Supplementing Inosine to Blood Collection Tubes Adds a Glycolytic Inhibitory Effect

At present, sodium fluoride (NaF)-supplemented tubes are used as usual blood collection tubes that can measure glucose and glycated hemoglobin (HbA1c) simultaneously. However, blood collection with NaF tubes show a decrease in glucose levels at room temperature within 4 h after blood collection [1]. The American Diabetes Association (ADA) guidelines recommend immersion of blood samples collected with heparin-lithium (Hp-Li) in ice water within 30 min or the use of tubes containing NaF and citrate buffer (FC) [2]. However, immediate immersion in ice water is difficult in clinical practice and citric acid is difficult to dissolve and is not suitable for HbA1c measurements because of hemolysis. Therefore, we aimed to develop blood collection tubes that are easier for clinical use than FC tubes and minimize blood glucose decline, compared with NaF tubes.

We collected 2 mL of whole blood each tube. Immediately after blood collection, each tube was agitated on a mix rotator at 3000 rpm (1470 g) for 5 min and then stored under storage conditions until immediately before measurement. The aliquoted blood collection tubes were stored at room temperature (25°C) or refrigerated (4°C). Then, the aliquoted whole blood was plasma-separated by centrifugation immediately and 2, 4, 24, and 48 h after collection. Glucose was measured immediately after separation, as well as HbA1c.

Changes in blood glucose levels in the NaF, FI, and FC tubes are shown in Figure 1A–D. Blood glucose changes were significantly attenuated to a greater degree in FI tubes than in NaF tubes at 4°C and 25°C conditions 4 and 24 h after sampling, whereas the blood glucose-preserving abilities of FI tubes were significantly inferior to those of FC tubes, except the case when stored at 4°C for 4 h. Regarding the criteria required from ADA (within 6.1% of the baseline blood glucose levels), the storage of FI blood collection tubes at 4°C did not reduce blood glucose levels by > 6.1%, which is the criteria required from ADA, in any sample up to 48 h, whereas the storage of FI blood collection tubes at room temperature reduced blood glucose levels by > 6.1% in 1 of 10 cases after 24 h (Figure S2), which were much superior to the NF tubes (Figures S1 and S2).

In addition, the enzymatic and immunoassay methods showed a significant difference with a p value of 0.005 in FC blood tubes, whereas no significant difference was observed for the FI blood tubes (Figure 1E,E). Concordantly, no significant difference in hemolytic Hb levels was observed in the NaF and FI tubes, but not in FC tubes, compared with Hp-Li tubes (Figure S4).

Regarding the mechanism, although the detail mechanisms will be published somewhere else, the inhibitory effects on ATP and glucose uptake decline in erythrocytes were considered. We believe that inosine addition to the NaF blood collection tube would contribute to measuring the blood glucose levels exactly, without disturbing HbA1c measurements.

Y.K. and M.K. designed the study. M.K. supervised the study. Y.K., M.O., N.N., and M.K. carried out the research. Y.K. wrote the manuscript. S.A.-O., T.Y., H.I., and M.K. reviewed the manuscript draft. All authors accept responsibility for the entire content of this manuscript and have approved its submission.

This study was approved by the ethics committee of the Graduate School of Medicine at the University of Tokyo (Approval no. 2020063NI).

Healthy participants provided written informed consent.

The authors declare no conflicts of interest.