Effectiveness of citrate buffer-fluoride mixture in Terumo tubes as an inhibitor of in vitro glycolysis

Abstract

Introduction: Glycolysis affects glucose determination in vitro. The placement of sample tubes in ice-water slurry with plasma separation within 30 minutes is recommended, or alternatively the use of a glycolysis inhibitor. The aim of our two-steps study was to evaluate which Terumo tube is best for glucose determination in routine clinical setting.

Materials and methods: In the first study, blood from 100 volunteers was collected into lithium heparin (LH), NaF/Na heparin (FH) and NaF/citrate buffer/Na2EDTA (FC-Mixture) tubes. LH sample was treated as recommended and considered as reference, while FH and FC-Mixture samples were aliquoted, maintained at room temperature (RT) for 1, 2 and 4 hours; centrifuged and plasma analysed in triplicate. In the second study, samples from 375 volunteers were collected in LH, FH and FC-Mixture tubes and held at RT before centrifugation from 10 to 340 minutes, depending on each laboratory practice. Samples were analysed in one analytical run.

Results: In the first study, FH glucose concentrations were 5.15 ± 0.66 mmol/L, 5.05 ± 0.65 mmol/L and 5.00 ± 0.65 mmol/L (P < 0.001) in tubes stored at RT for 1, 2 and 4 hours, respectively. Mean biases in all time points exceeded the analytical goal for desirable bias based on biological variation criteria. FC-Mixture glucose concentrations were 5.48 ± 0.65 mmol/L, 5.46 ± 0.6 mmol/L and 5.46 ± 0.64 mmol/L in tubes stored at RT for 1, 2 and 4 hours, respectively. Mean biases for FC-Mixture glucose in all time points reached optimal analytical goals. In the second study, the biases for LH and FH glucose compared to reference FC-Mixture glucose exceeded the preset analytical goals, regardless of the blood collection to centrifugation time interval.

Conclusions: FC-mixture tubes glucose concentrations were preserved up to 4h storage at RT. We confirmed that NaF alone does not allow immediate glycolysis inhibition in real life pre-centrifugation storage conditions (up to 340 minutes). FC-Mixture should be used exclusively for glucose determination in laboratories unable to implement the recommended blood samples' treatment.

Keywords: citrate acidification; glucose; pre-analytical phase; sodium fluoride; stability.

Figure 1

Comparison of glucose concentrations in sample tubes evaluated, stored at room temperature, with reference glucose concentrations using Bland-Altman plots. Reference glucose concentrations were obtained from lithium-heparin tubes placed in ice-water slurry and centrifuged within 30 minutes, according to ADA-NACB guidelines.
A – Comparison of reference glucose with sodium fluoride-sodium heparin (FH) glucose stored for 1 hour. B - Comparison of reference glucose with FH glucose stored for 2 hours. C - Comparison of reference glucose with FH glucose stored for 4 hours. D - Comparison of reference glucose with sodium fluoride-citrate buffer-sodium EDTA (FC-Mixture) glucose stored for 1 hour. E - Comparison of reference glucose with FC-Mixture glucose stored for 2 hours. F - Comparison of reference glucose with FC-Mixture glucose stored for 4 hours. Solid line (mean) – mean difference. Dashed lines (SD) – standard deviation. Dotted line – line of equality.

Figure 2

Median glucose concentrations in evaluated tubes stored at room temperature at different time periods between blood drawing and centrifugation.
LH - lithium-heparin tube. FH - Sodium-fluoride and sodium-heparin containing tube. FC-Mixture – Sodium-fluoride, citrate buffer and sodium EDTA containing tube.*Wilcoxon pair-wise test: P < 0.001.