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

Nontransferrin-bound iron (NTBI) is a heterogeneously speciated plasma iron, typically detectable when transferrin saturation (TfSat) exceeds 75%. Here, we examine factors affecting NTBI levels by a recently discovered direct chelator-based (CP851) fluorescent bead-linked flow-cytometric assay (bead-NTBI), compared with the established indirect nitrilotriacetate (NTA) assay in 122 iron-overloaded patients, including 64 on recent iron chelation therapy and 13 healthy volunteers. Both methods correlated (r = 0.57, P < 0.0001) but with low agreement, attributable to 2 major factors: (1) the NTA method, unlike the bead method, is highly dependent on TfSat, with NTBI under-estimation at low TfSat and over-estimation once Tf is saturated, (2) the bead method detects <3-fold higher values than the NTA assay in patients on recent deferiprone-containing chelation due to greater detection of chelate complexes but lower values for patients on deferasirox. The optimal timing of sample collection relative to chelation dosing requires further study. Patients with splenectomy, high-storage iron, and increased erythropoiesis had greater discrepancy between assays, consistent with differential access by both methods to the NTBI pools associated with these clinical variables. The bead-NTBI assay has advantages over the NTA assay, being less dependent on TfSat, hence of less tendency for false-negative or false-positive values at low and high TfSat, respectively.