Abstract

Background: The five antigens of the Yt blood group system are located on the membrane GPI‐linked erythrocyte acetylcholinesterase (AChE‐E), which is implicated in a range of clinical issues, including sleep apnoea and neurotoxin poisoning. Conformational changes associated with different Yt antigens, as well as the storage of red cells, may affect AChE‐E activity. Direct analysis of AChE‐E presents unique challenges that may be overcome by electrochemical measurement using low‐cost electron‐mediated screen‐printed carbon electrodes (SPCEs) under suitable amperometric conditions. Aims: We present the electrochemical analysis of AChE‐E activity of five different Yt phenotypes, the effect on AChE‐E of red cell refrigeration over a four‐week timeframe and storage by freezing. The efficacy of electron‐mediated SPCEs is examined in relation to the potential interferences and electrode‐fouling. Methods: SPCEs containing the electron mediator (cobalt phthalocyanine) were manufactured on a plastic substrate, each in association with a screen‐printed Ag/AgCl counter/reference electrode in a two‐electrode format. Whole blood samples were initially washed to remove butyrylcholinesterase and resuspended in PBS. AChE‐E activity was assessed at 37⁰C using chronoamperometry at + 0 mV following the addition of acetylthiocholine chloride to the cell suspension. Ten Yt(a+b‐) and four Yt(a‐b+) donor samples were tested after collection and at two and four weeks storage at 4⁰C. Five Yt(a+b‐) and twenty Yt(a‐b+) were tested following freezing in liquid nitrogen. Samples with confirmed rare Yt phenotypes were evaluated to determine if the phenotype effected AChE‐E activity. The assay longevity and electrode viability were examined using eight of the samples representing the different Yt phenotypes over an extended duration of measurement. Results: SPCEs gave a consistent response in all samples tested without evidence of interference or fouling by the adherence of the cells to the electrode surface. The sensors were shown to operate continuously for a minimum of 60 minutes until the substrate became limiting. The reproducibility of the sensors (six replicates each of five samples) was 14% (RSD), which is typical for SPCEs in biological samples. An optimal assay duration of 120 seconds was selected. Compared to fresh samples, storage by freezing resulted in a loss of approximately 40% AChE‐E activity in Yt(a+b‐), whereas Yt(a‐b+) samples showed a 40% increase in enzyme activity. These differences occurred despite the almost identical conformation of the Yta and Ytb AChE‐E structure as studied by molecular dynamic calculations. Samples stored at 4⁰C showed a significant deterioration in AChE‐E activity within the first 14 days; a reduction of 35% for Yt(a+b‐) and 28% for Yt(a‐b+) (P < 0.01), followed by no significant change between 14 and 28 days of storage. The effect of rare Yt phenotypes was examined using stored frozen samples. AChE‐E activity differed markedly when compared to Yt(a+b‐); 82% higher in Yt(a‐b+) samples, 39% in YTLI– and 210% in YTOT–. An exception was YTEG–, which remained comparable to Yt(a+b‐). Summary/Conclusions: Mediated SPCEs were used effectively in the electrochemical measurement of AChE‐E activity, showing their potential for rapid low‐cost analysis of red cells. We showed that the activity of the enzyme was reduced with refrigeration, although the consequences of freezing were more varied in terms of Yt phenotype.