Colorectal Cancer CTCs Detection Using Two FCM Protocol Approaches.

BACKGROUND: The presence and quantification of circulating tumor cells (CTCs) could minimize the mortality among cancer patients by tailored, personalized, and targeted therapy and could also help in the field of investigation about new therapeutic targets. Identification of CTCs has been performed using molecular techniques and more advanced automated techniques such as CellSearch® and Amnis®. Our aim was to test the possibility of identifying CTCs in colorectal cancer patients using a lower cost and less complex flow cytometry-based method. METHODS: Besides the CellSearch® system for CTCs enumeration and Amnis® Imaging Flow Cytometers, which are both commercially available, we tested and developed two FCM protocol approaches after immunomagnetic enrichment for CTCs enumeration in the blood of colorectal cancer patients. The CTCs numbers were assessed at baseline before anesthesia and the curative surgery and day one after the curative surgery. Blood from healthy donors was used as negative control. The research was performed by the Cyflow® Space cytometer (Partec, Münster, Germany). RESULTS: In the patient group, the enumeration using the direct protocol with a threshold 3 cells/mL, showed a mean of CTCs before surgery of 32, while after surgery it was 20, with sensitivity (SN) of 49.2% and specificity (SP) of 58.3%. On the other hand, using the intracellular protocol with a threshold of 1 cell/mL in patients' group, the mean of CTCs before surgery was 65 and after surgery it was 60, with a sensitivity (SN) of 62.7% and specificity (SP) of 70%. In the intracellular protocol the most significant correlation identified was for the expression of CKs with adenocarcinomas (r = 0.256, p = 0.044), with the existence of lymph node infiltration (r = 0.380, p = 0.008), and with the stage of the disease (r = 0.391, p = 0.003). For the direct protocol, considerable correlation was found with the samples of the right colon (r = 0.369, p = 0.002). CONCLUSIONS: Our findings demonstrate that we established two FCM low cost protocol approaches to detect and enumerate CTCs in colorectal cancer patients. In both FCM protocols (direct and intracellular) we observed a statistically significant increase (p ˂ 0.05) of CTCs number in the patient group. No statistical significance (p ˂ 0.05) of CTCs before and after surgery in patient group in both protocols was observed.

High levels of IL-6 and IL-8 characterize early-on idiopathic pulmonary fibrosis acute exacerbations.

INTRODUCTION: Controversy exists about the pathogenesis of idiopathic pulmonary fibrosis acute exacerbations (IPF-AEs). According to one hypothesis IPF-AEs represent the development of any etiology diffuse alveolar damage (DAD) upon usual interstitial pneumonia (UIP), whilst other researchers argue that an accelerated phase of the intrinsic fibrotic process of unknown etiology prevails, leading to ARDS. Different cytokines might be involved in both processes. The aim of this study was to assess pro-inflammatory and pro-fibrotic cytokines in the peripheral blood from stable and exacerbated IPF patients. METHODS: Consecutive IPF patients referred to our department were included. Diagnoses of IPF and IPF-AE were based on international guidelines and consensus criteria. The interleukins (IL)-4, IL-6, IL-8, IL-10, and IL-13 as well asactive transforming growth factor-beta (TGF-ß) were measured in blood from both stable and exacerbated patients on the day of hospital admission for deterioration. Subjects were followed for 12months. Mann-Whitney test as well as Tobit and logistic regression analyses were applied. RESULTS: Among the 41 patients studied, 23 were stable, and 18 under exacerbation; of the latter, 12 patients survived. The IL-6 and IL-8 levels were significantly higher in exacerbated patients (p=0.002 and p=0.046, respectively). An increase in either IL-6 or IL-8 by 1pg/ml increases the odds of death by 5.6% (p=0.021) and 6.7% (p=0.013), respectively, in all patients. No differences were detected for the other cytokines. CONCLUSION: High levels of IL-6 and IL-8 characterize early-on IPF-AEs and an increase in the levels of IL-6 and IL-8 associates with worse outcome in all patients. However, as the most representative pro-fibrotic cytokines, TGF-ß, IL-10, IL-4 and IL-13 were not increased and given the dualistic nature, both pro-inflammatory and pro-fibrotic of IL-6 further studies are necessary to clarify the enigma of IPF-AEs etiopathogenesis.

Quantitative Measurement of L1 Human Papillomavirus Type 16 Methylation for the Prediction of Preinvasive and Invasive Cervical Disease.

Background: Methylation of the human papillomavirus (HPV) DNA has been proposed as a novel biomarker. Here, we correlated the mean methylation level of 12 CpG sites within the L1 gene, to the histological grade of cervical precancer and cancer. We assessed whether HPV L1 gene methylation can predict the presence of high-grade disease at histology in women testing positive for HPV16 genotype. Methods: Pyrosequencing was used for DNA methylation quantification and 145 women were recruited. Results: We found that the L1 HPV16 mean methylation (±SD) significantly increased with disease severity (cervical intraepithelial neoplasia [CIN] 3, 17.9% [±7.2] vs CIN2, 11.6% [±6.5], P < .001 or vs CIN1, 9.0% [±3.5], P < .001). Mean methylation was a good predictor of CIN3+ cases; the area under the curve was higher for sites 5611 in the prediction of CIN2+ and higher for position 7145 for CIN3+. The evaluation of different methylation thresholds for the prediction of CIN3+ showed that the optimal balance of sensitivity and specificity (75.7% and 77.5%, respectively) and positive and negative predictive values (74.7% and 78.5%, respectively) was achieved for a methylation of 14.0% with overall accuracy of 76.7%. Conclusions: Elevated methylation level is associated with increased disease severity and has good ability to discriminate HPV16-positive women that have high-grade disease or worse.

Changes in Arterial Stiffness and N-terminal pro-brain natriuretic peptide Levels after Endovascular Repair of Descending Thoracic Aorta.

BACKGROUND: Limited data exist concerning the biomechanical and central hemodynamic changes induced by endograft implantation in the descending thoracic aorta. The aim of this prospective ongoing study was to evaluate changes in arterial stiffness, assessed by pulse wave velocity (PWV; m/sec), and N-terminal pro-brain natriuretic peptide (NT-proBNP; pg/mL) levels in patients undergoing endovascular repair of descending thoracic aorta (thoracic endovascular aortic repair [TEVAR]). METHODS: Twenty-seven patients with thoracic aorta pathology who underwent elective TEVAR were included in the study. Blood samples were obtained preoperatively, 24 hr, 48 hr, and 6 months postoperatively, and serum levels of NT-proBNP were measured. PWV was determined before and 6 months after TEVAR. One-way analysis of variance by ranks was used to test the alterations in PWV (from baseline to 6 months) and NT-proBNP (along the 4 phases of evaluation). Post hoc analyses were appropriately performed. RESULTS: We recorded an increase in values of NT-proBNP from baseline (median = 96.1, interquartile range [IQR] = 82.7-117.9) to 24 hr postoperative (median = 201.6, IQR = 82.8-425.9), 48 hr postoperative (median = 317.0, IQR = 102.5-1,479.5), and 6 months postoperative (median = 144, IQR = 82.8-276.4). The Kruskal-Wallis H test showed a statistically significant increase (x2(3) = 11.17, P = 0.01) in NT-proBNP from baseline (rank mean = 22.19) toward the postoperative time points of evaluation (24 hr postoperative: 35.17 [change: +12.9, P = 0.02]; 48 hr postoperative: 42.64 [change: +20.5, P < 0.001]; 6 month postoperative: 34.91 [change: +12.7, P = 0.03]). An increase in PWV values was recorded from baseline (median = 11.9, IQR = 10.0-13.5) to 6 months postoperatively (median = 13.9, IQR = 11.9-16.4). That change achieved statistically significant level [x2(1) = 4.86, P = 0.03], with an increase in mean rank PWV (+7.5). CONCLUSIONS: Implementation of thoracic stent grafts may be associated with considerable increase of both arterial stiffness and NT-proBNP serum levels along time. Those results may indicate an adverse cardiac impact of TEVAR. However, the early and long-term effects of TEVAR on cardiovascular outcomes require further investigation.

The Profile of Circulating Matrix Metalloproteinases in Patients Undergoing Lower Limb Endovascular Interventions for Peripheral Arterial Disease.

BACKGROUND: Matrix metalloproteinases (MMPs) play a significant role in the development and progression of atherosclerotic vascular disease. We aimed to document the profile of circulating MMPs in peripheral arterial disease (PAD) patients undergoing lower limb endovascular revascularization. METHODS: A total of 46 patients (37 male; mean age 66 ± 11 years) undergoing elective lower limb percutaneous revascularization (angioplasty/stent) for symptomatic PAD were recruited from 2 vascular centers. Exclusion criteria were: acute limb ischemia, active infection and/or wet gangrene, liver disease, end-stage renal disease, and cancer. Patients having open revascularization or hybrid (open combined with endovascular) procedures were also excluded. Peripheral venous blood samples were taken on admission and 24 hrs after the procedure. Levels of MMP-2, MMP-3, MMP-7, and MMP-9 were measured along with tissue inhibitors of MMPs (TIMPs) 1 and 2. RESULTS: Compared to baseline values, there was a significant elevation in serum MMP-3 (P = 0.014) and MMP-7 (P = 0.008) levels, whereas serum MMP-9 showed a nonsignificant trend to increase (P = 0.169). On the other hand, no significant alterations were found 24 hrs after angioplasty/stenting with regard to the MMP-2 level and TIMP-1 and 2 levels. CONCLUSIONS: This study documented the periprocedural profile of circulating MMPs in patients undergoing angioplasty/stenting for PAD. The implications of increased MMP-3 and MMP-7 activity after peripheral endovascular interventions and their potential clinical relevance require further investigation.

Personalised management of women with cervical abnormalities using a clinical decision support scoring system.

OBJECTIVES: To develop a clinical decision support scoring system (DSSS) based on artificial neural networks (ANN) for personalised management of women with cervical abnormalities. METHODS: We recruited women with cervical abnormalities and healthy controls that attended for opportunistic screening between 2006 and 2014 in 3 University Hospitals. We prospectively collected detailed patient characteristics, the colposcopic impression and performed a series of biomarkers using a liquid-based cytology sample. These included HPV DNA typing, E6&E7 mRNA by NASBA or flow cytometry and p16INK4a immunostaining. We used ANNs to combine the cytology and biomarker results and develop a clinical DSSS with the aim to improve the diagnostic accuracy of tests and quantify the individual's risk for different histological diagnoses. We used histology as the gold standard. RESULTS: We analysed data from 2267 women that had complete or partial dataset of clinical and molecular data during their initial or followup visits (N=3565). Accuracy parameters (sensitivity, specificity, positive and negative predictive values) were assessed for the cytological result and/or HPV status and for the DSSS. The ANN predicted with higher accuracy the chances of high-grade (CIN2+), low grade (HPV/CIN1) and normal histology than cytology with or without HPV test. The sensitivity for prediction of CIN2 or worse was 93.0%, specificity 99.2% with high positive (93.3%) and negative (99.2%) predictive values. CONCLUSIONS: The DSSS based on an ANN of multilayer perceptron (MLP) type, can predict with the highest accuracy the histological diagnosis in women with abnormalities at cytology when compared with the use of tests alone. A user-friendly software based on this technology could be used to guide clinician decision making towards a more personalised care.

Increased levels of circulating platelet derived microparticles in Crohn's disease patients.

OBJECTIVE: Platelet activation is a consistent feature in inflammatory bowel disease. However, the role of circulating platelet derived microparticles (PDMPs) and the effects of disease activity and treatment on their levels has not been clarified yet in this disorder. MATERIAL AND METHODS: Using flow cytometry, we measured platelet derived microparticles and platelet derived microparticles expressing Annexin V in platelet rich plasma from 47 Crohn's disease and 43 ulcerative colitis patients and 24 healthy controls. RESULTS: Crohn's disease patients have greater PDMPs (0.31% ± 0.07% versus 0.14% ± 0.04%, p = 0.02) and PDMPs expressing Annexin V (27% ± 2.6% versus 14.6% ± 2.7%, p = 0.002) levels in comparison with healthy controls; however, both microparticles levels are not related with disease activity. Crohn's disease patients on 5-ASA therapy show lower levels of PDMPs in comparison with those on no 5-ASA (0.30% ± 0.07% versus 0.32% ± 0.09%, p = 0.048). Ulcerative colitis patients have similar PDMPs and PDMPs expressing Annexin V levels, compared to healthy controls (p = 0.06 and p = 0.2, respectively) and there is no correlation of both microparticles expression with disease activity. 5-ASA has no effect on both microparticles levels in ulcerative colitis patients. Anti-TNF-α treatment has no effect on study's microparticles expression in Crohn's and ulcerative colitis patients. CONCLUSIONS: Circulating levels of platelet derived microparticles are increased only in Crohn's patients, but they do not correlate with disease activity. 5-ASA treatment is associated with lower levels of PDMPs only in Crohn's, while anti-TNF-α treatment does not influence expression of microparticles in inflammatory bowel disease patients.

Inflammatory response and renal function following endovascular repair of the descending thoracic aorta.

PURPOSE: To evaluate inflammatory response and renal function after thoracic endovascular aortic repair (TEVAR) of lesions in the descending thoracic aorta. METHODS: Thirty-two consecutive patients treated with TEVAR from January 2010 to August 2013 were enrolled in this prospective study. Two were excluded owing to dissecting thoracic aortic aneurysm (TAA) extending into the renal arteries with renal failure in one and a saccular TAA in which a multilayer flow-modulating stent was implanted in the other. This left 30 patients (28 men; mean age 68.8±5.9 years) with 28 TAAs, an aortic dissection, and an aortic ulcer for the analysis. Temperature and serum levels of white blood cells (WBCs), C-reactive protein (CRP), interleukin-10 (IL-10), IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), creatinine, urea, and cystatin C were measured preoperatively and at 24 and 48 hours postoperatively. RESULTS: Statistically significant increases in temperature and serum levels of WBCs, CRP, IL-10, and IL-6 were observed 24 and 48 hours postoperatively compared to baseline (all p<0.05). The number of endografts and the coverage of the celiac or subclavian artery did not affect the magnitude of the inflammatory response. No significant differences were observed concerning serum levels of IL-8, TNF-α, creatinine, or cystatin C from baseline to 24 or 48 hours postoperatively. CONCLUSION: Endograft implantation in the thoracic aorta may propagate an inflammatory response during the early postoperative period. No clinical adverse events related to the increased inflammatory response were observed. Renal function does not seem to be deteriorated after TEVAR in the descending thoracic aorta.

Volume of new-onset thrombus is associated with the development of postimplantation syndrome after endovascular aneurysm repair.

OBJECTIVE: The objective of this study was to evaluate the impact of contrast medium volume, inferior mesenteric artery (IMA) patency, and pre-existing as well as new-onset thrombus on the inflammatory response after elective endovascular aneurysm repair (EVAR). METHODS: The study included 87 patients undergoing elective endovascular repair of asymptomatic infrarenal aneurysms between January 2011 and November 2011. The patency of the IMA was determined by preoperative computed tomography angiography; the volume of the contrast medium used during the procedure was measured, and the volumes of chronic mural as well as new-onset thrombus were calculated from the preoperative and postoperative computed tomography angiograms with dedicated imaging software. The results were correlated to the occurrence of postimplantation syndrome (PIS) as well as to the main clinical and laboratory components of PIS: fever; increased white blood cell (WBC) count and C-reactive protein (CRP) level; decreased platelet count; and increased interleukin (IL)-6, IL-8, and IL-10 levels. RESULTS: Postoperatively, a statistically significant increase was recorded in the mean values of body temperature, WBCs, CRP, IL-6, IL-8, and IL-10, and a statistically significant decrease was recorded in the number of platelets. After adjustment for endograft type, the volume of new-onset thrombus was found to be significantly correlated with the peak postoperative temperature (ß = .307; P < .05) and the increase in WBC count (ß = .271; P < .05), CRP level (ß = .484; P < .001), and IL-6 level (ß = .288; P < .05). On the contrary, the volume of chronic mural thrombus, the patency of the IMA, and the volume of contrast medium were not found to significantly affect any parameter of the PIS. PIS occurred in 34 patients (39%). Multiple logistic regression analysis showed that both the volume of new-onset thrombus and the type of endograft were independently associated with the development of PIS. CONCLUSIONS: The volume of new-onset thrombus is associated with the development of PIS after EVAR, whereas chronic mural thrombus appears to be an inert material. IMA patency and contrast medium volume are irrelevant to the inflammatory response after EVAR.

Differential effects of stent-graft fabrics on arterial stiffness in patients undergoing endovascular aneurysm repair.

PURPOSE: To evaluate the differential effects of endograft fabric types on pulse wave velocity (PWV), a valid, clinically feasible marker of arterial stiffness, in patients undergoing endovascular repair (EVAR) of abdominal aortic aneurysms (AAAs). METHODS: As part of a registered study (ClinicalTrials.gov identifier NCT00636766), 118 consecutive men (mean age 71 ± 8 years) with AAA undergoing elective EVAR were divided into groups according to the type of fabric in the implanted endografts: 46 had polytetrafluoroethylene (PTFE)-covered stent-grafts and 72 received stent-grafts covered with polyester fabric. Patients with Marfan syndrome, collagen-related disorders, and end-stage renal failure were excluded. PWV, clinical characteristics (comorbidities, body mass index, blood pressure, lipids, and glucose profile), novel biomarkers [interleukin (IL)-6, IL-8, IL-10, and osteoprotegerin (OPG)] were determined at baseline and prospectively after 12 months. RESULTS: At baseline, standard multiple regression analysis revealed the independent association of mean blood pressure, OPG, and AAA diameter with PWV after adjustment for age (R(2)=0.729, p=0.036). At follow-up, serum levels of both IL-8 and IL-10 were significantly increased, while OPG decreased in both groups (p<0.05). However, between-group comparison revealed a more adverse effect of polyester covered endografts on serum IL-8 (p<0.001) and OPG (p=0.048) levels. At the same time, PWV was considerably increased in both polyester (+4.12±0.33 m/s, p<0.001) and PTFE (+2.82 ± 0.25 m/s, p=0.003) groups; the effect was more pronounced in the former group (p=0.033). In multivariate analysis, the polyester endograft type emerged as an independent determinant of PWV change after EVAR (R(2)=0.460, p=0.040). CONCLUSION: Both endograft types increased PWV, while recipients of polyester covered stent-grafts showed greater PWV elevation. Further investigation will clarify the inflammatory response and the differential clinical impact of endograft types.

Liver regeneration: immunohistochemical study of intrinsic hepatic innervation after partial hepatectomy in rats.

BACKGROUND: We examined the intrinsic hepatic innervation after partial hepatectomy (PH) in rats and the presence and pattern of neural sprouting in regenerating liver. METHODS: Male Wistar rats (age 9-13 weeks-w, weight 204-356 g), were submitted to two-thirds PH. Rats were sacrificed at postoperative days (d) 1, 3, 5, 7, at 2 and 4 w, and at 3 and 6 months (m) (6-7 animals/group, control group n = 4). Immunohistochemistry for the pan-neural marker protein gene product 9.5 (PGP9.5) and growth-associated protein 43 (GAP-43), a marker of regenerating nerve axons, was performed on tissue sections from the R1 lobe of the regenerating liver. Portal tracts (PTs) with immunoreactive fibers were counted in each section and computer-assisted morphometric analysis (Image Pro Plus) was used to measure nerve fiber density (number of immuno-positive nerve fibers/mm2 (40x)). RESULTS: Immunoreactivity for PGP9.5 was positive in all groups. The number of PGP9.5 (+) nerve fibers decreased from 0.32 +/- 0.12 (control group) to 0.18 +/- 0.09 (1d post-PH group), and gradually increased reaching pre-PH levels at 6 m (0.3 +/- 0.01). In contrast, immunoreactivity for GAP-43 was observed at 5d post-PH, and GAP-43 (+) PTs percentage increased thereafter with a peak at 3 m post-PH. GAP-43 (+) nerve fiber density increased gradually from 5d (0.05 +/- 0.06) with a peak at 3 m post-PH (0.21 +/- 0.027). At 6 m post-PH, immunoreactivity for GAP-43 was not detectable. CONCLUSIONS: Following PH in rats: 1) nerve fiber density in portal tracts decreases temporarily, and 2) neural sprouting in the regenerating liver lobes starts at 5d, reaches peak levels at 3 m and disappears at 6 m post-PH, indicating that the increase in hepatic mass after PH provides an adequate stimulus for the sprouting process.

Expression of HPV-related biomarkers and grade of cervical intraepithelial lesion at treatment.

OBJECTIVES: New human papillomavirus (HPV)-related biomarkers may allow better identification of clinically significant lesions that warrant excision and, conversely, identification of the false positive cases that have been overreported by traditional techniques. The aim of this study was to investigate how the expression of several HPV-related biomarkers correlates to the severity of the lesion at treatment. DESIGN: Prospective observational study. SETTING: University Hospital (2009-2011). POPULATION: All women submitted for excisional treatment. METHODS: A liquid-based cytology sample was obtained before treatment and was tested for HPV typing, mRNA E6 & E7 with NASBA or flow cytometry and p16. All women had histological diagnosis in the form of excisional cone (gold standard). MAIN OUTCOME MEASURES: Correlation of HPV biomarker positivity rates to the grade of the lesion at treatment histology. RESULTS: Two hundred women were recruited: 23 were found to have negative histology (11.5%), 79 (39.5%) CIN1, 50 (25.0%) CIN2 and 48 (24.0%) CIN3. All biomarkers (HPV DNA typing, HR HPV, single HPV 16/18, mRNA E6 & E7 expression and p16) revealed an increased linear positivity rate with increasing severity and grade of the lesion (chi-squared test for trend p < 0.05). This was stronger for HPV (all and high-risk) followed by mRNA with NASBA, flow cytometry, HPV 16/18 and ultimately p16 immunostaining. CONCLUSIONS: The linear correlation between various HPV-related biomarkers and the grade of the lesion suggests that these biomarkers may prove to be useful in the prediction of CIN grade and, as a result, the need for treatment.

The diagnostic accuracy of E6 & 7 mRNA detection as a primary screening test for the detection of severe cervical lesions.

PURPOSE: This prospective accuracy study aimed to assess the diagnostic accuracy of nucleic acid sequence amplification (NASBA) and flow cytometry for E6/7 human papillomavirus (HPV) mRNA detection as a primary screening test compared to cytology in the triage of severe cervical intraepithelial neoplasia (CIN) lesions. METHODS: 1083 women referred to our outpatient gynecology clinics for a routine Pap test were recruited. Residual material of the Pap smears was tested by NASBA and by flow cytometry for E6/7 mRNA expression. Biopsy results were used as reference standards. The accuracy indices of both techniques and of NASBA type-16 HPV were assessed for the detection of CIN2+ lesions and were compared to cytology. RESULTS: An increased lesion severity was associated with increased positivity rates of both NASBA and flow cytometry tests (x(2), p<0.001). A positive correlation between NASBA and flow cytometry was identified when these methods were examined with the Phi coefficient (value 0.369, 95% confidence interval [95%CI] : 0.307-0.426). Furthermore, NASBA (89.7 vs 57.7%, p<0.0005) and flow cytometry (77.3 vs 57.7%, p<0.0005) exhibited higher specificity rates than cytology. However, their sensitivity rates did not exceed those of cytology (NASBA:69.8 vs 84.6%, p=0.051; flow cytometry: 69.12 vs 84.6%, p=0.043). CONCLUSIONS: Both NASBA and flow cytometry exhibited increased specificity for the triage of CIN2+ lesions. However, their relatively lower sensitivity and higher positivity rates when compared to cytology do not make them ideal for a primary screening test. Hence, the role of mRNA detection in the screening for severe cervical lesions remains to be clarified.

The impact of endograft type on inflammatory response after endovascular treatment of abdominal aortic aneurysm.

OBJECTIVE: To evaluate the impact of endograft type on the inflammatory response after elective endovascular repair of abdominal aortic aneurysms. METHODS: From January 2011 to November 2011, we included 100 consecutive patients who underwent elective abdominal aortic aneurysm endovascular repair. Thirteen patients were excluded from the analysis: four with cancer, three with autoimmune disease, two because of recent infection, two who were receiving long-term anti-inflammatory medication, and two because of recent surgery. Temperature, white blood cell count, platelet count, and serum concentrations of cytokines (interleukin [IL]-6, IL-8, and IL-10) were measured preoperatively, 24 hours postoperatively, and 48 hours postoperatively. The study sample was divided into four groups with respect to the type of endograft used: group A, n = 28 (Anaconda; Sulzer Vascutek, Bad Soden, Germany); group B, n = 26 (Zenith; Cook Inc, Bloomington, Ind); group C, n = 23 (Excluder; W. L. Gore and Assoc, Flagstaff, Ariz); and group D, n = 10 (Endurant; Medtronic, Minneapolis, Minn). Endograft configurations included bifurcated grafts only. RESULTS: Epidemiologic characteristics, atherosclerotic risk factors, type of anesthesia, mean blood loss during surgery, and baseline serum levels of cytokines did not differ among the four groups. Mean elevated temperature was more pronounced postoperatively in group A. Serum levels of IL-6 and IL-10 were significantly higher 24 hours and 48 hours postoperatively compared with preoperative levels in all groups. Patients in group C showed the smallest increase in levels of serum IL-6 and IL-10 at 24 hours and 48 hours postoperatively. Mean difference in cytokine levels after aneurysm exclusion was greater for group A vs group C (P < .01) compared with group A vs B (P < .05). No differences in the mortality and morbidity rates were observed among the four groups. CONCLUSIONS: Endograft type appears to influence the inflammatory response after endovascular aortic repair. The postimplantation syndrome was apparent during the first 24 hours and decreased afterward. Anaconda and Zenith endografts induced a more intense inflammatory response. A "milder" inflammatory activation was observed in patients with an Excluder endograft. The postimplantation syndrome was not associated with perioperative adverse clinical events showing a benign course. The possible long-term sequelae of postimplantation syndrome require further investigation.

Prevalence of human papillomavirus in saliva and cervix of sexually active women.

OBJECTIVE: Human papillomavirus (HPV) is strongly associated with cervical cancer and possibly with some oropharyngeal cancers. However, the relation between oral and cervical HPV infection is not fully understood. This study evaluates the prevalence rate and type-concordance of HPVs in these areas. METHODS: HPV DNA typing was performed in saliva and cervical specimens of 43 sexually active women, with the use of general consensus PCR and nested PCR (NPCR) tests. RESULTS: The prevalence rate of HPV DNA in cervical and saliva samples was 51.2% and 11.6% with general PCR, and 60.5% and 44.2% with NPCR, respectively. The probability of HPV DNA detection with general PCR in saliva was about 8 times lower compared to the cervix (P<0.001, OR: 0.13, 95% CI: 0.04-0.37), but showed no difference when the more sensitive NPCR method was applied (P=0.139, OR: 0.52, 95% CI: 0.22-1.21). The distribution of HPV variants according to their oncogenic potential revealed no statistically significant difference, regardless to the PCR method used for both sites. All general PCR HPV DNA positive saliva specimens belonged to women with cytology findings (n=5). These women had also 8.5 times higher risk for presenting with positive HPV detection in saliva with the NPCR method (P=0.009, OR=8.50, 95% CI: 1.74-39.70). CONCLUSIONS: Women with genital HPV infection are at higher risk for asymptomatic oral HPV infection. Prophylactic HPV-vaccination may reduce the burden of HPV-related diseases in both cervix and oropharynx.

Detection of herplex simplex virus-1 and -2 in cardiac myxomas.

The etiology of sporadic cardiac myxomas remains elusive. The tendency for these lesions to recur following resection, their immunopathological characteristics, along with their histological and molecular profile, may implicate the presence of an infective agent in this type of tumor. In this study, we investigated the presence of herpes simplex virus (HSV) DNA in a cohort of cardiac myxomas in a tertiary referral centre. Twenty-nine formalin-fixed paraffin-embedded (FFPE) sporadic cardiac myxomas were obtained, 17 of which were shown to be informative. These were compared to 19 macroscopically and microscopically normal heart tissue specimens. The detection of HSV-1 and -2 genomic sequences was achieved with the use of a combined nested PCR-Restriction Fragment Length Polymorphism methodology. The presence of HSV-1 and/or -2 DNA was demonstrated in 6 of 17 (35%) informative sporadic cardiac myxomas, whereas no HSV DNA was detected in normal heart tissues (P < 0.01). The existence of HSV-1/2 DNA in sporadic cardiac myxomas, along with its absence from normal heart tissues, reinforces the possibility that HSV infection might be involved in the development of these lesions. Our findings raise the point of anti-HSV medication postsurgically with a potential benefit in reducing the rate of recurrences.

Identification of women for referral to colposcopy by neural networks: a preliminary study based on LBC and molecular biomarkers.

Objective of this study is to investigate the potential of the learning vector quantizer neural network (LVQ-NN) classifier on various diagnostic variables used in the modern cytopathology laboratory and to build an algorithm that may facilitate the classification of individual cases. From all women included in the study, a liquid-based cytology sample was obtained; this was tested via HPV DNA test, E6/E7 HPV mRNA test, and p16 immunostaining. The data were classified by the LVQ-NN into two groups: CIN-2 or worse and CIN-1 or less. Half of the cases were used to train the LVQ-NN; the remaining cases (test set) were used for validation. Out of the 1258 cases, cytology identified correctly 72.90% of the CIN-2 or worst cases and 97.37% of the CIN-1 or less cases, with overall accuracy 94.36%. The application of the LVQ-NN on the test set allowed correct classification for 84.62% of the cases with CIN-2 or worse and 97.64% of the cases with CIN-1 or less, with overall accuracy of 96.03%. The use of the LVQ-NN with cytology and the proposed biomarkers improves significantly the correct classification of cervical precancerous lesions and/or cancer and may facilitate diagnosis and patient management.

Evaluation of the role of the new INNOVANCE PFA P2Y test cartridge in detection of clopidogrel resistance.

Light transmittance aggregometry (LTA) has been extensively used in monitoring clopidogrel therapy. However, the availability of simple and rapid point-of-care platelet function assays is of great clinical importance. Thus, the manufacturer of the Platelet Function Analyzer (PFA)-100 System has recently produced the INNOVANCE PFA P2Y test cartridge. We assessed the ability of this new test to reliably detect clopidogrel resistance. We enrolled 90 consecutive patients with coronary artery disease receiving chronic clopidogrel maintenance therapy in combination with aspirin. Twenty healthy volunteers served as controls. Clopidogrel resistance was simultaneously analysed by the INNOVANCE PFA P2Y test cartridge, ADP-induced LTA, the flow-cytometric vasodilator-stimulated phosphoprotein (VASP)-phosphorylation assay and the multiple electrode aggregometry (Multiplate). Agreement among the four platelet function methods by two was assessed using Cohen's kappa coefficient. According to the cut-off points for clopidogrel resistance proposed by the literature, agreement was fair between INNOVANCE PFA-100 P2Y and LTA (74.4%) and Multiplate (75.6%), while poor agreement was noticed in VASP assay (63.3%). Based on cut-off points indicating a higher thrombotic risk, agreement between the PFA-100 System and the other three methods did not significantly differ compared to the previous cut-offs (72.2%, 71.1% and 55.1%, respectively). The INNOVANCE PFA-100 P2Y test seems to be comparable to other established platelet function assays in detecting clopidogrel resistance. However, the modest agreement among platelet function methods makes the performance of platelet function testing crucial with more than one technique in order to reliably identify poor responders to clopidogrel treatment.

The diagnostic accuracy of two methods for E6&7 mRNA detection in women with minor cytological abnormalities.

OBJECTIVE: To examine the diagnostic accuracy of nucleic acid sequence based amplification (NASBA) and flow cytometry for E6&7 human papillomavirus (HPV) mRNA detection in the triage of minor cytological abnormalities. DESIGN: Prospective diagnostic accuracy study. SETTING: Gynecology outpatient clinics of a university hospital. POPULATION: 472 women with low-grade squamous intraepithelial lesion (LSIL) or atypical squamous cells of undetermined significance (ASCUS). METHODS: Residual material of the liquid-based smears was tested by NASBA and by flow cytometry for E6&E7 mRNA expression. Histological diagnosis was used as reference standard. MAIN OUTCOME MEASURES: Accuracy indices of the two techniques and of type 16-specific NASBA for the detection of cervical intraepithelial neoplasia (CIN) 2+ and CIN3+, accuracy indices at age >35 years, correlation between NASBA and flow, comparison between integrated and episomal high-risk HPV infection for risk of CIN2+. RESULTS: Both tests showed increased positivity rates with increasing severity of the lesion (p < 0.05, chi-squared test for trend). There was a positive correlation between NASBA and flow results (phi coefficient = 0.325). NASBA-positive cases were more likely to have CIN2+ than were NASBA-negative/DNA-positive for types 16, 18, 31, 33, 45 (25/73 vs. 4/52, p= 0.0004; Fisher's exact test). In the LSIL group the NASBA accuracy indices for CIN3+ were: sensitivity 75%, specificity 78.7% and positivity rate 20.8%, and for flow 77.8%, 64.5% and 35.9%, respectively. CONCLUSIONS: NASBA has favorable specificity and positivity rates for triaging LSIL prior to colposcopy. A relatively low sensitivity warrants cytological surveillance of the NASBA-negative LSILs. Flow cytometry does not perform as well overall.

Assessment of static telecytological diagnoses' reproducibility in cervical smears prepared by means of liquid-based cytology.

OBJECTIVE: This study evaluated the accuracy and reproducibility of telecytological diagnoses proffered on the basis of digitized images from cervical smears prepared by means of liquid-based cytology. MATERIALS AND METHODS: Representative digital cytological images from a total of 404 cervical smears (benign, 135; atypical squamous cells of undetermined significance, 92; low-grade squamous intraepithelial lesion, 62; high-grade squamous intraepithelial lesion, 87; squamous cell carcinoma, 26; and adenocarcinoma, 2) were uploaded to the CytoTrainer e-learning telecytology platform (developed in the Department of Cytopathology, "ATTIKON" University General Hospital, Athens, Greece) with specific password-protected accounts and were reviewed remotely by four independent board-certified cytopathologists (checking round). Their reports were recorded and classified. After 12 and 24 months, the same representative digital images were transferred in random order to the same cytopathologists and were reviewed again (first and second review rounds, respectively). The cytopathologists' first and second round diagnoses were recorded and compared with their initial ones. RESULTS: Statistical evaluation of cytological diagnoses detected no significant difference in diagnostic accuracy among checking and review diagnoses. The overall interobserver agreement was almost perfect with κ values of 0.79-0.97, whereas intraobserver agreement ranged from almost perfect to perfect with κ values of 0.76-1 in all diagnostic rounds. CONCLUSIONS: Digital images of cervical smears can be used for rapid and accurate diagnosis, by diminishing turnaround times and improving small cytology departments' quality indices. Diagnoses made by using static telecytological systems can be as reliable as those made by using conventional microscopy, under the conditions that representative images are taken and that standard cytological diagnostic criteria are applied. Telecytology can be used as an alternative method for the cytologic diagnosis of cervical smears, particularly in quality assurance programs.