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Components of total measurement error for hemoglobin [A.sub.1c].

The Diabetes Control and Complications Trial (DCCT) and the UK Prospective Diabetes Study (UKPDS), undertaken in people with type 1 and 2 diabetes, respectively (1, 2), established the significance of glycohemoglobin (gHb), and in particular hemoglobin [A.sub.1c] ([HbA.sub.1c], as a prognostic indicator for long-term micro- and macrovascular complications. However, the [HbA.sub.1c] measured during the DCCT and UKPDS represents a gHb fraction characterized by its retention time on cation-exchange HPLC rather than its unique chemical structure (deoxyfructosylhemoglobin). Accordingly, the DCCT [HbA.sub.1c] procedure represents a selective, but not a specific assay method (3).

Because no definitive or reference method exists for quantification of [HbA.sub.1c] (4), the American Diabetes Association (ADA), in collaboration with the Association of Clinical Chemists, implemented the National Glycohemoglobin Standardization Program (NGSP) (5) to standardize [HbA.sub.1c] values determined by methods different from that used in the DCCT. The ADA (6) now states that their recommended [HbA.sub.1c] thresholds, with respect to patient management goals, are valid only for NGSP-certified methods.

The variability of [HbA.sub.1c] measurements depends on both analytical and biological variation. However, because [HbA.sub.1c] concentrations are used for individual patient management, only analytical imprecision and within-person biological variation ([s.sub.i.sup.2]) are relevant. Whereas the NGSP (5) states that within-person [HbA.sub.1c] variance is negligible, previous studies (7, 8) have reported [s.sub.i] estimates of 0.17-0.79, and investigations measuring gHb (9) or [HbA.sub.1] (10) have reported values between 0.45 and 1.03. Accordingly, both analytical and within-person variability, but particularly the latter, increase measurement uncertainty and, therefore, the potential for clinical misinterpretation at ADA-specified [HbA.sub.1c] thresholds.

The following definitions are provided to avoid ambiguity with respect to terminology: Within-person variance ([s.sub.i.sup.2]): the degree of random fluctuation of values around a persons homeostatic set-point for a particular biological analyte. For people with diabetes, the [HbA.sub.1c] homeostatic set-point is controlled by dietary and/or pharmacologic treatment, and not by the normal physiologic mechanism.

Repeatability ([s.sub.o]): closeness of agreement between successive results obtained with the same method on identical test material and under the same conditions (same operator, same apparatus, same laboratory, and same time).

Reproducibility ([s.sub.x]): closeness of agreement between individual results obtained with the same method on identical test material but under different conditions (different operator, different apparatus, different laboratory, and/or different time).

Analytical variance ([s.sub.a.sup.2]): comprises both within- ([s.sub.o.sup.2]) and between-assay components of variance.

Total measurement variance ([s.sub.E.sup.2]): comprises both biological and analytical variance.

Serial [HbA.sub.1c] measurements were made in a cohort of 26 diabetic patients, in stable metabolic control, taking part in a 48-week multicenter trial (11). The mean number of specimens per patient was 7.2 (range, 6-9). All gHb determinations were performed with an affinity microcolumn assay (12), and results were converted to [HbA.sub.1c] percent-equivalents based on an algorithm originally derived by comparison (n = 186) with a HPLC method (13). During the trial period (2 years), [s.sub.a] for the affinity column method was 0.47 at a mean [HbA.sub.1c] concentration of 9.6%. The standard error of the estimate ([s.sub.e]), calculated by nonparametric regression ([HbA.sub.1c], vs time) (14), was used to determine long-term variability associated with each patient's serial [HbA.sub.1c], measurements. The corresponding mean [s.sub.e] was determined as the root mean square of the individual estimates (15).

Four different blood samples (~100 [micro]L of each in a sealed ampoule), spanning [HbA.sub.1c] concentrations of ~6-13%, were hand-delivered on the same day to the five pathology laboratories performing physician-referred [HbA.sub.1c] assays in this State. The protocol (16) requires that all samples be analyzed in duplicate within a single analytical run. Three laboratories used Bio-Rad Variant HPLCs (NGSP-certified), and two used Pharmacia Mono S column HPLC systems (13) traceable to the Bio-Rad Diamat HPLC method. The cooperative trial method was defined as ion-exchange HPLC.

Routine data analysis was performed using SPSS for Windows, Release 10.0.7 (SPSS Inc.) and the Cbstat program ( The procedures described by Steiner (16) were used to calculate the corresponding estimates of repeatability and reproducibility for the interlaboratory study.

The individual estimates of [s.sub.e] were not statistically related to the respective baseline [HbA.sub.1c] concentrations (Kendall tau-b, 0.039; P = 0.8). The overall mean [s.sub.e] was 0.65, and given [s.sub.e.sup.2] = [s.sub.i.sup.2] + [s.sub.a.sup.2], the within-person standard deviation [s.sub.i] = [square root of ([s.sub.e.sup.2] - [s.sub.a.sup.2])] = 0.44.

For the interlaboratory study, the respective mean [HbA.sub.1c] values for the four samples were 5.8%, 7.8%,10.4%, and 12.8%, and no laboratory showed consistently high or low [HbA.sub.1c] values, based on a method that ranks the sum of replicates. Similarly, no abnormal data were identified within the four samples when we used the Dixon test, and experimental variation between laboratories and between replicates was homogeneous. ANOVA established a significant variance ratio between laboratories ([F.sub.4,12] = 6.5; P <0.05) and for laboratory-sample interaction ([F.sub.12,20] = 30.7; P <0.01). Solving standard ANOVA equations (16), we calculated the between-laboratory ([s.sub.L.sup.2]) and laboratory-sample interaction ([s.sub.LS.sup.2]) variances as [0.143.sup.2] and [0.12.sup.2], respectively. The reproducibility ([s.sub.x]), or variation arising from different operators, instruments, and laboratories is then given by:

[s.sub.x.sup.2] = [s.sub.L.sup.2] + [s.sub.LS.sup.2] + [s.sub.O.sup.2] (1)

and calculated as 0.19. Accordingly, among the five laboratories, 57% of total variance is between-laboratory, 40% is attributable to laboratory-sample interaction, and 3% is attributable to repeatability. The total error variance associated with the five [HbA.sub.1c] assays is therefore given by:

[s.sub.E.sup.2] = [s.sub.x.sup.2] + [s.sub.i.sup.2] (2)

and is calculated as [0.48.sup.2], of which [s.sub.i.sup.2] contributes 84%. The design of the present cooperative trial, however, did not allow an estimate of long-term repeatability.

Knowledge of [s.sub.E] allows estimation of the range within which the true value lies at a reported [HbA.sub.1c] value, assuming that biological variability is that of a typical patient. Moreover, because [s.sub.x.sup.2] [much less than] [s.sub.i.sup.2], total error can be decreased more by analyzing additional specimens on the same patient than by performing more assays on the same specimen. This is highlighted in Table 1, which summarizes confidence ranges at different probabilities for analysis of one and two specimens. Accordingly, from Table 1, to be 80% confident that the ADA goal of <7.0% has been achieved (single specimen), the measured [HbA.sub.1c] concentration should be <6.4%. A 95% confidence for the same goal requires a mean [HbA.sub.1c] concentration (two specimens) <6.3%. Alternatively, to be 90% confident (single specimen) that the ADA >8.0% intervention threshold has been exceeded, a measured [HbA.sub.1c] concentration [greater than or equal to]8.7% is necessary.

Although the goal of the NGSP is to minimize bias between the DCCT and other [HbA.sub.1c] methods, and thereby allow uniform application of DCCT-derived [HbA.sub.1c] results, measurement uncertainty at ADA clinical decision-making thresholds has not been thoroughly addressed. In particular, failure to acknowledge the magnitude of within-person variation produces a significant underestimation of total measurement error. Our estimate of [s.sub.i] (0.44) is remarkably similar to the value of 0.41 reported previously by Hyltoft Petersen et al. (7), although both the experimental design and [HbA.sub.1c] methodology were different. In contrast, Kolatkar et al. (8) reported lower values of 0.17 and 0.29 for 3- and 12-month study periods, respectively, where all patients were intensively treated and had [HbA.sub.1c] concentrations maintained at <7.0%.

Our findings for the five HPLC methods indicated that reproducibility was much less than within-person biological variation. However, the confidence intervals around a measured [HbA.sub.1c] concentration were still wide (Table 1) in comparison with the small difference (1%) between the ADA [HbA.sub.1c] management thresholds. The potential impact of measurement uncertainty on [HbA.sub.1c] thresholds has been discussed previously by Lytken Larsen et al. (17).

The most recently posted results for the College of American Pathologists [HbA.sub.1c] survey (18) show reproducibility values for certified methods between 0.19 and 0.85; the most common method (n = 335), the Abbott IMx (uncertified), had [s.sub.x] = 0.54. Only HPLC methods had [s.sub.x] <0.25, whereas all multianalyte methods had [s.sub.x] [greater than or equal to]0.43. Minimal analytical performance has been proposed as 0.75[s.sub.i] (19), which based on our results is 0.33. However, the actual DCCT [HbA.sub.1c] procedure had a repeatability of 0.15 on masked split-duplicate specimens, whereas the long-term internal quality control showed a [s.sub.a] of ~0.40 (20).

In summary, the degree of within-person biological variation associated with [HbA.sub.1c] determinations significantly increases the total measurement error. If a [HbA.sub.1c] assay of high reproducibility is not used, the dispersion range of true [HbA.sub.1c] values around the mean true biological set-point will be so wide that the ADA management thresholds may become unworkable. Although the NGSP has significantly reduced intermethod bias, only some HPLC methods currently meet the required analytical performance (19).

We thank P. Charles, M. Haywood, Dr. M. Whiting, Dr. S. Sykes, and D. Moore for taking part in the [HbA.sub.1c] interlaboratory study.


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(5.) National Glycohemoglobin Standardization Program (NGSP) 2001 [Online article]. (accessed April 9, 2001).

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(8.) Kolatkar NS, Cembrowski GS, Callahan PL, Etzwiler DD. Intensive diabetes management requires very precise testing of glycohemoglobin [Letter]. Clin Chem 1994;40:1608-10.

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(12.) Phillipou G, James RK, Farrant RK, Frith RG, Phillips PJ. Economical and reliable determination of glycated hemoglobin using affinity columns. In: Ryall RG, ed. Glycated proteins in diabetes mellitus. Adelaide: Adelaide University Press, 1990:42-8.

(13.) Philcox JC, Haywood MR, Rofe AM. Hemoglobin [A.sub.1c] by HPLC with the Pharmacia Mono S HR 5/N cation-exchange column: influence of sample protein load on optimal chromatographic conditions. Clin Chem 1992;38: 1488-90.

(14.) Woosley JT. A simple method for non-parametric linear regression analysis. Clin Chem 1986;32:203.

(15.) Gluer CC, Blake G, Lu Y, Blunt BA, Jergas M, Genant HK. Accurate assessment of precision errors: how to measure the reproducibility of bone densitometry techniques. Osteoporos Int 1995;5:262-70.

(16.) Steiner EH. Planning and analysis of results of collaborative tests. In: Youden WJ, Steiner EH, eds. Statistical manual of the Association of Official Analytical Chemists. Arlington, VA: AOAC, 1975:66-88.

(17.) Lytken Larsen M, Fraser CG, Hyltoft Petersen P. A comparison of analytical goals for haemoglobin [A.sub.1c] assays derived using different strategies. Ann Clin Biochem 1991;28:272-8.

(18.) College of American Pathologists (CAP) Survey Data: updated 1/01 [Online article], 2001. (accessed April 9, 2001).

(19.) Fraser CG, Hyltoft Petersen P. Analytical performance characteristics should be judged against objective quality specifications. Clin Chem 1999;45: 321-3.

(20.) Anonymous. Feasibility of centralized measurements of glycated hemoglobin in the Diabetes Control and Complications Trial: a multicenter study. The DCCT Research Group. Clin Chem 1987;33:2267-71.

George Phillipov * and Patrick J. Phillips Endocrinology, The Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia; * author for correspondence: fax 61-8-8222-6021, e-mail
Table 1. Confidence ranges, at different probabilities, around a
reported [HbA.sub.1c] concentration.

 One Two
Probability, % specimen specimens

95 [+ or -] 0.94 (a) [+ or -] 0.71
90 [+ or -] 0.79 [+ or -] 0.60
80 [+ or -] 0.61 [+ or -] 0.47
60 [+ or -] 0.40 [+ or -] 0.31

(a) Implies that 95% of values should be within 0.94 above or below
a reported [HbA.sub.1c] value.
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Title Annotation:Technical Briefs
Author:Phillipov, George; Phillips, Patrick J.
Publication:Clinical Chemistry
Article Type:Clinical report
Date:Oct 1, 2001
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