GlycoMark Assay Overview
GlycoMark® provides a quantitative measurement of 1,5 anhydroglucitol (1,5 –AG) in either serum or EDTA plasma.
A comprehensive evaluation of the GlycoMark assay by Nowatzke W. et al. notes that GlycoMark is stable and linear from 0.49 to 110 ug/ml. The intra- and interassay imprecision ranged from 1.3 to 3.8% and 0.79% to 3.7%, respectively. Similar results are reported by the manufacturers using the Hitachi 917. Sensitivity (package insert) is estimated at 0.2 ug/ml, (mean 1,5-AG concentration plus one standard deviation).
The GlycoMark enzymatic assay was developed by Nippon Kayaku, Inc. for use on automated open chemistry analyzers. There are two reactions in the method. Reaction 1 is a sample pretreatment performed by the addition of glucokinase (GK) to convert glucose to glucose 6-phosphate in the presence of adenosine triphosphate (ATP), pyruvate kinase (PK) and phosphoenol pyruvate (PEP). The purpose of this reaction is to alter glucose so it can not react in the primary assay for 1,5-AG (reaction 2).
Reaction 2 uses pyranose oxidase (PROD) to oxidize the second hydroxyl of 1,5-anhydroglucitol. The amount of hydrogen peroxide generated in this reaction is directly related to serum 1,5-AG concentrations and is detected by colorimetry using peroxidase (POD). (Note: A schematic of these reactions in presented on the package insert.
The GlycoMark assay requires the reagent kit (NK-8300), controls (NK-8330) and a standard (NK-8320). The reagent kit consists of two offset vials, R1 (20 ml) and R2 (10 ml), suitable for deployment on an open automated chemistry analyzer. Note: R1 and R2 contain sodium azide and proper precautions should be followed. The Controls kit contains three 2ml vials) of each low (Control L) and high control (Control H). The 50 ug/ml calibration standard kit contains three 5 ml vials. A saline reagent blank (0.9% sodium chloride in deionized water) is required but not supplied.
Studies have shown individuals of differing ages, genders and ethnic origins indicate that the only differences in GlycoMark normal values occur between genders.
A comprehensive evaluation of the GlycoMark assay is described by Nowatzke W. et al. (Evaluation of an assay for serum 1,5-anhydroglucitol [GlycoMark] and determination of reference intervals on the Hitachi 917 analyzer Clinca Chimica Acta 2004;350:201-9). This study showed that the assay was stable and linear from 0.49 to 110 ug/ml. The intra- and interassay imprecision ranged from 1.3 to 3.8% and 0.79% to 3.7%, respectively. Similar intra- and interassay precision results are reported by the manufacturers using the Hitachi 917. (nonparametric 2.5th to 97.5th percentiles) as 10.2-33.8 ug/ml (males) and 5.9-31.8 ug/ml (females). The GlycoMark package insert reports a reference interval of 10.7 -32.0 ug/ml (males) and 6.8-29.3 ug/ml (females).
Note: Each laboratory should determine its reference range for its own instrument
The GlycoMark assay is kinetic. Parameters for a variety of other instruments are available upon request. The following is a description of the Hitachi 917 procedure:
All reagents are placed undiluted directly on the analyzer. The reaction occurs at 37oC. At zero time, 4 ul of standard, control, reagent blank or serum or EDTA plasma sample is added to 120ul of R1. At 5 minutes, 60 ul of R2 is added and the absorbances at 546 nm (primary) and 700 nm (secondary) are immediately obtained. At 10 minutes, both absorbances are again determined. The absorbance at 700 nm is subtracted from that at 546 nm as a background correction for both the 5 and 10 minute readings. Finally the 5 min corrected value is subtracted from the 10 minute value. The 1,5-anhydroglucitol concentrations are then determined from standard curve (usually linear) established from the kinetic change in absorbance of the reagent blank and the 50 ug/ml 1,5-AG standard.
