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• Diagnosis of cystinuria is important in the long-term management of patients • Quantitative chromatographic methods are time-consuming and costly • FTIR and chemometric analysis identified cystinuric patients with a sensitivity of 1.0 • ATR-FTIR as screening test may reduce time and cost analysis for cystinuria diagnosis Cystinuria is an inborn congenital disorder characterised by a defective cystine metabolism resulting in the formation of cystine stones. The Brand's test, used for diagnosis, requires dangerous substances, so has been replaced with high-performance liquid chromatography with fluorimetric detection (HPLC-FL). However, this technique requires the use of complex equipment. Infrared spectroscopy, universally used for stone analysis, recently was employed to detect insoluble cystine in urine. The aim of this study is to evaluate Infrared Spectroscopy combined with chemometric analysis as screening method to identify those patients requiring confirmation by HPLC-FL analysis. We examined 24 h urine specimens from 57 patients. The quantitative analysis was performed by HPLC-FL. The infrared spectroscopic urine sediment analysis was performed with an ATR accessory (ATR-FTIR). Urine is centrifuged, the supernatant is discarded, and the sediment is dried on to the ATR prism surface. Statistical analysis was performed using a custom-made software developed in MATLAB environment. The HPLC-FL determination showed a normal excretion of cystine in 49 samples and an abnormal excretion in the remaining 8 samples. The ATR-FTIR analysis combined with a statistical approach gives a sensitivity of 1.0 and a specificity of 0.82 were obtained. The introduction of the ATR-FTIR technique in our clinical laboratory setting may reduce time and cost analysis for diagnosis of cystinuria. [ABSTRACT FROM AUTHOR]

• Large Genomic Rearrangements (LGRs) represent a relevant proportion of BRCA variants. • The detection of BRCA mutations is relevant to PARPi therapy administration. • The demand of BRCA test performed on tumor tissue is steadily increasing. • In somatic test the evaluation of LGRs represents a great challenge. • We showed how the ddPCR is a useful method for assessing somatic BRCA LGRs. With the introduction of Olaparib as target therapy for High Grade Serous Ovarian Cancer (HGSOC) patients with germline and somatic BRCA1/2 mutations, the genetic test performed on tumor tissue has become important like the germline test. In somatic testing the evaluation of Large Genomic Rearrangements (LGRs) represents the main challenge. We describe a droplet digital PCR (ddPCR) assay for the evaluation of target BRCA1 LGRs on blood and formalin-fixed paraffin-embedded (FFPE)/Fresh Frozen Tissue (FFT) samples. We analyzed blood, FFPE and FFT samples in a validation setting of n = 78 HGSOC patients. We applied the ddPCR to BRCA1 exons 2, 20 and 21 as some of the most common BRCA1 exons involved in LGRs in our cohort of patients. The ddPCR custom assays allowed the identification of LGRs in all sample types, including FFPE specimens. Moreover, we were able to clearly detect LGRs accounted as somatic event. The introduction of ddPCR in a comprehensive workflow, encompassing both germline and somatic tests, represents an improvement in BRCA1/2 testing. ddPCR can overcome challenges related to BRCA testing, especially on FFPE analysis. Finally, ddPCR represents a promising alternative strategy to the established standard methods currently used in clinical setting. [ABSTRACT FROM AUTHOR]

Background The aim of this study was to assess the ability of a Next Generation Sequencing (NGS)-based approach in the detection of BRCA1/2 Large Genomic Rearrangements (LGRs). Methods We investigated 287 consecutive unrelated Italian women affected by breast or ovarian cancer. A NGS pipeline with a reliable Copy number Variation (CNV) prediction algorithm was applied. In addition, all samples were investigated using MAQ (Multiplex Amplicon Quantification) assay, while the MLPA (Multiplex Ligation-dependent Probe Amplification) technique was used as alternative method if necessary. Results Small pathogenic variants were identified in 80 samples. During NGS analysis, 11 samples were identified by Amplicon Suite Software (SmartSeq, Novara, Italy) as positive for large deletions or duplications. However, MAQ assay provided positive results in 19 patients. In particular, BRCA2 exon 25 deletion, not revealed by NGS CNV prediction algorithm, was detected in 8 unrelated women as false positive result. Sequencing analysis showed IVS24-113T/G (c.9257-113T>G) variant in 6 of these 8 patients, while the novel IVS24-129G>A (c.9257-129G>A) variant was detected in only two samples. Discussion The prevalence of large rearrangements in BRCA1/2 genes was the 12% of all disease-causing mutations detected in our patients. In particular, BRCA1 rearrangements were the 14.5% of all BRCA1 causing variants identified. Differently, BRCA2 large deletions were only the 6.9% of all mutations occurring in this gene. While MAQ assay showed 2,8% of false positive results, our integrative NGS-based approach fully satisfied the sensitivity and specificity parameters required on the BRCA1/2 LGRs detection. The workflow represents a robust and easy-to-use method for full BRCA1/2 screening, which can be easily implemented in routine diagnostic testing. [ABSTRACT FROM AUTHOR]

• Arginine metabolism is important to evaluate organism oxidative stress. • LC-MS/MS methods are considered gold standard for small molecules determination. • Developed method is easy to implement in research and routine purpose. Arginine metabolism is involved in the regulation of several biological processes. Many liquid chromatography tandem-mass spectrometry methods for the determination of arginine and its metabolites have been developed but they are time consuming and imply long pre-analytical procedures. The purpose of this study was to develop a rapid method for the simultaneous determination of arginine, citrulline, ornithine, symmetric and asymmetric dimethylarginine and monomethylarginine in human plasma. The pre-analytical procedure consisted in a simple deproteinization. The chromatographic separation was performed using hydrophilic interaction liquid chromatography. Analytes detection was performed with a triple quadrupole equipped with electrospray ion source operating in positive ion mode. Mass spectrometry experiments were conducted in multiple reaction monitoring mode. Recovery ranged from 92.2 to 108.0%. The within-run imprecision and between-run imprecision ranged from 1.5 to 6.8 % and 3.8 to 11.9%, respectively. Carry over and matrix effect did not affect quantitative analysis. Extraction recovery was between 95 and 105 %. Stability after pre-analytical procedure was tested and all the metabolites were stable after 48 h at 4 °C. In conclusion, our novel method allow a rapid and easy determination of arginine and its metabolites both for research and clinical routine use. [ABSTRACT FROM AUTHOR]

Abstract: Background: The development of MALDI Imaging Mass Spectrometry is a promising technique in the investigation of biological molecular repertoire. We have pursued an analytical assessment of this technique in its application to proteome analysis. Methods: A specific statistical method of analysis has been developed to enable data processing in the absence of internal standards, by defining similarity scores. Results: The investigated linear mode MALDI-TOF set-up allows to obtain data variations comprised within the 30% of variation when assaying tissues samples from the same animal, while the 60% of variation was highlighted in the inter-mice series assaying syngenic animals. Conclusions: This analytical assessment represents the first step of a process that should validate the utilisation of this technique in the clinical practice. [Copyright &y& Elsevier]