A-115 Iron assay improvement: The need for thiourea addition in Ferene/Ferrozine reagent cartridge

Abstract

Abstract Background Iron assay is often ordered along with ferritin, transferrin, and iron binding capacity in patients suspected of having iron deficiency anemia or iron overload. A ferene/Ferrozine chromogen-based iron assay is more common in College of American Pathology accredited laboratories across U.S. Recently, a product recall/field safety notice was issued by a major manufacturer for several lots of ferene based iron reagent due to unacceptable positive bias in patient iron results. However, the reason(s) underlying this positive bias was not provided. In this report, we investigated the possible causes for the recall of specific lots of iron assay reagents. Methods The performance of 2 lots of recalled iron test kits was compared against 2 good lots (Sentinel Diagnostics) on the Alinity c analyzer utilizing approved calibrator lots, quality control (QC) materials (Bio-Rad Laboratories), and excess/leftover de-identified patient samples. Spectral scan (in the visible wavelength) of good and recalled lots of reagents, R1 and R2 was performed on the Tecan microplate reader. Trace elements in R1 and R2 reagents were estimated with Quantitative Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Spiking experiments in calibrator, discarded patient samples, and reagents were undertaken to further elucidate the cause and prevention of positive bias seen with recalled reagent lots. Graphs and statistical analysis were generated using Graphpad prism 10.1. Results Iron levels in QC materials and de-identified patient samples (n=42) tested with 2 recalled lot of reagents on Alinity c analyzer showed a positive bias of 15-36% compared to unimpacted lots of reagents. Calibrator and blank absorbance of recalled lots was much higher than the unimpacted lots. Percentage change in the basal absorbance of recalled vs unimpacted R2 reagent measured from 500 to 600 nm in 25 nm increments and corrected to 700 nm ranged from 150 to 250%. No such changes were noted with both R1 reagents. Interestingly, ICP-MS heavy metal panel analysis revealed that R2 reagent of the recalled lots had unanticipated presence of Cu (∼22 υg/dL) and lead (7.5 υg/L), which was not present in R1 reagents. Increased concentrations of Cu spiking (100, 200, and 400 υγ/dL) in R2 reagent from recalled lots discernibly increased the Cal Factor values as well as patient iron results relative to unspiked reagent. Further, the presence of Cu over time deteriorated the accuracy of R2 reagent reflecting on very high rise in Cal Factor and patient iron values. Interestingly, TU addition to R2 reagent from unimpacted lot prevented the Cu-induced spurious rise in Cal Factor and iron measurements in the patient samples. Further, TU exhibited sustained preventive effect on Cu-induced iron measurement changes over time. Conclusions Based on our investigation, we conclude that copper contamination of R2 reagent is the possible cause for the positive bias in the recalled iron assay reagent lots. We recommend the addition of TU to Ferene containing R2 reagent pack as a simple and effective step to prevent Cu-induced false elevation in iron values.