DOI 10.1515/cclm-2013-1059 Clin Chem Lab Med 2014; 52(7): 1061–1068
Huan Chan, Helen Lunt*, Harmony Thompson, Helen F. Heenan, Christopher M.A. Frampton and Christopher M. Florkowski
Plasma glucose measurement in diabetes: impact and implications of variations in sample collection procedures with a focus on the first hour after sample collection Abstract Background: Previous studies of participants with plasma glucose concentrations at or near the glucose reference range demonstrate glucose loss following delayed separation and extraction of plasma from the cellular components of blood, of ≤ 7% per hour. We aimed to assess pre-analytical glucose loss in diabetic subjects, focusing on the first hour after sample collection. Methods: Venous blood was collected from diabetes clinic attendees, into a series of lithium heparin PST™ (plasma separator tube) and fluoride oxalate Vacutainers™. Baseline (reference) plasma glucose measurements were undertaken on samples prepared under refrigerated conditions. The remaining samples underwent a series of controlled pre-analytical delays in sample preparation, at room temperature. Plasma glucose was measured using the hexokinase method. Results: Median baseline glucose (mmol/L) for the 62 participants was 10.6 (range 3.4–31.1). Using lithium heparin PST™ tubes, mean glucose loss (95% CI) was 0.16 (0.09–0.23) after 30 min delay in plasma preparation and 0.28 (0.21–0.34) after 60 min delay. Glucose loss was independent of both baseline glucose and also individual cellular count. Fluoride failed to inhibit glucose loss within the first hour after sample collection. Immediate plasma centrifugation of PST™ tubes, followed by delayed plasma extraction (median delay 92 min), produced a mean glucose loss of 0.02 mmol/L (–0.05–0.09). Conclusions: Samples collected into lithium heparin PST™ tubes show pre-analytical glucose loss at 1 h that is independent of baseline glucose and cellular count. Furthermore, immediate plasma separation using these tubes attenuates glucose loss across a wide range of glucose concentrations. Keywords: analytical sample preparation methods; glycolysis; glucose; hyperglycemia.
*Corresponding author: Helen Lunt, Diabetes Centre Christchurch, 550 Hagley Avenue, Christchurch 8011, New Zealand, Phone: +643 3640860, Fax: +643 3640171, E-mail: [email protected] Huan Chan and Helen F. Heenan: Canterbury District Health Board, Diabetes Centre Christchurch, Christchurch, New Zealand Harmony Thompson: School of Medicine, University of Otago Christchurch, Christchurch, Canterbury, New Zealand Christopher M.A. Frampton: Department of Medicine, University of Otago Christchurch, Christchurch, Canterbury, New Zealand Christopher M. Florkowski: Clinical Biochemistry Unit, Canterbury Health Laboratories, New Zealand
Introduction Accurate estimation of plasma glucose is essential for optimal clinical practice and high quality diabetes research [1–4]. Pre-analytical error in plasma glucose measurement secondary to glycolysis affects accuracy and is therefore of concern for both clinicians and researchers across a range of disciplines [3–8]. The current recommendation is that plasma should be separated from whole blood within 30 min of collection [2]. There is, however, a paucity of information about pre-analytical loss of glucose at the time point of 60 min or earlier. Information about early glycolysis is also of increasing relevance in relation to routine processing of samples, as the target for laboratory turnaround time is now often set at 60 min or less [9]. An earlier study reported pre-analytical error 1 h or more after sample collection, calculated from the mean of aggregated data across multiple time points, to be 5%–7% per hour [10]. This study and also similar findings from more recent studies [11–13] were based predominantly on samples with glucose concentrations at or near the laboratory reference range. There are very limited published data that describe pre-analytical glucose loss across a wide range of glucose values [14]. We therefore aimed to assess pre-analytical glucose loss in samples obtained from diabetic participants with a range of glucose values, focusing primarily
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1062 Chan et al.: Pre-analytical loss of glucose in diabetes on the first hour (time points 0 min, 30 min and 60 min) after sample collection. We chose to utilize a “real world” operational setting; samples were collected from patients attending a diabetes clinic and all measurements were undertaken using routine laboratory analyses. There were four secondary aims that were additional to the study’s main aim of describing pre-analytical glucose loss across a range of glucose values within an hour of sample collection. First, we aimed to assess the impact of cellular count on the rate of glycolysis. Whole blood samples with a high cellular count, e.g., leukocytosis associated with leukemia [15], and also high hematocrit levels [16], have the potential to decrease measured glucose levels prior to analysis, sometimes even causing pseudohypoglycemia secondary to glucose consumption by metabolically active cells [17]. It is, however, unknown whether small differences in cellular count within the physiological range of hematological values, e.g., those typically seen in ambulatory patients with diabetes, impact on the rate of glycolysis. Second, we aimed to determine if earlier publications showing that the glycolysis inhibitor fluoride had no effect on pre-analytical glycolysis within the first hour after sample collection [18], applied across a wide range of glucose values. Third, we aimed to determine the effect of immediate centrifugation of samples collected into tubes containing plasma separator gel such as the lithium heparin PST™ on pre-analytical glucose loss, as quantitative information about the impact of plasma cell separator tubes on analyte stability across a wide range of glucose values is sparse [4, 12, 18]. Finally, we aimed to explore the impact of early pre-analytical glycolysis on possible misclassification in a “real world” setting that was relevant to patients with established diabetes. Many previous studies have explored the effect of pre-analytical glucose loss on diabetes misclassification following administration of an oral glucose tolerance test, but this example is not relevant in the presence of established diabetes. In the current study we chose to assess the effect of pre-analytical loss of glucose on Error Grid assessment of capillary glucose meter performance, using plasma glucose as the reference value [19].
Materials and methods Participants Non-fasting participants aged 18 years and over, with established diabetes of any diagnostic type, were recruited from patients attending a diabetes outpatient clinic. Frail patients, those on oxygen and patients known to have experienced previous difficulties with
venesection, were excluded. Participants donated a single sample of 38 mL venous blood collected from the antecubital fossa and also a finger stick capillary sample.
Prescribed variations in sample collection procedures A venous sample was taken from the antecubital fossa using a 21 g (gauge) or 23 g BD winged infusion set or a 21 g or 22 g BD Vacutainer™ eclipse blood collection needle and hub for blood collection. The whole blood sample was placed into nine Vacutainers™; four lithium heparin PST™, four fluoride oxalate, one ethylene-diaminetetraacetic acid (EDTA). The order of draw was lithium heparin PST™ (green top), EDTA (purple top) followed by fluoride oxalate (gray top) tubes. It was ensured that as much blood as possible was taken up by the Vacutainer™ tubes and that there was no under-fill of the tubes. The blood was thoroughly mixed by gently inverting the tube 8–10 times. The processing of plasma samples is summarized in Figure 1. The lithium heparin PST™ and fluoride oxalate blood collection tubes were centrifuged at 1300 g (relative centrifugal force) for 15 min. The time that each step in sample processing occurred, including laboratory processing, was recorded on the study database. Given that the kinetics of glucose transport in red cells are well known to be dependent on temperature [20], the temperature at the clinic room bench where sample collection and processing occurred was also recorded. Further details of the prescribed pre-analytical delays outlined in Figure 1 are given below:
Lithium heparin PSTTM and fluoride oxalate tubes for optimal sample preparation Immediately after blood collection, test tubes were placed upright in ice slurry and then centrifuged in a pre-cooled refrigerated centrifuge (+4 oC) and the time of introduction to the centrifuge recorded. Plasma was separated into labelled plastic test tubes (3–4 mL Kahn tubes), then sent immediately to the laboratory.
Lithium heparin PSTTM and fluoride oxalate tubes for 30 min sample preparation Vacutainer™ tubes were left upright on the bench top at room temperature for 30 min after collection and then centrifuged at room temperature. Plasma was separated into labeled plastic test tubes (3–4 mL Kahn tubes), then sent immediately to the laboratory.
Lithium heparin PSTTM and fluoride oxalate tubes for 60 min sample preparation Vacutainer™ tubes were left upright on the bench top at room temperature for 60 min after collection and then centrifuged at room temperature. Plasma was separated into labeled plastic test tubes (3–4 mL Kahn tubes), then sent immediately to the laboratory.
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Chan et al.: Pre-analytical loss of glucose in diabetes 1063
Figure 1 Sampling protocol. FL, fluoride oxalate; PST, lithium heparin PSTTM.
Lithium heparin PSTTM and fluoride oxalate tubes treated as routine samples Immediately after sample collection, the lithium heparin PST™ tube was centrifuged at room temperature. The fluoride oxalate tube was treated as a routine clinic blood sample and did not undergo any preanalytical processing. In order to mimic routine conditions, tubes were left to lie flat after initial centrifugation (lithium heparin PST™ tubes) or from the time of initial sample collection (fluoride oxalate tubes). Tubes were taken to the laboratory within 2 h of blood collection.
Laboratory measurements All plasma samples were analyzed in an accredited laboratory using the Abbott C8000 analyzer (Abbott Diagnostics, North Chicago, IL, USA), utilizing the hexokinase method for measurement of plasma glucose (inter assay coefficient of variation
Huan Chan, Helen Lunt*, Harmony Thompson, Helen F. Heenan, Christopher M.A. Frampton and Christopher M. Florkowski
Plasma glucose measurement in diabetes: impact and implications of variations in sample collection procedures with a focus on the first hour after sample collection Abstract Background: Previous studies of participants with plasma glucose concentrations at or near the glucose reference range demonstrate glucose loss following delayed separation and extraction of plasma from the cellular components of blood, of ≤ 7% per hour. We aimed to assess pre-analytical glucose loss in diabetic subjects, focusing on the first hour after sample collection. Methods: Venous blood was collected from diabetes clinic attendees, into a series of lithium heparin PST™ (plasma separator tube) and fluoride oxalate Vacutainers™. Baseline (reference) plasma glucose measurements were undertaken on samples prepared under refrigerated conditions. The remaining samples underwent a series of controlled pre-analytical delays in sample preparation, at room temperature. Plasma glucose was measured using the hexokinase method. Results: Median baseline glucose (mmol/L) for the 62 participants was 10.6 (range 3.4–31.1). Using lithium heparin PST™ tubes, mean glucose loss (95% CI) was 0.16 (0.09–0.23) after 30 min delay in plasma preparation and 0.28 (0.21–0.34) after 60 min delay. Glucose loss was independent of both baseline glucose and also individual cellular count. Fluoride failed to inhibit glucose loss within the first hour after sample collection. Immediate plasma centrifugation of PST™ tubes, followed by delayed plasma extraction (median delay 92 min), produced a mean glucose loss of 0.02 mmol/L (–0.05–0.09). Conclusions: Samples collected into lithium heparin PST™ tubes show pre-analytical glucose loss at 1 h that is independent of baseline glucose and cellular count. Furthermore, immediate plasma separation using these tubes attenuates glucose loss across a wide range of glucose concentrations. Keywords: analytical sample preparation methods; glycolysis; glucose; hyperglycemia.
*Corresponding author: Helen Lunt, Diabetes Centre Christchurch, 550 Hagley Avenue, Christchurch 8011, New Zealand, Phone: +643 3640860, Fax: +643 3640171, E-mail: [email protected] Huan Chan and Helen F. Heenan: Canterbury District Health Board, Diabetes Centre Christchurch, Christchurch, New Zealand Harmony Thompson: School of Medicine, University of Otago Christchurch, Christchurch, Canterbury, New Zealand Christopher M.A. Frampton: Department of Medicine, University of Otago Christchurch, Christchurch, Canterbury, New Zealand Christopher M. Florkowski: Clinical Biochemistry Unit, Canterbury Health Laboratories, New Zealand
Introduction Accurate estimation of plasma glucose is essential for optimal clinical practice and high quality diabetes research [1–4]. Pre-analytical error in plasma glucose measurement secondary to glycolysis affects accuracy and is therefore of concern for both clinicians and researchers across a range of disciplines [3–8]. The current recommendation is that plasma should be separated from whole blood within 30 min of collection [2]. There is, however, a paucity of information about pre-analytical loss of glucose at the time point of 60 min or earlier. Information about early glycolysis is also of increasing relevance in relation to routine processing of samples, as the target for laboratory turnaround time is now often set at 60 min or less [9]. An earlier study reported pre-analytical error 1 h or more after sample collection, calculated from the mean of aggregated data across multiple time points, to be 5%–7% per hour [10]. This study and also similar findings from more recent studies [11–13] were based predominantly on samples with glucose concentrations at or near the laboratory reference range. There are very limited published data that describe pre-analytical glucose loss across a wide range of glucose values [14]. We therefore aimed to assess pre-analytical glucose loss in samples obtained from diabetic participants with a range of glucose values, focusing primarily
Brought to you by | University of California - San Francisco Authenticated Download Date | 12/12/14 12:47 PM
1062 Chan et al.: Pre-analytical loss of glucose in diabetes on the first hour (time points 0 min, 30 min and 60 min) after sample collection. We chose to utilize a “real world” operational setting; samples were collected from patients attending a diabetes clinic and all measurements were undertaken using routine laboratory analyses. There were four secondary aims that were additional to the study’s main aim of describing pre-analytical glucose loss across a range of glucose values within an hour of sample collection. First, we aimed to assess the impact of cellular count on the rate of glycolysis. Whole blood samples with a high cellular count, e.g., leukocytosis associated with leukemia [15], and also high hematocrit levels [16], have the potential to decrease measured glucose levels prior to analysis, sometimes even causing pseudohypoglycemia secondary to glucose consumption by metabolically active cells [17]. It is, however, unknown whether small differences in cellular count within the physiological range of hematological values, e.g., those typically seen in ambulatory patients with diabetes, impact on the rate of glycolysis. Second, we aimed to determine if earlier publications showing that the glycolysis inhibitor fluoride had no effect on pre-analytical glycolysis within the first hour after sample collection [18], applied across a wide range of glucose values. Third, we aimed to determine the effect of immediate centrifugation of samples collected into tubes containing plasma separator gel such as the lithium heparin PST™ on pre-analytical glucose loss, as quantitative information about the impact of plasma cell separator tubes on analyte stability across a wide range of glucose values is sparse [4, 12, 18]. Finally, we aimed to explore the impact of early pre-analytical glycolysis on possible misclassification in a “real world” setting that was relevant to patients with established diabetes. Many previous studies have explored the effect of pre-analytical glucose loss on diabetes misclassification following administration of an oral glucose tolerance test, but this example is not relevant in the presence of established diabetes. In the current study we chose to assess the effect of pre-analytical loss of glucose on Error Grid assessment of capillary glucose meter performance, using plasma glucose as the reference value [19].
Materials and methods Participants Non-fasting participants aged 18 years and over, with established diabetes of any diagnostic type, were recruited from patients attending a diabetes outpatient clinic. Frail patients, those on oxygen and patients known to have experienced previous difficulties with
venesection, were excluded. Participants donated a single sample of 38 mL venous blood collected from the antecubital fossa and also a finger stick capillary sample.
Prescribed variations in sample collection procedures A venous sample was taken from the antecubital fossa using a 21 g (gauge) or 23 g BD winged infusion set or a 21 g or 22 g BD Vacutainer™ eclipse blood collection needle and hub for blood collection. The whole blood sample was placed into nine Vacutainers™; four lithium heparin PST™, four fluoride oxalate, one ethylene-diaminetetraacetic acid (EDTA). The order of draw was lithium heparin PST™ (green top), EDTA (purple top) followed by fluoride oxalate (gray top) tubes. It was ensured that as much blood as possible was taken up by the Vacutainer™ tubes and that there was no under-fill of the tubes. The blood was thoroughly mixed by gently inverting the tube 8–10 times. The processing of plasma samples is summarized in Figure 1. The lithium heparin PST™ and fluoride oxalate blood collection tubes were centrifuged at 1300 g (relative centrifugal force) for 15 min. The time that each step in sample processing occurred, including laboratory processing, was recorded on the study database. Given that the kinetics of glucose transport in red cells are well known to be dependent on temperature [20], the temperature at the clinic room bench where sample collection and processing occurred was also recorded. Further details of the prescribed pre-analytical delays outlined in Figure 1 are given below:
Lithium heparin PSTTM and fluoride oxalate tubes for optimal sample preparation Immediately after blood collection, test tubes were placed upright in ice slurry and then centrifuged in a pre-cooled refrigerated centrifuge (+4 oC) and the time of introduction to the centrifuge recorded. Plasma was separated into labelled plastic test tubes (3–4 mL Kahn tubes), then sent immediately to the laboratory.
Lithium heparin PSTTM and fluoride oxalate tubes for 30 min sample preparation Vacutainer™ tubes were left upright on the bench top at room temperature for 30 min after collection and then centrifuged at room temperature. Plasma was separated into labeled plastic test tubes (3–4 mL Kahn tubes), then sent immediately to the laboratory.
Lithium heparin PSTTM and fluoride oxalate tubes for 60 min sample preparation Vacutainer™ tubes were left upright on the bench top at room temperature for 60 min after collection and then centrifuged at room temperature. Plasma was separated into labeled plastic test tubes (3–4 mL Kahn tubes), then sent immediately to the laboratory.
Brought to you by | University of California - San Francisco Authenticated Download Date | 12/12/14 12:47 PM
Chan et al.: Pre-analytical loss of glucose in diabetes 1063
Figure 1 Sampling protocol. FL, fluoride oxalate; PST, lithium heparin PSTTM.
Lithium heparin PSTTM and fluoride oxalate tubes treated as routine samples Immediately after sample collection, the lithium heparin PST™ tube was centrifuged at room temperature. The fluoride oxalate tube was treated as a routine clinic blood sample and did not undergo any preanalytical processing. In order to mimic routine conditions, tubes were left to lie flat after initial centrifugation (lithium heparin PST™ tubes) or from the time of initial sample collection (fluoride oxalate tubes). Tubes were taken to the laboratory within 2 h of blood collection.
Laboratory measurements All plasma samples were analyzed in an accredited laboratory using the Abbott C8000 analyzer (Abbott Diagnostics, North Chicago, IL, USA), utilizing the hexokinase method for measurement of plasma glucose (inter assay coefficient of variation
