Modified SureSelectQXT Target Enrichment Protocol for Illumina Multiplexed Sequencing of FFPE Samples.

BACKGROUND: Personalised medicine is nowadays a major objective in oncology. Molecular characterization of tumours through NGS offers the possibility to find possible therapeutic targets in a time- and cost-effective way. However, the low quality and complexity of FFPE DNA samples bring a series of disadvantages for massive parallel sequencing techniques compared to high-quality DNA samples (from blood cells, cell cultures, etc.). RESULTS: We performed several experiments to understand the behaviour of FFPE DNA samples during the construction of SureSelectQXT libraries. First, we designed a quality checkpoint for FFPE DNA samples based on the quantification of their amplification capability (qcPCR). We observed that FFPE DNA samples can be classified according to DIN value and qcPCR concentration into unusable, or low-quality (LQ) and good-quality (GQ) DNA. For GQ samples, we increased the amount of input DNA to 150 ng and the digestion time to 30 min, whereas for LQ samples, we used 50 ng of DNA as input but we decreased the digestion time to 1 min. In all cases, we increased the cycles of the pre-hyb PCR to 10 but decreased the cycles of the post-hyb PCR to 8. In addition, we confirmed that using half of the volume of reagents can be beneficial. Finally, in order to obtain better results, we designed a decision flow-chart to achieve a seeding concentration of 12-14 pM for MiSeq Reagent Kit v2. CONCLUSIONS: Our experiments allowed us to unveil the behaviour of low-quality FFPE DNA samples during the construction of SureSelectQXT libraries. Sequencing results showed that, using our modified SureSelectQXT protocol, the final percentage of usable reads for low-quality samples was increased more than three times allowing to reach median depth/million reads values of 76.35. This value is equivalent to ~ 0.9 and ~ 0.7 of the values obtained for good-quality FFPE and high-quality DNA respectively.

High sensitivity cardiac troponin T and interleukin-6 predict adverse cardiovascular events and mortality in anticoagulated patients with atrial fibrillation.

UNLABELLED: There are limited data on the prognostic role of biomarkers in anticoagulated patients with atrial fibrillation (AF). We evaluated the prognostic value of high sensitivity TnT (hsTnT) and high-sensitivity interleukin-6 (hsIL6) in a large cohort of AF patients taking oral anticoagulant therapy (OAC) as both biomarkers have been associated with adverse cardiovascular events. METHODS: We studied 930 patients (51% male; median age 76) with permanent/ paroxysmal AF who were stabilized (for at least 6 months) on OAC (INRs 2.0-3.0). Plasma hsTnT and hsIL6 levels were quantified by electrochemiluminescense immunoassay at baseline. Patients were followed-up for up to 2 years, and adverse events (thrombotic and vascular events, mortality and major bleeding) were recorded. RESULTS: At follow-up, 96 patients (3.97%/year) died whilst 107 had an adverse cardiovascular event (3.14%/year). On multivariate analysis, high hsTnT and high hsIL6 remained significantly associated with prognosis even after adjusting for CHADS2 score: HR 2.21 (1.46-3.35, P<0.001) for high hsTnT and 1.97 (1.29-3.02, P=0.002) for high hsIL6, for adverse cardiovascular events. For all-cause mortality, the HRs were 1.79 (1.13-2.83, P=0.013) and 2.48 (1.60-3.85, P<0.001), respectively. The integrated discrimination index (IDI) values of clinical scores (CHADS2 and CHA2 DS2-VASc) were improved by the addition of hsTnT and/or hsIL6 (all P<0.05). CONCLUSION: In a large 'real world' cohort of anticoagulated AF patients, both hsTnT and hsIL6 levels provided prognostic information that was complementary to clinical risk scores for prediction of long-term cardiovascular events and death, suggesting that these biomarkers may potentially be used to refine clinical risk stratification in AF.

Strontium levels in different causes of death: diagnostic efficacy in drowning.

Trace element determination can be applied in forensic medicine to diagnose the cause of death. Drowning is the second leading cause of death from unintentional injury. Despite the many diagnostic methods used, the post-mortem diagnosis of drowning continues to be one of the most difficult in forensic pathology. Strontium is a highly sensitive marker of water aspiration in a liquid medium rich in this metal. The aims of this study were to confirm the diagnostic value of strontium in cases of drowning compared with other causes of death, to analyse factors that could affect its concentration and to ascertain the sensitivity and specificity of strontium in right and left ventricles and peripheral serum for the post-mortem diagnosis of drowning. We studied 120 cadavers selected from medico-legal autopsies with different causes of death. Strontium (Sr) levels were measured in the serum (left and right ventricles and peripheral vein) of all cadavers and, in the case of drowning, in the water medium itself, by using Zeeman AAS. Our results confirm the usefulness of blood Sr levels for diagnosing seawater and freshwater drowning, although great care should be exercised in the latter case.

[The Langerhans cell histiocytosis with thymic localization as initial and exclusive place]. / Histiocitosis de células de Langerhans con afectación tímica como manifestación única de la enfermedad.

The Langerhans' cell histiocytosis (LCH), also known as Histiocitosis X it is an illness not very frequent granulomatosus etiology not clarified yet, that it can have different manifestations and localizations, however the thymic localization as initial and exclusive place gives presentation HCL it is quite unusual. The present case is presented a patient that debuted with a clinical unspecific, where the tests give image they put she gives apparent a mass in previous mediastinum and that after the pathologic and immunohistochemical analysis they evidenced a proliferation Langerhans s cells and eosinophils it being positive for CD1a and S-100 confirming the diagnosis of the LCH.

[Calcifications in renal adenocarcinoma. Image assessment in 17 cases]. / Estudio de las calcificaciones en el adenocarcinoma renal. Valoración de las imágenes en 17 casos.

Of 60 renal adenocarcinoma tumors that had been diagnosed at our hospital between 1984 and 1990, 17 had presented intratumoral calcification. These 17 tumors with calcification were studied to assess the sensitivity of different techniques relative to detection, localization and morphologic characterization of the calcifications. We can conclude from the study that the use of CT and US not only raised detection rate (28%), but also permitted more precise evaluation of the calcified tumor mass. Furthermore, tumor spread beyond the calcified area was found to be a sign of malignancy.

[Wunderlich disease. First manifestation of a renal adenocarcinoma]. / Enfermedad de Wünderlich. Primera manifestación de un adenocarcinoma renal.

We present a case of the classical Wünderlich syndrome or circumscribed extracapsular renal hematoma in a 54-year-old male patient who had consulted for an inguinal mass and hemiscrotal ecchymosis. The patient had referred an episode of intense pain in the left renal fossa and the clinical picture of syncope 3-4 days earlier. Surgery disclosed severe perirenal hemorrhage from an undiagnosed renal adenocarcinoma that had ruptured.

[Pyogenic hepatic abscesses: role of ultrasonography in their diagnosis and treatment]. / Abscesos hepáticos piógenos: valor de la ultrasonografía en su diagnóstico y tratamiento.

We present 26 cases of pyogenic hepatic abscess diagnosed by echography, 14 of which were treated by percutaneous drainage guided by ultrasound. An analysis is made of the clinical and radiological findings, as well as the differential diagnosis of focal hepatic lesions. Due to the lack of clinical specificity in many patients, we recommend fine needle biopsy directed by ultrasound to confirm the suspicion of pyogenic hepatic abscess and isolation of noncausative microorganisms. We think that the treatment of choice in these patients is percutaneous drainage guided by echography.

Computed tomography in hepatic echinococcosis.

Computed tomography (CT) was used to evaluate 50 cases of hydatid disease of the liver. It was definite in 49 cases and negative in one case. Pre- and postcontrast scans were performed. CT may reveal the exact location and extension of cysts and possible complications. However, a false-negative case was found in a hydatid cyst located in a fatty liver.