investigated in white blood cell DNA from 142 members of 18 families. The genomic marker was found informative in 88% of cases. Two recombinations between the marker and the disease locus were observed in 79 meioses. In 42 of the subjects at risk the results of DNA analysis and renal ultrasonography were compared. In 36 subjects the tests confirmed each other (18 were positive). In the other six subjects (all under 20 years of age and four under 10) only DNA analysis could diagnose the inheritance of cystic disease in the absence of demonstrable cysts. The findings indicate that in the population of the Po river delta the presymptomatic detection of adult polycystic kidney disease by 3'HVR linkage analysis is feasible in 88% of cases with approximately 95% reliability.
The authors summarize their first experiences with DNA analysis of defective low density lipoprotein receptor (LDLR) gene of the familial hypercholesterolemia heterozygotes that were selected from the III. Medical Clinic of the 1st Medical Faculty in Prague patients group. First genotype studies of unrelated FH individuals were performed by restriction fragment length polymorphism (RFLP) method. Relative allele frequencies of PvuII (0.69) and StuI (0.91) restriction enzymes agree with the world literature datas, in the ApaLI (0.69) case the higher value may be caused by, for the present, small number of analyzed patients. Possibilities of DNA analysis for pedigree FH diagnosis were demonstrated on the PvuII restriction enzyme case. By the use of polymerase chain reaction (PCR) DNA diagnosis of the familial defective apolipoprotein (Apo) B-100 (exon 26) was performed. 43 unrelated FH individuals were screened and none defective ApoB-100 gene was recorded.
Mitochondrial DNA analysis of a maggot crop can identify the corpse a maggot has been feeding on. Analysis of the crop could be useful in a criminal investigation if maggots are found at a suspected crime scene in the absence of a body, or if there is a question of whether a maggot used in postmortem interval estimations moved onto the corpse from another food source. Such analysis can also resolve a chain of custody dispute if it has been suggested that larval samples have been switched. When recovering DNA from a maggot crop, the analyst must be careful to avoid external contamination. We investigated the effects of three simple wash methods developed to reduce external contamination. Maggots raised on pig liver and intentionally contaminated with cow blood were washed using water, 20% bleach, or a solution containing the enzyme DNase. Only washing the maggots in 20% bleach reduced the amount of vertebrate DNA amplified from the maggots' exteriors. No wash method affected the ability to recover DNA from the maggot crops.
Flow cytometric DNA content analyses were performed on samples of 54 patients with neuroblastoma. DNA aneuploidy was detected in 55.6% of the 54 patients. A high incidence of DNA aneuploidy was observed in patients with prognostically favorable variables such as age (less than 1 year), clinical stage (I, II, or IVs), and primary site (extraadrenal sites). DNA aneuploidy was predominant in surviving patients, even in those with unfavorable variables. In patients 1 year old or more, the survival rate among those with DNA aneuploidy was 58.8% compared with 28.6% in patients with diploidy. Likewise, in patients with advanced stage (III or IV) neuroblastoma, the survival rate among those with DNA aneuploidy was 63.2% compared with 30.4% in patients with DNA diploidy. It is concluded that DNA content analysis is of value in predicting the prognosis of patients with neuroblastoma.
The use of flow cytometric DNA analysis as an adjunct to cytology in peritoneal fluid evaluation was studied. One hundred ninety-five fluids from 193 gynecologic patients were subjected to both DNA analysis and cytologic examination. It was found that 117/195 (60%) had invasive malignancies (50 ovarian, 48 endometrial, 17 cervical, and 2 miscellaneous); 34/117 (28%) patients with malignancies were positive by cytology, and 10/117 (8.5%) were positive (aneuploid) by DNA analysis. Of 34 cytologically positive cases, 7 (21%) were DNA positive, 25 (74%) were DNA negative, and in 2 (6%) insufficient cells were obtained. Only 3 fluids (3%) from malignancies were positive by flow cytometry and negative by cytology (1 stage I ovarian cancer, 1 stage I endometrial cancer, and 1 stage III ovarian cancer). No false-positive cytology and one probable false-positive flow result was obtained. If only those patients with histologically documented peritoneal involvement are considered, 29/43 (65%) had positive cytology and 8/43 (19%) had a positive flow result. We conclude that: (1) the high false-negative rate of flow cytometry (79%) versus cytology in this study may be related to a high percentage of diploid cancers, specimen preparation, or histogram interpretation, and (2) flow cytometry rarely adds to cytologic evaluation and is probably best reserved for use only in selected cases.
A study was performed on a nonconsecutive series of 51 patients in order to assess the feasibility, reliability, and usefulness of flow cytometric (FCM) DNA analysis of samples obtained from benign and malignant hepatic tumours by means of ultrasound-guided fine-needle aspiration (UG-FNA). Cytological and often histological confirmation of the nature of the lesion was obtained in all cases from an expert pathologist. For FCM DNA analysis in 32 cases, it was also possible to use samples obtained at surgery from the actual tumours. There were no post UG-FNA complications, either early or late. It was possible to perform FCM DNA analysis on 6/7 (85.7%) of the benign tumour aspirates and all 44 (100%) coming from the malignant tumours. All the benign tumours showed a DNA-diploid pattern, while the DNA content was aneuploid in 91% of the malignant tumours. Apart from one case, the results of the FCM DNA analysis of the samples removed at surgery were the same as those obtained from the aspirates (97%). FCM DNA analysis on UG-FNA samples from hepatic tumours is a fairly simple, reproducible, well-tolerated technique; it does not involve risks if performed by skilled operators and, since it can be easily repeated even on small tumours, it is a suitable method for monitoring hepatic metastases during chemotherapy.
PURPOSE: This study was designed to determine the reliability of DNA analysis in predicting outcome of patients with colorectal carcinoma and compare the prognostic efficacy of DNA analysis using two parameters vs. a single parameter. METHODS: Two hundred forty-eight patients with colon and rectal carcinoma were entered in the study over a period of 84 months. Thirty-one percent of carcinomas were found in the rectum. A total of 23.6 percent of cancers were found in the sigmoid colon. Majority of the patients were 60 years or older. Male to female ratio was equal. The colon bearing carcinoma was sent in a fresh state after resection for flow cytometry and DNA analysis. In this study, analysis was made, comparing two parameters (aneuploidy and S-phase fraction over 20 percent) with single parameters (aneuploidy) for staging and as a prognostic indicator in patients with colon and rectal carcinoma. RESULTS: The combined presence of aneuploidy and S-phase fraction over 20 indicated a poorer prognosis in patients with colon and rectal carcinoma. The prediction of patient outcome was more accurate with DNA analysis using two parameters than using a single parameter alone (P value = 0.00003). CONCLUSION: In our analysis, the odds of survival are over 3.7 times greater for aneuploidy than aneuploidy plus S-phase fraction over 20 percent.
OBJECTIVES: To examine DNA content abnormalities in patients with Barrett's esophagus (BE) who progress to esophageal adenocarcinoma, using image cytometric DNA analysis (ICDA) of formalin-fixed tissues. METHODS: Studies were performed on archived biopsies of BE patients' undergoing endoscopic surveillance before developing adenocarcinoma. A comparison group consisted of BE patients' free of cancer during a follow-up period of over 9 yr. Tissue sections were analyzed for the degree of dysplasia and for DNA content abnormalities, using image cytometry. Additional patients were also analyzed in a cross-sectional study of 56 BE cases with and without dysplasia, including 12 cases of adenocarcinoma. RESULTS: Five patients developed adenocarcinoma during follow-up and earlier biopsies obtained before cancer diagnosis showed specialized intestinal metaplasia (SIM) followed by low-grade dysplasia (LGD) in one, SIM followed by high-grade dysplasia (HGD) in one, LGD in two, and HGD in one case. All five showed some DNA abnormality at baseline or in interval biopsies. In the comparison group, five of seven patients showed normal diploid DNA at baseline and on follow-up biopsies. One patient initially had diploid DNA, but developed aneuploidy 11 yr later. Another case initially had aneuploidy, but was diploid on follow-up. Overall, DNA abnormalities were found in 13% of cases with SIM without dysplasia, 60% with LGD, 73% with HGD, and 100% with adenocarcinoma. CONCLUSIONS: (i) Image cytometric DNA analysis is a useful method to examine DNA abnormalities in formalin-fixed tissues and may be more sensitive in predicting progression to adenocarcinoma than HGD. (ii) Histological dysplasia of any grade and DNA abnormalities, help identify BE patients at high risk for adenocarcinoma.
Quantitative analysis of cellular DNA content may be clinically useful for several solid tumors. The technology for this analysis by flow cytometry or image analysis has existed for several years but has not been widely used, except in a handful of specialized research institutions. Recently, however, relatively inexpensive image analyzers intended for use by hospital pathologists have been introduced that can analyze DNA content from cytology or imprint specimens which are readily obtainable from solid tumors. We report here an assessment of this technology for analysis of tumor imprint specimens, using flow cytometry of tissue blocks as the standard for comparison. We used image analysis equipment on Feulgan-stained imprint preparations from 31 tumors and compared the histograms with those obtained by flow cytometric analysis of archival tissue blocks from the same tumors. The ploidy descriptors (diploid, tetraploid, and aneuploid) were concordant for the two methods in 27 specimens, with three specimens yielding discordant results and one specimen considered unevaluable by image analysis. The image analysis method using imprints appeared to have several advantages over flow cytometry, including lower instrument cost, no need to dissociate paraffin blocks or fresh tissue, and ability to analyze very small samples. Somewhat lower resolution of the histograms, extremely localized tissue sampling, and possibly greater risk of occasionally obtaining unevaluable preparations were disadvantages. Microcomputer-based image analysis performed on imprints appeared to be a viable alternative to flow cytometric analysis of tissue blocks for quantitative DNA analysis of tumor specimens.
Advances in genetic engineering influence to an increasing extent a number of medical disciplines. DNA analysis can be used in the diagnosis of hereditary diseases, in investigations of malignant processes, in forensic medicine and for detection of infectious pathogens. Two main methodical approaches to DNA analysis, Southern's method and procedures based on primer directed enzymatic amplification of DNA by the PCR method, resolve the complicated detection of slight changes in the vast volume of human genetic information. Cystic fibrosis may serve as an example of a serious hereditary disease the diagnosis of which improved greatly after introduction of DNA analysis. The diagnosis of this disease is nowadays possible by direct analysis of mutations and indirectly by investigations of the link between the disease and DNA polymorphisms.
Although DNA analysis based on the polymerase chain reaction (PCR) offers potential advantages for screening newborns for sickle cell disease, few data are available concerning the reliability of PCR-based tests for such screening. We describe a protocol for detecting the A, S, and C alleles of the beta-globin gene in dried blood from phenylketonuria screening cards. This method is based on PCR and detection with allele-specific oligonucleotide probes. Results of a blind comparison of PCR analysis of the dried blood with hemoglobin electrophoresis of whole-blood samples agreed for 80 of 81 samples. The single discrepancy is probably not attributable to a failure of the PCR method, but rather to limitations of the electrophoresis method. The PCR method should be a highly accurate means of detecting beta-globin alleles in routine genetic screening with dried blood already collected for (e.g.) phenylketonuria screening.
Abnormal DNA content has been considered as an additional criterion for determining the biological behavior of a tumor. Flowcytometric DNA analysis was done on 121 patients with thyroid carcinoma encountered during the period between 1975 and 1987. Tumor tissues were sampled from paraffin-embedded blocks and the histology of thyroid carcinoma found to consist of 91 papillary, 23 follicular, 2 medullary, 1 squamous cell and 4 anaplastic carcinomas. The incidence of aneuploidy in thyroid carcinoma was 7.4 per cent (9 patients) while that of diploidy was 92.6 per cent (112 patients). The aneuploid specimens consisted of 6 papillary, 1 follicular, 1 medullary and 1 anaplastic carcinomas and, of 4 anaplastic carcinoma patients with subsequent death within 6 months, only 1 was aneuploid. As an indicator of proliferative potential, S-phase fraction (SPF) was also determined by flow cytometry, but this could not be used as an independent prognostic factor. The aneuploid patients showed a significantly decreased survival rate (p less than 0.01). Thus, although DNA measurement proved useful for predicting the survival of aneuploid patients, there is some discrepancy between DNA content and the biological behavior of the tumor.
Preliminary data from this institution suggested that flow cytometric DNA analysis was an objective prognostic indicator in archival localized squamous cell carcinoma of the tongue. Technical improvements were made, including analysis of tumor, normal tissue, and a combination of the two; standardized cursor placement; mathematic determination of tetraploid populations; and development of a statistical analysis. A larger number of patients (60) with this disease were reviewed. DNA content was related to disease-free survival, local recurrence, regional metastasis, and incidence of second primary tumors. There was no significant difference between aneuploid and diploid tumors with respect to the variables analyzed. We believe these technical improvements will enhance flow cytometric DNA analysis of paraffin-embedded tissues. However, in this retrospective review of localized squamous cell carcinoma of the tongue, DNA analysis was not a valuable prognostic indicator. Only prospective studies will address this issue.