Negative interference of icteric serum on a bichromatic biuret total protein assay

Abstract

Background Bilirubin is stated to be a negative interferent in some biuret assays and thus could contribute to pseudohypoproteinemia in icteric samples. Objective The purpose of the study was to evaluate the magnitude of and reason for a falsely low total protein concentration in icteric serum when the protein concentration is measured with a bichromatic spectrophotometric biuret assay. Methods Commercially available bilirubin was dissolved in 0.1 M NaOH and mixed with sera from 2 dogs to achieve various bilirubin concentrations of up to 40 mg/dL (first set of samples) and 35 mg/dL (second set of samples, for confirmation of first set of results and to explore the interference). Biuret total protein and bilirubin concentrations were determined with a chemistry analyzer (Cobas 6000 with c501 module). Line graphs were drawn to illustrate the effects of increasing bilirubin concentrations on the total protein concentrations. Specific spectrophotometric absorbance readings were examined to identify the reason for the negative interference. Results High bilirubin concentrations created a negative interference in the Cobas biuret assay. The detectable interference occurred with a spiked bilirubin concentration of 10.7 mg/dL in one set of samples, 20.8 mg/dL in a second set. The interference was due to a greater secondary‐absorbance reading at the second measuring point in the samples spiked with bilirubin, which possibly had converted to biliverdin. Conclusion Marked hyperbilirubinemia is associated with a falsely low serum total protein concentration when measured with a bichromatic spectrophotometric biuret assay. This can result in pseudohypoproteinemia and pseudohypoglobulinemia in icteric serum.

Pseudo‐hypoproteinemia in a hyperbilirubinemic dog with immune‐mediated hemolytic anemia

“…Previous studies have documented decreased TP biuret with unconjugated bilirubin values as low as 14 mg/dL, or minimal differences in TP biuret with bilirubin up to 30 mg/dL . A significant negative and proportional interference of TP biuret with hyperbilirubinemia near 10 mg/dL has been previously observed . Therefore, it is reasonable to assume that the high bilirubin concentration of 81 mg/dL in this dog contributed to the low TP biuret , although intravenous fluid administration may have also contributed to the decreased TP biuret .…”

Serum proteins and their diagnostic utility in veterinary medicine: a review

“…Despite this disadvantage, the biuret assay is still frequently used because of its simple analytical procedure and the easy preparation of reagents. Further, compared with other copper-based assays this method is less susceptible to chemical interference (Gupta and Stockham 2014). Moreover, many of the assay kits developed for automated use in wet biochemical analysers, as well as dry chemistry analysers, are based on this principle.…”

Protein characterization using electrophoresis and immunofixation; a case‐based review of dogs and cats

“…These methods can have variable performance based on the number and type of protein species present in the sample, and minor interference can be caused by peptides and free amino acids . Additionally, icterus can cause interference with the biuret method . A survey revealed that only 2.4% of 842 participating human laboratories used refractometry‐based total protein quantification for SPE .…”