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Clinical Chemistry 48: 1565-1570, 2002;
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(Clinical Chemistry. 2002;48:1565-1570.)
© 2002 American Association for Clinical Chemistry, Inc.

Modification of Fully Automated Total Iron-binding Capacity (TIBC) Assay in Serum and Comparison with Dimension TIBC Method

Hachiro Yamanishi1,3a, Shigeru Iyama3, Yoshihisa Yamaguchi3, Yuzuru Kanakura3 and Yoshinori Iwatani1,2

1 Division of Laboratory Science, Course of Health Science, Graduate School of Medicine, and
2 Department of Clinical Laboratory Science, School of Allied Health Sciences, Faculty of Medicine, Osaka University, 1-7 Yamada-oka, Suita, Osaka 565-0871, Japan.

3 Laboratory for Clinical Investigation, Osaka University Hospital, 2-15 Yamada-oka, Suita, Osaka 565-0871, Japan.

aAddress correspondence to this author at: Laboratory for Clinical Investigation, Osaka University Hospital, 2-15 Yamada-oka, Suita, Osaka 565-0871, Japan. Fax 81-6-6879-6635; e-mail yamaha{at}hp-lab.med.osaka-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: We previously reported the development of a fully automated assay for total iron-binding capacity (TIBC) in serum, using a multipurpose automated analyzer. However, this method requires four different reagents and is thus useful only with a limited number of available analyzers. We simplified our original assay and compared the analytical performance of the modified method with that of a commercial, fully automated TIBC assay (Dimension® TIBC assay).

Methods: We simplified our original method to require only three reagents. Calibration was also altered and was performed with human transferrin standard solutions. An advantage of this method is that it does not require separation of excess unbound iron after the first step of transferrin saturation. Unbound iron is eliminated by formation of a complex with the chromogenic reagent ferrozine in the second step. Iron dissociated from transferrin by acidic pH reacts with ferrozine to form a colored complex in the final step, and the increase in absorbance at 570/660 nm is directly proportional to the TIBC measured. TIBC values were determined for 49 healthy individuals and 148 patients with this modified TIBC assay and with a commercial, fully automated TIBC method (Dimension clinical chemistry system), and calculation of TIBC based on the sum of the serum iron and unsaturated iron-binding capacity was performed for 97 patients.

Results: The within-run CVs for the modified TIBC assay and the Dimension TIBC assay were <4.8% and <2.4%, and the between-run CVs were 1.2% and 1.7%, respectively. The dilution curves were linear for TIBC values up to at least 180 µmol/L with both methods. TIBC values obtained by our method were linearly correlated with serum transferrin concentrations (r = 0.984; Sy|x = 3.18 µmol/L; P <0.001). The correlation between the values obtained with the present method (y) and those obtained with the Dimension TIBC method (x) was y = 1.04x + 1.19 µmol/L (r = 0.985; Sy|x = 2.47 µmol/L), and with the calculation method (x) was y = 1.18x + 2.62 µmol/L (r = 0.976; Sy|x = 3.27 µmol/L).

Conclusions: Our modified, fully automated TIBC assay performed similarly to the Dimension TIBC assay and is adaptable for use with many multipurpose automated analyzers.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Transferrin, which possesses two high-affinity binding sites for ferric iron (1), is the primary serum iron-transport protein (2). Total iron-binding capacity (TIBC) indicates the maximum amount of iron necessary to saturate all available transferrin iron-binding sites. Therefore, TIBC correlates well with transferrin concentration (3). Measurements of TIBC, serum iron, and the ratio of serum iron to TIBC (transferrin saturation) are widely used for the clinical diagnosis and monitoring of treatment for iron-deficiency anemia and chronic inflammatory diseases (4)(5) as well as for screening tests for other clinical purposes (6).

Fully automated methods have been developed for the determination of serum iron concentrations and are widely used in the clinical laboratory (7), but a fully automated method for direct TIBC analysis has been lacking. One reason is that there are manual procedures that involve centrifugation or pretreatment of serum samples to remove unbound iron after transferrin saturation. Another reason is that TIBC can also be obtained by calculation of the sum of the serum iron concentration and unsaturated iron-binding capacity (UIBC), which is determined automatically. This calculation method is used routinely as a substitute for the direct determination of TIBC, but it requires two separate procedures. Moreover, when either serum iron or UIBC values are below the detection limit, the TIBC value cannot be calculated.

We have developed a fully automated TIBC method for use with a multipurpose clinical chemistry analyzer, as reported previously (8). In addition, a commercial, fully automated TIBC method has been developed (9). More recently, a direct TIBC assay that measures decreases in absorbance when transferrin extracts iron from iron-chromazurol B complexes at neutral pH has been reported (10). Our original TIBC method requires four reagents and therefore is compatible with only a limited number of analyzers. In addition, the iron concentration of the saturation reagent for transferrin has to be particularly precise because the calibration factor is generated on the basis of this value.

We modified our fully automated TIBC method to make it more applicable for use with more automated analyzers by simplifying reagent preparation, and we describe the method herein. We compared the performance of our modified TIBC assay with that of the commercial, fully automated TIBC method (Dimension® TIBC assay) (9), which we also describe herein.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
serum samples
Sera from 49 healthy individuals who are our laboratory staff and from 148 patients (52 with leukemia, 18 with myelodysplastic syndrome, 27 with anemia, 15 with malignant lymphoma, 26 with cancer, and 10 with cirrhosis) were used in the present study. Patients were informed that blood samples were being used for research purposes and that their privacy would be protected.

reagents
Analytic-grade sodium dihydrogen phosphate, manganese sulfate, copper sulfate, thiourea, Triton X-100, and iron-standard solution (assigned concentration, 1004 mg/L) were purchased from Wako Pure Chemical Industries. Sodium hydrogen carbonate, citric acid, and L-ascorbic acid were purchased from Katayama Chemical. Ferrozine was purchased from Dojindo Laboratories. Tris was purchased from Sigma Chemical Co., and lipemic turbidity, hemoglobin, and bilirubin conjugate were purchased from International Reagent Co. Hemolysates were prepared from human whole-blood samples supplemented with EDTA. TIBC Flex® reagent cartridges and TIBC calibration reagents for the Dimension clinical chemistry system were obtained from Dade Behring Co. The calibration reagent was a liquid bovine serum albumin-based product. Level 1 calibrator contained no detectable transferrin. Levels 2 and 3 contained human transferrin, and the assigned TIBC values were 101.1 and 203.0 µmol/L, respectively. The assigned TIBC values of the calibrators were traceable to NIST iron standard Standard Reference Material 937.

preparation of reagents for modified tibc assay
The dilution buffer contained 300 mmol/L Tris, 150 mmol/L sodium hydrogen carbonate, and 4.2 g/L Triton X-100 (pH 8.4). Reagent 1 (R1) contained the iron-standard solution, which was diluted 500-fold with dilution buffer. Reagent 2 (R2) contained 10 mmol/L ferrozine and 32.6 mmol/L L-ascorbic acid in 50 mmol/L Tris buffer (pH 4.0). Reagent 3 (R3) was composed of 600 mmol/L citric acid and 25.6 mmol/L thiourea.

apparatus
The modified automated TIBC assay was carried out with the Model 7070 automated analyzer from Hitachi (also known as the Model 911). The Dimension TIBC assay was carried out with the Dimension clinical chemistry system (Dade Behring).

assay principles
Modified fully automated TIBC assay.
Our TIBC assay consisted of three reaction steps. In the first step, serum transferrin was saturated by ferric iron under alkaline conditions:

Unbound iron was then reduced to Fe2+ by ascorbic acid and eliminated by formation of a complex with ferrozine, which was used as a chromogenic reagent (11):

The final step involved dissociation of Fe3+ from transferrin and the formation of ferrozine-Fe2+ complex proportional to TIBC under acidic conditions (pH 4.5). The increase in absorbance at 570 nm (primary wavelength) and 660 nm (reference wavelength) was measured:

Calibration was performed with level 1 and 2 calibration reagents for the Dimension TIBC assay.

Dimension fully automated TIBC assay.
The Dimension TIBC assay was developed on the basis of our original automated TIBC assay. It therefore also consisted of three reaction steps. One difference from our original method is in the use of ferene (12)(13) as a chromogen. The ferrous complex formed by the use of ferene exhibits a single peak with maximum absorbance at 593 nm. Changes in absorbance are measured bichromatically at 600 nm (primary wavelength) and 700 nm (reference wavelength) in the Dimension TIBC assay. TIBC analysis was performed with the Dimension clinical chemistry system according to the manufacturer’s instructions.

assay procedure for the modified tibc assay
The calibration solution or a serum sample (50 µL) was mixed with 250 µL of R1 and incubated at 37 °C. After 4.9 min, 50 µL of R2 was added, and the mixture was allowed to incubate for 4.9 min. One hundred microliters of R3 was then added, and the absorbance change was recorded for 6.3 min at 570 and 660 nm.

serum iron-mode method
Serum iron concentrations were measured with the use of dilution buffer instead of R1 and by changing the dispensing points for R2 and R3. The calibration was performed with iron-standard solution (36.0 µmol/L).

calculation of tibc by serum iron and uibc
We also calculated TIBC from the sum of serum iron and UIBC measured by colorimetric methods (Wako Pure Chemical Industries) on the Model 7070 automated analyzer (Hitachi). Bathophenanthroline was used as the chromogen in these assays.

transferrin determination
Serum transferrin concentrations were determined by a nephelometric assay with the Behring Nephelometer II analyzer (Dade Behring Marburg GmbH).

statistical analyses
Normally distributed data are reported as the mean ± SD; data not normally distributed are reported as medians, ranges, and the 5 and 95 percentiles. Differences between the TIBC values obtained by the modified TIBC assay and the Dimension TIBC assay were assessed with the Student t-test. Correlations were assessed by Deming regression analysis. A significance level of 0.05 was used for all statistical tests, and two-tailed tests were applied.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
optimization studies for the modified tibc method
The maximal overall reaction rate was achieved when the ferrozine concentration was >0.6 mmol/L and the L-ascorbic acid concentration was >2.2 mmol/L. Thus, 1.11 mmol/L ferrozine and 3.62 mmol/L L-ascorbic acid were used in the modified TIBC assay. The pH of the solution of serum sample or calibration reagent and R1 was 8.40 ± 0.01, and the pH after addition of R2 was 8.39 ± 0.01. The pH of the final mixture after addition of R3 was 3.90 ± 0.02.

For analysis of the dissociation of iron from transferrin after the addition of R3, serum iron concentrations were measured by the serum iron-mode method, and the results were compared with those from the commercial direct colorimetric method. The correlation between values obtained with our method (y) and the colorimetric method (x) was: y = 0.987x + 0.86 µmol/L (r = 0.998; Sy|x = 0.49 µmol/L; n = 28). A typical time course of the modified TIBC assay is shown in Fig. 1 .



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  		Figure 1. Typical time course of overall reaction.

(A), time course of zero calibrator ({circ}) and high calibrator (x; assigned value, 101.1 µmol/L). (B), time course of human serum sample (•). After addition of the calibrator or a serum sample to R1 (0 min), R2 is added at 4.9 min, and R3 is added at 9.8 min. The absorbance change ({Delta}A) is recorded from 9.8 min to 16.1 min.

evaluation of other methods
The precision of the modified TIBC assay and the Dimension TIBC assay was assessed with four pooled human sera (A, B, C, and D) for within-run precision and with two serum-based, commercially available chemistry controls (E and F) for between-run precision. The results are shown in Table 1 . There were no significant differences between the TIBC values obtained by these TIBC methods.


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  		Table 1. Precision studies.

For determination of the detection limit, zero calibration reagent (level 1) was measured 10 times, and the mean ± SD value was calculated. The result obtained with the modified TIBC assay was 2.32 ± 0.72 µmol/L, and that by the Dimension TIBC assay was 2.45 ± 0.34 µmol/L. The detection limits of the modified TIBC assay and the Dimension TIBC assay, defined as the mean TIBC value for the zero calibrator + 3 SD, were 4.48 and 3.47 µmol/L, respectively. To study the linearity of both methods, we assayed calibration reagent (level 3) diluted serially up to fivefold with physiologic saline. The dilution curves were linear for TIBC values up to at least 180 µmol/L with both methods.

At TIBC concentrations of 23.0–56.5 µmol/L, we found no interference by bilirubin (0–210 mg/L), Cu2+ (0–200 µmol/L), Mn2+ (0–100 µmol/L), or lipemic material (2473 formazine turbidity units) added to pooled human sera. However, there were negative effects of hemolysis on the modified TIBC assay, and slight positive effects were observed on the Dimension TIBC assay (Fig. 2 ). The interference of hemolysis was more significant in the modified TIBC assay. Similar interference was identified when hemoglobin, added to the human pooled sera, was measured by both methods. Interference by ferritin iron was also examined. The TIBC in sera from patients with hyperferritinemia (942–4526 µg/L), before and after adsorption of ferritin by an immunoadsorption method (14), was measured by both methods. No interference by ferritin iron was observed.



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  		Figure 2. Effect of increased hemoglobin concentrations on the modified and Dimension TIBC assays.

{circ}, modified TIBC assay; •, Dimension TIBC assay. Mean measured TIBC values (SD) were 17.7 (4.5), 35.7 (3.1), and 65.7 (3.5) µmol/L for the modified method and 25.8 (1.5), 40.4 (0.75), and 72.5 (1.5) µmol/L for the Dimension method.

The medians and ranges for TIBC in healthy individuals and patients determined by our method are shown in Table 2 . The 5 and 95 percentiles of the TIBC values were 48.8 and 79.5 µmol/L, respectively, in the healthy individuals and 25.7 and 73.3 µmol/L, respectively, in the patients. When the TIBC values of the non-patient group were transformed to gaussian distribution, the mean TIBC value was 61.9 µmol/L (SD, 8.77 µmol/L), and the 95% confidence interval was 45.9–80.1 µmol/L. The correlation between values obtained with our TIBC assay (y) and serum transferrin concentration in g/L (x) was: y = 26.0x + 0.80 µmol/L (r = 0.984; Sy|x = 3.18 µmol/L; n = 59). As shown in Fig. 3 , the correlation between values obtained with the modified TIBC assay (y) and the Dimension TIBC assay (x) was: y = 1.04x + 1.19 µmol/L (r = 0.985; Sy|x = 2.47 µmol/L; SEslope = 0.018; n = 97), and with the calculation method (x) was: y = 1.18x + 2.62 µmol/L (r = 0.976; Sy|x = 3.27 µmol/L; SEslope = 0.026; n = 97). The correlation between values of the Dimension TIBC (y) and the calculation method (x) was: y = 1.15x + 0.58 µmol/L (r = 0.989; Sy|x = 2.19 µmol/L; SEslope = 0.018; n = 97). Table 3 shows the TIBC values of the calibration reagents and human pooled sera determined by our method, the Dimension TIBC assay, and the calculation method. The values obtained by the original method and the calculation method were lower than those obtained by the modified method and the Dimension TIBC assay. The ratio of the assigned value of level 2 calibration reagent to the calculated TIBC value was 1.12. The slope of the regression line between the values of the modified TIBC assay (y) and the calculation method (x), corrected with this ratio, was 1.06.


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  		Table 2. TIBC values in healthy individuals and patients with various diseases.



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  		Figure 3. Correlation between the modified TIBC method and other TIBC methods.

(A), the present method (y) vs the Dimension method (x). The equation for the line is: y = 1.04x + 1.19 µmol/L (r = 0.985; P <0.001). (B), the present method (y) vs the calculation method (x). The equation for the line is: y = 1.18x + 2.62 µmol/L (r = 0.976; P <0.001). Dashed line indicates y = x.


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  		Table 3. Values obtained by each TIBC method.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TIBC values can be calculated from the sum of measured serum iron and the UIBC. However, we believe that the total amount of iron that is really bound to transferrin should be measured as the TIBC, and that the UIBC should be calculated as the difference of TIBC minus serum iron. Thus, we attempted to develop a fully automated TIBC assay. We found that it was possible to reduce the number of reagents and to simplify the preparation of the saturation reagent for transferrin by use of human transferrin standard solutions as calibration reagent in our modified TIBC assay.

No differences were identified between the assay precision of the modified TIBC method and the Dimension TIBC method. The detection limit of the modified method was approximately equivalent to that of the Dimension method and was sensitive enough for clinical use because 5 µmol/L TIBC is theoretically comparable to 0.2 g/L transferrin (15).

The TIBC values obtained by our method correlated highly with serum transferrin concentrations. Theoretically, the factor for conversion from transferrin (g/L) to TIBC (µmol/L) is 25.0 [i.e., TIBC (µmol/L) = 25.0 x transferrin (g/L)] (15)(16). The slope of the regression line between the values obtained with our TIBC assay and serum transferrin concentrations is consistent with this theoretic ratio. These results suggest that serum transferrin was saturated by ferric iron completely in the first step of our TIBC assay. The results obtained with our modified TIBC assay correlated highly with those of the Dimension assay. There was also a good correlation between the values obtained with the modified TIBC assay and the calculated TIBC method; however, a proportional bias (slope = 1.18; SEslope = 0.026) was identified in the regression line. We obtained a similar result when we compared the Dimension TIBC assay and the calculation method.

A proportional bias has also been reported between the new direct TIBC assay and the calculated TIBC (10). It has been reported that the ratio of TIBC to transferrin concentration obtained from the relationship between the two measurements was lower than the theoretically expected ratio of TIBC to transferrin concentration (15). It was suggested that this bias may be attributable to differences in the calibration reagent used, and the authors showed that two-point calibration of UIBC with protein-based calibration reagents yielded superior results (15). Direct colorimetric methods for serum iron and UIBC are calibrated with physiologic saline (zero calibration) and one-point iron-standard solution (36 µmol/L Fe3+ in 0.9 g/L H2SO4). In contrast, our TIBC method and the Dimension method are calibrated with a protein-based zero calibration reagent and human transferrin-containing, protein-based calibration reagents. The values obtained with our original TIBC assay, which was calibrated with aqueous iron-standard solution, were lower than those obtained with our modified assay (Table 3Up ). In our previous study, we did not find a significant proportional bias in correlations between the original TIBC assay and the calculated TIBC (8). These results suggest that TIBC values obtained by direct methods calibrated with human transferrin-containing calibration reagents should be compared with those obtained by the calculation method, which is also calibrated with protein-based calibration reagents.

In conclusion, our modified fully automated TIBC assay showed analytical performance similar to that of the Dimension method and is applicable for use with many automated analyzers. This method could be useful for the diagnosis of iron metabolism abnormalities in the clinical laboratory.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Morgan EH. Transferrin: biochemistry, physiology and clinical significance. Baum H Gergely J eds. Molecular aspects of medicine 1981:1-123 Pergamon Press Oxford. .
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