Influence of hematocrit and localisation of punch in dried blood spots on levels of amino acids and acylcarnitines measured by tandem mass spectrometry
Introduction
The integration of electrospray injection tandem mass spectrometry (ESI-MS/MS) into newborn screening programs allows the detection of numerous metabolites that are relevant for the diagnosis of many inborn errors of metabolism (IEM) [1], [2], [3], [4], [5]. It has become the mainstay for screening of IEM, including amino acidopathies, defects of fatty acid oxidation and organic acidopathies [1], [2], [3], [4], [5].
Newborn screening by ESI-MS/MS is based on the semi-quantitative analysis of amino acids (AA) and acylcarnitine ester (AC) using stable isotopically labelled internal standards in whole blood dried on filter cards. Recently, analysis of GAA, a characteristic metabolite for guanidinoacetate-methyl-transferase deficiency, which represents an inborn error in creatine synthesis, was included in our newborn screening program. Diagnosis of IEM is suspected when certain AA, GAA or AC concentrations exceed population dependent cut-off limits. These limits typically reflect the 99th to 99.5th percentile [2], [3].
Most IEM detected by ESI-MS/MS in newborn screening programs show characteristic metabolite profiles that unequivocally exceed the respective cut-off limits. While this may be true for some disorders, diagnosis may be difficult for others. In addition metabolite levels depend on the metabolic status, dietary intake and maturity of the infants which may pose a diagnostic challenge requiring confirmatory tests such as further biochemical testing, measurement of enzyme activity in fibroblasts or mutation analysis [1], [2], [4].
There may be additional factors that have a profound effect on the analysis of metabolites. Hematocrit, for example, has a considerable effect on blood viscosity, and thereby may affect flux and diffusion properties of the blood put on filter paper used for newborn screening. In addition there may be a significant difference of metabolite concentrations between central and peripheral areas within the dried blood spot, due to chromatographic effects. Neonates screened on the third day of life show considerable interindividual variability of hematocrit levels and may therefore be particularly amenable to changes of metabolite concentrations [6], [7], [8]. This may be particularly true for preterm infants and those who are critically ill on the day of screening [8].
Consequently, we devised a study to investigate whether hematocrit or the location of the punch within the dried blood spot has any effect on concentrations of AA, free carnitine (FC), total carnitine (TC), AC and guanidinoacetate (GAA) measured by ESI-MS/MS.
Section snippets
Samples
Heparinised blood (50 ml) was taken from a healthy adult volunteer following written informed consent. Blood was spun down immediately and the plasma was separated. Plasma was then added in respective amounts to the corpuscular elements so that samples with defined hematocrit levels (20%, 30%, 40%, 50%, 60%) were obtained. Forty dried blood spots were made for each hematocrit level using standard filter cards (Schleicher, SS 2992). Blood spots were generated by dispensing blood into the centre
Results
Levels of most AA and GAA increased significantly with increasing hematocrit (p < 0.001), while the effect of hematocrit on aspartate and tyrosine was less pronounced and not statistically significant (Table 1).
Total carnitine, FC, some long chain AC (C16, C18, C18:1, C18:2) as well as some medium and short chain AC (C2, C3, C4, C4DC, C5OH) correlated with hematocrit (p < 0.001) (Table 1, Table 2). For other AC species (C6, C8, C10, C12, C14) a significant correlation was not observed (data not
Discussion
Newborn screening programs employing tandem mass spectrometry rely on population based cut-off values for different metabolites in dried blood filter cards that are typically sampled on day of life three [3], [4]. In addition, this analytical set-up is used for selective metabolic diagnosis and retrospective analysis of stored filter cards [11], [12], [13]. For analysis a 3 mm disk is punched from the filter card, the metabolites eluted and typically derivatised to yield corresponding butyl
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