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Letters to the Editor |
1 Laboratory for Clinical Investigation, Osaka University Hospital, 2-15 Yamada-oka, Suita, Osaka 565-0871, Japan
2 Division of Laboratory Science, Course of Health Science, Graduate School of Medicine, and
3 Department of Clinical Laboratory Science, School of Allied Health Sciences, Faculty of Medicine, Osaka University, 1-7 Yamada-oka, Suita, Osaka 565-0871, Japan
aAuthor for correspondence. Fax 81-6-6879-6635; e-mail yamaha{at}hp-lab.med.osaka-u.ac.jp.
To the Editor:
We reported recently that total iron-binding capacity (TIBC) values calculated from serum iron and unsaturated iron-binding capacity (UIBC) values were significantly lower than those obtained by a direct and fully automated TIBC assay (1)(2). We also reported that slopes of regression lines for calculated TIBC values plotted against serum transferrin (TRF) were 
7% lower than the theoretical ratio of TIBC to TRF (TIBC/TRF = 25.1 µmol/g). We found that this could be attributed to underestimation of UIBC values by colorimetric methods. One possible reason for underestimation of UIBC values was insufficient saturation of TRF. We modified the assay conditions of a colorimetric method for UIBC measurement to improve the correspondence between TIBC values converted from TRF and those calculated from serum iron and UIBC.
Both serum iron and UIBC were determined by colorimetric methods (Wako Pure Chemical Industries) with a Hitachi Model 7070 automated analyzer. Serum TRF concentrations were determined by a nephelometric assay on a Behring Nephelometer II analyzer (Dade Behring). UIBC values were determined by four modified methods (Table 1
). UIBCA was measured by the original method, in which assay conditions were set by the manufacturer. Incubation time for saturation of TRF was extended from 5 min to 10 min for the measurement of UIBCB. The ratio of iron provided to saturate TRF per serum sample (iron/serum) was increased from 0.195 µmol/L to 0.26 µmol/L for the measurement of UIBCC by decreasing sample volume from 20 µL to 15 µL. Finally, both incubation and the iron/serum ratio were increased for the measurement of UIBCD. TIBC values were calculated as the sum of serum iron and each UIBC value and designated Cal-TIBCA, Cal-TIBCB, Cal-TIBCC, and Cal-TIBCD, respectively. TIBC values converted from serum TRF concentrations with the theoretical TIBC/TRF ratio were designated Con-TIBC. We also calculated Cal-UIBC values as the difference between Con-TIBC and serum iron values. Correlations were assessed by principal component regression analysis. The 95% confidence interval (95% CI) for the slope of a regression line was estimated by the bootstrap method. A significance level of 0.05 was used for all statistical tests, and a two-tailed paired t-test was applied.
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Comparisons of the Cal-TIBC values and Con-TIBC and TRF concentrations are shown in Table 1
. The within-run CVs for the modified UIBC assays were 2.6–3.9%. The values obtained by the UIBCB method and the UIBCD method were significantly higher than those obtained by the UIBCA method (P <0.001), whereas there was no significant difference between the values obtained by the UIBCA method and the UIBCC method (P = 0.893). The Cal-TIBCB and Cal-TIBCD values were significantly higher than the Cal-TIBCA values (P <0.001), whereas there was no significant difference between the Cal-TIBCA and Cal-TIBCC values (P = 0.893). All slopes of the regression lines for correlations between the Cal-TIBC and Con-TIBC values were <1.0, and the 95% CIs for the slopes did not include 1.0. However, the slope of the regression line for the Cal-TIBCB values plotted against the Con-TIBC values was closest to 1.0, and the 95% CI for the slope included the theoretical TIBC/TRF ratio. The slope of the correlation between the Cal-TIBCC and Con-TIBC values was lower than that between the Cal-TIBCA and the Con-TIBC values, but the slope of the correlation between the Cal-TIBCD values and the Con-TIBC values improved slightly. The regression equation for the comparison of UIBCB (y) and Cal-UIBC [x; mean (SD), 42.0 (17.2) µmol/L] was: y = 0.952x + 0.42 µmol/L (n = 185; r = 0.987; 95% CI for the slope, 0.927–0.975).
The rate of iron binding to TRF is affected by factors such as reaction temperature, pH, ionic strength, and anions. It has been reported that TRF binds iron with HCO3- as a coligand in the presence of O2 and that the concentrations of HCO3- and O2 affect the kinetics of the binding process (3). Insufficient saturation of TRF by the effects of these factors leads to underestimation of the UIBC value.
The UIBCB values were significantly higher than the UIBCA values, whereas there was no difference between the UIBCA values and the UIBCC values. These results suggest that an extended incubation time for the saturation of TRF ensures more reliable UIBC values but the increase in iron/serum ratio does not. The significant difference between the UIBCD and UIBCA values was not attributable to the increase in the iron/serum ratio but to the extended incubation time. Larger UIBC values contributed to an improved correlation between the Cal-TIBC and Con-TIBC values; i.e., the upper limit of the 95% CI for the slope between the Cal-TIBCB values and the Con-TIBC values was slightly <1.0, and the 95% CI of the slope (23.9–25.4) included the theoretical TIBC/TRF ratio of 25.1. These results show that the standard incubation time for saturation of TRF should be increased to improve the agreement between the Cal-TIBC and Con-TIBC values. Although available ranges of incubation times for the TRF-saturation step differ among commercial analyzers, it is essential that the incubation time for saturation of TRF be set as long as is needed.
References
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