A case report: Use of FT-IR analysis to improve Colovesical fistula diagnosis.

Colovesical fistula (CVF) is an abnormal connection between the colon and the urinary bladder. Faecaluria, reported in 40-70% of cases, is virtually pathognomonic for CVF. During the 5th day of recovery in an 84 years old subject, the passage of cloudy, malodorous urine with visible debris was observed. According to the pathognomonic character of faecaluria, the sample was signed to the laboratory for biochemical and microbiological investigation, able to define the type and origin of materials. Following clinical requirements, both biochemical pathways and instrumental procedures able to confirm or exclude the presence of faecal components in urine were considered. No biochemical compound or component addressing faecal compounds in urine results available between laboratory tests. The brown powder component of the pellet was identified as Keratin, with 90% overlapping with the reference spectrum of the compound. FT-IR analysis on urine pellet can be proposed as a simple, non-invasive, and fast method to improve the diagnostic course of CVF.

KDIGO 2012 Clinical Practice Guideline CKD classification rules out creatinine clearance 24 hour urine collection?

OBJECTIVES: The recent guideline for the evaluation and management of Chronic Kidney Disease recommends assessing GFR employing equations based on serum creatinine; despite this, creatinine clearance 24-hour urine collection is used routinely in many settings. In this study we compared the classification assessed from CrCl (creatinine clearance 24h urine collection) and e-GFR calculated with CKD-EPI or MDRD formulas. DESIGN AND METHODS: In this retrospective study we analyze consecutive laboratory data: creatinine clearance 24h urine collection, serum creatinine and demographic data such as sex and age from 15,777 patients >18 years of age collected from 2011 to 2013 in our laboratory at Careggi Hospital. The results were then compared to the estimated GFR calculated with the equations according to the recent treatment guidelines. Consecutive and retrospective laboratory data (creatinine clearance 24h urine collection, serum creatinine and, demographic data such as sex and age) from 15,777 patients >18 years of age seen at Careggi Hospital were collected. RESULTS: Comparison between e-GFR calculated with CKD-EPI or MDRD formulas and GFR according CrCl determinations and bias [95% CI] were 11.34 [-47,4/70.1] and 11.4 [-50.2/73] respectively. The concordance for 18/65 years aged group when compared with e-GFR classification between MDRD vs CKDEPI, MDRD vs CrCl and CKD-EPI vs CrCl were 0.78, 0.34, and 0.41 respectively, while in the 65/110years aged group the concordance Kappas were 0.84, 0.38, and 0.36 respectively. CONCLUSIONS: The use of CrCl provides a different classification than the estimation of GFR using a prediction equation. The CrCl is unreliable when it is necessary to identify CKD subjects with decrease of GFR of 5ml/min/1.73m(2)/year.

Relationship between hypertension, diabetes and proteinuria in rural and urban households in Yemen.

Little information is available on the meanings of proteinuria in low-resource settings. A population-based, cross-sectional survey was performed in Yemen on 10 242 subjects aged 15-69 years, stratified by age, gender and urban/rural residency. Hypertension is defined as systolic blood pressure (BP) of 140 mm Hg and/or diastolic BP of 90 mm Hg, and/or self-reported use of antihypertensive drugs; diabetes is diagnosed as fasting glucose of 126 mg dl(-1) or self-reported use of hypoglycaemic medications; proteinuria is defined as +1 at dipstick urinalysis. Odds ratios (ORs) for associations were determined by multivariable logistic regression models. Prevalence (weighted to the Yemen population aged 15-69 years) of hypertension, diabetes and proteinuria were 7.5, 3.7 and 5.1% in urban, and 7.8, 2.6 and 7.3% in rural locations, respectively. Proteinuria and hypertension were more prevalent among rural dwellers (adjusted ORs 1.56; 95% confidence limit (Cl) 1.31-1.86, and 1.23; 1.08-1.41, respectively), diabetes being less prevalent in rural areas (0.70; 0.58-0.85). Differently from hypertension and diabetes, proteinuria was inversely related with age. Most importantly, 4.6 and 6.1% of urban and rural dwellers, respectively, had proteinuria in the absence of hypertension and diabetes. The approach of considering kidney damage as a consequence of hypertension and diabetes might limit the effectiveness of prevention strategies in low-income countries.

The impact of fine-tuning of optical recognition system on database reliability.

Although optical reading systems are important tools to transfer data from a paper form to electronic databases, the impact of system fine-tuning on the final error rate is not usually considered. At the end of a multi-step process involving paper form design training of operators, and fine-tuning procedure, the final rate of error can be reduced from 0.65% to 0.05%. Fine-tuning should be introduced as a standard procedure while using optical reading systems.

Evaluation of diagnostic accuracy of screening by fecal occult blood testing (FOBT). Comparison of FOB Gold and OC Sensor assays in a consecutive prospective screening series.

We evaluated a new immunological fecal occult blood testing assay (FOB Gold, Sentinel = SENT) compared to the assay currently employed in the Florence screening program (OC-Hemodia, Eiken = OC). A total of 4,133 subjects were screened with both tests and underwent colonoscopy if positive (100 ng/mL Hb cutoff) to either test: 190 (4.59%) were positive (OC =140 (3.4%); SENT = 131 (3.2%)). The relative sensitivity for 7 cancers was 100% with OC and 67.9% with SENT, and for 48 high-risk adenomas (HRAs) it was 77.0% with OC and 66.6% with SENT. The positive predictive value (PPV) for cancer+HRA was 31.4% for OC and 28.2% for SENT and the specificity was 97.7 for both. The differences were not statistically significant. Adding SENT to OC increased the positivity rate by 32% and the cancer+HRA detection rate by 25%, and decreased the PPV by 10%. Both tests were performed on the same tubes in 1,601 cases, and in 18 of 47 cases they differed on different tubes but not on the same tube, suggesting inhomogeneous Hb content or varying fecal matrix influence in different samples. SENT has practical advantages for screening [corrected] (fully automated, high output, requires no dedicated instrument), a comparable specificity and a lower sensitivity, though the latter difference may be partially ascribed to differences in sampling and not to the assay itself [corrected] Because of the statistical insignificance of the differences, further studies are needed for confirmation.

The c-erbB-2 oncogene amplification by competitive PCR in aneuploid cell clones flow sorted from breast cancer samples.

The amplification of c-erbB-2 oncogene has been reported to have clinical relevance as a prognostic index in breast cancer. However, controversies still remain about its interpretation, mainly due to the inaccuracy of methods used for this purpose and to the unpredictable variability of the ratio between cancer and normal cells. Accurate quantitative assay, combined with strategies for selection or enrichment of tumor cell populations, could shed a new light on the relationships between molecular alterations and their clinical relevance. In this study, amplification of c-erbB-2 was measured by competitive PCR in 21 aneuploid breast cancers using a multiple DNA competitor both in whole homogenized cancer cells and in aneuploid enriched clones obtained after flow cytometry cell sorting. Most breast cancers (10/12) carrying c-erbB-2 oncogene amplification showed a significant increase in copy number in sorted aneuploid clones, and 2/9 apparently not amplified in basal samples were found to be amplified after being sorted for the aneuploid population. A general concordance between amplification and c-erbB-2 overexpression was found. The mean degree of amplification in sorted aneuploid clones is increased in breast cancers with the highest levels of c-erbB-2 protein overexpression. These data indicate that in breast cancers the amplification of c-erbB-2 oncogene is mainly associated with aneuploid cells.

Preoperative evaluation of tumor ploidy in endometrial carcinoma: An accurate tool to identify patients at risk for extrauterine disease and recurrence.

BACKGROUND: Tumor ploidy is a strong prognostic factor in patients with endometrial carcinoma, but generally is evaluated only after surgery. The availability of a simple and reliable method to determine tumor ploidy before any treatment is initiated could be helpful in the selection of patients at high risk for advanced primary disease and subsequent recurrence, with several possible benefits. The objectives of the current study were: 1) to test the accuracy of flow cytometric determination of tumor ploidy from preoperative outpatient endometrial biopsies compared with standard postoperative evaluation from the surgical specimen and 2) to correlate this preoperative parameter with the local recurrence and extrauterine tumor spread. METHODS: Tumor ploidy from both preoperative biopsy material and the macroscopic surgical specimens was evaluated prospectively in 50 consecutive patients with endometrial carcinoma. DNA analyses were performed in a blind fashion. Patients were followed for a median of 26 months (range, 16-46 months). RESULTS: In 9 of 50 cases (18%) an aneuploid tumor was found by the standard postoperative analysis. All 9 aneuploid tumors (100%) also were identified correctly by the preoperative test on biopsy material. Occult extrauterine tumor spread was found in 10 patients (20%). The incidence rate of aneuploidy among these tumors was 50% compared with 10% in surgical International Federation of Gynecology and Obstetrics Stage I tumors (P = 0.01). The recurrence rate was 55.5% (5 of 9 tumors) in the aneuploid group and 2.4% (1 of 41 tumors) in the diploid group (P < 0.001). The disease free survival rates of patients with diploid and aneuploid tumors were 97.5% and 44.4%, respectively (P < 0.0001). CONCLUSIONS: Preoperative tumor ploidy determination based on outpatient endometrial biopsy is as accurate as the standard postoperative evaluation in patients with endometrial carcinoma. Tumor aneuploidy confirms the usefulness of this method in selecting patients at risk for occult extrauterine tumor diffusion and recurrence.

Reticulocytes and reticulated platelets: simultaneous measurement in whole blood by flow cytometry.

Reticulated platelets are a fraction of newly released circulating elements characterized by a residual amount of RNA. It has been suggested that the reticulated platelet count, providing an estimate of thrombopoiesis in the same way as erythrocyte reticulocyte count is a measure of erythropoiesis, may be useful in the study of thrombocytopenic disorders. Reticulated red cells and platelets can be analyzed by flow cytometry using specific stains for nucleic acids such as Thiazole Orange and Auramine-O. The aim of our work was to perform the simultaneous evaluation of reticulated elements in whole blood using a standard flow cytometer and to correlate the results obtained with a dedicated cytometer. A group of 14 patients with abnormal absolute reticulocyte counts (range 1.1-11%) and a group of 41 patients showing a platelet discrimination error when analyzed with a dedicated flow cytometer (Sysmex R1000) were enrolled. Linear amplification of both scatter and fluorescence was used to perform reticulocyte count. A gate was set on platelet dimensions, and logarithmic amplification of scatter and fluorescence was used to count reticulated platelets. A good correlation was obtained both for results of reticulocyte count (r2 = 0.9825) and for reticulated platelets (r2 = 0.8717) between our method and those using dedicated instruments. These data show that reticulated platelet count may be easily introduced in clinical laboratories that routinely perform reticulocyte count by flow cytometry.

Expression of the retinoblastoma-related gene Rb2/p130 correlates with clinical outcome in endometrial cancer.

PURPOSE: The retinoblastoma gene is the prototype of tumor-suppressor genes and has been shown to be involved in the pathogenesis and progression of several human malignancies. In this study, we determined the relation between the expression of a newly discovered retinoblastoma-related gene Rb2/p130 and outcome in patients with endometrial carcinoma. PATIENTS AND METHODS: pRb2/p130 expression was determined immunohistochemically in specimens of endometrial carcinoma (stages I to IV) from 100 patients who underwent surgery as the first treatment. The pRb2/p130 status was analyzed in relation to the length of disease-free survival and disease-specific survival. RESULTS: Decreased levels of pRb2/p130 in endometrial cancer cells was significantly associated with a decreased probability of remaining disease-free after treatment (P = .003) and with decreased probability of survival (P < .0001). In a multivariate analysis, pRb2/p130 status (P = .004), tumor stage (P = .009), and ploidy status (P = .02) were independent predictors of clinical outcome. The risk of dying of disease was increased substantially (risk ratio, 4.91; 95% confidence interval, 1.66 to 14.54) among patients with decreased levels of pRb2/p130 in tumor cells. CONCLUSION: In patients with endometrial carcinoma who did not receive radiotherapy or chemotherapy before surgery, the presence of decreased levels of pRb2/p130 in tumor cells is associated with a significantly increased risk of recurrence and death of disease, independent of tumor stage and ploidy status.

Flow cytometric detection of circulating activated platelets in primary antiphospholipid syndrome. Correlation with thrombocytopenia and anticardiolipin antibodies.

Platelet activation has been suggested to play a crucial role in the pathogenesis of haemostatic disorders in antiphospholipid syndrome (APS). In 16 patients with primary APS (PAPS) we investigated by flow cytometry the presence of circulating activated platelets as defined by the surface expression of activation-dependent glycoproteins CD62 and CD63. In addition, the relationships among activated platelets, thrombocytopenia, antiphospholipid antibodies (aPL) and platelet associated IgG (PalgG) were evaluated. Compared to normal subjects CD62, but not CD63 expression, was found significantly increased in patients. All thrombocytopenic subjects showed a percentage of CD62 expressing platelets above the cut off. In thrombocytopenics a significantly increased percentage of CD62 and higher levels of aCL IgG were found compared to PAPS patients with normal platelet count. No correlation was found between activated platelets and both lupus anticoagulant antibodies and PalgG. Our data demonstrate that circulating activated platelets are detectable by flow cytometry in the majority of PAPS patients and suggest the existence of a relationship among activated platelets, thrombocytopenia and aPL levels.

Flow cytometric measurement of DNA content in human solid tumors: a comparison with cytogenetics.

The aims of this study were: (1) to test the accuracy of flow cytometery (FC) in the measurement of DNA content in human solid tumors, (2) to correlate the FC DNA-index (DI) with the chromosome modal number (CMN) provided by cytogenetic analysis (CG), and (3) to investigate the most frequent pitfalls in FC histograms classification. FC and CG analyses were performed in parallel on 113 samples of human solid tumors of different origin. FC provided an evaluable histogram in 110 out of 113 cases (97%), whereas a successful CG culture was obtained in 79 out of 113 samples (72%). In the 79 cases evaluable by both FC and CG, a concordant ploidy status was found in 66 cases (84%) (47 diploid and 19 aneuploid) (P < 0.001, chi-square test). In the 19 concordant aneuploid tumors a close correlation between the CMN and the DI was found (y = 0.019 x + 0.151; r = 0.860). Concerning the 13 discordant cases, 11 (85%) were classified as aneuploid by FC and as diploid by CG, while 2 cases (15%) were CG aneuploid (1 near-diploid and 1 tetraploid) and FC diploid. The current study suggests that FC is a reliable method for the measurement of tumor DNA content of the studied solid tumors. Special attention should be paid to the improvement of DNA histograms quality, in order to reduce the difficulties in the detection of near-diploid and near-tetraploid cell populations. Multiple sampling should be warranted whenever possible.

Prognostic value of flow cytometric deoxyribonucleic acid index in endometrial carcinoma: comparison with other clinical-pathologic parameters.

OBJECTIVE: The aims of the current study were to verify the impact of flow cytometric deoxyribonucleic acid index on clinical outcome in endometrial carcinoma and to assess whether its value is independent from the other clinical-pathologic features. STUDY DESIGN: In a prospective series 74 cases of endometrial carcinoma with surgery performed at our institution were studied. Flow cytometry was performed on fresh tumor samples. The median follow-up period was 31 months (range 8 to 52). Disease-free survival and actuarial survival were the end points of the study. RESULTS: Among the 74 patients (53 with diploid and 21 with aneuploid tumors) there were 14 recurrences and 10 deaths caused by the disease. The recurrence rate was 7.5% for the diploid and 47.6% for the aneuploid tumors (p < 0.001). Mortality was 3.8% for diploid and 38% for aneuploid cases (p < 0.001). Disease-free survival was 89.1% in the former group and 36.3% in the latter. Actuarial survival (Kaplan-Meier method) was 94.7% and 49.5%, respectively. Deoxyribonucleic acid index, stage (International Federation of Gynecology and Obstetrics), and grade of differentiation were significantly correlated with survival, whereas age, depth of myometrial invasion, and histologic type were not. In a multivariate analysis (Cox proportional hazards) deoxyribonucleic acid index was the strongest independent predictor of clinical outcome, followed by International Federation of Gynecology and Obstetrics stage, whereas grade of differentiation yielded no independent prognostic information. CONCLUSIONS: The flow cytometric deoxyribonucleic acid index is an important independent prognosticator, and its determination should be included in the standard management of endometrial cancer.

Estrone 3-glucuronide chemiluminescence immunoassay (LIA) and 17beta estradiol radioimmunoassay (RIA) in the monitoring of superovulation for in vitro fertilization (IVF): correlation with follicular parameters and oocyte maturity.

Many works in the literature of the last years had reported that urinary approach to superovulation study is a suitable method to evaluate ovarian response to pharmacological stimulation. Before applying urinary determination of hormonal levels with a chemiluminescence immuno assay (LIA) method in early morning urine (EMU) samples, we had studied the correlation of RIA-LIA procedures with reference to follicular volumes at hCG day and to recovered oocyte maturity; in fact follicular growth and oocyte morphological features are the main parameters to evaluate a successful induced cycle. In our department the IVF cycles are daily monitored with RIA seric E2 and LIA E1-3G determination, besides ultrasound examination of follicular growth. We have studied E2 and E1-3G levels on the hCG administration day and their correlation with follicular areas and volumes; moreover, we have evaluated hormonal values on oocyte pick-up day with reference to recovered oocyte number and maturity. We have assumed as good timing for oocyte pick-up when more than 50% of recovered oocytes were of good quality (maturity score 4). We have observed that the highest pre ovulatory E1-3G value is consistent with the best timing for oocyte pick-up; it's possible to obtain a conversion coefficient follicular volumes and urinary E1-3G excretion. We have not found significant differences between plasmatic and urinary estrogenic parameters. It is important to remember the advantages connected by a not isotopic and not invasive method. The absence of discomfort for the patients may be a decisive factor to choose the monitoring method and LIA procedure may represent a valid alternative to RIA.

Measurement of glucuronometabolites of 17 beta-estradiol and progesterone in early morning urine samples: a promising new method for the evaluation of hormonal responses of Buserelin-hMG treatment in an IVF program.

The success of IVF is related to the number and quality of embryos transferred. In our IVF program we have used two different protocols for superovulation induction. In this study a group of 35 patients was randomized into two groups; the first group (15 patients) was treated with pure FSH and hMG, in the other (20 patients) the normal pituitary function was inhibited with Buserelin before the stimulation with hMG. Ovarian stimulation was monitored by ultrasound examination and endocrine parameters. The RIA determination of 17-beta estradiol (17-E2) plasmatic levels is one of the most widely used techniques in IVF cycle monitoring; however many serious disadvantages are connected with the use of RIA (short half-life of the reagents; hazard of handling radioactive materials; frequent venipunctures for the patients). For these reasons, we propose a chemiluminescence immuno assay (LIA) method for the measurement of glucuronometabolites of 17-E2 and progesterone in early morning urine samples: this technique allows to follow hormonal levels for all the cycle, avoiding patient discomfort. We have found a highly significant correlation between serum and urinary hormonal values. We noted a marked variability in the individual estrogenic response and a significant difference in the response of the two groups. The mean peak on hCG administration day was 6.4 +/- 3.5 micrograms/h for urinary E1-3G and 1270 +/- 678 pg/ml for 17-E2 in Buserelin-hMG group: these hormonal levels appear lower than without ovarian desensitization; (in FSH-hMG group we have, on the same day, 9.7 +/- 3.2 micrograms/h for E1-3G and 1630 +/- 730 pg/ml for serum 17-E2).(ABSTRACT TRUNCATED AT 250 WORDS)