Annals of Clinical & Laboratory Science 39:150-154 (2009)
© 2009 Association of Clinical Scientists
Changes in Total CO2 Measurement According to Reagent Cassette Rotation in Chemistry Autoanalyzers
Hee-Jung Chung1,
Woochang Lee1,
Sail Chun1,
So Young Kang2,
Woo In Lee2,
Hae-Il Park3 and
Won-Ki Min1
1 Departments of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, 2 The East-West Neo Medical Center, Kyung Hee University College of Medicine, and 3 College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
Address correspondence to Won-Ki Min, M.D., Ph.D., Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, 388-1 Pungnap-2dong, Songpa-gu, Seoul 138-736, Republic of Korea; tel 82 2 3010 4503; fax: 82 2 478 0884; e-mail wkmin{at}amc.seoul.kr.
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Abstract
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The quality control (QC) failure rate in the serum total carbon dioxide (TCO2) test increases at a higher rate than in other tests over time after calibration. The causes of the increased QC failure rate in the TCO2 test were examined. Using a TBA200RF analyzer (Toshiba Medical Systems), the TCO2 of the QC material was measured at 2-hr intervals and was found to decrease by up to 16.5% at 10 hr after calibration. In contrast, using the P-module and D-module analyzers (Roche Diagnostics), the TCO2 of the QC material did not change significantly during 10 hr after calibration. When the TCO2 level of the QC material was measured hourly over 5 hr with the TBA200FR analyzer while the reagent bottle was rotated at 0, 80, 120, 160, or 200 rpm, the rate of decline of TCO2, increased over time after calibration and with increasing reagent cassette rotation. Therefore, in a clinical laboratory using an automated analyzer with a rotating reagent cassette, it is necessary to set a limit to the calibration time interval in order to satisfy the QC goal.
Keywords: carbon dioxide assay, clinical chemistry automated analyzers, quality control
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Introduction
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The serum total carbon dioxide (TCO2) test measures bicarbonate (HCO3–) and all other forms of carbon dioxide, such as carbonic acid (H2CO3), carbamino compound, carbonate (CO32–), and dissolved CO2. Serum TCO2 testing is useful and important in assessing acid-base imbalance [1–4]. The Third Evaluation of the College of American Pathologists Survey CHM, 2007, indicated that 5,138 institutions performed TCO2 assays [5]. This suggests that TCO2 is a basic test conducted in most laboratories. TCO2 reagents for open-system autoanalyzers are available from a number of manufacturers. However, as there are no guidelines for calibration time intervals, each laboratory sets and applies calibration time intervals based on experimental observations. The Department of Laboratory Medicine of Asan Medical Center performs serum TCO2 tests using the Toshiba 200FR (Toshiba Medical Systems, Tokyo, Japan) analyzer and carbon dioxide L3K assay liquid reagent (Diagnostic Chemical Ltd., Oxford, CT, USA). Quality control (QC) of TCO2 tests is performed with a target of 43.6 ± 2.4 (coefficient of variance, CV: 5.5%) for Level 1 QC material (Bio-Rad Laboratories, Hercules, CA, US), and 19.6 ± 1.1 (CV: 5.5%) for Level 2 QC material. Calibration is routinely performed every morning before the tests are begun, and QC is performed immediately after calibration, followed by once every 3 hr.
In this laboratory, TCO2 tests showed a significantly higher QC failure rate than other assays, and the QC failure rate was noted to increase over time after calibration. This study was performed to determine the cause of the increase in QC failure rate in TCO2 tests over time.
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Materials and Methods
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TCO2 was measured by enzymatic assay using 3 different clinical chemistry automated analyzers: (a) model TBA200FR (Toshiba Medical Systems, Tokyo, Japan), (b) Roche modular analytics P-module (Roche Diagnostics, Mannheim, Germany), and (c) Roche modular analytics D-module. The CO2-L (Roche Diagnostics) liquid reagent was used for the analyses. Two concentrations of QC material were used from the same lot of ammonia/ethanol/CO2 control (Roche Diagnostics). Total imprecision was determined according to Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) protocols [6] using the 3 automated analyzers. The 2 concentrations of QC material were measured in duplicate in 2 runs each day for 20 days.
Changes in TCO2 measurement over time after calibration.
The 3 instruments, TBA200FR, P-module, and D-module, were calibrated at 08:00 am. The 2 concentrations of QC material were measured with 5 replicates at 0, 2, 4, 6, 8, and 10 hr after calibration. The TBA200FR and D-module were used under normal working conditions in the clinical laboratory. The P-module was used under resting conditions, which means that the reagent cassette was held stationary after calibration.
Change in TCO2 using reagent rotated at 0–200 rpm over time.
The TCO2 reagent was aliquoted into the reagent bottles of TBA200FR and rotated at 5 different velocities (0, 80, 120, 160, and 200 rpm) by the plate rotator. During the rotations, the 2 concentrations of QC material were measured 5 times using 2 ml of reagent, which was retrieved from the rotating reagent bottles every hr, from 0 to 5 hr.
Statistics.
Statistical analyses were performed using SPSS (version 13.0, SPSS, Inc., Chicago, IL, USA) and EP evaluator (version 7, David G. Rhoads, Inc., Kennett Square, PA, USA), and p <0.05 was taken to indicate statistical significance.
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Results
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Precision of TCO2 measurements in 3 different chemistry analyzers (Table 1
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The within-run and total CVs of TCO2 for the TBA200FR analyzer were 2.1% and 2.4% for Control Abnormal, and 2.3% and 2.7% for Control Normal, respectively. For the P-module analyzer, the within-run and total CVs were 1.5% and 2.4% for Control Abnormal, and 2.3% and 3.3% for Control Normal, respectively. For the D-module analyzer, the within-run and total CVs were 1.2% and 1.3% for Control Abnormal, and 2.4% and 3.2% for Control Normal, respectively.
Changes in TCO2 measurements over time after calibration (Table 2 and Fig. 1).
For the TBA200FR analyzer, compared to values obtained immediately following calibration, at 2, 4, 6, 8, and 10 hr after calibration the Control Abnormal measurement was decreased by 2.5, 4.8, 9.3, 11.6, and 12.2%, respectively, and the Control Normal measurement was decreased by 2.9, 5.5, 14.0, 15.5, and 16.5%, respectively. The changes were statistically significant in TBA200FR from 4 hr post-calibration in both QC materials. For the P-module analyzer, the Control Abnormal measurement changed by –0.6% to +2.4%, and the Control Normal by –0.2% to +2.4%, which was not statistically significant in either QC material. For the D-module analyzer, the Control Abnormal measurement changed by –0.6% to +0.6% and the Control Normal measurement by –2.1% to +1.3% with a similar time course, which was not statistically significant in either QC material. The differences among the instruments were statistically significant from 4 hr post-calibration in Control Abnormal and from 6 hr post-calibration in Control Normal as shown in Fig. 1.
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Table 2. Changes in TCO2 concentrations over time lapsed since calibration of the automated analyzer instruments.
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Fig. 1. TCO2 measured in QC materials over time lapsed since calibration in each analyzer. TCO2 levels are expressed relative to those measured just after calibration. Differences among the 3 instruments were significant from 4 hr post-calibration in Control Abnormal (upper panel) and from 6 hr post-calibration in Control Normal (lower panel), computed by one-way ANOVA and Student t-test.
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Changes in TCO2 measurements with reagent rotation at 0–200 rpm over time (Table 3 and Fig. 2).
After 1 hr of rotation, the Control Abnormal measurement decreased by 0.1% to 4.7% and the Control Normal measurement by 3.1% to 9.1% in comparison to those observed before rotation. The Control Abnormal and Control Normal measurements decreased by 0.6% to 9.8% and 6.4% to 18.0% after 2 hr; by 0.6% to 9.8% and 6.4% to 18.0% after 3 hr; by 0.8 to 12.5% and 7.1 to 20.3% after 4 hr, and by 1.1 to 13.7% and 7.6 to 21.5% after 5 hr, respectively. The differences were significant from 2 hr of rotation at 120, 160, and 200 rpm and from 3 hr of rotation at 80 rpm in both QC materials. At each time interval, the TCO2 measurement decreased with increasing velocity of rotation. The timewise differences among various velocities were statistically significant from 2 hr of rotation in both QC materials as shown in Fig. 2.
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Table 3. Change in TCO2 concentration according to the rotation velocity of the reagent bottle and the duration of rotation.
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Fig. 2. TCO2 measured in QC materials over time using reagent rotated at various velocities. TCO2 levels are expressed relative to that measured before rotation. Timewise differences among various velocities were statistically significant from 2 hr of rotation in both the Control Abnormal (upper panel) and the Control Normal (lower panel), computed by one-way ANOVA and Student t-test.
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Discussion
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The results of this study indicated that, in the TBA200FR autoanalyzer, the QC measurement of TCO2 decreased over time after calibration, reaching as much as 16.5% at 10 hr after calibration. We performed an experiment using rotation of the reagent bottle to determine the cause of this decrease in TCO2 measurement after calibration of the TBA200FR instrument. The results indicated that the magnitude of the decline in TCO2 measurements became greater with increasing reagent rotation, reaching as much as 21.5% for rotation at 200 rpm for 5 hr.
In measuring TCO2, the substrate phosphoenolpyruvate (PEP) and HCO3– in the sample react with each other via PEP carboxylase, resulting in the production of oxaloacetate [7]. As the oxaloacetate consumes NADPH analog, the absorbance decreases at 415 nm [7]. The concentration of HCO3– is then calculated by a kinetic assay based on the principle that the decrease in absorbance is proportional to the concentration of HCO3– in the sample [7]. If CO2 from the air is dissolved in the reagent, the titer of PEP reactive with HCO3– in the sample decreases, causing a decrease in the measurement of TCO2.
Rotation of the reagent bottle at 0, 80, and 200 rpm for 5 hr produced TCO2 measurements of 0.8, 1.5, and 2.1 mmol/L, respectively. These observations indicate that the measurement increased proportionally with the rotation velocity of the reagent bottle. Accordingly, it was concluded that CO2 in the air is dissolved in the reagent through the exposed aperture of the reagent bottle for pipetting.
In the TBA200FR instrument, the reagent bottles are mounted on a reagent cassette. The reagent cassette is rotated according to the analyte, and the corresponding reagent bottle on the cassette comes to the position of the probe. Then, the probe pipettes and samples the reagent and performs the test. Normal working status in our clinical laboratory entails an average of 1680 tests/hr/instrument for the TBA200FR. The estimated maximum rotation velocity for the reagent cassette is less than 20 rpm but the actual rotation of the reagent cassette can change direction depending on the analyte, so the swirling of reagent is more severe than with one-way rotation. The decrease in TCO2 measurement of the QC material in a normally working TBA200FR was similar to that observed for reagent rotated at 80 rpm at room temperature. This can be explained by the temperature dependence of Henry’s constant, denoting that the solubility of gases decreases with increasing temperature. Accordingly, CO2 is dissolved more readily in the reagent cassette (at 4.0 to 8.0°C), than at room temperature.
In the D-module analyzer with the reagent bottle fixed without rotation, and the P-module analyzer with the reagent cassette held stationary, the QC material measurement maintained the range of the total CV until 10 hr after calibration. For the TBA200FR analyzer with the reagent cassette held stationary, the measurement results of the 2 concentrations of QC materials with 5 replicates at 2-hr intervals over 10 hr after calibration were within ± 1.8%, the range of the total CV (data not shown). When a sealed reagent bottle was used, the Control Abnormal and Control Normal measurements of TCO2 decreased by 3.4 and 4.5% (1.1 and 0.9 mmol/L), respectively after 5 hr of rotation at 200 rpm. These changes had no statistical significance when compared to those measured before the rotation. All these observations support that, for the TBA200FR instrument, the decrease in TCO2 measurement after calibration is due to dissolution of CO2 in the reagent with the rotation of the reagent cassette.
The same experiment as the "changes in TCO2 measurement over time after calibration" described in the Methods and Materials section was performed using another reagent, CO2 L3K assay liquid reagent (Diagnostic Chemical Ltd.). When the TCO2 of the QC materials was measured with the L3K assay reagent in the TBA200FR instrument, the results indicated a decrease over time after calibration, similar to those observed with the CO2-L reagent (Roche Diagnostics) (data not shown).
When the TCO2 test is performed using an autoanalyzer with a rotating reagent cassette, the rate of decline in TCO2 measurements over time after calibration increases with increasing rotation velocity of the reagent cassette. Therefore, clinical laboratories using an autoanalyzer with a rotating reagent cassette for TCO2 testing must set a limit for the calibration time interval in order to satisfy the QC goal. Manufacturers of chemistry automated analyzers with a rotating reagent cassette need to develop a device to block air exposure in the TCO2 reagent bottle.
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References
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- Clinical and Laboratory Standards Institutes. Blood Gas and pH Analysis and Related Measurements. CLSI document C46-A., CLSI Press, Wayne, PA, 2001.
- Anon. IFCC reference measurement procedure for substance concentration determination of total carbon dioxide in blood, plasma or serum. Clin Chem Lab Med 2001;39:283–288.[Medline]
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- Anon. Comprehensive Chemistry Survey Set C-A. College of American Pathologists, Chicago, IL, 2007.
- National Committee for Clinical Laboratory Standards. Evaluation of Precision Performance of Quantitative Measurement Methods. NCCLS document EP5-A2. NCCLS Press, Villanova, PA, 2004.
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Abstract
Introduction
