Seminal suPAR Levels as Marker of Abacterial Male Accessory Gland Inflammation in Hypogonadism.

BACKGROUND: Recent evidences suggest that hypogonadism is an important risk factor for lower urinary tract symptoms and benign prostatic hyperplasia. Several papers have discussed the role of chronic inflammation in the development of BPH, which may be modulated by the hypogonadal state. Soluble Urokinase-type Plasminogen Activator Receptor (suPAR), known protein marker of systemic inflammation, can be assayed in the seminal plasma and represents a reliable and sensitive marker of inflammation for the Male Accessory Gland Inflammation (MAGI). OBJECTIVE: The aim of this study has been to investigate if seminal suPAR is elevated in MAGI with hypogonadism and if suPAR represent a useful marker of abacterial inflammation in hypogonadism. METHODS: We included in the study twenty male patients aged between 25 and 55 year-old with secondary postsurgical hypogonadism. The same patients were also evaluated after a 3-month of Testosterone Replacement Therapy (TRT), to evaluate the effect of androgen replacement therapy on suPAR. Ten fertile men have been enrolled as a control group in the protocol. SuPAR concentrations were assayed on seminal plasma using an Enzyme-Linked Immunosorbent Assay (ELISA) kit. RESULTS: Hypogonadic patients presented significantly increased levels of seminal suPAR respect to controls (86.1±36.8 vs 55.2±20.0 ng/mL, p<0.05). TRT in hypogonadic patients has been associated with a significant reduction of suPAR levels as reported in the control group (50.9±22.91 vs 86.1±36.8 ng/ml p<0.05). CONCLUSIONS: These results confirm the role of suPAR as a protein marker of MAGI and support the hypothesis that hypogonadism induces a state of inflammation in male accessory glands which is involved in male infertility. Moreover demonstrated that testosterone treatment probably exerts a positive effect on MAGI and infertility as documented by reduction of suPAR levels in hypogonadic treated patients.

A STARD-compliant prediction model for diagnosing thrombotic microangiopathies.

Aim of the study was the definition of a predictive model for the initial diagnosis of thrombotic microangiopathies (TMA). We retrospectively collected data on all adult patients admitted to the Gemelli Hospital from 2010 to 2014. ICD-9 codes from primary diagnoses were used for TMA diagnosis. Demographic and laboratory characteristics on admission of patients with TMA were then compared with a random sample of 500 patients with other diagnoses. The prediction model was externally validated in a cohort from another hospital. Overall, 23 of 187,183 patients admitted during the study period received a primary diagnosis of TMA. LDH (OR 1.26, 95% CI 1.05, 1.63) and platelets (OR 0.96, 95% CI 0.94, 0.98) were the only independent predictors of TMA. The AUROC of the final model including only LDH and platelets was 0.96 (95% CI 0.91, 1.00). The Hosmer-Lemeshow (HL) test (p = 0.54) suggested good calibration. Our model also confirmed good discriminatory power (AUROC 0.72 95% CI 0.60, 0.84) and calibration (HL test p = 0.52) in the validation sample. We present a simple prediction model for use in diagnosing TMA in hospitalized patients. The model performs well and can help clinicians to identify patients at high risk of TMA.

Enhanced liver fibrosis test as a reliable tool for assessing fibrosis in nonalcoholic fatty liver disease in a clinical setting.

BACKGROUND: Liver fibrosis is the main determinant and predictor of the clinical course of nonalcoholic fatty liver disease (NAFLD). To date, a liver biopsy is still considered the gold standard for staging fibrosis. The aim of this study was to investigate the diagnostic accuracy of the commercial enhanced liver fibrosis (ELF) test manufacturer's cutoff value (≥9.8) in identifying severe fibrosis for adult patients with histologically confirmed NAFLD. METHODS: We tested the ELF test in a clinical practice, prospective cohort of 82 consecutive patients who consecutively underwent percutaneous liver biopsy. RESULTS: All stages of liver fibrosis were represented in our cohort, and severe fibrosis was present in 15 of 82 patients (18.3%). The stage of fibrosis was significantly associated with ELF score (Spearman's rho = 0.483, p<0.001). The commercial ELF test manufacturer's cutoff identified severe fibrosis with good sensitivity (86.7%; 95% confidence interval [95% CI], 0.69-1.04) and high specificity (92.5%; 95% CI, 0.86-0.99), with a positive predictive value of 72% and negative predictive value of 97%. CONCLUSIONS: Our data could support the use of the ELF test in clinical practice.

Identification and Characterization of a New BRCA2 Rearrangement in an Italian Family with Hereditary Breast and Ovarian Cancer Syndrome.

INTRODUCTION: Many studies document the involvement of BRCA1/2 gene rearrangements in genetic predisposition to breast and ovarian cancer. Large genomic rearrangements (LGRs) of BRCA1 account for 0-27% of all disease-causing mutations in various populations, while LGRs in BRCA2 are rarer. Here, we describe a novel BRCA2 LGR, involving the duplication of exons 4-26, in an Italian family with hereditary breast and ovarian cancer (HBOC) syndrome. OBJECTIVE: Our purpose was to provide an effective characterization of this variant using a combination of different methods able to establish the exact breakpoints of the duplication. METHODS: A multiplex amplicon quantification (MAQ) assay was used as the primary screening method in the detection of LGRs. Array comparative genomic hybridization (CGH), reverse transcriptase polymerase chain reaction (RT-PCR) and long-range PCR were used for the careful characterization of the rearrangement and breakpoint regions. The Repeat Masker program was employed to identify Alu sequences at breakpoint junctions. RESULTS: Array CGH and long-range PCR strategies revealed that the BRCA2 exons 4-26 duplication (g.12016_87170dup) involved exactly 75,154 bp nucleotides between intron 3 and intron 26 of the gene. Given that no Alu repeats were found at the junction sites, we support the hypothesis that the new duplication could be the result of a microhomology-mediated event (MH) involving very short homologous sequences at an upstream breakpoint. DISCUSSION: LGR investigation is mandatory in BRCA1/2 routine testing in order to provide a complete result for a targeted therapeutic decision. Nevertheless, the characterization and classification of novel BRCA1/2 variants represents a crucial step in the support of genetic counselling. Our results, including a comprehensive co-segregation analysis, indicate that the novel duplication identifed has a pathogenic role and would be considered a causing-disease variant in genetic and oncologic counselling.

Characterization of a new BRCA1 rearrangement in an Italian woman with hereditary breast and ovarian cancer syndrome.

BACKGROUND: We report a novel BRCA1 LGR, involving the complete duplication of exon 3, in an Italian patient with a strong family history of breast and ovarian cancer. Our purpose is to provide an effective characterization of this LGR using a combination of different methods able to establish the exact breakpoints of the duplication. METHODS: MAQ assay was used as primary screening method in LGRs detection. Array CGH, RT-PCR, and Long-PCR were used for a careful characterization of rearrangement and breakpoint regions. The Repeat Masker program was employed to identify Alu sequences at breakpoint junctions. RESULTS: RNA analysis showed that this in tandem duplication of exon 3 causes an in frame insertion of 18 amino acids within the protein. Array CGH and Long-PCR strategies revealed that the duplication (g.100411_102863dup) involves exactly 2.452 nucleotides between intron 2 and intron 3 of the gene. In addition, while an Alu Sx sequence was identified at upstream breakpoint, no Alu repeats were found at downstream junction. This supports the hypothesis that the new duplication was the result of a non-homologous recombination event between Alu and Non-Alu sequences. CONCLUSION: Our strategy, which combines a comprehensive set of methodologies, has been able to characterize the new BRCA1 duplication confirming, as previously reported, that MAQ assay represents a reliable and effective method for a primary screening of BRCA rearrangements. We underline the relevance of incorporating quantitative methods for BRCA genes dosage testing into routine diagnostic practice.

Competitive PCR-High Resolution Melting Analysis (C-PCR-HRMA) for large genomic rearrangements (LGRs) detection: A new approach to assess quantitative status of BRCA1 gene in a reference laboratory.

AIM OF THE STUDY: Evaluation of copy number variation (CNV) in BRCA1/2 genes, due to large genomic rearrangements (LGRs), is a mandatory analysis in hereditary breast and ovarian cancers families, if no pathogenic variants are found by sequencing. LGRs cannot be detected by conventional methods and several alternative methods have been developed. Since these approaches are expensive and time consuming, identification of alternative screening methods for LGRs detection is needed in order to reduce and optimize the diagnostic procedure. The aim of this study was to investigate a Competitive PCR-High Resolution Melting Analysis (C-PCR-HRMA) as molecular tool to detect recurrent BRCA1 LGRs. MATERIAL AND METHODS: C-PCR-HRMA was performed on exons 3, 14, 18, 19, 20 and 21 of the BRCA1 gene; exons 4, 6 and 7 of the ALB gene were used as reference fragments. RESULTS: This study showed that it is possible to identify recurrent BRCA1 LGRs, by melting peak height ratio between target (BRCA1) and reference (ALB) fragments. Furthermore, we underline that a peculiar amplicon-melting profile is associated to a specific BRCA1 LGR. All C-PCR-HRMA results were confirmed by Multiplex ligation-dependent probe amplification. CONCLUSIONS: C-PCR-HRMA has proved to be an innovative, efficient and fast method for BRCA1 LGRs detection. Given the sensitivity, specificity and ease of use, c-PCR-HRMA can be considered an attractive and powerful alternative to other methods for BRCA1 CNVs screening, improving molecular strategies for BRCA testing in the context of Massive Parallel Sequencing.

CYP21A2 intronic variants causing 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase gene (CYP21A2). Most of CYP21A2 mutations result from intergenic recombinations between CYP21A2 and closely linked CYP21A1P pseudogene. Rare mutations not generated by gene conversion account for 5-10% of 21-hydroxylase deficiency alleles. Intronic variants represent only a little part of these but their effect on the protein is generally deleterious. The aim of this paper is to provide a comprehensive literary review regarding all intronic CYP21A2 pathological variants reported to date. In addition, we describe three novel causing disease variants in our patients affected by the classic form of CAH: IVS4-1G>A, IVS5-8T>A, IVS8-2A>G. In silico analysis revealed that all these substitutions affect the splicing process leading to a non-functional protein. Based on these results, we are able to classify them as pathological variants according to the patient's phenotype.

Fecal but not serum calprotectin is a potential marker of GVHD after stem cell transplantation.

Gastrointestinal graft-versus-host disease (GvHD) represents a life-threatening complication after stem cell transplantation. Differential diagnosis between gut GvHD and other causes of diarrhea after HSCT is still subjected to endoscopy and histological findings. The research for a reliable biomarker for gut GvHD might allow an early diagnosis of this condition and a consequent prompt treatment that could reduce unfavorable outcomes. Recently, fecal calprotectin was reported as reliable marker of gut involvement. We would evaluate if serum instead of fecal calprotectin could be considered a possible biomarker of gut GvHD. Serum calprotectin was measured in a cohort of 54 patients submitted to allogeneic stem cell transplantation using ELISA assay. For a subset of 21 patients, calprotectin serum levels were compared with fecal calprotectin detection. Contrary to fecal calprotectin, we found only a trend to high level of serum calprotectin for GvHD development and gut involvement, but statistical difference was not reached. Fecal but not serum calprotectin could be considered as possible biomarker for gut GvHD.

Comparison of Fully Automated and Semiautomated Systems for Protein Immunofixation Electrophoresis.

BACKGROUND: In order to establish a diagnosis of monoclonal gammopathy, it is necessary to detect and identify monoclonal components. To confirm the immunological nature of the proteins, the next step is to define their composition in heavy and light chains using immunofixation. The purpose of this study was to compare two different instruments, one semiautomated and the other fully automated for serum and urine immunofixation. METHODS: We selected 150 sera and 100 urines from patients admitted for routine analysis, which were analyzed by immunofixation to characterize monoclonal components. RESULTS AND CONCLUSION: Comparison study showed a difference in the identification of small monoclonal components and hypogammaglobulinemia, in serum and urine, between the two analyzers. We also observed a difference in the length of the electrophoretic pattern that is of considerable importance as it leads to a better resolution of the gamma region, allowing to identify even the smallest monoclonal component that can be easily hide in an oligoclonal pattern. For this reason, there is need to ameliorate commercial immunofixation assays. It is essential to improve data harmonization and standardize measurement procedures in order to guarantee a correct diagnosis for the right patient care.

Follow-up of IgD-κ multiple myeloma by monitoring free light chains and total heavy chain IgD: A case report.

Immunoglobulin (Ig)D-κ multiple myeloma (MM) is a rare neoplastic disease characterized by an aggressive and rapidly progressing course, which constitutes only a very small proportion of all MM cases. In the present report, the clinical case of a 51-year-old Caucasian woman diagnosed with IgD-κ MM is described. The patient underwent different chemotherapeutic treatments subsequently to a single autologous stem cell transplantation. Despite the inherent difficulty of monitoring IgD levels and performing serum immunofixation electrophoresis, the clinical outcome of the patient was almost uniquely monitored by measuring the levels of κ and λ free light chains (FLCs) and total heavy chain IgD. The data suggest the non-invasive potential and usefulness of FLCs evaluation for early detection of stringent complete remission, follow-up and early detection of disease relapse. In addition, this diagnostic procedure has successfully been employed for the therapeutic monitoring of the present patient, and may represent a very helpful, non-invasive tool for the follow-up of IgD myeloma patients without the requirement of serial bone marrow aspirate.

Simultaneous quantification of 17-hydroxyprogesterone, androstenedione, testosterone and cortisol in human serum by LC-MS/MS using TurboFlow online sample extraction.

BACKGROUND: The simultaneous quantification of a steroid hormones panel provides more clinical information than a single steroid assay. Traditionally, steroids have been quantified with immunoassays which are characterized by high rate of positive results. Aim of this work, was to develop a TurboFlow-LC-MS/MS method for the simultaneous quantification of 17-hydroxyprogesterone, androstenedione, cortisol and testosterone in serum. METHODS: To 100µL of serum, 100µL of internal standard solution in methanol were added; after centrifugation the supernatant was injected in the TurboFlow for further purification. Steroids were determined using a TSQ Vantage operating with an atmospheric pressure chemical ionization source. Method was fully validated and results compared with immunoassay methods. RESULTS: Limit of quantification ranged from 0.02ng/mL to 1ng/mL. The precision was lower than 11% and accuracy ranged from 93.5 to 121.6%. The correlation was acceptable for all analytes except for low levels of testosterone. However, the Bland-Altman plots display a positive bias for androstenedione and 17-hydroxyprogesterone, and a negative bias for cortisol and testosterone. CONCLUSIONS: TurboFlow analysis provides a simple and effective clean-up procedure minimizing the interference of the matrix. The presented method is selective, precise, and sensitive being suitable in a clinical laboratory.

Urinary Albumin Detection: Comparison of Two Different Methods.

BACKGROUND: Monitoring urinary albumin is a useful method in clinical practice for the management of diabetic nephropathy, chronic kidney disease, and hypertension. Currently there are neither standardized methods nor reference material for the determination of urinary albumin; for this reason it is useful to compare different assays used in clinical laboratory. OBJECTIVES: The aim of this study is to verify analytical performance of an immunoturbidimetric assay on Roche Cobas 8000 platform and to compare urinary albumin results with those obtained by immunonephelometry on Siemens Dade Behring BN II Nephelometer. RESULTS: The method comparison showed a good linear relationship, confirmed by Passing-Bablok and Bland-Altman plots. The turbidimetric assay meets the requirements of accuracy and precision for the practice of medical diagnostics and clinical use. CONCLUSIONS: The present study can contribute to the methods standardization and harmonization of urinary albumin assay.

Validation of an LC-MS/MS Method for Urinary Lactulose and Mannitol Quantification: Results in Patients with Irritable Bowel Syndrome.

Aim. Lactulose/mannitol ratio is used to assess intestinal barrier function. Aim of this work was to develop a robust and rapid method for the analysis of lactulose and mannitol in urine by liquid chromatography coupled to tandem mass spectrometry. Lactulose/mannitol ratio has been measured in pediatric patients suffering from irritable bowel syndrome. Methods. Calibration curves and raffinose, used as internal standard, were prepared in water : acetonitrile 20 : 80. Fifty µL of urine sample was added to 450 µL of internal standard solution. The chromatographic separation was performed using a Luna NH2 column operating at a flow rate of 200 µL/min and eluted with a linear gradient from 20% to 80% water in acetonitrile. Total run time is 9 minutes. The mass spectrometry operates in electrospray negative mode. Method was fully validated according to European Medicine Agency guidelines. Results and Conclusions. Linearity ranged from 10 to 1000 mg/L for mannitol and 2.5 to 1000 mg/L for lactulose. Imprecision in intra- and interassay was lower than 15% for both analytes. Accuracy was higher than 85%. Lactulose/mannitol ratio in pediatric patients is significantly higher than that measured in controls. The presented method, rapid and sensitive, is suitable in a clinical laboratory.

Intraoperative high-dose calcium stimulation test in patients with sporadic medullary thyroid carcinoma is highly accurate in predicting lateral neck metastases.

BACKGROUND: Intraoperative measurement of calcitonin is not highly accurate in predicting the completeness of the operative resection after total thyroidectomy combined with central neck dissection (TT-CND) in patients with medullary thyroid carcinoma (MTC). We evaluated whether an intraoperative, high-dose calcium stimulation test (IO-CST) after TT-CND can predict lateral neck involvement. METHODS: Eleven patients who underwent primary operation for sporadic MTC were included. High-dose (25 mg/kg) calcium gluconate was administered after TT-CND with calcitonin measured at 2, 5, and 10 minutes after the calcium gluconate infusion. RESULTS: There were 2 males and 9 females (mean age, 51 years; range, 18-88). Three patients showed lateral neck metastases. At a mean follow-up of 7.0 months (range, 2-10), 1 patient showed distant metastases and 1 a slightly increased calcitonin level. After IO-CST, serum calcitonin increased in all the 3 patients with lateral neck metastases, and it remained unchanged or decreased in the other patients without lateral neck metastases. Percent variation of serum calcitonin after IO-CST was 92% in patients with lateral neck metastases and -3.1 ± 4.9% in patients without lateral neck metastases. CONCLUSION: Calcitonin measurement after IO-CST in patients with sporadic MTC can be highly accurate in predicting lateral neck nodes involvement. These results could represent a stimulus toward the development of a quick calcitonin assay.

Nasal lavage levels of granulocyte-macrophage colony-stimulating factor and chronic nasal hypereosinophilia.

BACKGROUND: The aim of the present study was to measure levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in nasal lavage of patients affected by chronic eosinophilic sinonasal inflammation to clarify the relationship with eosinophilic tissue infiltration and clinical features. METHODS: Between November 2012 and June 2013, we selected 70 patients with chronic eosinophilic inflammation (average age 41.8 years) who were classified into the following groups: persistent allergic rhinitis (group 1), noninfectious non-allergic rhinitis with eosinophilia syndrome (group 2) and chronic rhinosinusitis with polyps (group 3). Finally, we enrolled 20 healthy subjects as controls (group 4). All patients underwent symptoms score questionnaire based on a visual analogue scale, nasal endoscopy and/or computed tomography (CT) scan, and allergy testing. Nasal cytology by scraping of the mucosa and GM-CSF assays in nasal lavage were performed in all subjects. RESULTS: Detectable levels of GM-CSF were found in 34 of 70 (48.57%) patients, with an average concentration of 2.67 ± 0.8 pg/mL, whereas in controls only 1 of 20 individuals showed detectable GM-CSF levels. Eosinophil infiltration was significantly higher in patients with detectable GM-CSF compared to those with undetectable levels (49.4% vs 39.2%, respectively; p < 0.05). Furthermore, significant weakly-moderate correlation was found between GM-CSF levels and percentage of eosinophil infiltration in tissue (p < 0.05). Correlation between symptom scores and GM-CSF levels was significant only in group 2, which showed higher average concentrations of GM-CSF compared to groups 1 and 3 (2.9 pg/mL vs 1.6 pg/mL and 1.8 pg/mL, respectively; p < 0.05). CONCLUSION: Our data confirm that GM-CSF is more frequently detectable in nasal lavages of patients affected by chronic sinonasal eosinophilic inflammation than in controls. Statistical analyses revealed a significant weakly-moderate correlation between GM-CSF levels in nasal lavage of all patients and percentage of eosinophil infiltration of nasal mucosa.

Lactose intolerance genetic testing: is it useful as routine screening? Results on 1426 south-central Italy patients.

Adult-type hypolactasia is a widespread condition throughout the world, causing lactose malabsorption. Several studies suggested that the identification of C/T-13910 and G/A-22018 mutations, located upstream the gene encoding the lactase-phlorizin hydrolase (LPH), is a useful tool for the differential diagnosis of hypolactasia. We evaluated the frequencies of C/T-13910 and G/A-22018 variants in a central-south Italian population and the usefulness of lactase deficiency genetic testing in the clinic practice. The genomic DNA of 1426 patients and 1000 healthy controls from central-south Italy was isolated from peripheral whole blood and genotyped for the C/T-13910 and G/A-22018 polymorphisms by high-resolution melting analysis (HRMA) and sequencing. The frequencies of genotypes in the 1426 patients analysed were as follows: 1077 CC/GG (75.5%), 287 CT/GA (20.1%), 24 TT/AA (1.7%), 38 CC/GA (2.7%). Only 64 out of 1426 (4.5%) performed also L-BHT test, 29 of which were negative for L-BHT also in presence of different genotypes. Among the 35 individuals with L-BHT positive, 34 were CC/GG and only one CT/GA. Although lactose genetic test is a good predictor of persistence/non-persistence lactase in specific population, its use in the central-south Italy population should be limited given the high prevalence of the CCGG diplotype in normal individuals.

A preliminary Quality Control (QC) for next generation sequencing (NGS) library evaluation turns out to be a very useful tool for a rapid detection of BRCA1/2 deleterious mutations.

BACKGROUND: Recent advances in next generation sequencing (NGS) technology have enabled comprehensive and accurate screening of the entire genomic region of BRCA1/2 genes and, to date, many studies report the effectiveness of these technologies. Here we show that Gene Scan (GS) labeling Quality Control (QC), performed before massive parallel pyrosequencing, coupled with Multiple Amplicon Quantification software (MAQ-S) analysis is a rapid and powerful tool in the detection of deleterious BRCA mutations carried by different patients. METHODS: GS labeling QC assay was performed according to the manufacturers' instructions and MAQ-S software was employed for analysis results. RESULTS: GS labeling QC was able to detect 14 different BRCA frameshift mutations in our patients. In addition, two novel BRCA mutations (c.1893_1894insTTAAGCCCACAAAT in BRCA1 gene and c.9413_9414insT in BRCA2 gene) were identified. CONCLUSION: We prove that a simple QC step may represent a valid and useful tool for a rapid detection of frameshift mutations in BRCA genes. For this reason, we recommend using this approach before massive parallel sequencing.