Abstract
Background: The laboratory diagnosis of alpha-1 antitrypsin deficiency (AATD) consists of alpha-1 antitrypsin (AAT) plasma levels quantification and AAT genotype and phenotype determination. Phenotyping helps to confirm the presence of the most common S and Z alleles and to detect rare variants. The classical method used to determine phenotype is IsoElectric Focusing (IEF), based on the separation of proteins according to their charge. In our lab, AATD diagnosis is lead on dried blood spot (DBS), drops of blood that have been dried onto a special filter paper, since 2006, in order to allow the centralization of AATD diagnosis in Italy.
Objectives: We adapted a semi-automatic system for the determination of AAT phenotypes on DBS and we compared this method to the standard IEF performed with home made polyacrylamide gels1.
Methods: The new method uses the Hydragel 18 A1AT Isofocusing® kit on the Sebia Hydrasys® System. The procedure consists of samples runs on ready-to-use agarose gels and immunofixation with an AAT specific antiserum labeled with peroxidase.
Results: The Hydragel 18 A1AT Isofocusing® kit has been conceived to work with serum samples. To make the kit work with DBS samples we introduced some changes in the protocol, in particular regarding dilutions. We ran 100 DBS samples both with the old and the new method and we obtained superimposable results. Moreover, the new method made easier the identification of rare variants and clearer the Z-AAT pattern than old method.
Conclusions: The new semi-automatic IEF method applied to DBS resulted to be easier, faster and more reproducible than the old manual procedure.
1.Ferrarotti I, et al. Transl Res. 150:267-74, 2007.
- Copyright ©the authors 2016