Removal of interfering substances

If the methods described above to correct the interference do not work or cannot be applied, then interference can be resolved by removing the interferents and re-assaying the treated specimen. Because bilirubin in plasma is mostly bound to plasma proteins, wherever possible, free drug measurement or assay with protein-free ultrafiltrate (e.g., digoxin) can avoid bilirubin interference. An example is the removal of bilirubin interference in the enzymatic acetaminophen assay as described earlier (8). Some commercial assays (TDx Digoxin from Abbott Diagnostics, Abbott Park, IL, USA) routinely use sulfo-salicylic acid precipitation of proteins to make protein-free specimens, which are then assayed in the analyzer.

Hemoglobin and blood substitutes, however, are soluble and cannot be removed this way. A report in 1997 described a synthetic solid phase anionic polyelectrolyte, Hemoglobind®, which, when used with a hemolytic sample could effectively remove most hemoglobin or hemoglobin derivatives (27). The authors reported the removal of hemoglobin of 91% at 1000mg/dL, 86% at 1500mg/dL, and 84% at 2000mg/dL of hemoglobin. In this simple protocol, 250 ^L of the solid agent is added to the 500 ^L of the hemolyzed specimen, centrifuged at 3000 x g for 10min, and the supernatant is used for the assay. However, the method could remove only up to 0.6g/dL Hemolink.

Because the interfering triglyceride particles, chylomicron and VLDL, have lower density than the serum, they can be removed by ultra-centrifugation (they remain in the supernatant and the remaining serum in bottom layers).

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