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Title: Development of Preparation Methods for Glycated Biomolecules
Authors: Wanutchaya Duanginta
วณัชยา ดวงอินตา
Wanvisa Treebuphachatsakul
วันวิสาข์ ตรีบุพชาติสกุล
Naresuan University
Wanvisa Treebuphachatsakul
วันวิสาข์ ตรีบุพชาติสกุล
Keywords: Reference material
Processed blood material
Quality control material
Issue Date: 2022
Publisher: Naresuan University
Abstract:         Blood materials are required for quality assurance and control of hemoglobin A1C (HbA1C) measurements. This study presents an optimal in vitro glycation condition for preparing blood materials for HbA1C with the desired high HbA1C content and commutable are needed for performance evaluations to ensure the quality of HbA1C results. Whole blood in CPDA-1 from blood bank was washed with 0.85% normal saline, erythrocytes were incubated with various concentrations of D-glucose in phosphate buffer saline at 37 °C for up to 120 hours for in vitro glycation.  Processed blood materials from in vitro glycation were tested for homogeneity and stability following ISO Guide 35. Twenty-five clinical blood samples and nine blood materials were commutability tested using six methods in reference and clinical laboratories, as well as two point-of-care devices. Unweighted means based on commutability were used to characterize HbA1C in blood materials. Incubating erythrocytes with 400 mM D-glucose for 15 hours at 37 °C resulted in a significant (p<0.001) increase (p<0.001) in HbA1C in blood materials, with a remaining Hct ranging from 38% to 42%. Hemoglobin A1C in blood materials was stable at 3.8±0.8 °C for 70 days and stable at 8.1 to 23.5 °C during transportation. Six out of nine blood samples had commutable HbA1C measurement results between enzymatic and turbidimetric immunoassays, whereas three out of nine were non-commutable. The commutability of HbA1C in blood materials showed a variance and dependence on the preparation methods, level of HbA1C, and measurement principles. An optimal condition for in vitro glycation by incubation of erythrocytes with 400 mM D-glucose for 15 hours at 37 °C was able to generate HbA1C material with intact erythrocytes that is sufficiently stable and commutable between enzymatic and turbidimetric immunoassay. Therefore, this condition is suitable for the preparation of blood material for HbA1C immunoassays.
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