Treatment of multiple adjacent RT 1 gingival recessions with the modified coronally advanced tunnel (MCAT) technique and a collagen matrix or palatal connective tissue graft: 9-year results of a split-mouth randomized clinical trial.

OBJECTIVES: To evaluate t he long-term outcomes following treatment of RT 1 multiple adjacent gingival recessions (MAGR) using the modified coronally advanced tunnel (MCAT) with either a collagen matrix CM or a connective tissue graft (CTG). MATERIAL AND METHODS: Sixteen of the original 22 subjects included in a randomized, controlled split-mouth clinical trial were available for the 9-year follow-up (114 sites). Recessions were randomly treated by means of MCAT + CM (test) or MCAT + CTG (control). Complete root coverage (CRC), mean root coverage (MRC), gingival recession depth (GRD), probing pocket depth (PD), keratinized tissue width (KTW), and thickness (KGT) were compared with baseline values and with the 12-month results. RESULTS: After 9 years, CRC was observed in 2 patients, one in each group. At 9 years, MRC was 23.0 ± 44.5% in the test and 39.7 ± 35.1% in the control group (p = 0.179). The MRC reduction compared to 12 months was - 50.1 ± 47.0% and - 48.3 ± 37.7%, respectively. The upper jaw obtained 31.92 ± 43.0% of MRC for the test and 51.1 ± 27.8% for the control group (p = 0.111) compared to the lower jaw with 8.3 ± 46.9% and 20.7 ± 40.3%. KTW and KGT increased for both CM and CTG together from 2.0 ± 0.7 to 3.1 ± 1.0 mm (< 0.0001). There were no statistically significant changes in PD. CONCLUSION: The present results indicate that (a) treatment of MAGR using MCAT in conjunction with either CM or CTG is likely to show a relapse over a period of 9 years, and (b) the outcomes obtained in maxillary areas seem to be more stable compared to the mandibular ones. CLINICAL RELEVANCE: The mean root coverage at 12 months could not be fully maintained over 9 years. On a long-term basis, the results seem to be less stable in the mandible as compared to maxillary areas.

Analysis of microRNA expression in brush cytology specimens improves the diagnosis of pancreatobiliary cancer.

BACKGROUND/OBJECTIVES: Malignant pancreatobiliary strictures are in many cases clinically indistinguishable and present a major problem to endoscopy specialists. Intraductal sampling procedures such as brush cytology are commonly used for diagnosis with a sensitivity that is low for a diagnostic test used in daily clinical practice. MicroRNA (miR) alterations detected in many cancers are disease-specific, which can be utilized in clinical applications. The aim of the present study was to analyze whether determination of miR expression levels in intraductal brush cytology specimens is a feasible approach to improve the diagnosis of pancreatobiliary cancer. METHODS: Brush cytology specimens have been collected during endoscopic retrograde cholangio-pancreatography (ERCP) and analyzed by routine cytology and ancillary miR assays. Total RNA was extracted using the miRNeasy Mini Kit and the expression of miRs frequently dysregulated in pancreatobiliary cancer (miR-16, miR-21, miR-196a, miR-221) were analyzed by quantitative real-time PCR using RNU6B as internal control. RESULTS: Routine cytology resulted in no false positive diagnoses, however, the combined sensitivity remained at 53.8%. Expression (ΔCt values) of miR-16 (p = 0.0039), miR-196a (p = 0.0003) and miR-221 (p = 0.0049) showed a clear statistical significance between malignant and benign pancreatobiliary specimens (n = 35). Malignancy could be detected combining routine cytology and the miR-196a single marker expression levels with a sensitivity of 84.6% (92.9% in biliary strictures) with no false positives. CONCLUSIONS: The results offer the first direct demonstration that microRNAs are readily detectable in brush cytology specimens obtained during ERCP, and have the potential to help the cytological diagnosis of pancreatobiliary malignancy.

Molecular cloning, mRNA expression and biological activity of the pheromone biosynthesis activating neuropeptide (PBAN) from the European corn borer, Ostrinia nubilalis.

Pheromone biosynthesis activating neuropeptide (PBAN) is a member of the pyrokinin (FXPRLamide) insect neuropeptides. Here, we report the cloning of the gene Ostnu-PBAN from the E and Z pheromone strains of the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae), a major pest of maize. The Ostnu-PBAN genomic sequence is > 5 kb in length and consists of six exons. The deduced amino acid sequence revealed a 200-residue precursor protein including a signal peptide, a 24-amino acid (aa) diapause hormone, a 37-aa PBAN and three other FXPRLamide neuropeptides. Our in vivo assays suggest that the 37-aa synthetic Ostnu-PBAN is hormonally active in the pheromone gland. It restores sex pheromone production to normal levels in mated females and decapitated virgins of both E and Z cultures. The results of a real-time PCR analysis indicated that Ostnu-PBAN mRNA levels reached a plateau in the brain-suboesophageal ganglion complexes 1 day after eclosion, and mating did not affect the mRNA expression. Three size classes of Ostnu-PBAN mRNA (1.9, 2.0 and 2.1 kb) were obtained, differing only in the length of the 3' untranslated region. However, there was no correlation between sequence divergence and the pheromone composition, voltinism or geographical origin (Hungary, Slovenia, Sweden, Turkey) of ECB moths.

Plastic waste from marine environment: Demonstration of possible routes for recycling by different manufacturing technologies.

The increasing amount of marine plastic waste poses challenges including, not only the collection, but also the subsequent recyclability of the plastic. An artificial accelerated weathering procedure was developed, which modelled the marine environment and investigated the recyclability of weathered and non-weathered PET. Marine conditions were simulated for poly(ethylene terephthalate) (PET) bottle material and high-density polyethylene (HDPE) cap material. It consisted of 2520 h cyclical weathering, alternating the sample between a salt spray and a Xenon-chamber-this corresponds to roughly 3-4 years on the surface of an ocean. It was proved that the molecular weight of PET is a function of weathering time and can be described mathematically. Microscopic examination of the surface of the PET bottles and HDPE caps proved that these surfaces were damaged. After weathering, manufacturing tests were performed on the PET material by extrusion, injection moulding, 3D printing and thermoforming. Quantitative comparison between products manufactured by the same technology was performed in order to compare the qualities of products made from original PET, non-weathered PET waste, which was the example of classical recycling, and weathered PET. In the case of products made from weathered PET, certain mechanical and optical properties (e.g. impact strength and transparency) were significantly impaired compared to the original PET and the recycled, non-weathered PET. Certain other properties (e.g. strength and rigidity) did not change significantly. It was proved that the samples from weathered plastic material can be successfully recycled mechanically and used to manufacture plastic products.

Reversal of gene expression changes in the colorectal normal-adenoma pathway by NS398 selective COX2 inhibitor.

BACKGROUND AND AIMS: Treatment of colorectal adenomas with selective cyclooxygenase-2 inhibitors can contribute to the chemoprevention of colorectal cancer (CRC), but the molecular background of their effect is not fully understood. We analysed the gene expression modulatory effect of N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS398) on HT29 cells to be correlated with expression data gained from biopsy samples. METHODS: HT29 colon adenocarcinoma cells were treated with NS398, and global mRNA expression was analysed on HGU133Plus2.0 microarrays. Discriminatory transcripts between normal and adenoma and between adenoma and CRC biopsy samples were identified using HGU133Plus2.0 microarrays. The results were validated using RT-PCR and immunohistochemistry. RESULTS: Between normal and adenoma samples, 20 classifiers were identified, including overexpressed cadherin 3, KIAA1199, and downregulated peptide YY, glucagon, claudin 8. Seventeen of them changed in a reverse manner in HT29 cells under NS398 treatment, 14 (including upregulated claudin 8, peptide YY, and downregulated cadherin 3, KIAA1199) at a significance of P<0.05. Normal and CRC could be distinguished using 38 genes, the expression of 12 of them was changed in a reverse manner under NS398 treatment. CONCLUSION: NS398 has a reversal effect on the expression of several genes that altered in colorectal adenoma-carcinoma sequence. NS398 more efficiently inverted the expression changes seen in the normal-adenoma than in the normal-carcinoma transition.

The potential of digital microscopy in breast pathology.

The rapidly evolving field of digital microscopy supports the efficient exploitation of inherent information from stained glass slides to offer widespread utilization in breast histopathology. Digital image signals can be accurately measured, integrated into databases and shared through computer networks. Therefore, digital microscopy can boost telepathology-consultation, gradual- and postgradual teaching, proficiency testing, intra- and interlaboratory validation of biomarker screening interpretation, and automated image analysis of biomarker expression for both diagnostics and research applications. This is a brief highlight of the potential of digital microscopy in breast pathology applications.

Recent psychopathology, suicidal thoughts and suicide attempts in households with and without firearms: findings from the National Comorbidity Study Replication.

OBJECTIVE: To assess the relationship between firearm ownership and possible psychiatric confounders of the firearm-suicide relationship. METHODS: Multivariate logistic regression was used to estimate the association between living in a home with firearms and 12-month occurrence of major Diagnostic and statistical manual of mental disorders (DSM)-IV disorders and suicidal behaviour among respondents to the National Comorbidity Survey Replication, a household survey of 9282 adults aged 18+. Analyses controlled for sociodemographic characteristics including age, sex, race/ethnicity, educational attainment and poverty. RESULTS: Approximately one in three Americans reported living in a home with firearms. People living in a home with firearms were no more or less likely than people in homes without firearms to have recent (past year) anxiety disorders (OR = 1.0, 95% CI 0.8 to 1.2), mood disorders (OR = 0.9, 95% CI 0.7 to 1.1) or substance dependence and/or abuse (OR = 0.9, 95% CI 0.6 to 1.3). Past year suicidal ideation (OR = 0.8, 95% CI 0.5 to 1.3) and suicide planning (OR = 0.5, 95% CI 0.2 to 1.4) were also not associated with living in households with firearms. Having made a suicide attempt over the previous year was the only outcome more common among participants reporting that they currently lived in a home without [corrected] firearms. CONCLUSIONS: The previously reported association between household firearm ownership and heightened risk of suicide is not explained by a higher risk of psychopathology among gun-owning families. As there are Americans with suicidal ideation and/or significant and recent psychiatric disorders currently living in homes with firearms, future work should focus on understanding the impediments to effectively communicating the suicide risk associated with household firearms.

Correction to: Abstracts : 31st European Congress of Pathology.

The name of the co-author of MD-05-001 (page S62) should be presented as 'S. Vári-Kakas' instead of 'I.î Vári-Kakas' in the authorship group. The name has been corrected in the authorship group shown above.

Alteration of sarcoplasmic reticulum Ca2+ ATPase expression in lower limb ischemia caused by atherosclerosis obliterans.

Atherosclerosis is a disease caused by a build-up of fatty plaques and cholesterol in the arteries. The lumen of the vessels is obliterated resulting in restricted blood supply to tissues. In ischemic conditions, the cytosolic Ca2+ level of skeletal muscle may increase, indicating the alteration of Ca2+ removal mechanisms. Ca2+ is transported from cytosol into the sarcoplasmic reticulum by Ca2+ ATPase (SERCA), with its 1a isoform expressed in adult, while its 1b isoform in neonatal and regenerating fast-twitch skeletal muscle. To investigate the role of these isoforms in ischemic skeletal muscle, biopsies from musculus biceps femoris of patients who underwent amputation due to atherosclerosis were examined. Samples were removed from the visibly healthy and hypoxia-affected tissue. Significantly increased SERCA1a expression was detected under the ischemic conditions (246 ± 69%; p < 0.05) compared with the healthy tissue. Furthermore, the ratio of SERCA1a-positive fibers was slightly increased (46 ± 4% in healthy tissue and 60 ± 5% in ischemic tissue; p > 0.05), whereas SERCA2a did not change. In addition, in primary cultures derived from hypoxia-affected tissue, the diameter and fusion index of myotubes were significantly increased (30 ± 1.6 µm vs. 41 ± 2.4 µm and 31 ± 4% vs. 45 ± 3%; p < 0.05). We propose that the increased SERCA1a expression indicates the existence and location of compensating mechanisms in ischemic muscle.

Proton pump inhibitor co-therapy normalizes the increased cell turnover of the gastric mucosa both in NSAID and selective COX-2 users.

Proton pump inhibitor (PPI) co-therapy is considered the best strategy in preventing gastrointestinal complications during non-steroidal anti-inflammatory drug (NSAID) treatment, but there is limited information available on its effect on gastric mucosal cell kinetics. To evaluate the effect of PPI co-therapy on gastric mucosa we investigated epithelial cell proliferation, apoptosis, epithelial growth factor receptor (EGFR) and p53 expression in patients on chronic non-selective NSAID or cyclooxygenase-2 selective inhibitor (COX-2) treatment. Gastric biopsies of the antrum were taken from 10-10 patients on chronic NSAID and COX-2, therapy prior and after 6 months PPI co-therapy, and 10 controls without any treatment. Cell proliferation, apoptosis, EGFR and p53 expression were measured by immunohistochemistry. At least 600 glandular epithel cells were encountered and results were expressed as % of total cells counted. We found increased cell proliferation in patients on chronic COX-2 but not on NSAID therapy. Patients on either NSAID or COX-2 therapy had an increased p53 and decreased EGFR expression. PPI therapy reversed not only the increased cell proliferation and p53 expression, but also the suppressed EGFR expression when administered as co-therapy. The fewer gastrointestinal side effects observed during chronic COX-2 therapy may partially be the result of the higher cell proliferation. This effect is not mediated by the EGFR pathway. PPI co-therapy normalizes the disturbed cell kinetics irrespective of NSAID treatment used.

Circulating tumor cell clusters in the peripheral blood of colorectal cancer patients.

PURPOSE: Recently several reverse transcription-PCR techniques have been proven to be useful for the detection of circulating micrometastases. However, this way intact cell clusters that were found in animal experiments of prognostic value could not be detected. In this study, evaluation and modification of a commercial, cytokeratin-based, immunomagnetic cell separation method was performed for the detection of intact cell clusters in colorectal carcinoma patients. EXPERIMENTAL DESIGN: Thirty-two colon cancer patients (6 were in Dukes stage B, 13 in stage C, and 13 in stage D) and 20 healthy donor samples were evaluated. Immunomagnetic cell separation was performed from the buffy coat of peripheral blood samples (20 ml) using the Carcinoma Cell Enrichment Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), avoiding any filtering steps. The enriched cell fraction was cytocentrifuged and immunocytochemically labeled using a pancytokeratin antibody (MNF116; Dako). RESULTS: Of 20 healthy samples, 2 contained one cytokeratin-positive cell. Of 32 single samples from malignant cases, 24 showed cytokeratin-positive cells. Tumor cell clusters, mixed-cell doublets (one cytokeratin-positive and -negative cell), and mixed-cell clusters were detected in 22 of 24 patients. In six cases, cytokeratin-positive dendritic-like cells were detected. Follow-up data indicate that chemotherapy cannot destroy all of the circulating tumor cell clusters. CONCLUSIONS: Using the methods presented, we could detect circulating colon cancer cells and cell clusters in colon carcinoma patients. Similar cellular structures were described previously only in rats. Present data prove that such structures are present in human colorectal cancer, too.

Reliability of quantitative reverse-transcriptase-PCR-based detection of tumour cells in the blood between different laboratories using a standardised protocol.

Differences in methods of reverse-transcriptase (RT)-polymerase chain reaction (PCR)-based detection of tumour cells in the blood gives rise to conflicting results, and standardisation is urgently needed. This pilot study aimed to assess the variation of RT-PCR-based detection of tumour cells in blood between four different laboratories using a commercially available kit with a standardised protocol. This kit allows comparison of results from different laboratories and facilitates the investigation of the influence of pre-analytical parameters. All laboratories analysed identical sets of blood samples spiked with tumour cells in a concentration range of 1-100 tumour cells/ml. To study at which level variation was introduced, three kinds of sample sets were generated in which (i) tumour cell RNA was spiked in the RNA of mononuclear cells (MNC), (ii) tumour cells were spiked in isolated MNC, and (iii) tumour cells were spiked in blood. Real-time quantitative RT-PCR was used to detect and quantify cytokeratin 20 (CK20) expression, which is indicative for the presence of epithelial tumour cells. All laboratories were able to detect CK20 expression in all spiked-RNA samples with limited variation in expression levels between laboratories. There was a positive correlation between the amount of spiked tumour cell RNA and CK20 expression level. RT-PCR analysis of spiked-MNC samples resulted in more variation in the CK20 expression levels between laboratories, however again all spiked samples were reported to be positive by all of the laboratories. The evaluation of spiked-blood samples gave rise to considerable quantitative and qualitative variation between the laboratories. Our results underline the importance and need for standardisation and extended quality control studies in the field of pre-analytics.

Review article: Deoxyribonucleic acid-based diagnostic techniques to detect Helicobacter pylori.

Helicobacter pylori is an important cause of many gastrointestinal disorders, ranging from chronic gastritis to gastric lymphoma and adenocarcinoma. The deoxyribonucleic acid-based assays have the potential to be a powerful diagnostic tool given its ability to specifically identify H. pylori deoxyribonucleic acid. Markers used to include general H. pylori structures and pathogenetic factors like ureaseA, cagA, vacA, iceA. Deoxyribonucleic acid or bacterial ribonucleic acid for polymerase chain reaction assays can be collected from gastric biopsy, gastric juice, stool, buccal specimens. Polymerase chain reaction can yield quantitative and genotyping results with sensitivity and specificity that approaches 100%. A clear trend in the direction of the determination of quantitative H. pylori infection by real-time polymerase chain reaction can be observed. Fluorescent in situ hybridization is suggested for routine antibiotic resistance determination. To identify the organism, deoxyribonucleic acid structure and its virulence factors may be feasible by using oligonucleotide microarray specifically recognizing and discriminating bacterial deoxyribonucleic acid and various virulence factors. Deoxyribonucleic acid-based H. pylori diagnosis yields higher sensitivity, however, specificity requires sophisticated labour environment and associated with higher costs.

Digital slide and virtual microscopy based routine and telepathology evaluation of routine gastrointestinal biopsy specimens.

AIMS: To evaluate a recently developed digital slide and virtual microscope system, and to compare this method with optical microscopy on routine gastrointestinal biopsy specimens in both local and remote access modes. METHODS: A fully computer controlled commercial microscope was used. The scanning program included object detection, autofocus, and image compression algorithms. The overall hard disk space for a gastric biopsy was between 30 and 50 MB and the scanning time was between 20 and 40 minutes. Haematoxylin and eosin stained routine gastric (61) and colon (42) biopsy specimens were selected, scanned, and evaluated by two specialists on an optical (OM) and virtual microscope (VM). RESULTS: The overall concordance of VM and OM with the consensus diagnosis was 95.1% and 97%, respectively. Clinically important concordance was 96.1% and 98% for VM and OM, respectively. The two methods showed concordance in 92% of cases and clinically important concordance in 94.1% of cases. The reasons for discordance were image quality (one case), interpretation difference (three cases), and insufficient clinical information (three cases). Remote evaluation of the digital slides through the Internet has the advantages of the previously used static and dynamic telepathology methods. CONCLUSIONS: Diagnostic concordance was found between OM and VM. The digital slide and the virtual microscope can be alternative techniques in the computerisation of the histology laboratory and in teleconsultation services after further evaluation of time and storage constraints.

Determination of Helicobacter pylori cagA, vacA genotypes with real-time PCR melting curve analysis.

OBJECTIVES: genotypes of Helicobacter pylori are the focus of interest because they play a prominent role in mucosal injury. The purpose of this study was to determine cagA and vacA genotypes of H. pylori using real-time polymerase chain reaction (PCR) method with a double strain DNA binding SYBR Green I.dye, and to compare this with those of two immunohistochemical methods. METHODS: forty-three paraffin-embedded biopsy tissue samples were examined by histology, epidermal growth factor receptor (EGFR), proliferating cell nuclear antigen (PCNA) immunohistochemistry and melting curve analysis of real-time PCR. RESULTS: the presence of cagA gene was associated with a significantly higher frequency of gastritis (P=0.003) than that of vacA gene with intestinal metaplasia (P=0.045). Significant difference was found between the presence of cagA gene and EGFR expression in intestinal metaplasia cases in comparison with cagA negative samples (P=0.0418). Statistically significant difference was detected between increased cell proliferation and the presence of gastritis. CONCLUSIONS: this method seems to be suitable for H. pylori genotype determination. Sensitivity, speed and simplicity are key areas in the development of PCR assays for H. pylori. Results supported the notion that infection with cagA positive H. pylori strain causes more augmentated cell proliferation in the stomach mucosa.

Effect of Helicobacter pylori infection and eradication on gastric epithelial cell proliferation and apoptosis.

OBJECTIVES: the effect of Helicobacter pylori infection on gastric epithelial cell proliferation and apoptosis is still controversial. Our aim was to evaluate the effect of H. pylori infection on cell kinetic parameters in normal gastric epithelium, gastritis with/without intestinal metaplasia and gastric cancer. PATIENTS AND METHODS: antral biopsies were taken from 121 patients (61 women, 60 men, mean age 58.5+/-14.3 years of age) who underwent routine gastroscopy for upper gastrointestinal symptoms. Sections were scored for normal epithelia (n=15), gastritis without intestinal metaplasia (n=74), gastritis with intestinal metaplasia (n=24), and gastric adenocarcinoma (n=8). Fifty-two patients had H. pylori positive gastritis, and success of H. pylori eradication therapy was controlled in 12 cases, all with intestinal metaplasia. To characterize cell proliferation and assess apoptosis, immunohistochemistry [Proliferating Cell Nuclear Antigen (PCNA)], histochemistry [Argyrophil Nucleolar Organizer Regions (AgNOR)], and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridinetriphosphate (dUTP) nick end-labeling (TUNEL) were used, respectively. RESULTS: both cell proliferation and apoptosis is was higher in chronic gastritis when compared with normal epithelia, but neither PCNA LI (54.79+/-19.1 vs. 53.20+/-20.7) nor AgNOR counts (291.43+/-44.3 vs. 277.8+/-57.54) were different in H. pylori positive versus negative chronic gastritis. A significant positive correlation (P<0.05) was found in this group between PCNA and AgNOR techniques. Apoptosis was significantly higher (P<0.05) in H. pylori positive cases only when intestinal metaplasia was not present. Cell proliferation in intestinal metaplasia decreased to the activity of normal epithelium after successful eradication of H. pylori but remained high if eradication therapy failed. CONCLUSIONS: epithelial cell proliferation does not depend on H. pylori status in chronic gastritis. H. pylori increases apoptosis only in the absence of intestinal metaplasia.

Fibronectin determination in pregnancy.

The levels of fibronectin were determined by immunoturbidimetric assay in two populations: (a) plasma of healthy nonpregnant and pregnant women, and in amniotic fluid of healthy pregnant females; (b) plasma of umbilical cord blood of healthy newborns and of newborns with sepsis. Fibronectin concentrations of amniotic fluid showed a significant decrease during pregnancy, but the changes of plasma fibronectin levels were not significant in this period. In newborn sepsis, the levels of plasma fibronectin were significantly decreased. We did not find a significant difference between the fibronectin concentration of umbilical cord blood of premature infants compared to mature infants.

DNA cytometry and morphometry by TV based image analysis system (TAS) in the diagnosis of gastric carcinoma.

Cytological smears from gastric biopsies were studied by two types of TAS. Böcking's algorithm and a great number of form factors were used to separate the intact cells from the tumourous ones. The numerically expressed density features and morphometric parameters are able to indicate the possibility of malignant transformation even in dysplasia. These values become much higher in tumours and lead to unequivocal diagnosis. Both types of TAS render valuable help in assessing cases not reliably diagnosed by routine staining procedures.

Elevated nascent DNA content of peripheral blood mononuclear cell compartment in lymphoma patients.

The kinetics of DNA synthesis and the DNA pattern of isolated peripheral blood mononuclear cells from control subjects and lymphoma patients prior to drug treatment were studied as a possible tool in the early diagnosis of lymphoma. Thymidine and [H3]-dTTP incorporation represented the measure of replicative DNA synthesis in permeable murine thymocytes and HT-168 human melanoma cells as described earlier. The kinetic parameters of nucleotide incorporation were compared with the ploidity parameters of the Feulgen-stained smears examined by the DNASK TV-cytophotometric system. For better evaluation and visualization of the S-phase population, the method of silver impregnation of the Nucleolar-Organizer-Region in combination with the Feulgen technique was used. Significant differences were observed among the five determined parameters of healthy and lymphoma patients. Our results allow to explain some of the facts related to T-cell function deficiency in lymphoma.

Quantitative DNA and morphometric analysis of gastroscopic brush smears by TV image analysis.

OBJECTIVES: To determine quantitative nuclear morpho-and densitometric classifiers and classification techniques for analysis of gastric, Feulgen-stained brush smears. DESIGN: TV image analysis-based quantitative DNA and morphometric analysis of gastric brush smears in a prospective study. PATIENTS AND METHODS: Ninety-eight (11 normal, 77 gastritis (17 with intestinal metaplasia) and 10 adenocarcinoma) Feulgen-Schiff-stained gastric brush smears were analysed by TV image analysis. The classification of the smears was based on parallel histological examination. For standards, DNA content of lymphocyte cell cultures was determined by the image and by flow cytometry. From every nucleus, six morphometric (surface, layers, minimum diameter, maximum diameter, perimeter and form factor) and six densitometric (integrated optical density (IOD), average density, sigma density, minimum and maximum density and density range) parameters were simultaneously determined. The smear parameters (object cells CV, DNA index, 2c deviation index, 5c exceeding rate, G1 -S-G2 ratio) were analysed together with the mean and SD values of the nuclear parameters by discriminant analysis and back-propagation neural networks. RESULT: The normal smears were all diploid and their S + G2 ratio was 15.24+/-7.75% (mean +/- SD). The gastritis smears were all diploid with a proliferation fraction of 20.89+/-6.75%. The tumours were aneuploid in eight of the ten cases with 5c exceeding rate > 6.23%, the S + G2 fraction ratio was 34.72+/-10.12%. The mean nuclear surface area was 46+/-20, 58+/-20 and 74+/-22 microm2 in normal, gastritis and malignant groups, respectively. Significant differences (P<0.05) were found in nuclear surface, minimum and maximum diameter, and perimeter parameters. Using linear discriminant analysis, 100% of the non-malignant cases and 70% of the tumour cases were correctly classified. Using 30 non-malignant and five malignant cases as a training set, the neural networks classified 95% of the remaining cases correctly. The DNA index increased significantly (P<0.05) in Helicobacter pylori-positive cases compared to the negative ones. In gastritis with intestinal metaplasia, the proliferation ratio decreased significantly (P<0.05). CONCLUSIONS: The image analysis is a useful tool for quantitative gastric cytology. The combination of nuclear morphometric parameters and neural network classifiers with multivariate quantitative DNA analysis is suggested for gastric brush smear quantitative cytology analysis.