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AIM: To analyze the accuracy of the sentinel lymphatic node biopsy (SLNB) and to investigate predictive factors for sentinel node (SN) status and prognostic factors for recurrence-free survival (RFS) and disease-specific survival (DSS) in patients with melanoma. MATERIAL AND METHODS: Between June 1997 and June 2017, 440 consecutive patients, who underwent SLNB by a single surgical team, were prospectively included. Descriptive and survival analysis were performed. RESULTS: 119 of 440 patients (26%) had positive SN. SLNB's false-negative rate was 6.3%. Breslow thickness, Clark´s level, ulceration and histological subtype were statistically significant predictive factors of SN metastases. In a multivariate analysis, positive SN (HR = 2.21, p = 0.01), deeper Breslow thickness (HR = 2.05, p = 0.013), male gender (RR = 2.05, p = 0.02), and higher Clark's level (HR = 2.30, p = 0.043) were significantly associated with decreased RFS; and positive SN (HR = 2.58, p < 0.001), deeper Breslow thickness (HR = 2.57, p = 0.006) and male gender (HR = 1.93, p = 0.006) were associated with lower DSS. CONCLUSION: SLNB is a reliable and reproducible procedure with high sensitivity (93.7%). Positive SN metastases, Breslow thickness and male gender were statistically associated with poorer outcomes. Male gender was an independent prognostic factor of tumor thickness or SN status.

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Introducción. La incidencia del melanoma cutáneo ha aumentando durante las últimas décadas en muchos países, pero desconocemos si esto también ha sucedido en Madrid. Hemos analizado los cambios epidemiológicos ocurridos en el melanoma cutáneo durante un período de 15 años. Pacientes y métodos. Se obtuvieron de forma retrospectiva los datos referentes a todos los pacientes con melanoma cutáneo atendidos en el Hospital Gregorio Marañón de Madrid entre los años 1996-2010, dividido en 3 periodos: 1996-2000; 2001-2005, y 2006-2010. Resultados. Se diagnosticaron histológicamente un total de 969 melanomas. La edad media en el momento del diagnóstico fue de 58,5 años. El espesor tumoral medio fue de 1,61mm. El tipo histológico más frecuente fue el melanoma de extensión superficial. Las diferencias estadísticamente significativas (p<0,05) entre los 3 periodos del estudio fueron: mayor edad media al diagnóstico (p<0,001); mayor número de melanomas en cabeza y cuello (p<0,001); mayor número de melanomas en tronco en el sexo femenino (p<0,001); aumento del melanoma tipo lentigo maligno (p<0,001); disminución del espesor medio (índice de Breslow) (p<0,001); mayor número de melanomas intraepidérmicos (p<0,001). Sin embargo, la proporción de melanomas gruesos (>2mm de Breslow) se mantuvo por encima del 20%, siendo más frecuente este subtipo de melanomas en varones y en mayores de 65 años. Conclusiones. El melanoma cutáneo en España se diagnostica con un espesor tumoral medio cada vez más fino, aunque se siguen diagnosticando melanomas de gran espesor especialmente en varones y pacientes de más de 65 años(AU)

Background. The incidence of malignant melanoma has increased over recent decades all over the world; however, we are not aware if this also occurs in Madrid. Our objective was to analyze epidemiological changes in cutaneous malignant melanomas diagnosed over a 15-year period. Patients and methods. Retrospective analysis of data of patients with primary cutaneous melanomas attended at Hospital Gregorio Marañón, Madrid, Spain, between 1996 and 2010, divided into three periods: 1996-2000; 2001-2005; 2006-2010, were obtained. Results. In total, 969 melanomas were histologically diagnosed. The mean age at the moment of diagnosis was 58.5 years old. The mean tumor thickness was 1.61mm. The most common histological type was surface-spreading melanoma and the most common site was the trunk. There were statistically significant differences (P<.05) between the three periods of the study: Older age at diagnoses (P<.001); larger number of head and neck melanomas (P<.001); more melanomas on trunk in women (P<.001); increase of lentigo maligna melanoma (P<.001); thinner mean tumor thickness (Breslow index) (P<.001); larger number of melanomas in situ (P<.001). However, thick melanomas (tumor thickness over 2mm) ratio was still over 20% in all periods, especially in males and in those over 65 years old. Conclusions. Diagnosis of cutaneous melanoma in Spain is made increasingly with a thinner mean tumor thickness, although thick melanomas are still diagnosed in men and in individuals over 65 years(AU)

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BACKGROUND: The incidence of malignant melanoma has increased over recent decades all over the world; however, we are not aware if this also occurs in Madrid. Our objective was to analyze epidemiological changes in cutaneous malignant melanomas diagnosed over a 15-year period. PATIENTS AND METHODS: Retrospective analysis of data of patients with primary cutaneous melanomas attended at Hospital Gregorio Marañón, Madrid, Spain, between 1996 and 2010, divided into three periods: 1996-2000; 2001-2005; 2006-2010, was obtained. RESULTS: In total, 969 melanomas were histologically diagnosed. The mean age at the moment of diagnosis was 58.5 years old. The mean tumor thickness was 1.61 mm. The most common histological type was surface-spreading melanoma and the most common site was the trunk. There were statistically significant differences (P < .05) between the three periods of the study: older age at diagnoses (P < .001); larger number of head and neck melanomas (P < .001); more melanomas on trunk in women (P < .001); increase of lentigo maligna melanoma (P < .001); thinner mean tumor thickness (Breslow index) (P < .001); larger number of melanomas in situ (P < .001). However, thick melanomas (tumor thickness over 2 mm) ratio was still over 20% in all periods, especially in males and in those over 65 years old. CONCLUSIONS: Diagnosis of cutaneous melanoma in Spain is made increasingly with a thinner mean tumor thickness, although thick melanomas are still diagnosed in men and in individuals over 65 years.

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The Spanish Quality Assurance Program applied to the process of donation after brain death entails an internal stage consisting of a continuous clinical chart review of deaths in critical care units (CCUs) performed by transplant coordinators and periodical external audits to selected centers. This paper describes the methodology and provides the most relevant results of this program, with information analyzed from 206,345 CCU deaths. According to the internal audit, 2.3% of hospital deaths and 12.4% of CCU deaths in Spain yield potential donors (clinical criteria consistent with brain death). Out of the potential donors, 54.6% become actual donors, 26% are lost due to medical unsuitability, 13.3% due to refusals to donation, 3.1% due to maintenance problems and 3% due to other reasons. Although the national pool of potential donors after brain death has progressively decreased from 65.2 per million population (pmp) in 2001 to 49 pmp in 2010, the number of actual donors after brain death has remained at about 30 pmp. External audits reveal that the number of actual donors could be 21.6% higher if all potential donors were identified and preventable losses avoided. We encourage other countries to develop similar comprehensive approaches to deceased donation performance.

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BACKGROUND: Human embryonic stem cells (hESCs) have opened up a new area of research in biomedicine. The efficiency of hESC derivation from frozen poor-quality embryos is low and normally achieved by plating embryos on mouse or human foreskin feeders (HFFs). We attempted to optimize embryo survival and hESC derivation. METHODS: Three conditions were tested on frozen poor-quality embryos: (i) embryo treatment with the Rho-associated kinase (ROCK) inhibitor, Y-27632; (ii) use of human mesenchymal stem cells (hMSCs) as feeders; and (iii) laser drilling (LD) for inner cell mass (ICM) isolation. Two hundred and nineteen thawed embryos were randomly treated with (n = 110) or without (n = 109) 10 microM Y-27632. Surviving embryos that developed to blastocyst stage (n = 50) were randomly co-cultured on HFFs (n = 21) or hMSCs (n = 29). ICM isolation was either by whole-blastocyst culture (WBC) or WBC plus LD. RESULTS: Embryo survival was 52% higher with Y-27632. hMSCs appeared to facilitate ICM outgrowth and hESC derivation: three hESC lines were derived on hMSCs (10.3% efficiency) whereas no hESC line was derived on HFFs. ROCK inhibition and ICM isolation method did not affect hESC efficiency. The lines derived on hMSCs (AND-1, -2, -3) were characterized and showed typical hESC morphology, euploidy, surface marker and transcription factor expression and multilineage developmental potential. The hESC lines have been stable for over 38 passages on hMSCs. CONCLUSION: Our data suggest that Y-27632 increases post-thaw embryo survival and that hMSCs may facilitate the efficiency of hESC derivation from frozen poor-quality embryos.

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The optimization of human embryonic stem (hES) cell line derivation methods is challenging because many worldwide laboratories have neither access to spare human embryos nor ethical approval for using supernumerary human embryos for hES cell derivation purposes. Additionally, studies performed directly on human embryos imply a waste of precious human biological material. In this study, we developed a new strategy based on the combination of whole-blastocyst culture followed by laser drilling destruction of the trophoectoderm for improving the efficiency of inner cell mass (ICM) isolation and ES cell derivation using murine embryos. Embryos were divided into good- and poor-quality embryos. We demonstrate that the efficiency of both ICM isolation and ES cell derivation using this strategy is significantly superior to whole-blastocyst culture or laser drilling technology itself. Regardless of the ICM isolation method, the ES cell establishment depends on a feeder cell growth surface. Importantly, this combined methodology can be successfully applied to poor-quality blastocysts that otherwise would not be suitable for laser drilling itself nor immunosurgery in an attempt to derive ES cell lines due to the inability to distinguish the ICM. The ES cell lines derived by this combined method were characterized and shown to maintain a typical morphology, undifferentiated phenotype, and in vitro and in vivo three germ layer differentiation potential. Finally, all ES cell lines established using either technology acquired an aneuploid karyotype after extended culture periods, suggesting that the method used for ES cell derivation does not seem to influence the karyotype of the ES cells after extended culture. This methodology may open up new avenues for further improvements for the derivation of hES cells, the majority of which are derived from frozen, poor-quality human embryos.

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Human embryonic stem cells (hESCs) represent a promise for future strategies of tissue replacement. However, there are different issues that should be resolved before these cells can be used in cellular therapies; among others, the rejection of transplantable hESCs as a result of HLA incompatibility between donor cells and recipients. The hESCs exhibit a weak HLA class I expression on the cell surface, but today the responsible mechanisms are unknown. We have analyzed the level expression of HLA class I heavy chain, beta2-microglobulin (beta2-m), and antigen-processing machinery (APM) components (TAP1, TAP2, LMP2, LMP7, and Tapasin) using the HS293 hESC line by real-time quantitative RT-PCR. This analysis has revealed a low expression of beta2-m, HLA-B, and Tapasin, and an absence of expression of: TAP1, TAP2, LMP2, and LMP7 genes in the HS293 hESC line respect to the embryoid bodies (EBs) and the induced stem cells with IFNgamma (with significant differences, p<0.05). The lack or loss of HLA class I molecules due to the down-regulation of the APM components has been frequently found in tumors of different histology as specific mechanisms of immune-evasion. We described for the first time in this report that the hESCs shared similar mechanisms with respect to tumor cells responsible for the weak HLA class I expression on the cell surface.

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Mitomycin C (MMC) treatment has been used to arrest cell proliferation but not much is known about the effect of MMC on human foreskin fibroblasts (HFF) used as feeders for human embryonic stem cells (hESC). We tested the ability of MMC to stop the proliferation of HFF and to induce apoptosis. MMC inhibited the proliferation of HFF at 10 microg/ml over 2.5h of MMC treatment showing a decrease in the proliferation index measured by Ki-67 and S and G2/M phases related to active HFF. A low percentage of cells showed necrotic or apoptotic features using different lengths of incubation. Over time, the majority of cells remained in a mitotically inactive state. The percentage of apoptotic cells increased from day 2 to day 10, at the same time as the necrotic ones increased. The HS181 hESC line grew in an undifferentiated state on inactive HFF throughout the study.

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Cell line cross-contamination is a phenomenon that arises as a result of the continuous cell line culture. It has been estimated that around 20% of the cell lines are misidentified, therefore it is necessary to carry out quality control tests for the detection of this issue. Since cell line cross-contamination discovery, different methods have been applied, such as isoenzyme analysis for inter-species cross-contamination; HLA typing, and DNA fingerprinting using short tandem repeat and a variable number of tandem repeat for intra-species cross-contamination. The cell banks in this sense represent the organizations responsible for guaranteeing the authenticity of cell lines for future research and clinical uses.

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Human embryonic stem cells (hESCs) have an unlimited capacity to proliferate by a self-renewal process and can be differentiated in the three germ layers, opening doors to new clinical therapies to replace missing or damaged cells. The number of research groups and projects using human stem cells has increased largely in the last 5 yr. The creation of stem cell banks is another important step to support the advance of research in this field. Banks must be operated within the strict regulatory famework of good manufacturing practices and good laboratory practices that assure the highest quality standards and must implement a quality system that complies with international quality systems standards. It may also be appropriate to aim at an accreditation in order to assure correct laboratory practices at all times. Stem cell banks should receive the lines previously derived by other groups and hESCs should be provided for groups that justify their use in a research project previously approved by an ethical committee. The assays generally accepted as typical of hESCs together with the microbiological analysis should be performed in order to assure a consistent, reliable, and safe line for the researchers. In this article, the Andalusian Stem Cell Bank proposes a model of a stem cell banking process in order to create a flow diagram of hESC lines and, following the international initiatives in stem cells research, to achieve the full characterization of cells and a standardization of protocols that would simplify the hESCs culture.

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AIM: The aim of this study was to evaluate the diagnostic efficacy of an electronic system for the determination of working length, in comparison with two radiological methods (conventional film and digital radiography). METHODOLOGY: The study sample consisted of 28 root canals belonging to 20 human mandibular teeth. A comparison was made between the working length measurements obtained by two radiological methods (conventional film and digital radiography) and an apex locator, using as gold standard the observation of the file position within the root following selective grinding of the root tissue. RESULTS: The electronic method was satisfactory in 67.8% of cases, versus 50.6% and 61.4% for the conventional and digital radiological methods. respectively. No statistically significant differences occurred between the techniques according to the chi-squared and Kruskal-Wallis nonparametric tests. CONCLUSIONS: None of the techniques was totally satisfactory in establishing the true working length. There were no differences between the techniques investigated.

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Propósito: La biopsia del ganglio centinela en pacientes con carcinoma mamario posee actualmente un elevado índice de falsos negativos. Inicialmente, es aconsejable completar este procedimiento con una linfadenectomía axilar.Material y métodos: Presentamos una paciente de 74 años, con un carcinoma ductal infiltrante TI NOMO localizado en la mama derecha, en la que se llevó a cabo una linfadenectomía axilar tras la biopsia del ganglio centinela.Resultados: La biopsia del ganglio centinela fue negativa al igual que la linfadenectomía axilar, excepto un ganglio donde existía colonización por un linfoma folicular. El estudio de extensión para el linfoma fue negativo y se agregó como único tratamiento adyuvante tamoxifeno 20 mg/día.Conclusiones: No hay una clara relación de riesgo entre linfoma y carcinoma mamario. Sin embargo, gracias a completar la biopsia del ganglio centinela con una linfadenectomía axilar, se diagnosticó de forma fortuita un linfoma folicular localizado, en una paciente con carcinoma mamario (AU)

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We assessed the diagnostic usefulness of helical CT scan of the thorax in the setting of chronic thromboembolic pulmonary hypertension by prospectively comparing the results of helical CT scan to those of the pulmonary angiogram (gold standard). We studied 40 patients with diagnosis of pulmonary hypertension of diverse etiology (mean age: 40.7 +/- 12 y.o.; mean systolic pulmonary artery pressure: 91 +/- 33 mmHg)). Thirty of these patients fulfilled the diagnostic criteria of chronic thromboembolic pulmonary hypertension and the other ten were used as controls. Diagnosis in control patients included: primary pulmonary hypertension (4); patent ductus arteriosus (2); atrial septal defect (1); rheumatic valve disease (1); ischemic heart disease (1); and acute pulmonary embolism (1). Both helical CT scan and pulmonary angiogram were part of the routine diagnostic work up of these patients, and were, performed and interpreted almost simultaneously (within one week) by a different group of investigators in a blind manner. Only the diagnostic accuracy of the method regarding central (major arteries) vascular lesions was evaluated. Helical CT scan had an overall sensitivity of 100% (29/29), and a specificity of 91% (10/11). Positive predictive and negative predictive values were 96.6% (29/30) and 100% (10/10), respectively. Overall diagnostic accuracy was 97.5% (39/40). We conclude that helical CT scan of the thorax is an excellent alternative approach for the diagnosis of major arteries lesions in the setting of chronic thromboembolic pulmonary hypertension.

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OBJECTIVES: Certain tooth groups pose problems as to radiologic determination of root canal morphology. For example, the premolars may have superimposed roots that give the appearance of a single canal when 2 are actually present. This study examined the effect of X-ray tube inclination so as to visualize the complete root canal system in premolars. STUDY DESIGN: One hundred extracted premolars were divided into 4 groups of 25 teeth each: 1MxP (first maxillary premolar), 2MxP (second maxillary premolar), 1MdP (first mandibular premolar), and 2MdP (second mandibular premolar). A total of 10 radiographs were made of each tooth in the horizontal and vertical planes (horizontal angles of 0, 20, and 40 degrees, then vertical angles of 0, 15, and 30 degrees). Logistic regression analysis was used for the statistical analysis of the results--the dependent variable being the number of canals visualized. RESULTS: On varying the horizontal angle (20 and 40 degrees), the number of root canals observed in the case of 1MxP, 2MxP, and 1MdP coincided with the actual number of canals present. In the case of 2MdP, only the 40 degree horizontal angle correctly identified the number of canals regardless of the vertical angle (although only 4% had more than 1 canal). CONCLUSIONS: Varying the horizontal angle improved the visualization of additional (superimposed) canals in premolars. Changing of the vertical angle had no statistically significant influence except for the first mandibular premolars.

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