CCQM-K11: Key Comparison on the Determination of Total Glucose in Human Serum Final Report May 2003 Michael Welch, Lorna Sniegoski, Reenie Parris, and Willie May Analytical Chemistry Division Chemical Science and Technology Laboratory National Institute of Standards and Technology Gaithersburg, MD USA Gwi Suk Heo Korea Research Institute of Standards and Science Daejeon, South Korea Andre Henrion Physikalisch-Technische Bundesanstalt Braunschweig, Germany INTRODUCTION The accuracy and traceability for clinical laboratory measurements is becoming increasingly important. New regulations such as the EU IVD Directive are requiring that in vitro diagnostic products be traceable to measurement standards of a higher metrological order. To begin to address the need for higher order standards for clinical laboratory measurements, the Consultative Committee on the Amount of Substance (CCQM) is conducting a series of exercises to document the capabilities of NMIs in this area. Because there are hundreds of health status markers being measured in clinical laboratory settings, it is not possible to perform comparison exercises for all of them. To better focus the exercises, the Organic Working Group of CCQM decided to perform studies for three important health status markers that are representative of the measurement challenges associated with the quantitative determination of well-defined organic substances in blood. In 2000-2001, a Key Comparison for the determination of cholesterol in serum was completed and the report is now available on the BIPM website 1. Cholesterol is present at relatively high concentrations in serum, is of very low polarity, and is predominantly esterified with fatty acids in blood. In 2001, two more Key Comparisons were conducted, one for serum glucose and one for serum creatinine 2. Glucose is also present at relatively high concentrations in serum. It is highly water soluble and is partially associated with serum proteins. In contrast, creatinine, a small product of protein metabolism, is present at much lower concentrations. In addition, creatine, the open-ringed analog is also present. Without proper handling, creatine and creatinine can interconvert, leading to biased results. Because the array of challenges in accurately measuring these three analytes in human serum covers a wide range of Page 1 of 14
challenges associated with measuring other organic molecules with molecular weights less than 500 daltons, a laboratory that can demonstrate competence for all three of these measurands has a basis for claiming competence for measurements of other organic compounds in the same range of concentrations and molecular weights in human serum. Glucose is a six-carbon monosaccharide. It serves as the major source of energy for cells in the body and its concentration in blood is carefully regulated in healthy individuals through production of insulin that acts to stimulate absorption of glucose by the cells in the liver, muscle, and adipose tissue. In diabetics, either insulin production or activity is reduced, leading to elevated levels of glucose in blood and toxicity. Blood glucose is measured to determine if diabetes is present and if so, to what extent is the glucose out of normal bounds. The nature and timing of treatments depend upon these measurements, so accuracy in these measurements is important to proper diagnosis and treatment. Routine clinical laboratory measurements of blood glucose generally use enzymatic methods based upon hexokinase, glucose oxidase, or other enzymes that act on glucose. Such methods may not be specific to glucose and method-method differences can be large. Thus, there is a need for higher order methods to provide an accuracy base to which the routine methods can be compared. This study examined how well NMIs could use isotope dilution mass spectrometry, a technique suitable for primary methods, to measure glucose in human serum. This report describes the 2001 Key Comparison on measurement of serum glucose. This project was approved by the CCQM after completion of a successful pilot study in 2000 (Table 1). The materials used for this study were from the same two lots of frozen serum materials used for the IMEP-17 interlaboratory comparison. The materials had been prepared under the direction of A. Uldall (DEKS, DK). Participants in this CCQM study received samples directly from the Institute for Reference Materials and Measurements (IRMM). The pilot laboratory for this study, NIST, sent instructions and forms to the participants. The NMIs that agreed to participate were: Korea Research Institute of Standards and Science (KRISS) [S. Korea] National Institute of Standards and Technology (NIST) [USA] Pilot Laboratory Physikalisch-Technische Bundesanstalt (PTB) [Germany] Results were received from KRISS, NIST, and PTB. All of the participants used methods based upon isotope dilution combined with gas chromatography/mass spectrometry (GC/MS). SUMMARY OF PILOT STUDY A pilot study (CCQM-P8) for the determination of serum glucose was organized by NIST in 2000. Samples from two frozen human serum pools were sent to four participating institutions. These pools were unknown to the participants, except for the pilot laboratory, and were two levels of SRM 965 Glucose in Frozen Human Serum, levels I Page 2 of 14
and III. Level I (Material A) is unfortified while Level III (Material B) was spiked with glucose to achieve an elevated level. Processing of the serum was minimized to keep the matrix as close to fresh serum as possible. This material is stored at 80 C to maintain the glucose levels. The glucose in these materials had been previously determined and the stability verified using the NIST ID/MS method for glucose. Each participant also received a sample of SRM 917, Glucose, to use for calibration. The participants were free to use whatever methods they chose. Results were received from all four participants, all of whom used ID/GC/MS-based methods. The results are shown in Table 1. Results were in excellent agreement among all of the participants and with the certified values for SRM 965, the material used in the exercise. Reported uncertainties are extremely close among the participants and are about the same as the uncertainty in the certified values. Based upon the excellent agreement of this study, the CCQM decided to proceed with a Key Comparison. All of the participants used methods based upon isotope dilution GC/MS, so that was the only technique approved for results to be included in the Key Comparison Reference Value (KCRV). PROTOCOL FOR KEY COMPARISON The CCQM-K11 study utilized two frozen serum materials provided as part of the IMEP- 17 study. One of these materials had a glucose level in the normal range for humans while the other had a level representative of an elevated concentration. Both materials came as unknowns to all of the participants, including the pilot laboratory, although an approximate target value was provided for both materials. All three NMIs (KRISS, NIST, PTB) used ID/GC/MS-based methods. All followed the published method 3 developed at NIST that utilizes of a butylboronate-acetate derivative of glucose for the sample preparation. NIST uses a bracketing procedure for calibration while the other two use a single calibration standard whose ratio closely matches that of the standard. The measurement equation used to calculate the glucose mass fraction in mg/g of serum is dependent upon how the calibration is performed. If bracketing is used as in the published NIST method 3, the equation is as follows: C = [(I Sam - I Lo ) x (W Hi W Lo ) + W Lo ] M Lab (I Hi - I Lo ) x M Ser Where: C I Sam I Lo W Hi W Lo M Lab is the mass fraction of glucose in the serum sample; is the unlabeled/labeled ion intensity ratio measured for serum sample; is the unlabeled/labeled ion intensity ratio for the lower ratio calibration standard; is the unlabeled/labeled mass ratio for the higher ratio calibration standard; is the unlabeled/labeled mass ratio for the lower ratio calibration standard; is the mass of the labeled glucose added to the serum sample; Page 3 of 14
I Hi M Ser is the unlabeled/labeled ion intensity ratio for the higher ratio calibration standard; is the mass of serum sample In addition to the ion intensity measurements, the other critical measurement is determining the mass of the reference compound used to prepare the calibration standards. This measurement requires careful weighing of a material that has a known purity and associated uncertainty. In contrast to the pilot study, no calibration material was supplied for the Key Comparison by the organizers. Thus, each participant was responsible for selecting a calibration material that had a stated purity and uncertainty, which were to be incorporated in calculations of concentration and uncertainty. All of the participants used SRM 917b Glucose from NIST with a certified purity of 99.7 mass % ± 0.2 mass %. RESULTS FROM KEY COMPARISON The results are shown in Tables 2a and 2b and Figures 1 and 2. The quality of results was very different for the two materials. For Material I, the agreement among the participants was quite good (range of the means = 0.89%). In contrast, the range for Material II was 3.96%, with the high and low results about equally spaced from the median result. For comparison, results for glucose in these two materials from an unnamed European reference laboratory using an ID-GC/MS method were 0.7877 mg/g ± 0.0115 mg/g for Material I and 1.4905 mg/g ± 0.0216 mg/g for Material II. This Material I result falls outside the results of the three participants and doubles the range, but the Material II result falls well within the range seen for Material II among the three participants. Key Comparison Reference Values (KCRV) and associated uncertainties: Based on guidelines established by the CCQM Organic Working Group, Key Comparison Reference Values are to be established based on results from study participants that had their method(s) validated through participation in the preceding Pilot Study. All three participants had their methods validated in the Pilot Study. There were no outliers in the data submitted for Material I, consequently all of the eligible data was used to calculate the KCRV. For both Material I, it is recommended that the KCRV be assigned as the mean ± U of the eligible results. That calculation yields a KCRV of 0.7762 ± 0.0088 mg/g for Material I, corresponding to a 95% confidence interval of 0.7674 to 0.7850 mg/g. Because of the lack of agreement among the participants for the concentration of glucose in Material II, it was decided not to calculate a KCRV, and therefore not include these results in the BIPM Appendix B database. Even for Material I, it is likely that systematic biases contribute significantly to the total biases of some of the participants results. Because all of the eligible results are included in calculating the KCRV, systematic biases in individual results would bias the KCRV. Therefore the KCRV may not be the best estimate of the true mass fraction of glucose in the materials. However, even with results with systematic biases included in the calculations, the true mass fractions should fall within the 95% confidence intervals. Page 4 of 14
The Table of Equivalence for Material I, which enumerates the relationships among the results of the participants in this Key Comparison is shown in Table 3. The graph of equivalence is shown in Figure 1. A graph of the results for Material II is shown in Figure 2. Tables 4a-4c describe the uncertainty calculations for each of the participants. Major components of uncertainty for all participants include measurement imprecision (type A), and the uncertainty in the purity of the primary reference compound (SRM 917b for all participants) as a type B factor. Other significant factors used by individual NMIs include uncertainty in sample preparation (KRISS), uncertainty in the equilibration between labeled and unlabeled forms (NIST), and risk of systematic deviation in the measurement procedure (PTB). The end result was that all three NMIs had expanded uncertainties U that were very similar in magnitude to each other for both materials analyzed in this Key Comparison. DISCUSSION Based upon results from the pilot study, good agreement was expected among the participants. Good agreement was achieved for Material I, but the agreement was unsatisfactory for Material II, considering that all three participants used ID-GC/MSbased methods. It is known that Material I was unfortified with glucose, while material II was fortified. That would not be expected to make any difference in the results, but at present there are no satisfactory explanations for the discrepant results for Material II. It is possible that there were differences in the materials received by the three organizations, however QC data on the materials did not show any evidence of inhomogeneity at the level that would be required here. It is interesting to note that for three of the four materials measured in the pilot study and Key Comparison, the concentration order was the same, with PTB having the highest result and KRISS having the lowest result. This suggests that there may be some systematic effects that are biasing some of the results. Very low results for a laboratory might be explained if the material had been thawed during shipping, for example, but assuming the two materials were kept together, the laboratory should see similar effects for both materials, which was not the case in this study. Various options have been considered. Among them are: A. All of the results can be accepted and put in the BIPM database and the study closed. This would send a mixed signal as to the capabilities of the NMIs, because of the large difference in scatter for the results for the two materials. B. Only Material I results would be included in the BIPM database and the study closed. This would provide only a single data point for glucose concentration and would not show capabilities for the NMIs at concentrations of concern for diagnosis and treatment. Page 5 of 14
C. A new study could be undertaken involving a new frozen human serum material that will become NIST SRM 965a. This would require a significant additional effort by the participants. At the 2002 CCQM Meeting in Sevres, a decision was made to go with option B. No further studies on the measurement of serum glucose are planned at this time. CONCLUSIONS AND HOW FAR THE LIGHT SHINES? All three participants demonstrated that they were able to determine glucose in human serum with good accuracy in the pilot study and key comparison, with the exception of one material in the Key Comparison. These results combined with those of CCQM-K6 1, Determination of Cholesterol in Human Serum, and CCQM-K12 2, Determination of Creatinine in Human Serum, were chosen to provide evidence for the capabilities of participating NMIs to measure a wide range of relatively small (non-protein) organic analytes in Human Serum. Cholesterol was chosen, and studied previously because it represents a lipophilic serum analyte. Glucose is highly water-soluble and also associates strongly with proteins. Creatinine is very polar, present at much lower levels than cholesterol and glucose, and its determination requires considerable care to assure separation from creatine, without interconversion between creatinine and creatine. Two NMIs, NIST and PTB, have participated in all three of these key comparisons with consistently good agreement with the KCRVs and the other participants. The results from this suite of key comparisons provide supporting evidence for measurement claims of these two NMIs related to a range of well-defined, small organic molecules in human serum. For other NMIs to make similar claims, they may wish to participate in bilateral studies or subsequent key comparisons to document their capabilities across this suite of compounds. REFERENCES: 1 CCQM K-6 Determination of cholesterol in serum. Report available at: http://kcdb.bipm.org/appendixb/appbresults/ccqm-k6/ccqm-k6_final_report.pdf 2 CCQM K12 Determination of creatinine in human serum. Report to be available at the BIPM Key Comparison database: http://kcdb.bipm.fr/bipm-kcdb/appendixb/ 3 White V, E., Welch, M.J., Sniegoski, L.T., Schaffer, R., Hertz, H.S., Cohen, A., The accurate determination of serum creatinine by isotope dilution mass spectrometry two methods Biomed. Mass Spectrom., 9, 395-405 (1982). Page 6 of 14
Table 1a. Results for the Pilot Study - CCQM-P8 Material A in mg/g Material A Lab Mean U Diff from cert(%) PTB 1.002 0.011 0.23 KRISS 0.9948 0.0095-0.49 NIST 0.9982 0.0100-0.15 MHH* 0.9930 0.0100-0.68 CCQM mean 0.9983 0.0063 Cert value 0.9997 0.0082 Diff from Cert (%) -0.14 Range (%) 0.91 Table 1b. Results for Pilot Study - CCQM-P8 Material B in mg/g Material B Lab Mean U Diff from cert(%) PTB 2.896 0.033 0.66 KRISS 2.852 0.032-0.87 NIST 2.8933 0.0319 0.56 MHH* 2.865 0.03-0.41 CCQM mean 2.8804 0.0342 Cert value 2.8771 0.0324 Diff from Cert (%) 0.12 Range (%) 1.53 *Medizinische Hochschule, Hanover, Germany Page 7 of 14
Table 2a. Results for CCQM-K11 Material I in mg/g Laboratory Mean Result mg/g Std. Uncertainty mg/g degrees of freedom k Exp. Uncertainty mg/g KRISS 0.7753 0.0034 2.9 3.180 0.0109 NIST 0.7801 0.0041 357 1.967 0.0081 PTB 0.7732 0.0025 2 4.303 0.0108 Mean 0.7762 Std dev of mean 0.00204 Degr. of freedom 2 k 4.303 U 0.0088 Rel U (%) 1.13 Range (%) 0.89 KCRV 0.7762 mg/g ± 0.0088 mg/g Table 2b. Results for CCQM-K11 Material II in mg/g Participant Mean U KRISS 1.4559 0.0249 NIST 1.4744 0.01532 PTB 1.5146 0.01818 Mean 1.4945 Std dev of mean 0.01641 Degr. of freedom 2 k 4.303 U 0.0706 Rel U (%) 4.72 Range (%) 3.93 Page 8 of 14
Table 3. MATRIX OF EQUIVALENCE - Material I Measurand: Amount of substance of glucose in human serum Unit: milligrams/gram KCRV KRISS NIST PTB D i U i D ij U ij D ij U ij D ij U ij KRISS -0.0009 0.011-0.0048 0.011 0.0021 0.012 NIST 0.0039 0.009 0.0048 0.011 0.0069 0.010 PTB -0.0030 0.010-0.0021 0.012-0.0069 0.010 Page 9 of 14
Figure 4a. Uncertainty reporting form for KRISS Material I Parameter Value u i v ci [ci*u)^2 (ci*ui)^4/ v W sample (g) W is-sol, sample (g) W is-sol, std (g) W s-sol, std (g) C s-sol (mg/g) (ui include only the uncertainty of purity) 1.0256 0.00006 large 0.3871 0.00006 large included in standard deviation of 0.9992 0.00006 large measurement results of 4 ampoules (σ L ) 1.0107 0.00006 large 1.9593 0.0078 2 0.3954 9.602E-06 4.61E-11 0.4000 (due to preparation repeatability) included in uncertainty of C s-sol 0.1000 (due to uncertainty of purity of reference material) not included in σ L AR sample,c 1.2482 0.0025 3 AR std,c 1.2052 0.0024 3 included in σ L σ L (u i =σ L ) 0.0014 3 1 2.071E-06 1.43E-12 C x, concentration of glucose in serum (mg/g) 0.7747 0.0034 2.9 *Combined uncertainty of the measurement result, u c =[(σ L ) 2 + (c i u i ) 2 ] 1/2. ci and u i are the sensitivity coefficient and the standard uncertainty of C s-sol. The effective degrees of freedom of the combined uncertainty u c is obtained from the Welch-Satterthwaite formula. 4. Estimation of Uncertainty for Material II Parameter Value u i v ci [ci*u)^2 (ci*ui)^4/ v W sample (g) W is-sol, sample (g) W is-sol, std (g) W s-sol, std (g) C s-sol (mg/g) (ui include only the uncertainty of purity) 1.0290 0.00006 large 0.7191 0.00006 large 0.9992 0.00006 large 1.0107 0.00006 large 1.9593 0.0059 2 0.7415 1.9E-05 1.8E-10 included in standard deviation of measurement results of 4 ampoules (σ L ) 0.4000 (due to preparation repeatability) included in uncertainty of C s-sol 0.1000 (due to uncertainty of purity of reference material) not included in σ L AR sample,c 1.2643 0.0013 3 AR std,c 1.2052 0.0012 3 included in both σ L σ L (u i =σ L ) 0.0078 3 1 6.155E-05 1.26E-09 Page 10 of 14
Concentration of glucose in serum (mg/g) 1.4528 0.0090 4.5 Figure 4b. Uncertainty reporting form for NIST CCQM K-11 Relative Uncertainty Uncertainty type Matl 1 Matl 2 d.f. Steps in Process A B Purity of reference standard X 0.100 0.100 inf Equilibration X 0.500 0.500 inf GC/MS measurements X 0.1450 0.13950 2 combined rel std uncertainty 0.530 0.529 Calculated degrees of freedom 357 412.5 k-factor 1.967 1.966 Relative expanded uncertainty (%) 1.043 1.039 Mean value 0.7801 1.474 mg/g Abs. expanded uncertainty 0.0081 0.015 mg/g Page 11 of 14
Figure 4c. Uncertainty Reporting Form For PTB Material 1 Contributions to Uncertainty Type Relative Standard Degrees of (describe) (A or B) Uncertainty (%) Freedom Standard deviation (CV) of the mean calculated for the 3 vials investigated 1) A 0.286 2 Purity of neat glucose reference material (NIST-SRM 917 a) 2) B 0.115 Risk of systematic deviation in the calibration of the balance 2) B 0.009 Risk of systematic daviation in the measurement procedure (not to be detected) 2) B 0.100 Combined Relative Standard Uncertainty (%) 0.324 Calculated Degrees of Freedom 2 Coverage Factor (k) 4.303 Relative Expanded Uncertainty (%) 1.4 Mean Result in mg/g 0.77317 Expanded Uncertainty (U) in mg/g 0.01082 1) This includes all type A components caused by weighing in, spiking, sample preparation, GC/MSmeasurement and heterogeneity 2) Supposed to be rectangularly distributed. Page 12 of 14
Figure 4c continued. (PTB) Material II Contributions to Uncertainty Type Relative Standard Degrees of (describe) (A or B) Uncertainty (%) Freedom Standard deviation (CV) of the mean calculated for the 3 vials investigated 1) A 0.232 2 Purity of neat glucose reference material (NIST-SRM 917 a) 2) B 0.115 Risk of systematic deviation in the calibration of the balance 2) B 0.009 Risk of systematic daviation in the measurement procedure (not to be detected) 2) B 0.100 Combined Relative Standard Uncertainty (%) 0.278 Calculated Degrees of Freedom 2 Coverage Factor (k) 4.303 Relative Expanded Uncertainty (%) 1.2 Mean Result in mg/g 1.51461 Expanded Uncertainty (U) in mg/g 0.01818 1) This includes all type A components caused by weighing in, spiking, sample preparation, GC/MSmeasurement and heterogeneity 2) Supposed to be rectangularly distributed. Page 13 of 14
Figure 1. Plot of Degree of Equivalence for CCQM-K11 Material I CCQM-K11 MATERIAL I EQUIVALENCE mg/g 0.02 0.01 0.01 0.00-0.01-0.01-0.02 KRISS NIST PTB NMI Figure 2. CCQM-K11 Material II No Degree of Equivalence Established CCQM-K11 Material II 1.56 1.52 mg/g 1.48 1.44 1.4 KRISS NIST PTB NMI Page 14 of 14