Second international round robin for the quantification of serum non-transferrin-bound iron and labile plasma iron in patients with iron-overload disorders

Abstract

N on-transferrin-bound iron and its labile (redox active) plasma iron component are thought to be potentially toxic forms of iron originally identified in the serum of patients with iron overload. We compared ten worldwide leading assays (6 for non-transferrinbound iron and 4 for labile plasma iron) as part of an international interlaboratory study. Serum samples from 60 patients with four different iron-overload disorders in various treatment phases were coded and sent in duplicate for analysis to five different laboratories worldwide. Some laboratories provided multiple assays. Overall, highest assay levels were observed for patients with untreated hereditary hemochromatosis and β-thalassemia intermedia, patients with transfusion-dependent myelodysplastic syndromes and patients with transfusion-dependent and chelated β-thalassemia major. Absolute levels differed considerably between assays and were lower for labile plasma iron than for non-transferrin-bound iron. Four assays also reported negative values. Assays were reproducible with high between-sample and low withinsample variation. Assays correlated and correlations were highest within the group of non-transferrin-bound iron assays and within that of labile plasma iron assays. Increased transferrin saturation, but not ferritin, was a good indicator of the presence of forms of circulating nontransferrin-bound iron. The possibility of using non-transferrin-bound iron and labile plasma iron measures as clinical indicators of overt iron overload and/or of treatment efficacy would largely depend on the rigorous validation and standardization of assays. Second international round robin for the quantification of serum non-transferrin-bound iron and labile plasma iron in patients with iron-overload disorders

Laboratory methodologies for indicators of iron status: strengths, limitations, and analytical challenges

“…The measurement of serum NTBI is fraught with technical difficulties related to the determination of heterogeneous chemical forms of circulating NTBI, as demonstrated in an international round robin by considerable method differences (40-fold variation) and large analytical variation (4.4–193%) (56). A second international round robin of current leading analytical assays for NTBI and LPI indicated good assay reproducibility, but still relatively poor correlation and agreement among assays (57). Recently, a new NTBI assay system utilizing a conventional automated analyzer was reported to have good linearity, reproducibility, and comparability with HPLC (58).…”

Clinical and methodological factors affecting non-transferrin-bound iron values using a novel fluorescent bead assay

“…A range of NTBI methods used previously differ considerably in their detection principles and reported reference ranges 18 . The most long-standing and frequently reported NTBI method 19 involves iron capture from NTBI by a high concentration of a low affinity/specificity iron chelator, nitrilotriacetic acid (NTA, 80 mM), followed by ultrafiltration and detection of NTA-iron by high-performance liquid chromatography2, 19 or spectrophotometrically 3 .…”

“…Another approach is measuring NTBI indirectly by quantifying the redox-active subset of NTBI, which has been termed the ‘labile plasma iron’ assay 20 . A further approach is measuring the directly chelatable iron with a fluorophore-labeled high-affinity chelator,18, 21, 22 but background fluorescence in plasma may interfere with data interpretation. Most recently, an adaptation of this approach was described, using a high-affinity fluorescent chelator CP851, covalently linked to magnetic beads with fluorescence signal separated flow-cytometrically from plasma autofluorescence 23 .…”

Should every patient with MDS get iron chelation – probably yes.

“…This iron overload brings about genomic instability, 4,8,9 mitochondrial damage and mutation in mitochondrial genome, 10,11 and by producing an unbalanced immune system, leads to rapid progression of MDS 12 to accelerated phase of leukemogenesis. If our second argument and Angelucci et al 1 argument are correct the iron accumulation even in a minimal amount in crucial cellular compartments without necessarily producing massive iron overload could be damaging and mutagenic.…”

“…Unfortunately, the specific diagnostic tools are not, so far, available but transferrin saturation is an accurate biomarker of absence of NTBI and LPI. 9 Drs Kanjaksha Ghosh and Kinjalka Ghosh also discuss the possible interaction between toxic iron species and MDS progression. This is an attractive new issue requiring in deep laboratory and clinical studies [10][11][12][13][14][15] as for the relationship between iron toxicity and hemopoietic stem cell recovery after hemopoietic cell transplant and transplant outcome.…”