engleski

Determination of N-glycan profile from dried blood spots

The development of diagnostic methods for glycan analysis could provide interesting possibilities for disease diagnosis, prognosis and monitoring. Dried blood spots (DBS) technology could significantly simplify and cheapen the development and research of those methods and their potential routine application. This study examines whether it is possible to compare the N-glycan profile obtained from DBS with the plasma N-glycan profile obtained directly from plasma samples, and whether it is possible to get an equally satisfactory glycan profile from blood cards that are two years old. For that purpose DBS were eluated from blood cards with one out of three different solutions (lysis buffer, PBS or water), after which N-linked glycans were released by N-glycosidase F, labeled with a fluorescent marker, and subjected to hydrophilic interaction high performance liquid chromatography (HILIC). Additional experiments were carried out to test the reproducibility of the method and its potential application in diagnosis. The results showed that the N-glycan profile can be obtained from DBS (including two year old DBS) with all three types of solutions used in the experiment, although with differences in resolution between glycan profiles. Red blood cell lysis buffer proved to be the best choice for DBS elution. The additional experiment showed that acetonitrile does not help with the elution of glycoproteins from cellulose paper. It also showed that glycan profiles obtained from blood spots stored on regular filter paper were equally as good as those stored on non-FTA blood cards. The method showed acceptable repeatability for complex biological samples and it could be used in diagnosis. However, there is still room for optimization. Once the method becomes fully optimized, the advantages of DBS technology will be numerous.

glycosylation; N-glycan profile; dried blood spots; HPLC

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