Comparación de dos métodos para la cuantificación de acilcarnitinas mediante espectrometría de masas en tándem en muestras de sangre impregnadas en papel para el diagnóstico de los defectos de la β-oxidación mitocondrial de los ácidos grasos

Abstract

The main objective of the Neonatal Screening Programs (PCN) is the identification of newborns (NBs) with serious diseases before the appearance of symptoms and for which there is an effective treatment that would avoid serious consequences on the NB. Technological advances have allowed the implementation of tandem mass spectrometry (MS/MS) in PCN, allowing the detection, among others, of defects in the β-oxidation of fatty acids (DOAG), which are congenital errors of metabolism (ECM) with autosomal recessive inheritance. The early detection of these disorders is carried out through the quantification of acylcarnitines in blood samples impregnated on paper and allows the establishment of treatments at an early age that improves their prognosis. Methodology: Two methodologies based on MS/MS are used. In one of them, the derivatization (butylation) of the acylcarnitines extracted from the paper support is carried out by means of an extractor solution, while the other consists of the quantification of the acylcarnitines directly after the extraction process. Our laboratory has used both methods: one based on the derivatization of acylcarnitines, using a mass spectrometer model API 3200 and another, based on the underivatized method and in which the measurements were made in a mass spectrometer model TQD. Results: The first part of this work consists of checking the validity of both methods for the quantification of acylcarnitines in blood samples impregnated on paper (S&S 903). For this, samples of unimpregnated S&S 903 filter paper and quality controls belonging to the Newborn Screening Quality Assurance Program of the Center for Disease Control and Prevention are analyzed. It is found that the method with derivatization results in higher detection and quantification limits, greater sensitivity and poorer precision, compared to the method without derivatization. Regarding bias, the method with derivatization, in general values are obtained higher than the reference value while, when it is not derivatized, the results are lower, with the exception of C0. The coefficients of determination obtained by both methods are statistically equal to and greater than 0.99. The correlation between both work methods is strong and fits a linear regression model. In the second part of this study, the results of 167,563 NBs from the Region of Murcia and the Autonomous City of Melilla were studied, whose blood samples impregnated on paper were received between 02/09/2007 and 01/28/2016. It is found that all acylcarnitines have a dependence on the age of the NB at which the samples were taken. In general, all acylcarnitines show good stability except C2 and C3. Likewise, the concentrations of most acylcarnitines are dependent on the weight of the newborn at birth, as well as the type of feeding (maternal or artificial) of the NB at the time of taking the sample. All these results are independent of the method used to quantify acylcarnitines. During the study period, 13 patients with DOAG confirmed by molecular studies were detected: 2 CUD, 4 SCADD, 3 MCADD, 1 VLCAD, 2 LCHADD and 1 MADD, yielding an overall incidence of 1/12,889. Conclusion: both methods are equally valid to be used in a PCN intended for the early detection of DOAG, each method presenting advantages and disadvantages with respect to the other, which will have to be taken into account by the laboratories that want to implement them. The diagnostic capacity of both techniques for the detection of ECM related to DOAGs are similar