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Separation of ß₂-Transferrin by Denaturing Gel Electrophoresis to Detect Cerebrospinal Fluid in Ear and Nasal Fluids

¹ Department of Otorhinolaryngology, Head and Neck Surgery, University of Kiel, Kiel, Germany.
² Department of Pathology, University of Zürich, Zürich, Switzerland.
³ Department of Otorhinolaryngology, University of Marburg, Marburg, Germany.

^aAddress correspondence to this author at: Department of Otorhinolaryngology, Head and Neck Surgery, University of Kiel, Arnold-Heller-Strasse 14, D-24105 Kiel, Germany. Fax 49-431-597-2272; e-mail gorogh{at}hno.uni-kiel.de.

Background: Cerebrospinal fluid (CSF) leakage is a critical condition with a substantial risk of meningitis. We investigated the use of transferrin isoform analysis as a diagnostic marker for detection of CSF leakage in fluid samples.

Methods: We analyzed 241 samples from patients with CSF leakage, most commonly presenting as otorrhea or rhinorrhea, by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent Western blotting and immunostaining for transferrin. Tears, saliva, nasal fluid, and ear secretions (20 samples each) were analyzed in parallel, and normal human serum served as a control in each experiment. We compared the minimum volume of added CSF that could be detected in secretions by our assay with the minimum volume detected by the prostaglandin-D synthase (ß-trace) test. CSF was admixed with blood in different proportions to determine the influence of blood contamination on the transferrin pattern.

Results: In all CSF samples, ß₁- and ß₂-transferrin were present in nearly equal amounts. In tears and ear secretions, ß₂-transferrin migrated in the gel in the same manner as in CSF, but its concentration was noticeably lower than that of ß₁-transferrin, a difference that allowed a clear distinction from the transferrin pattern of CSF. In saliva, both transferrin isoforms were also present but could be distinguished from those of other fluids by electrophoretic migration pattern rather than relative concentrations. With the ß-trace test, a minimum of 5 µL of CSF was needed for detection, whereas our ß₂-transferrin assay yielded a signal of comparable intensity with a minimum of 2 µL of CSF.

Conclusion: Analysis of the transferrin microheterogeneity pattern by SDS-PAGE for the identification of CSF leakage is a highly sensitive and specific method that merits consideration as a routine technique.