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Technical Briefs |
1 Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, Clinical Chemistry Branch, Atlanta, GA; 2 Battelle Memorial Institute, Atlanta, GA
aaddress correspondence to this author at: Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, Clinical Chemistry Branch, 4770 Buford Hwy. NE, MS F25, Atlanta, GA 30341-3724; fax 770-488-4192, e-mail hav2{at}cdc.gov
Self-monitoring of blood glucose is used to assist patients with diabetes in achieving and maintaining blood glucose concentrations close to those found in individuals without diabetes. This goal requires that blood glucose monitors meet analytical performance criteria that are appropriate for patient care (1)(2)(3). The lack of accuracy of monitors and the resulting variability among monitors limit their use for assessing and maintaining glycemic control (4)(5).
No suitable means exists for establishing calibration traceability between a glucose monitor and a reference method. In the absence of a whole-blood glucose reference material, the recommended approach to establishing traceability is to perform split-sample comparisons with patient samples and a reference method (6). This can be achieved by following the Clinical and Laboratory Standards Institute (NCCLS) EP9-A2 (7) guideline procedure. Because the capillary whole blood required by these monitors usually is available only in small amounts and because glucose is not stable in this matrix, such split-sample comparison studies are difficult to perform, particularly when specimens need to be transported elsewhere (8). Capillary collection devices have been used in method-comparison studies to characterize glucose monitors (8)(9)(10)(11)(12)(13), but data on the stability and reproducibility of glucose measurements of blood in these capillary collection devices are limited (8)(9). In this study, we assessed a capillary collection procedure in combination with a gas chromatography–mass spectrometry (GC/MS) reference method for measuring glucose in whole blood.
The specimen collection procedure and measurement of glucose by GC/MS were based on a previously published method (9). The specimen collection procedure consisted of filling a 1-µL heparinized glass capillary with specimen and transferring it into a microcentrifuge tube containing 500 µL of internal standard solution, which is an aqueous solution of [13C6]glucose (Cambridge Isotope Laboratories) at a concentration of 15 mmol/L (270 mg/dL). The tube was closed and shaken vigorously to remove the specimen from the capillary. The measurement of glucose was then performed (9) with protein precipitation, derivatization of glucose, and analysis of the glucose derivatives by GC/MS.
Calibration was performed with Standard Reference Material 917b [D-Glucose (Dextrose)] from NIST. The within- and among-day variabilities of this method were assessed with use of frozen serum pools (NIST SRM 965) and EDTA-whole blood that was aliquoted immediately after collection and frozen at –70 °C until analysis. The frozen specimens were processed immediately as described above after thawing and homogenizing.
The method was linear within the tested glucose concentration range from 2 to 30 mmol/L (36 to 540 mg/dL) glucose and had a detection limit of 0.1 mmol/L (1.8 mg/dL) glucose. The mean recovery determined by addition of calibrators to SRM 965 was 100.3%. The differences between the measured and assigned values for the three "levels" of the NIST SRM 965 were 0.35%, 0.27%, and 0.85% at 5.68 mmol/L (100 mg/dL), 11.10 mmol/L (200 mg/dL), and 16.36 mmol/L (300 mg/dL), respectively. The within-day imprecision (CV) was 0.52% for EDTA-whole blood and 0.41%, 0.57%, and 0.40% for NIST SRM 965 at the three concentrations listed above (five replicates per concentration), respectively. The among-day imprecision (over 20 days) was 0.88% for whole blood and 1.6%, 1.2%, and 0.93%, respectively, for the three levels of NIST SRM 965.
To assess the stability of glucose, we stored a 1-µL freshly collected venous EDTA-whole blood specimen under three different conditions: in a glass capillary, after transfer of the specimen from the glass capillary into water (500 µL), and after transfer of the specimen from the glass capillary into internal standard solution (500 µL). The durations tested were 0, 0.5, 1, 2, 6, and 8 h at 20 °C for each condition. At the end of the storage time, the samples were processed as described above. The glucose concentrations in EDTA-whole blood stored at room temperature decreased faster (50% within 8 h and 90% after 24 h; Fig. 1
) than in EDTA-whole blood diluted in water (15% and 20% after 8 and 24 h, respectively). The reasons for this improved stability are not fully understood. Diluting the sample with water lyses cells and changes the immediate environment of glycolytic enzymes, including the pH and ionic strength. This may decrease enzyme activity and slow glucose degradation to a degree that differences between the aqueous solution and internal standard solution become apparent only after 8 h. The use of internal standard corrects for any loss of glucose during storage of diluted EDTA-whole blood. We concluded that diluting whole blood in aqueous internal standard solution enables the storage of this specimen for later blood glucose measurements.
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This finding was further assessed in a second experiment in which the stability of capillary whole-blood glucose after transfer into the internal standard solution was assessed at 20, 4, and –20 °C. Three sets of samples (one set per storage temperature, each set with six capillaries collected from the same finger puncture) were prepared by collecting 1 µL of capillary whole blood from one finger puncture, transferring the specimen from the glass capillary into the internal standard solution (500 µL), and storing the resulting samples for 0, 0.25, 0.5, 1, 2, 3, 6, 12, 24, and 48 h at the described temperature. At the end of the storage time, the samples were processed as described above.
The glucose concentrations obtained at different time points were compared with those in samples processed immediately (time 0), and the percentage of glucose remaining was calculated (Table 1
). To assess the possible loss of glucose over time, we performed a polynomial regression using all data points obtained at each temperature. ANOVA was used to test whether a time point would be predictive for a certain loss. The obtained P values were 0.084, 0.926, and 0.134 for 20, 4, and –20 °C, respectively. We conclude that no statistically significant loss over time was detected at any of the tested storage temperatures. The CVs for data from all time points (0 h to 48 h; n = 60) were 2.0%, 2.3%, and 1.9% for 20, 4, and –20 °C, respectively.
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To establish traceability of results of glucose monitors and to assess accuracy in routine clinical settings, reference methods are needed that are characterized with whole blood, e.g., the method of Hannestad and Lundblad (9). We adopted and modified this method by using smaller volume glass capillaries, which allow the collection of replicate samples from one finger puncture, as required by the NCCLS EP9-A2 procedure. The capillary collection procedures (9) require 50 µL of specimen per measurement. Such a volume is difficult to obtain in replicate because of clotting during blood collection and the limited amount of available specimen from one finger puncture. Using 1-µL glass capillaries, we were able to collect six capillaries in <1 min without observing problems with clotting or insufficient amounts of specimen. Furthermore, whereas larger volume capillaries require manual rinsing of the capillary to transfer the blood sample into the internal standard solution, small-volume capillaries fit into a microcentrifuge vial, allowing transfer of the sample by simply shaking the tube.
The observed rapid and profound glycolysis in whole blood is similar to previous study findings (14)(15)(16)(17). However, as shown in this study, diluting the specimen with an aqueous solution of stable-isotope-labeled internal standard overcomes measurement problems associated with analyte loss even when the sample solution is stored over a period of 48 h. This allows samples to be collected and transported for measurement at a different site.
The described specimen collection procedure, including the use of stable-isotope-labeled internal standards, in combination with the GC/MS method provides a means to establish traceability in glucose monitors using patient samples, as recommended by ISO 17511, and offers a mechanism for calibration verification for manufacturers and external quality assessment programs. The GC/MS method allows the measurement of glucose in 1 µL of capillary whole blood, EDTA-whole blood, or serum, with use of primary calibrators and secondary reference materials.
Acknowledgments
We thank Dr. Ulf Hannestad, University Hospital, Linköping, Sweden, for help and assistance in setting up the GC/MS method.
References
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, undiluted sample; 
, sample diluted in water (1:500 by volume); 
, sample diluted in internal standard solution {1:500 by volume; 15 mmol/L (270 mg/dL) [13C6]glucose}.