[Statistical power in medical research. What position should be taken when research results are not significant?] / Potencia estadística de una investigación médica. ¿Qué postura tomar cuando los resultados de la investigación no son significativos?

The original idea of rejecting studies with low power and authorising them if their power is sufficiently high is reasonable and even an obligation, although in practice this reasoning is heavily constrained by the fact that the power of a study depends on several factors, rather than a single one. Furthermore, there is no threshold separating 'high' power values from 'low' power values'. However, if the result is very significant, considering how powerful it was it makes little sense after the study has been carried out. It is only possible to take advantage of the result. Situations in which this result is not statistically significant warrant further consideration. Consideration of the power may be useful in these circumstances. This article focuses on the position that should be adopted in these cases, and it shows that in order to draw reasonable conclusions about the effect size of the population, calculating the confidence interval is more useful than calculating the power, and its interpretation is more easily understood by physicians who lack training in statistical analysis.

TITLE: Potencia estadística de una investigación médica. ¿Qué postura tomar cuando los resultados de la investigación no son significativos?La idea original de rechazar estudios con baja potencia y autorizarlos si es suficientemente alta es razonable e incluso obligada, aunque en la práctica este razonamiento se ve muy limitado por el hecho de que la potencia de un estudio depende de varios factores y, por tanto, no es única. Además, no hay un valor frontera que separe los valores 'altos' de potencia de los 'bajos'. Pese a esto, una vez realizado el estudio, si su resultado es muy significativo, no tiene sentido preguntarnos por la potencia que tenía. Sólo cabe aprovechar su resultado. Consideración aparte merece el caso en que dicho resultado no sea estadísticamente significativo. Entonces sí puede ser pertinente considerar su potencia. A continuación, se hace una reflexión sobre qué postura adoptar en estos casos y se muestra que, para sacar conclusiones razonables sobre el efecto poblacional, el cálculo de su intervalo de confianza es más útil que el cálculo de la potencia y su interpretación más fácilmente entendible por el médico sin formación en análisis estadístico.

[The enormous difference between not rejecting a null hypothesis and stating that it is true]. / La abismal diferencia entre no rechazar la hipótesis nula y afirmar que es cierta.

Assuming that a hypothesis is true because insufficient evidence has been found to reject it is a very common error when interpreting the p-value of a test in biomedical research. For example, a value of p = 0.28 obviously does not mean the null hypothesis should be ruled out, but if we understand what it means (which is not a mathematical issue, but instead a purely logical one) that it is equally obvious that it cannot be stated that it is true. If the samples in a comparison of a new drug with an old one show that the new one has a higher healing percentage and the p-value of the test is 0.0004, for example, the scientific community concludes that the new one is better. However, if for example the p-value of the test is 0.14, the scientific community does not conclude that the new one is as good as the old one. It merely concludes that the new one has not been shown to outperform the other one. It is therefore possible that an extension of the study with more cases may demonstrate that the new one is better.

TITLE: La abismal diferencia entre no rechazar la hipótesis nula y afirmar que es cierta.Un error muy frecuente al interpretar el valor de p del test en la investigación biomédica consiste en asumir que una hipótesis es cierta porque no se ha encontrado suficiente evidencia para rechazarla. Es obvio que un valor de p = 0,28, por ejemplo, no invita a rechazar la hipótesis nula, pero, si se entiende lo que indica (que no es un tema matemático, sino puramente lógico), resulta igualmente obvio que no autoriza a afirmar que es cierta. Si al comparar un nuevo fármaco frente al antiguo encontramos en las muestras que el nuevo presenta mayor porcentaje de curaciones y, por ejemplo, el valor de p del test es = 0,0004, la comunidad científica concluye que el nuevo es mejor. Pero si, por ejemplo, el valor de p del test es = 0,14, la comunidad científica no concluye que el nuevo es igual al antiguo. Sólo concluye que no queda demostrado que el nuevo aventaje al otro. Por ello, queda abierta la puerta a que en una ampliación del estudio con más casos pueda demostrarse que el nuevo es mejor.

[The p-value of the test is not a 'mathematical index', it is simply a relative frequency]. / El valor de p del test no es un 'índice matemático', es simplemente una frecuencia relativa.

Leading scientific journals in fields such as medicine, biology and sociology repeatedly publish articles and editorials claiming that a large percentage of doctors do not understand the basics of statistical analysis, which increases the risk of errors in interpreting data, makes them more vulnerable to misinformation and reduces the effectiveness of research. This problem extends throughout their careers and is largely due to the poor training they receive in statistics - a problem that is common in developed countries. As stated by H. Halle and S. Krauss, '90% of German university lecturers who regularly use the p-value in tests do not understand what that value actually measures'. It is important to note that the basic reasoning of statistical analysis is similar to what we do in our daily lives and that understanding the basic concepts of statistical analysis does not require any knowledge of mathematics. Contrary to what many researchers believe, the p-value of the test is not a 'mathematical index' that allows us to clearly conclude whether, for example, a drug is more effective than a placebo. The p-value of the test is simply a percentage.

TITLE: El valor de p del test no es un 'índice matemático', es simplemente una frecuencia relativa.Las revistas científicas más importantes en campos como medicina, biología y sociología publican reiteradamente artículos y editoriales denunciando que un gran porcentaje de médicos no entiende los conceptos básicos del análisis estadístico, lo que favorece el riesgo de cometer errores al interpretar los datos, los hace más vulnerables frente a informaciones falsas y reduce la eficacia de la investigación. Este problema se extiende a lo largo de toda su carrera profesional y se debe, en gran parte, a una enseñanza deficiente en estadística que es común en países desarrollados. En palabras de H. Halle y S. Krauss, 'el 90% de los profesores universitarios alemanes que usan con asiduidad el valor de p de los test no entiende lo que mide ese valor'. Es importante destacar que los razonamientos básicos del análisis estadístico son similares a los que realizamos en nuestra vida cotidiana y que comprender los conceptos básicos del análisis estadístico no requiere conocimiento matemático alguno. En contra de lo que muchos investigadores creen, el valor de p del test no es un 'índice matemático' que nos permita concluir claramente si, por ejemplo, un fármaco es más efectivo que el placebo. El valor de p del test es simplemente un porcentaje.

De-escalating axillary management after neoadjuvant chemotherapy in breast cancer: The ratio of positive sentinel lymph nodes matters.

BACKGROUND: De-escalation of axillary surgery in breast cancer (BC) patients diminishes sequelae without compromising cancer outcomes. Surgical management of the axilla is challenging after neoadjuvant treatment. We aimed to identify the factors associated with residual axillary disease amenable to lymphadenectomy in patients with positive sentinel lymph node biopsy (SLNB). METHODS: We conducted a retrospective observational study in Hospital 12 de Octubre (Spain). We included BC patients with positive SLNB who underwent axillary dissection after neoadjuvant chemotherapy. Univariate and multivariate logistic regression models were performed to identify independent predictors of residual axillary disease. We estimated the ratio of positive nodes in SLNB and assessed the diagnostic validity of this ratio in relation to residual axillary disease. RESULTS: We included 103 patients in the study. Residual axillary disease was identified in 54 patients (52.4%). Clinically node positive status at diagnosis (OR = 18.3, 95%CI: 4.0-83.6) and a ratio of positive nodes in SLNB ≥0.5 (OR = 6.5, 95%CI 41.7-23.7) were associated with residual axillary disease. The sensitivity and negative predictive value of a ratio of positive nodes in SLNB ≥0.5 were 87% (95%CI 75.1%-94.6%) and 75% (95%CI 55.1%-89.3%), respectively. CONCLUSIONS: In our study, for patients with positive SLNB after neoadjuvant chemotherapy, stage N+ at diagnosis and a ratio of positive nodes in SLNB ≥0.5 were independent risk factors of positive residual axillary disease. This ratio is a feasible measure with a good diagnostic validity for residual axillary disease and could be used as a guiding factor in the surgical management of these patients.

Clinical study types and guidance for their correct post-pandemic interpretation.

Randomized clinical trials (RCTs) are key to the advancement of medicine and microbiology, but they are not the only option. Observational studies provide information on long-term efficacy and safety, are less expensive, allow the study of rare events, and obtain information more quickly than RCTs. On the other hand, they are more vulnerable to confounding factors. Prospective exploratory pilot studies share many aspects with RCTs but are not subject to supervision by external commissions or mandatory registration. Multitesting can pervert the balance of publications in favor of the desired effect. Bonferroni's reasoning shows that if 10 studies are performed with an ineffective antibiotic, the probability that at least one will show P <0.05 might be 40%. Scenarios in which there is intensive pressure to perform research, such as the recent pandemic, might result in many research teams trying to study the effect of an antimicrobial. Even if the drug has no efficacy, if 100 research teams conduct a study to assess its usefulness, it might be virtually certain that at least one will get a P value <0.05. If the other studies (with P >0.05) are not published, the scientific commu nity would consider that there is strong evidence in favor of its usefulness. In conclusion, RCTs are a very good source of clinical information, but are not the only one. The systematic registration of all research can and should be applied to all types of clinical studies.

[The dramatic loss of statistical power when dichotomising continuous variables]. / La dramática pérdida de potencia estadística al dicotomizar variables continuas.

A very common practice in medical research, during the process of data analysis, is to dichotomise numerical variables in two groups. This leads to the loss of very useful information that can undermine the effectiveness of the research. Several examples are used to show how the dichotomisation of numerical variables can lead to a loss of statistical power in studies. This can be a critical aspect in assessing, for example, whether a therapeutic procedure is more effective or whether a certain factor is a risk factor. Dichotomising continuous variables is therefore not recommended unless there is a very specific reason to do so.

TITLE: La dramática pérdida de potencia estadística al dicotomizar variables continuas.Una práctica muy habitual en la investigación médica, durante el proceso de análisis de los datos, es dicotomizar variables numéricas en dos grupos. Dicha práctica conlleva la pérdida de información muy útil que puede restar eficacia a la investigación. A través de varios ejemplos, se muestra cómo con la dicotomización de variables numéricas los estudios pierden potencia estadística. Esto puede ser un aspecto crítico que impida valorar, por ejemplo, si un procedimiento terapéutico es más efectivo o si un determinado factor es de riesgo. Por tanto, se recomienda no dicotomizar las variables continuas si no existe un motivo muy concreto para ello.

[Statistical power in medical research. What stance should be taken when the research results are significant?] / Potencia estadística en investigación médica. ¿Qué postura tomar cuando los resultados de la investigación son significativos?

When researchers request funding and authorisation from financial institutions to carry out their project, one of the first questions they are asked is: what is the statistical power of the study you are proposing? If the researcher answers, for example, 90%, and the evaluator is satisfied, it is certain that he/she is not really familiar with the subject. The power of a study is not unique. It depends on certain parameters and what happens is that, in most cases, by introducing a slight variation in the values of these parameters, the power takes on an acceptable value. If this is not the case and the study is carried out anyway, and its results are very significant, there is no room to question its success by arguing that the power of the study was very low. It is just the time to celebrate.

TITLE: Potencia estadística en investigación médica. ¿Qué postura tomar cuando los resultados de la investigación son significativos?Cuando el investigador pide subvención y autorización a entidades financieras para llevar a cabo su proyecto, entre las primeras cuestiones que le plantean está: ¿qué potencia estadística tiene este estudio que usted propone? Si el investigador responde, por ejemplo, el 90%, y el evaluador se da por satisfecho, es seguro que no conoce realmente el tema. La potencia de un estudio no es única. Depende de determinados parámetros y ocurre que, en la mayoría de los casos, variando ligeramente los valores de esos parámetros, la potencia toma un valor aceptable. Si no es así, y a pesar de ello se lleva a cabo el estudio, y sus resultados son muy significativos, no ha lugar a cuestionar el éxito encontrado argumentando que el estudio tenía poca potencia. Tan sólo es momento de celebrarlo.

[Myths and reality about calculating sample size]. / Mitos y realidad en el cálculo del tamaño muestral.

When we decide to conduct a study, one of the first questions that arises is what number of individuals should be included in the sample for it to be 'representative' and for the study to be 'valid'? As in other areas of life, there are many matters for which there is no 'right' amount and different quantities are valid. The same applies here. When asked the question 'How many euros did this bicycle cost?', the answer is a definite number. But the question 'How many euros do I need to buy a bicycle?' can be answered in many different ways, depending on the size and other characteristics of the bicycle. Statistics textbooks contain formulas relating sample size to certain parameters and most doctors believe that one of these will give them the 'right' size for their research, and that by using them their choice of sample size will be justified in the eyes of potential reviewers. This document reflects on the true value of these formulas and how researchers should make proper use of them. It is necessary to show errors and simulations that benefit no one and hinder many by taking up large amounts of time and energy.

TITLE: Mitos y realidad en el cálculo del tamaño muestral.Resumen. Cuando decidimos hacer un estudio, una de las primeras cuestiones que se plantea es ¿qué número de individuos debo incluir en la muestra para que sea 'representativa' y el estudio sea 'válido'? Como en otros ámbitos de la vida, hay muchas cuestiones para las que no hay una cantidad 'adecuada' y son válidas diferentes cantidades. Aquí ocurre lo mismo. La pregunta '¿cuántos euros costó esta bicicleta?' tiene como respuesta un número concreto. Pero la pregunta '¿cuántos euros necesito para comprar una bicicleta?' admite muchas cifras distintas como respuesta, dependiendo del tamaño y otras características de la bicicleta. Los libros de estadística contienen fórmulas que relacionan el tamaño de la muestra con ciertos parámetros y la mayoría de los médicos cree que una de ellas les dará el tamaño 'adecuado' para su investigación, y que usándolas queda 'justificado el tamaño de la muestra' ante posibles revisores. En este documento se hace una reflexión sobre el verdadero peso que tienen dichas fórmulas y cuál debe ser el uso adecuado que el investigador haga de ellas. Es necesario mostrar errores y simulaciones que no benefician a nadie y perjudican a muchos restando tiempo y energía.

Top ten errors of statistical analysis in observational studies for cancer research.

Observational studies using registry data make it possible to compile quality information and can surpass clinical trials in some contexts. However, data heterogeneity, analytical complexity, and the diversity of aspects to be taken into account when interpreting results makes it easy for mistakes to be made and calls for mastery of statistical methodology. Some questionable research practices that include poor analytical data management are responsible for the low reproducibility of some results; yet, there is a paucity of information in the literature regarding specific statistical pitfalls of cancer studies. In addition to proposing how to avoid or solve them, this article seeks to expose ten common problematic situations in the analysis of cancer registries: convenience, dichotomization, stratification, regression to the mean, impact of sample size, competing risks, immortal time and survivor bias, management of missing values, and data dredging.

Most advisable strategy in search of asymptomatic target organ damage in hypertensive patients.

OBJECTIVE: To evaluate the diagnostic potential of seven examinations in order to define the most suitable strategy for target organ damage (TOD) search in hypertensive patients. METHODS: This is a descriptive, cross-sectional study. 153 consecutive treated and essential hypertensive patients were enrolled. Patients with established cardiovascular or chronic renal disease (stage ≥4) were excluded. TOD search was assessed by: glomerular filtration rate (GFR), albumin/creatinine ratio (ACR), electrocardiogram (ECG), echocardiogram (ECO), ankle-brachial index (ABI), pulse wave velocity (PWV), and carotid ultrasound (intima media thickness and presence of plaques). The rationale of our strategy ought to determine the performance of applying a set of the most widely available tests (GFR, ACR, ABI, ECG) and advise about the optimal sequence of the remaining tests. RESULTS: The sample was 64.4±7.9 years old, 45.8% males. 82.6% of the sample had any TOD at all. The resulting algorithm found a 37% TOD in relation to GFR, ACR, ABI and ECG values. Adding carotid ultrasound added up to 70% of the studied population and properly classified (TOD+/TOD-) 89% of the cohort. When performing PWV, 78% of the patients had been identified as TOD+ and 96% of the population was correctly identified. Contribution of ECO was minor. CONCLUSION: After running the more widely available explorations (GFR, ACR, ABI, ECG), a step-by-step strategy that included carotid ultrasound, PWV and ECO could be the best sequence for TOD search in asymptomatic hypertensive patients.

[Control of anticoagulation in patients with non-valvular atrial fibrillation in a primary care clinical practice setting in the different autonomous communities. PAULA study]. / Control de la anticoagulación en pacientes con fibrilación auricular no valvular en práctica clínica de atención primaria en las diferentes comunidades autónomas. Estudio PAULA.

AIMS: To determine the differences between regions in the level of control of patients with non-valvular atrial fibrillation treated with vitamin K antagonists, included in the PAULA study. METHODS: Observational, and coss-sectional/retrospective study, including 139 Primary Care physicians from 99 Health Care centres in all autonomous communities (except La Rioja). Anticoagulation control was defined as the time in therapeutic range assessed by either the direct method (poor control <60%), or the Rosendaal method (poor control <65%). RESULTS: A total of 1,524 patients were included. Small differences in baseline characteristics of the patients were observed. Differences in the percentage of time in therapeutic range were observed, according to the Rosendaal method (mean 69.0±17.7%), from 78.1%±16.6 (Basque Country) to 61.5±14% (Balearic Islands), by the direct method (mean 63.2±17.9%) from 73.6%±16.6 (Basque Country) to 57.5±15.7% (Extremadura). When comparing regions, in those where the Primary Care physicians assumed full control without restrictions on prescription, the percentage of time in therapeutic range by the direct method was 63.89 vs. 60.95% in those with restrictions (p=.006), by Rosendaal method, 69.39% compared with 67.68% (p=.1036). CONCLUSIONS: There are significant differences in the level of control between some regions are still inadequate. Regions in which the Primary Care physicians assumed the management of anticoagulation and without restrictions, time in therapeutic range was somewhat higher, and showed a favourable trend for better control. These findings may have clinical implications, and deserve consideration and specific analysis.

[Prevalence of multiple sclerosis in the Region of Murcia]. / Prevalencia de la esclerosis multiple en la Region de Murcia.

INTRODUCTION: Multiple sclerosis (MS) is a demyelinating inflammatory disease of the central nervous system with immune-mediated pathogenesis. Recent research points to an increase in its prevalence, and a number of studies relate Epstein-Barr virus (EBV) with its aetiology. AIMS: This study seeks to analyse the prevalence of MS in the Region of Murcia, and includes a description of the clinical characteristics at the time of onset of the disease, and of the EBV serological status of patients with MS. PATIENTS AND METHODS: We conducted a retrospective epidemiological study based on a sample consisting of the population living within the central-west healthcare area of the Region of Murcia (257,865 inhabitants). Clinical and serological data extracted from different sources were analysed. RESULTS: Prevalence of MS in the population under study: 88 cases/100,000 inhabitants. Prevalence of MS together with isolated demyelinating syndrome: 98.4 cases/100,000 inhabitants. Mean incidence of MS: 5.8 cases/100,000 inhabitants/year. At the onset of MS, 67.8% were females, 81.9% presented a relapsing-remitting course, the mean age was 31.4 years, the sensory system was the most frequently compromised (45.1%), onset was monofocal in 55.4% and the degree of disability on the Expanded Disability Status Scale was 2.1 points. The seroprevalence of EBV was 99.3%. The reactivation of EBV infection was related to the clinical activity of MS in 10 patients (45.4%). CONCLUSIONS: Currently, the prevalence of MS in the Region of Murcia is similar to that estimated in other Spanish autonomous regions. The study confirms the trend of increased prevalence observed over the last few decades.

TITLE: Prevalencia de la esclerosis multiple en la Region de Murcia.Introduccion. La esclerosis multiple (EM) es una enfermedad inflamatoria desmielinizante del sistema nervioso central con patogenia inmunomediada. Recientes estudios indican un aumento de su prevalencia, y numerosos trabajos relacionan el virus de Epstein-Barr (VEB) con su etiologia. Objetivo. Analisis de prevalencia de la EM en la Region de Murcia, incluyendo la descripcion de las caracteristicas clinicas en el momento del inicio de la enfermedad, y del estado serologico del VEB de los pacientes con EM. Pacientes y metodos. Estudio epidemiologico retrospectivo, tomando como muestra la poblacion residente en el area sanitaria centro-oeste de la Region de Murcia (257.865 habitantes). Se analizan datos clinicos y serologicos extraidos de diferentes fuentes. Resultados. Prevalencia de la EM en la poblacion estudiada: 88 casos/100.000 habitantes. Prevalencia de la EM junto con el sindrome desmielinizante aislado: 98,4 casos/100.000 habitantes. Incidencia media de la EM: 5,8 casos/100.000 habitantes/año. En el inicio de la EM, el 67,8% eran mujeres, el 81,9% presentaba un curso recurrente-remitente, la edad media era de 31,4 años, el sistema funcional mas frecuentemente afectado era el sensitivo (45,1%), el inicio fue monofocal en el 55,4% y el grado de discapacidad en la Expanded Disability Status Scale era de 2,1 puntos. La seroprevalencia del VEB fue del 99,3%. La reactivacion de la infeccion por VEB se relaciono con actividad clinica de EM en 10 pacientes (45,4%). Conclusiones. Actualmente, la prevalencia de la EM en la Region de Murcia es similar a la estimada en otras comunidades autonomas españolas. El estudio confirma la tendencia de incremento de prevalencia observada en las ultimas decadas.

The challenges of managing a complex stoma with NPWT.

This paper reports the clinical experience of a 71-year-old female with resection of the colon, who subsequently developed postoperative complications. A colostomy was carried out; necrosis of the colostomy edges and stoma retraction complicated optimal stoma appliance placement. It was possible to treat the resulting cavity with negative pressure wound therapy (NPWT) by isolating the stoma and treating the peristomal wound area. The wound was treated with NPWT for 30 days; at day 20, the patient was discharged to home to continue with NPWT for a further 10 days by community nurses, with regular follow-up visits in the outpatient clinic. The patient improved steadily and achieved complete closure of the wound.

Testing combinations of protease inhibitor and preservation solution to improve islet quality and yield.

UNLABELLED: Pancreas preservation using an oxygenated two-layer method (TLM) has been reported to improve islet yields, as has supplementation of Liberase with Pefabloc. We hypothesized that using both TLM and Pefabloc could enhance islet yield as compared with preservation in University of Wisconsin (UW) or Histidine-Tryptophan Ketoglutarate (HTK) solution. METHODS: Ninety-eight pancreata with no significant differences of age, body mass index, or cold ischemia time preserved randomly with UW (n = 40), TLM (n = 48), or HTK (n = 10) were processed with (n = 36) or without (n = 66) Pefabloc. RESULTS: The total islet equivalent (IEQ) from TLM-preserved pancreata processed with Pefabloc (n = 12) showed lower yields versus those processed without Pefabloc (n = 36): 216,120 +/- 27,906 vs. 301,427 +/- 21,447 IEQ (P < .05). Islets from 1 of 12 (8.33%) pancreata processed with Pefabloc in TLM were transplanted, in contrast with 15/36 TLM (41.67%) pancreata processed without it. Islet yields were not significantly different among pancreata preserved in UW and processed with Pefabloc (n = 17) versus without Pefabloc (n = 23): 342,693 +/- 45,588 versus 266,609 +/- 29,006 IEQ (P = .149). The number of transplants from UW-preserved pancreata was 3/17 (17.65%) when processed with Pefabloc and 4/23 (17.39%) without. Among the HTK group, there was no significant difference in islet yields between pancreata processed with (n = 7) versus without Pefabloc (n = 3): 248,227 +/- 65,294 versus 483,555 +/- 144,070 IEQ (P = .118). CONCLUSIONS: Pefabloc showed no benefit to improve islet yields. Pancreata preserved in TLM provided better transplant quality islets when processed in the absence of Pefabloc.

Total selenium and selenomethionine in pharmaceutical yeast tablets: assessment of the state of the art of measurement capabilities through international intercomparison CCQM-P86.

Results of an international intercomparison study (CCQM-P86) to assess the analytical capabilities of national metrology institutes (NMIs) and selected expert laboratories worldwide to accurately quantitate the mass fraction of selenomethionine (SeMet) and total Se in pharmaceutical tablets of selenised-yeast supplements (produced by Pharma Nord, Denmark) are presented. The study, jointly coordinated by LGC Ltd., UK, and the Institute for National Measurement Standards, National Research Council of Canada (NRCC), was conducted under the auspices of the Comité Consultatif pour la Quantité de Matière (CCQM) Inorganic Analysis Working Group and involved 15 laboratories (from 12 countries), of which ten were NMIs. Apart from a protocol for determination of moisture content and the provision of the certified reference material (CRM) SELM-1 to be used as the quality control sample, no sample preparation/extraction method was prescribed. A variety of approaches was thus used, including single-step and multiple-step enzymatic hydrolysis, enzymatic probe sonication and hydrolysis with methanesulfonic acid for SeMet, as well as microwave-assisted acid digestion and enzymatic probe sonication for total Se. For total Se, detection techniques included inductively coupled plasma (ICP) mass spectrometry (MS) with external calibration, standard additions or isotope dilution MS (IDMS), inductively coupled plasma optical emission spectrometry , flame atomic absorption spectrometry and instrumental neutron activation analysis. For determination of SeMet in the tablets, five NMIs and three academic/institute laboratories (of a total of five) relied upon measurements using IDMS. For species-specific IDMS measurements, an isotopically enriched standard of SeMet (76Se-enriched SeMet) was made available. A novel aspect of this study relies on the approach used to distinguish any errors which arise during analysis of a SeMet calibration solution from those which occur during analysis of the matrix. To help those participants undertaking SeMet analysis to do this, a blind sample in the form of a standard solution of natural abundance SeMet in 0.1 M HCl (with an expected value of 956 mg kg(-1) SeMet) was provided. Both high-performance liquid chromatography (HPLC)-ICP-MS or gas chromatography (GC)-ICP-MS and GC-MS techniques were used for quantitation of SeMet. Several advances in analytical methods for determination of SeMet were identified, including the combined use of double IDMS with HPLC-ICP-MS following extraction with methanesulfonic acid and simplified two-step enzymatic hydrolysis with protease/lipase/driselase followed by HPLC-ICP-IDMS, both using a species-specific IDMS approach. Overall, satisfactory agreement amongst participants was achieved; results averaged 337.6 mg kg(-1) (n = 13, with a standard deviation of 9.7 mg kg(-1)) and 561.5 mg kg(-1) (n = 11, with a standard deviation of 44.3 mg kg(-1)) with median values of 337.6 and 575.0 mg kg(-1) for total Se and SeMet, respectively. Recovery of SeMet from SELM-1 averaged 95.0% (n = 9). The ability of NMIs and expert laboratories worldwide to deliver accurate results for total Se and SeMet in such materials (selensied-yeast tablets containing approximately 300 mg kg(-1) Se) with 10% expanded uncertainty was demonstrated. The problems addressed in achieving accurate quantitation of SeMet in this product are representative of those encountered with a wide range of organometallic species in a number of common matrices.

Improvement of human islet cryopreservation by a p38 MAPK inhibitor.

The activation of p38 mitogen-activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum-free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS-p38IH and islet viability was not significantly different in the three groups. beta Cell numbers and function were the highest in islets cryopreserved with ICS-p38IH. Glucose-stimulated human C-peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

Multipotent progenitor cells isolated from adult human pancreatic tissue.

The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 mum contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide-positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus.

Inhibition of p38 mitogen-activated protein kinase protects human islets from cryoinjury and improves the yield, viability, and quality of frozen-thawed islets.

The development of an optimal islet cryopreservation method will permit transplantation of islets from multiple donors in a single procedure and contribute to alleviation of the islet shortage. In this study, we have improved human islet cryopreservation methods under serum-free conditions using an intracellular-based islet cryopreservation solution (ICS), especially supplemented with a p38 pathway inhibitor (p38IH) to suppress p38 mitogen-activated protein kinase (MAPK) activation. Three different solutions were compared for freezing and thawing of human islets (1) conventional RPMI1640 medium, (2) ICS, and (3) ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Islet cryopreservation with ICS-p38IH significantly improved islet recovery, viability, and quality after thawing of cryopreserved islets. This improvement may allow the use of cryopreserved islets in clinical islet transplantation.