Rafael Venta

Optimization of nucleated red blood cell (NRBC) recovery from maternal blood collected using both layers of a double density gradient

The isolation of fetal nucleated red blood cells (NRBC) from maternal blood represents a promising approach to non‐invasive prenatal diagnosis. However, the number of fetal NRBC in maternal circulation is quite low and therefore difficult to isolate. An enrichment procedure in which both layers from a double density 1.077/1.107 g/ml gradient are collected was optimized, followed by MACS selection using non‐commercial monoclonal antibodies. The influence of the delay in processing maternal blood on the NRBC distribution in both interfaces of the gradient was also studied in cord blood and peripheral maternal blood samples. A significant increase in the number of NRBC isolated from maternal blood was achieved by collecting both layers of the double density gradient compared with the previous protocol in which only the lower layer was recovered. Cord blood samples showed significant differences in the number of NRBC recovered when processed at 24 instead of within 3 h. This effect was also observed in the number of NRBC collected only from the upper layer of peripheral maternal blood samples. Therefore, in order to minimize the target cell losses, it is advisable to process the maternal blood samples as soon as possible. Copyright

Biological variation of cardiac troponin T in patients with end-stage renal disease and in healthy individuals

Background Serum cardiac troponin T concentrations are important predictors of cardiovascular and all-cause mortality in end-stage renal disease. In patients with end-stage renal disease, assessment of serial results is essential to distinguish between a cardiovascular event and chronic elevation. We employed a high-sensitivity serum cardiac troponin T assay to evaluate the long-term biological variation in end-stage renal disease patients and in healthy individuals; these biological variation data were used to define the reference change value and the analytical goals. Methods Serum samples were collected from 18 end-stage renal disease patients in steady-state conditions, one per month for 6 months, and from 11 healthy volunteers at weekly intervals over 5 weeks. Biological variation data were derived using analysis of variance. Results Baseline serum cardiac troponin T concentrations in end-stage renal disease patients were above the 99th percentile of the healthy population and increased with duration of haemodialysis. For end-stage renal disease patients, within-subject (CVI) and between-subject (CVG) coefficients of variation were 14.7 and 77.8%, respectively, whereas these were 5.9 and 30.4%, respectively, for healthy individuals. The derived two-tailed and one-tailed reference change values were 44.1 and 37.1%, respectively, for end-stage renal disease patients, and 21.6 and 18.2% for healthy subjects. Conclusions For appropriate clinical management of end-stage renal disease patients in the context of a cardiovascular event, regular monitoring of serum cardiac troponin T concentrations could be important in order to allow future comparison through reference change value. Biological variation data in end-stage renal disease patients were significantly higher than for healthy individuals; therefore, the use of proper reference change value data is recommended. Moreover, the observed CVI values provide demanding imprecision goals for current technology.

Biological variation of free plasma amino acids in healthy individuals

Abstract Background: Biological variation data for free plasma amino acids are lacking from the more comprehensive databases. Therefore, we determined the intra- and inter-individual components of variation in healthy subjects. These data were used to define desirable goals for imprecision, bias and total error, indices of individuality and reference change values. Methods: Plasma samples were collected from 11 volunteers at weekly intervals over 5 weeks. Free plasma amino acids were analyzed by reversed-phase HPLC and analytical and biological variation data were derived using ANOVA. Results: Intra-individual coefficients of variation ranged from 9.5% to 46.4%, with lower values among the essential amino acids. The mean inter-individual coefficient of variation was 46.6% and was higher than intra-individual values for all amino acids. Thus, indices of individuality were below 0.8. Reference change values ranged from 30.9% to 128.4% and total error values ranged from 15.2% to 61.0%. Conclusions: Plasma amino acids exhibit relatively low intra-individual coefficients of variation, with essential amino acids showing tighter homeostatic control. Analytical quality goals can be reasonably achieved with current methods. Reference intervals are of limited value in the detection of unusual results in an individual. Therefore, comparison of serial results by means of the reference change values is recommended. Clin Chem Lab Med 2010;48:99–104.

Isolation of fetal nucleated red blood cells from maternal blood in normal and aneuploid pregnancies

Abstract Fetal nucleated red blood cells (NRBC) have been widely reported in maternal blood during pregnancy. However, there is no consensus with regard to their presence in all pregnancies. Therefore, the usefulness of developing a fetal NRBC-based noninvasive method suitable for clinical prenatal diagnosis remains uncertain. Fluorescence in situ hybridization (FISH) method was used to evaluate the ability of one of our own monoclonal antibodies (mAb), 2B7.4, to isolate fetal NRBC from maternal blood by magnetic activated cell sorting (MACS). Our mAb was able to isolate from 25 to 822 NRBC from all of the 45 maternal blood samples included in this study. A correct diagnosis was achieved in 21 out of 24 pregnancies carrying trisomic fetuses (87.5%), with a fetal/maternal NRBC frequency of 8.4%. In contrast, a significantly lower percentage of fetal NRBC (0.2%) was observed in 22% of pregnancies carrying a chromosomally normal male fetus, that were correctly predicted. In conclusion, using 2B7.4 mAb we succeeded in isolating NRBC from the maternal blood samples, but most of the isolated cells were maternal in origin. Nevertheless, a higher number of fetal NRBC was found in the peripheral blood of pregnant women carrying aneuploid fetuses, which could allow development of a screening method for prenatal diagnosis of fetal aneuploidies.

Regression-based reference limits for urinary amino acids in a pediatric population.

Abstract Age-related variation of the urinary excretion of amino acids has been widely reported. Instead of the conventional reference intervals, continuous reference limits might be a more practical approach during periods of rapid changes such as infancy or childhood. Regression-based reference limits have been produced for the urinary excretion of 23 amino acids analyzed in 148 healthy individuals from 0 to 12 years of age. Urinary excretion was modeled as a function of age by two parametric procedures: the polynomial and the piecewise linear regression methods. Likewise, age-specific standard deviation was estimated by the regression of the absolute values of the residuals on age. Residual analysis was employed to select the best-fitting model and the 95% reference limits, and their 95% confidence intervals were calculated and plotted against the raw data. The urinary excretion of 19 amino acids decreased rapidly during the first year of life with a further slow decline thereafter. These amino acids fit better the piecewise model. The other four amino acids displayed a steady decrease in the urinary excretion from birth to puberty, and the excretion patterns fit better the quadratic or linear model. Fifteen amino acids showed a significant change in standard deviation with age. Regression-based reference limits differed consistently from conventional limits (<3 years) and narrower confidence intervals were obtained throughout the entire period studied. Avoiding partitioning gave rise to smoothly changing limits. Therefore, such alternative way of presenting amino acid reference limits may facilitate the follow-up of patients with inborn errors of amino acid metabolism.

Muscular contraction frequency does not affect plasma homocysteine concentration in response to energy expenditure- and intensity-matched acute exercise in sedentary males

Acute exercise seems to increase total plasma homocysteine (tHcy); since this variable associated with cardiovascular risk, it is important to understand the determinants of its response to all types of exercise. The aim of this study was to examine the impact of cycling at 2 different rates of muscle contraction on the complete tHcy kinetics. Eight young sedentary males were required to complete 2 isocaloric (400 kcal) acute exercise trials at 50% peak oxygen uptake on separate occasions at 50 or 80 rpm. Blood samples were drawn at different points before (4 h before exercise and immediately before exercise), during (10, 20, 30, 45, and 60 min during exercise), and after exercise (immediately and 19 h after exercise). Dietary and lifestyle factors were controlled during the research. Maximum tHcy occurred during exercise for both conditions (50 rpm: 11.4 ± 2.7 μmol·L-1; 80 rpm: 10.8 ± 3.2 μmol·L-1). From this point onwards tHcy declined until the cessation of exercise and continued descending below pre-exercise values at 19 h postexercise (p < 0.05). No hyperhomocysteinemia were observed at any sampling point in both trials. In conclusion, the different muscular contraction frequency during exercise has no impact on tHcy during an acute bout of exercise in sedentary individuals, when at least 400 kcal are spent during exercise and the nutritional status for folate, B12, and B6 is adequate. This information is relevant to further inform healthy exercise prescription, not only in terms of duration and intensity of exercise, but also taking into account frequency of contraction.

Relationship between breast arterial calcifications seen on screening mammograms and age-related macular degeneration.

the inferior quadrant. The Spectralis OCT classic glaucoma application and the Nsite Axonal Analytics application revealed significant thinning in the inferior and inferotemporal RNFL in patients with AD compared with controls. There is histopathologic evidence of retinal ganglion cell loss and optic nerve degeneration in patients with AD (Blanks et al. 1996). Thus, we postulate that axonal loss secondary to other pathologic changes that occur in the brain can be detected by scanning the RNFLand the optic nerve, as these nonmyelinated axons form the optical path that culminates in the occipital cortex. Although we believe the OCT could potentially provide a biomarker of AD, it should take into account a series of limitations: the relatively small sample size (despite being larger than that in previous studies) and the exclusion of subjects with mild cognitive impairment (preclinical stage of AD) or advanced dementia. Future prospective studies should attempt to segment the RNFL and macula to know which retinal layers are predominantly affected and should investigate Age Macular Disease-AD associations (by calculating amount of druse; measuring thickness of Bruch’s membrane and auto-fluorescence of retina in AD cases, etc.) (Ohno-Matsui 2011).We think this is an interesting and promising research field in an unfortunately increasingly common dementing disorder in the elderly. Time will tell whether we are onlymeasuring and comparing different parameters with the latest technologies or whether we are facing a new and accessible biomarker of disease.

Utilidad de la alfafetoproteína sérica materna como parámetro de riesgo del embarazo

Resumen Objetivo Demostrar que las embarazadas con valores elevados, sin justificacion aparente, de alfafetoproteina serica tienen un riesgo mayor de resultados perinatales adversos Sujetos y metodos Se estudiaron 43.424 gestantes desde el segundo trimestre del embarazo hasta el parto. Se calculo el riesgo relativo entre los valores de alfafetoproteina en suero materno (AFPSM) y los resultados perinatales siguientes: partos preterminos, muertes fetales anterior y posterior a la semana 28 de gestacion, y recien nacidos con bajo peso Resultados Se estudio la influencia de las concentraciones de AFPSM sobre 4 resultados perinatales adversos, observandose en todos ellos una diferencia significativa entre el grupo de gestantes considerado control (AFPSM 2,5 MDM). Se observo un mayor riesgo relativo en las gestantes con muerte fetal anterior a la semana 28 de gestacion Conclusiones Se ha comprobado que existe una relacion entre los valores elevados de AFPSM y el riesgo de un resultado perinatal adverso. Sin embargo, la AFPSM no se puede considerar un marcador de cribado adecuado, por su baja sensibilidad, para seleccionar gestantes con un riesgo elevado de un resultado adverso

Reducción de la prevalencia de los defectos de cierre del tubo neural mediante un programa regional de cribado con alfafetoproteína en suero materno

Resumen Objetivo Optimacion de un programa regional de cribado con alfafetoproteina serica materna con objeto de que la prevalencia de los defectos de cierre del tubo neural en el Principado de Asturias sea proxima a cero Sujetos y metodos Las participantes han sido gestantes pertenecientes a las diferentes areas de salud del Principado de Asturias independientemente de su procedencia, sanidad publica o privada. Se estudiaron a 63.163 embarazadas en 11 anos. Todas las tecnicas bioquimicas se realizaron en el Laboratorio de Bioquimica del Hospital San Agustin Resultados Se ha conseguido optimizar el programa de cribado con alfafetoproteina en suero materno con porcentajes muy bajos de falsos positivos y amniocentesis realizadas, el 1,9 y el 0,5%, respectivamente. Se ha observado que las anencefalias presentan valores de alfafetoproteina en suero materno significativamente mas elevados que las espinas bifidas abiertas, y se ha conseguido disminuir la prevalencia de este tipo de malformaciones en el Principado de Asturias de una media de 1,3% a practicamente cero Conclusiones La puesta en funcionamiento de un programa de estas caracteristicas requiere un equipo multidisciplinario para que los resultados obtenidos sean satisfactorios. Despues de varios anos de desarrollo los resultados obtenidos han convencido a los obstetras y medicos generalistas de la utilidad de incorporar dicho programa al protocolo del embarazo. Este programa ha servido, ademas, para obtener un conocimiento exhaustivo de este tipo de malformaciones en el Principado de Asturias