Bleomycin-detectable Iron Assay for Non-Transferrin-bound Iron in Hematologic Malignancies

Abstract

Background: A microwell modification of the bleomycin assay for determining non-transferrin-bound iron (NTBI) was evaluated and compared with a chelation method. Methods: The bleomycin assay reagent and sample volumes were halved, and measurements were done in microwell plates. Samples from patients treated for hematologic malignancies were studied. The chelation method was based on mobilization of NTBI with a chelator and measurement of the ultrafiltered iron-chelator complex. NTBI results were also compared with transferrin saturation and the distribution of transferrin iron forms by urea-polyacrylamide gel electrophoresis. Results: The bleomycin assay intraassay imprecision (CV) was 7.7% and 8.2% and the interassay imprecision was 18% and 9.8% for a low (0.2 μmol/L) and a high (1.5 μmol/L) control, respectively. Hemolysis increased measured NTBI. A detection limit of 0.1 μmol/L was established based on the interference of nonvisible hemolysis and on accuracy studies. In patient samples, NTBI exceeded the detection limits only when transferrin saturation was >80%. Compared with the chelation method, the bleomycin assay gave clearly lower NTBI concentrations. The chelation method also gave positive results at <80% transferrin saturation. The recovery of iron added as ferric nitrilotriacetate to serum was 33% by the bleomycin assay and 64% by the chelation assay. Conclusions: The microwell version of the bleomycin assay is reproducible. When hemolyzed samples were excluded, bleomycin-detectable iron was found only when the transferrin saturation was >80%, suggesting high specificity. Bleomycin-detectable iron constitutes only a portion of the NTBI measured by the chelation method.

Apotransferrin administration prevents growth ofStaphylococcus epidermidisin serum of stem cell transplant patients by binding of free iron

“…Transferrin saturation (%) was calculated by the formula : serum iron (WM)/serum transferrin (g l 31 )U3.98. NTBI was measured using the bleomycin-detectable iron assay adopted for small (25 Wl) serum volumes [25]. Brie£y, bleomycin was added to serum in the presence of ascorbic acid.…”

Non‐transferrin‐bound iron in haematological patients during chemotherapy and conditioning for autologous stem cell transplantation

“…Serum samples were collected coinciding with routine sampling, 0-3 d before the initiation of the chemotherapy referred to as day 0 sample, and during the first 20 d after the onset of the chemotherapy. The median number of samples per patient was 18.5 (range 16-21) in the aSCT group and 15.5 (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) in the Chemo group.…”

Effective binding of free iron by a single intravenous dose of human apotransferrin in haematological stem cell transplant patients

“…NTBI probably comprises heterogeneous iron complexes which may differ in their binding to chelating agents and whose proportions may vary in different clinical conditions (Breuer et al, 2000). The bleomycin assay used in the present study measures redox-active iron and this assay has given positive results in SCT patients only when transferrin saturation exceeds 80% (von Bonsdorff et al, 2002). The disappearance of bleomycin-detectable NTBI after the apotransferrin injection indicated that the redox-active iron in the serum of SCT patients was in a form that was effectively bound by transferrin.…”

“…The bleomycin assay gives clearly lower values for NTBI than other methods based on mobilization of NTBI with a chelator, and determination of the ultrafiltered iron-chelator complex (von Bonsdorff et al, 2002). We used the bleomycin assay because it has a high specificity for NTBI and does not measure ferritin-bound iron.…”