Comparison of bleomycin-detectable iron and labile plasma iron assays.

Non–transferrin-bound iron (NTBI) is detected in the plasma of patients with diseases in which the transferrin-binding capacity for iron is exceeded by a massive iron overload, such as in certain forms of thalassemia and hemochromatosis, repeated blood transfusions, or bone marrow failure. NTBI may occur in plasma as insoluble polynuclear ferrihydrate species, as Fe(III) citrate or acetate complexes, or bound to certain flavonoids, amino acids, albumin, or modified albumin. Excess iron is harmful because it causes oxidative stress, accumulates in and damages tissues, and enhances the growth of pathogens. Therefore, the measurement of NTBI is of clinical importance, because NTBI may be used as an indicator of systemic iron overload and iron toxicity (1, 2 ). We compared 2 NTBI assays: a labile plasma iron (LPI) assay that uses desferrioxamine as the iron chelator (3 ) and a microwell modification of a bleomycin-detectable iron (BDI) assay (2 ). The BDI assay is an indirect method based on the formation redox-active complexes between NTBI in the sample and the bleomycin reagent. The Fe(II)– bleomycin complex then degrades DNA in the sample mixture via a free-radical reaction, which can be measured photometrically. The detection limit of our BDI assay was 0.05 mol/L, and the interassay CV was 18% and 9.8% at 0.2 mol/L and 1.5 mol/L, respectively (2 ). The LPI assay is based on the measurement of the redox-active and readily chelatable fraction of serum NTBI. The assay measures iron-catalyzed radical generation in the presence of a low ascorbate concentration. Radical generation is measured with the fluorogenic redoxsensitive probe dihydrorhodamine 123 (Sigma-Aldrich), and ironcatalyzed radical generation is calculated by subtracting the radical generation in the presence of 100 mol/L of the iron chelator desferrioxamine (Novartis International) (3 ). In our laboratory, the detection limit of this assay was 0.07 mol/L, which is calculated as 3 SDs above the mean of a zero sample (n 8). The interassay CV was 11% and 12% at 0.3 mol/L (n 8) and 2.1 mol/L (n 8), respectively. The transferrin concentration was measured, and the percentage of transferrin saturation (TS) was calculated as previously described (2 ). We measured NTBI and TS in 78 serum samples from 5 leukemia patients undergoing allogeneic hematopoietic stem cell transplantation, as described in a previous report (4 ). The median serum NTBI concentration was 0.12 g/L (95% CI, 0.06 – 0.21 g/L) and 0.10 g/L (95% CI, 0.050.16 g/L) as measured with the BDI and LPI assays, respectively. The results were correlated according to the equation: y 1.33x 0.06 g/L, where x and y are the NTBI concentrations obtained with the BDI and LPI assays, respectively (Sy x 0.17, Deming regression). There was no statistically significant difference between the NTBI results obtained by the 2 methods (P 0.329, paired t-test). The median TS in the samples was 89.9% (95% CI, 83.1%–92.4%). The NTBI concentration measured by both methods was correlated with TS (P 0.0001). The NTBI concentrations in samples with TS 87% were low: For the BDI assay, all NTBI results except one (NTBI, 0.21 g/L; TS, 13.6%) were 0.1 mol/L. For the LPI assay, all NTBI results were 0.17 mol/L (Fig. 1). The exact biochemical nature of NTBI is unknown, and analytical approaches to measure NTBI vary with respect to testing principle and practical application. In 2005, an international roundrobin for the quantification of serum NTBI compared 8 methods in 6 different laboratories (5 ). Seven of the methods were chelating assays based on 5 different chelators, 1 Nonstandard abbreviations: NTBI, non–transferrinbound iron; LPI, labile plasma iron (assay); BDI, bleomycin-detectable iron (assay); TS, transferrin saturation. Fig. 1. Serum NTBI (S-NTBI) concentration as measured by the BDI ( ) and LPI ( ) assays and compared with the TS percentage. An increased NTBI concentration was detected in samples when the TS value was 87%. Clinical Chemistry 59:8 000 – 000 (2013) Letters to the Editor