title: 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 micromol/L) and a high (1.5 micromol/L) contrtrol, respectively. Hemolysis increased measured NTBI. A detection limit of 0.1 micromol/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.
authors.affiliation: Finnish Red Cross Blood Transfusion Service, Kivihaantie 7, FIN-00310 Helsinki, Finland. leni.bonsdorff@bts.redcross.fi
authors.forename: Leni
authors.initials: L
authors.lastname: von Bonsdorff
authors.forename: Enni
authors.initials: E
authors.lastname: Lindeberg
authors.forename: Leila
authors.initials: L
authors.lastname: Sahlstedt
authors.forename: Jari
authors.initials: J
authors.lastname: Lehto
authors.forename: Jaakko
authors.initials: J
authors.lastname: Parkkinen
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use; D001761:Bleomycin; D003131:Combined Modality Therapy; D003520:Cyclophosphamide / Q000627:therapeutic use; D004591:Electrophoresis, Polyacrylamide Gel; D019337:Hematologic Neoplasms / Q000097:blood* / Q000628:therapy; D018380:Hematopoietic Stem Cell Transplantation; D006461:Hemolysis; D006801:Humans; D007501:Iron / Q000097:blood* / Q000378:metabolism; D007502:Iron Chelating Agents; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014168:Transferrin / Q000378:metabolism*; D014916:Whole-Body Irradiation
chemical_list: D018906:Antineoplastic Agents, Alkylating; D007502:Iron Chelating Agents; D014168:Transferrin; D001761:Bleomycin; D003520:Cyclophosphamide; D007501:Iron