What can cause falsely increased serum total protein results when using the biuret reaction?

Falsely Increased Serum Total Protein Results with Biuret Reaction

Hemolysis of the specimen causes falsely increased serum total protein results when using the biuret reaction.

Mechanism of Interference in Hemolyzed Specimens

Hemolysis introduces hemoglobin into the serum, which directly interferes with the biuret reaction. When red blood cells rupture during specimen collection or processing, the released hemoglobin contributes to the total protein measurement in the following ways:

• Hemoglobin is itself a protein that forms complexes with copper in the biuret reagent, adding to the measured protein concentration
• At a hemoglobin concentration of 3 g/L, the apparent serum protein increases by approximately 0.4 g/L 1
• The chromogenic reaction between hemoglobin and the biuret reagent creates additional absorbance at the measurement wavelength

Other Potential Interferences with Biuret Reaction

While hemolysis is the correct answer, it's important to understand other factors that can affect serum total protein measurements:

1. Lipemia:

   • Lipids can cause a positive bias in total protein measurements
   • Lipemic samples can increase apparent protein values by approximately 5% 2
   • This interference occurs because serum blanking in short-term biuret procedures cannot adequately eliminate effects of lipid interferents

2. Bilirubin:

   • High bilirubin levels (hyperbilirubinemia) can cause a positive bias
   • However, bilirubin up to 300 mg/L does not interfere with properly performed reference methods 1

3. Other interfering substances:

   • Certain amino acids and dipeptides can cross-react in the biuret reaction 3
   • These compounds can form 5- or 6-member ring chelation complexes with copper
   • The reaction with amino acids produces a blue color rather than the purple color seen with larger peptides and proteins

Why the Other Options Are Incorrect

A. Lipids removed prior to analysis: Removing lipids would actually decrease interference and improve accuracy, not cause falsely elevated results.

B. Arterial blood used: The type of blood (arterial vs. venous) does not significantly affect total protein measurement by the biuret method.

D. Fasting specimen used: Fasting status does not cause falsely elevated total protein results with the biuret method.

Best Practices for Accurate Total Protein Measurement

To ensure accurate serum total protein results:

• Avoid hemolysis during specimen collection and processing
• Use proper calibration with appropriate standards (serum-based calibrators may be preferable to pure albumin calibrators) 2
• Be aware that pure albumin calibrators may cause a positive bias of approximately 3-5% in serum total protein assays 2
• Consider the potential impact of interfering substances in specific patient populations
• Use reference methods with appropriate reaction times (30-60 minutes at 25°C) for maximum accuracy 1

Clinical Implications

Falsely elevated total protein results can lead to:

• Misdiagnosis of conditions associated with hyperproteinemia
• Inappropriate clinical decisions based on inaccurate laboratory data
• Unnecessary additional testing to investigate apparent hyperproteinemia

Recognizing hemolysis as a common cause of falsely increased total protein results is essential for proper interpretation of laboratory data and appropriate clinical decision-making.