How pre-analytical conditions impact glucose measurement and (gestational) diabetes diagnosis: A real-world stability study and a call for harmonization.
Author(s): Nevraumont A, Deltombe M, Bayart JL
Publication: Clin Chim Acta, 2024, Vol. 562, Page 119875
PubMed ID: 39013524 PubMed Review Paper? No
Purpose of Paper
This paper compared the stability of glucose in case-matched lithium heparin, sodium-fluoride potassium oxalate (NaF/KOx), and sodium-fluoride EDTA-citrate (FC Mix) plasma during a 6 h post-centrifugation storage timecourse at room temperature and in NaF/KOx and FC Mix blood specimens that were stored for up to 24 h at room temperature or 4°C before centrifugation. The effect of delayed centrifugation was also assessed in matched NaF/KOx and FC Mix specimens collected from patients after an overnight fast and from pregnant women during an oral glucose tolerance test (OGTT).
Conclusion of Paper
The mean glucose concentration was lower in lithium heparin than NaF/KOx or FC Mix specimens when specimens were analyzed within 15 min of centrifugation, resulting in a clinically relevant bias in lithium heparin specimen relative to the FC Mix specimen. While not clinically relevant, the bias in glucose levels in the NaF/KOx specimen relative to the FC Mix specimen increased with post-centrifugation storage at room temperature (0.53% after <15 min, 0.94% after 2 h, 1.71% after 4 h, and 2.32% after 6 h).
Glucose levels were comparable in NaF/KOx and FC Mix specimens when each was centrifuged within 5 min. The concentration of glucose declined in the NaF/KOx specimens when centrifugation was delayed but remained stable in the FC Mix tubes, resulting in a clinically relevant bias after a 30 min delay to centrifugation at room temperature or 4°C. Importantly, this bias was observed in two different studies using different patients. When FC Mix tubes were used in a study of 701 specimens collected from fasting patients rather than NaF/Kox tubes, 149 additional patients had glucose levels above the threshold for impaired fasting glucose (≥5.55 mmol/L) and 18 additional patients had glucose levels greater than the threshold for diabetes (≥7.0 mmol/L). Similarly, when FC Mix tubes, as opposed to NaF/KOX tubes, were used to collect specimens from pregnant women during oral glucose tolerance testing, the number of specimens classified as pathological was higher in specimens with the longest delay to centrifugation. Consequently, the use of NaF/KOx tubes instead of FC Mix tubes resulted in 24 fewer women (of 109) diagnosed with gestational diabetes.
Studies
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Study Purpose
This study compared the stability of glucose in case-matched lithium heparin, sodium-fluoride potassium oxalate (NaF/KOx), and sodium-fluoride EDTA-citrate (FC Mix) plasma during 6 h of post-centrifugation storage at room temperature. Blood was collected from the antecubital vein of 20 healthy volunteers (10 men and 10 women, mean 35.1 years) into lithium heparin, NaF/KOx, and FC Mix tubes. Each tube was mixed by ten inversions and centrifuged at 2,870 g for 10 min within 5 min of venipuncture. Tubes were stored at room temperature and glucose was measured <15 min (referred to as 0 h), 2 h, 4 h and 6 h after centrifugation. Glucose was quantified using the Atellica CH Glucose Hexokinase 3 assay on an Atellica CH 930 analyzer. Clinically relevant bias was defined as a bias exceeding the EFLM desirable bias of 2.3%.
Summary of Findings:
When specimens were analyzed within 15 min of centrifugation, the mean glucose concentration was lower in specimens in lithium heparin than NaF/KOx or FC Mix tubes (4.82 versus 4.90 and 4.95, respectively). Further, the lithium heparin specimen had a clinically relevant mean absolute bias of 3.08% relative to the FC Mix specimen. The bias between the lithium heparin and FC Mix tube specimens remained relatively stable during post-centrifugation storage at room temperature (2.66% at 2 h, 3.18% at 4 h and 3.27% at 6 h). While not clinically relevant, the bias in glucose levels in the NaF/KOx specimen relative to the FC Mix specimen increased with post-centrifugation storage at room temperature (0.53% after <15 min, 0.94% after 2 h, 1.71% after 4 h, and 2.32% after 6 h).
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Carbohydrate Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Time at room temperature <15 min (0 h)
2 h
6 h
Biospecimen Acquisition Anticoagulant Sodium fluoride/potassium oxalate
Lithium heparin
Citrate/sodium fluoride EDTA
Biospecimen Acquisition Type of collection container/solution Lithium heparin tube
NaF/KOx tube
FC Mix tube
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Study Purpose
This study compared the stability of glucose in blood collected in sodium-fluoride potassium oxalate (NaF/KOx) and sodium-fluoride EDTA-citrate (FC Mix) tubes after a delay to centrifugation of 5 min, 30 min, 2 h, 4 h, 6 h or 24 h at room temperature or 4°C. For the initial study, blood was collected from the antecubital vein of five healthy volunteers (3 men and 2 women, mean 34.4 years) into NaF/KOx and FC Mix tubes. Tubes were then stored for 5 min, 30 min, 2 h, 4h, 6 h and 24 h both at room temperature and at 4°C before centrifugation at 2,870 g for 10 min and analysis. To verify the effects of delayed processing in the clinic, glucose levels were measured in case-matched blood specimens collected in NaF/KOx and FC Mix tubes; 701 patients undergoing fasting glucose analysis were used in the study (FPG study). Additionally, glucose was measured at the end of collection in matched NaF/KOx blood specimens from 109 pregnant women collected 0, 60 and 120 min after an oral glucose tolerance test (OGTT), which resulted in mean delays to centrifugation of 302.9, 242.9 and 182.9 min, respectively. Glucose was quantified using the Atellica CH Glucose Hexokinase 3 assay on an Atellica CH 930 analyzer.
Summary of Findings:
Glucose levels were comparable in NaF/KOx and FC Mix specimens when each was centrifuged within 5 min of venipuncture (4.90 mmol/L and 4.93 mmol/L, respectively), but when centrifugation was delayed, the concentration of glucose declined rapidly in the NaF/KOx specimens. The bias in the glucose levels in the NaF/KOx specimen relative to the promptly centrifuged FC Mix specimen were both clinically relevant after only a 30 min delay to centrifugation at room temperature or 4°C (4.90% and 3.06%, respectively), and this bias increased over the first 4 h of pre-centrifugation storage, after which it remained stable. In contrast, glucose levels remained relatively stable in FC Mix specimens during a pre-centrifugation delay of up to 24 h. In both tube types, glucose levels decreased more rapidly when the delay to centrifugation was at room temperature than 4°C. The mean bias in glucose levels between matched blood specimens collected in NaF/KOx and FC Mix tubes in the FPG study was 2.87 ± 1.85% in specimens centrifuged within 30 min but the mean bias was 8.59 ± 2.56% in specimens centrifuged 2-4 h after blood collection. When FC Mix tubes, rather than NaF/Kox tubes, were used in a study of 701 specimens collected from fasting patients, 149 additional patients (84% increase) had glucose levels above the threshold for impaired fasting glucose (≥5.55 mmol/L) and 18 additional patients (36.7%) had glucose levels greater than the threshold for diabetes (≥7.0 mmol/L). In the OGTT study, longer pre-centrifugation delays resulted in a higher number of FC Mix tube blood specimens classified as pathologic: 21 for the specimens with a mean delay of 302.9 min (0 h OGTT), 2 for the specimens with a mean delay of 242.9 min (1 h OGTT), and 6 for the specimens with a mean delay of 182.9 min (2 h OGTT); this trend was not observed among NaF/Kox blood specimens. Consequently, use of the FC Mix tube instead of the NaF/KOx tube would have resulted in 24 more women (150% increase) diagnosed with gestational diabetes.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Diabetes Type 2
- Pregnant
- Normal
- Not specified
Platform:
Analyte Technology Platform Carbohydrate Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution NaF/KOx tube
FC Mix tube
Biospecimen Acquisition Anticoagulant Citrate/sodium fluoride EDTA
Sodium fluoride/potassium oxalate
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage temperature Room temperature
4°C
Storage Storage duration 5 min
30 min
2 h
4 h
6 h
24 h
<30 min
2-4 h
Mean 302.9 min (0 h OGTT)
Mean 242.9 min (60 min OGTT)
Mean 182.9 min (120 min OGTT)