Image cytometry DNA ploidy analysis: Correlation between two semi-automated methods.

BACKGROUND AND OBJECTIVES: Quantitation of cell DNA content, DNA ploidy, has been established as a research and prognostic technique for decades. A variety of instruments have been used although only a few commercially available systems have established quality assurance and published outcome data. The aim of this study was to compare two automated systems. METHODS: Nuclear monolayers were obtained from 112 oral biopsies by enzyme digestion and Feulgen staining. These were scanned on both the Fairfield and the Ploidy Work Station (PWS) systems. The overall ploidy diagnosis, number of epithelial nuclei, coefficient of variation (CV) and 5c exceeding rate (5CER) were compared by quantile-quantile plots, t test, Wilcoxon and Spearman's tests. RESULTS: The PWS system identified more nuclei (p < 0.0001) at a lower CV (p < 0.0001). Using the PWS system, fewer samples were classified as indeterminate. No difference between 5CER was found between systems (p > 0.54). There was complete concordance between the two systems in terms of DNA ploidy diagnosis. CONCLUSIONS: The PWS system is comparable to the Fairfield system for determination of DNA ploidy and has advantages that may lead to improved performance.

Karyomegalic interstitial nephritis and DNA damage-induced polyploidy in Fan1 nuclease-defective knock-in mice.

The Fan1 endonuclease is required for repair of DNA interstrand cross-links (ICLs). Mutations in human Fan1 cause karyomegalic interstitial nephritis (KIN), but it is unclear whether defective ICL repair is responsible or whether Fan1 nuclease activity is relevant. We show that Fan1 nuclease-defective (Fan1(nd/nd)) mice develop a mild form of KIN. The karyomegalic nuclei from Fan1(nd/nd) kidneys are polyploid, and fibroblasts from Fan1(nd/nd) mice become polyploid upon ICL induction, suggesting that defective ICL repair causes karyomegaly. Thus, Fan1 nuclease activity promotes ICL repair in a manner that controls ploidy, a role that we show is not shared by the Fanconi anemia pathway or the Slx4-Slx1 nuclease also involved in ICL repair.

Quantification of lymph nodes in the central compartment of the neck: a cadaveric study.

Differentiated thyroid cancer (DTC) accounts for over 90 % of thyroid malignancies, and is frequently associated with central neck compartment nodal metastasis that requires a therapeutic central compartment neck dissection (CCND) for clinically evident nodes. Current knowledge on the expected lymph node yield from a CCND is limited, compared with data on the lateral neck. The aim of our study was to accurately quantify nodal yield from the cadaveric central neck compartment. Twenty-eight cadaveric necks were dissected and the central neck compartment was subdivided into four regions: pre-laryngeal (delphian), pre-tracheal, right and left para-tracheal regions. Each cadaver had a thyroid gland, which was also removed, and the CCND tissue in each compartment was processed and examined by a consultant histopathologist. Only lymphoid tissue with a defined microscopic fibrous capsule and subcapsular sinus was included in the node count. The median total lymph node count per cadaver was four (range 1-16), with a median of one node detectable in each para-tracheal region (range 0-7) and the pre-tracheal region (range 0-8). The median pre-laryngeal node count was 0 (range 0- 2). The average lymph node size across all compartments was 2.9 mm. This is the first European study to assess cadaveric central neck lymph nodes and establish baseline counts for nodal yield. If a prophylactic or therapeutic CCND is required during thyroid surgery, those involved in DTC management must recognise that there is a wide range, and low median yield of central neck compartment lymph nodes.

Predictive value of dysplasia grading and DNA ploidy in malignant transformation of oral potentially malignant disorders.

Dysplasia grading is widely used to assess risk of transformation in oral potentially malignant disorders despite limited data on predictive value. DNA ploidy analysis has been proposed as an alternative. This study examines the prognostic value for both tests used in a routine diagnostic setting to inform clinical management. A retrospective study of conventional dysplasia grading was conducted on 1,401 patients. DNA ploidy analysis was conducted on a subset of 273 patients and results correlated with clinical information, pathologic diagnosis, and outcome over 5 to 15 years. Malignant transformation occurred in 32 of 273 patients (12%) and, of these, 20 (63%) of preexisting index lesions were aneuploid. Of 241 patients not developing carcinoma, only 39 (16%) of index lesions were aneuploid. Epithelial dysplasia correlated with DNA ploidy status (P < 0.001). The overall positive predictive value for malignant transformation by DNA aneuploidy was 38.5% (sensitivity 65.2% and specificity 75%) and by severe dysplasia grade 39.5% (sensitivity 30% and specificity 98%). DNA diploid and tetraploid status had negative predictive value of 90% to 96%. Combining DNA ploidy analysis with dysplasia grading gives a higher predictive value than either technique alone. Each of three traditional dysplasia grades predicts a significantly different risk of carcinoma development and time to transformation. DNA ploidy analysis had equivalent predictive value and also detected additional risk lesions in the absence of dysplasia.

Investigation of isotopic linkages between precursor materials and the improvised high explosive product hexamethylene triperoxide diamine.

The results of isotope ratio mass spectrometry (IRMS) on hexamethylene triperoxide diamine (HMTD) and its precursor hexamethylenetetramine (hexamine) is presented. HMTD was prepared from hexamine using several different sources of hexamine under both controlled laboratory conditions and in field experiments that represent the less controlled conditions that are likely to be observed in forensic casework scenarios. Precursor and product carbon isotope δ values consistently fit a linear relationship regardless of precursor or conditions. The magnitude of the isotope fractionation observed is affected by the efficiency of the reaction, with greater yielding reactions giving rise to HMTD with δ values more similar to the precursor material than lower yielding reactions. Nitrogen isotope δ values comparing precursor with product show some linearity when the reaction conditions are carefully controlled; however, results indicate a poor fit with linearity when synthesis conditions are more variable. Despite the greater variation, the HMTD product consistently has a more positive δ value compared with the hexamine precursor. The results observed from these experiments suggest hexamine reacts to form HMTD in a 1:1 ratio. Having prepared multiple HMTD samples from various precursors using a range of experimental conditions, we have observed results that may be useful in forensic investigations of improvised explosive materials.

Results of four inter-laboratory comparisons provided by the Forensic Isotope Ratio Mass Spectrometry (FIRMS) network.

Between 2004 and 2008 the Forensic Isotope Ratio Mass Spectrometry (FIRMS) network organised four Inter-Laboratory Camparison excercises with the aim of harmonising and validating isotopic measurements to be used for forensic applications. The samples distributed comprised materials of potential forensic interest such as packaging and pharmaceuticals to be analysed for delta2H, delta13C, delta15N and delta18O composition. As many as 30 international laboratories participated in these comparisons. Results have shown overall improvements for both within- and inter-laboratory reproducibility with respect to delta13C and delta15N measurements. Only a small number of laboratories returned results for delta2H and delta18O analysis and these results highlighted a need for improvement in the reproducibility of these measurements and a need to address the exchange of hydrogen between samples and ambient moisture. Results also highlighted the importance of sample preparation procedures and the need to standardise both these procedures and calibration against Standard Reference Materials. Future Inter-laboratory Comparison exercises will assess the suitability of laboratories to submit data to national and international databases similar to those currently operated for fingerprints, DNA etc.

N(2): a potential pitfall for bulk (2)H isotope analysis of explosives and other nitrogen-rich compounds by continuous-flow isotope-ratio mass spectrometry.

Observations made during the (13)C isotope analysis of gaseous CO(2) in the simultaneous presence of argon in the ion source of the isotope ratio mass spectrometer prompted us to investigate what influence the simultaneous presence of nitrogen would have on both accuracy and precision of bulk (2)H isotope analysis of nitrogen-rich organic compounds. Initially an international reference material, IAEA-CH7, was mixed with silver nitrate in various ratios to assess the impact that N(2) evolved from the pyrolysis of nitrogen-rich organic compounds would have on measured delta(2)H-values of IAEA-CH7. In a subsequent experiment, benzoic acid was mixed with silver nitrate to mimic the N:H ratio of organic-rich nitrogen compounds such as cellulose nitrate and RDX. The results of both experiments showed a significant deterioration of both accuracy and precision for the expected delta(2)H values for IAEA-CH7 and benzoic acid when model mixtures were converted into hydrogen and nitrogen, and subsequently separated by gas chromatography using standard experimental conditions, namely a 60 cm packed column with molecular sieve 5 A as stationary phase held at a temperature of 85 degrees C. It was found that bulk (2)H stable isotope analysis of nitrogen-rich organic compounds employing published standard conditions can result in a loss of accuracy and precision yielding delta(2)H values that are 5 to 25 per thousand too negative, thus suggesting, for example, that tree-ring (2)H isotope data based on cellulose nitrate may have to be revised.

Investigation of isotopic linkage between precursor and product in the synthesis of a high explosive.

In criminal proceedings involving illegally manufactured substances, evidence demonstrating the manufacture is required for successful prosecution. In many circumstances the presence of manufacturing equipment and 'recipes' are clear indicators. However establishing chemical links between starting materials and products would be beneficial additional evidence in such circumstances. Isotope analysis is becoming more commonly employed in disputes over authenticity and provenance of materials such as food stuffs or pharmaceuticals [S. Benson, C. Lennard, P. Maynard, C. Roux, Forensic applications of isotope ratio mass spectrometry--a review, Forensic Sci. Int., 157 (2006) 1-22]. We report here the results of an isotope study investigating a link between hexamine used as a starting material and the product explosive RDX, manufactured using the Woolwich process. Isotopic composition of hexamine and RDX was analysed by stable isotope ratio mass spectrometry. Five different hexamine samples were selected from a group of eleven for use in this study. One of the five hexamine samples was used twice, providing data on the reproducibility of the synthesis. Carbon and nitrogen isotope analysis of the hexamine starting material and RDX products reveals a reproducible relationship between reactant and product.

Evaluation of preliminary isotopic analysis (13C and 15N) of explosives A likelihood ratio approach to assess the links between semtex samples.

Currently, the use of isotopic ratio as corroborative evidence in criminal trials is explored. Beyond the analytical challenges that have been reported elsewhere, the crucial issue of the interpretation of analytical results in a fair and balanced way remains poorly documented. The aim of this paper is to propose a likelihood ratio approach for the evaluation of stable isotope data acquired from semtex samples. It will also lead to recommendations in relation to the acquisition of normalised international data.