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Journal Scan

July 2019

Preanalytic error in plasma/serum glucose measurement examined

Summarized from Lippi G, Salvagno G, Lampus S et al. Impact of blood cell counts and volumes on glucose concentration in uncentrifuged serum and lithium-heparin blood tubes. Clin Chem Lab Med 2018; 56: 2125-31
Accurate estimation of in vivo blood glucose concentration is potentially compromised by in vitro glycolysis, the temperature- and time-dependent enzymatic metabolism of glucose by blood cells after blood is removed from the body. 

For more than 40 years it was recommended that in order to avoid the potential problem of falsely reduced glucose due to in vitro glycolysis, blood for estimation of plasma glucose should be collected into a tube containing sodium fluoride (NaF), an inhibitor of glycolysis, in addition to the anticoagulant additive required for blood plasma recovery. 

With more recent recognition that NaF fails to prevent glycolysis during the first few hours after blood is collected, and that glycolysis is immediately inhibited by acidification of blood to pH 6, expert recommendation has changed. 

Current recommendation for accurate estimation of plasma glucose is that blood should be collected into a tube containing a citrate buffer that ensures reduction of blood pH to 6.0; NaF alone should not be relied on to prevent in vitro glycolysis. 

Despite expert recommendation, many laboratories around the world generate and report serum and plasma glucose concentrations derived from blood collected into tubes used for general biochemical profiling that contain no additive to prevent in vitro glycolysis. The only strategy for prevention (or mitigation) of the deleterious effect of in vitro glycolysis, if such tubes are used, is to separate serum or plasma from blood cells by centrifugation immediately blood is collected (i.e. within 15-30 minutes of blood collection). Lowering the temperature of blood to 4 °C by the use of an ice slurry during the time between collection and serum/plasma separation also inhibits in vitro glycolysis. 

Recognition that in vitro glycolysis remains a potential problem if non-standard collection tubes are used for glucose estimation, prompted this recently published study by a group with long-established interest in preanalytic aspects of clinical laboratory testing. 

The aim of the study was to examine the effect that blood cell numbers have on the time-¬dependent decline in plasma and serum glucose concentration that occurs in uncentrifuged blood collected to blood tubes without glycolysis-inhibitor additive. 

The study population comprised 30 ostensibly healthy non-fasting volunteers recruited from laboratory personnel. Venous blood was sampled from each into three serum (clot activator and gel separator) tubes and three plasma lithium heparin tubes. A complete blood count (CBC) was performed on the first lithium heparin tube immediately after collection. 

Within 15 minutes of collection, the first lithium heparin tube and first serum tube were centrifuged; recovered plasma and serum samples were submitted for baseline glucose estimation. The other two sets of samples were left at room temperature for 1 and 2 hours, respectively, before centrifugation and glucose measurement. 

At baseline, median plasma glucose was slightly, but significantly higher than median serum glucose (4.72 mmol/L; IQR 4.46-5.26 versus 4.66 mmol/L; IQR 4.49-5.04).

After 1 hour storage before centrifugation, median plasma glucose fell from 4.72 to 4.41 mmol/L and after 2 hours, to 3.97 mmol/L. 

After 1 hour storage before centrifugation, median serum glucose fell from 4.66 mmol/L to 4.44 mmol/L and after 2 hours, to 4.14 mmol/L. 

The calculated mean rate of decline in glucose concentration (due to in vitro glycolysis) was greater in plasma samples than serum samples (0.33 mmol/L/hour versus 0.24 mmol/L/hour). In relative terms, glucose concentration decreased by 7 % and 5 % per hour in plasma and serum, respectively.

In univariate analysis, the absolute decrease in glucose of both plasma and serum samples was associated with gender (higher in men than in women), red blood cell (RBC) count; hematocrit; white blood cell (WBC); neutrophil count; and monocyte count. In multivariate analysis decease of glucose was shown to be independently associated with RBC, WBC, neutrophils and monocytes. No association was evident between decline in glucose and other hematological parameters (platelet count, erythrocyte volume and platelet volume). 

Application of the analysis of variance (ANOVA) tool confirms the association between the four hematological parameters (RBC, WBC, neutrophil and monocyte). As might be predicted, though never before demonstrated, the higher the blood cell count, the higher is the rate of decrease in glucose of uncentrifuged blood that contains no glycolysis inhibitor. 

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Chris Higgins

has a master's degree in medical biochemistry and he has twenty years experience of work in clinical laboratories.

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