Streamlined determination of lysophosphatidylcholines in dried blood spots for newborn screening of X-linked adrenoleukodystrophy.
BACKGROUND
Pre-symptomatic hematopoietic stem cell transplantation is essential to achieve best possible outcomes for patients with the childhood cerebral form of X-linked adrenoleukodystrophy (X-ALD). We describe a high-throughput method for measurement of C20-C26 lysophosphatidylcholines (LPCs) and biochemical diagnosis of X-ALD using the same dried blood spots (DBS) routinely used for newborn screening.


METHODS
LPCs are extracted from 3-mm DBS punch with methanol containing an isotopically labeled LPC as internal standard. This extract is transferred to a 96-well plate, evaporated and then reconstituted in mobile phase for flow injection analysis tandem mass spectrometry (FIA-MS/MS) in selected reaction monitoring mode for measurement of four different LPCs (C20, C22, C24, C26) and the internal standard (d4-C26-LPC). Analysis time is 1.5min per sample.


RESULTS
The mean CVs from the intra- and inter-assay experiments for LPCs were 6.3-15.1% for C20-LPC, 4.4-18.6% for C22-LPC and 4.5-14.3% for C24-LPC. Limits of detection were determined for C20-LPC (LOD=0.03μg/mL), C22-LPC (0.03μg/mL), C24-LPC (0.03μg/mL) and C26-LPC (0.01μg/mL). Reference ranges were established from DBS of 130 newborns and 20 adults. Samples of patients with X-ALD (n=16), peroxisomal biogenesis disorders (n=8), and X-ALD carriers (n=12) were analyzed blindly and all were correctly identified.


CONCLUSION
Analysis of LPC species by FIA-MS/MS is a fast, simple and reliable method to screen for X-ALD and other peroxisomal disorders in DBS. To maximize specificity, abnormal results can be verified by a 2nd tier assay using LC-MS/MS.
['Turgeon|Coleman T|CT|', 'Moser|Ann B|AB|', 'Mørkrid|Lars|L|', 'Magera|Mark J|MJ|', 'Gavrilov|Dimitar K|DK|', 'Oglesbee|Devin|D|', 'Raymond|Kimiyo|K|', 'Rinaldo|Piero|P|', 'Matern|Dietrich|D|', 'Tortorelli|Silvia|S|']
D000326:Adrenoleukodystrophy D000328:Adult D002853:Chromatography, Liquid D059788:Dried Blood Spot Testing D057166:High-Throughput Screening Assays D006801:Humans D007231:Infant, Newborn D057230:Limit of Detection D008244:Lysophosphatidylcholines D015997:Neonatal Screening D018901:Peroxisomal Disorders D012016:Reference Values D053719:Tandem Mass Spectrometry
Lysophosphatidylcholine Dried blood spot Newborn screening X-linked adrenoleukodystrophy Tandem mass spectrometry Peroxisomes