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 sample, which directly interferes with the biuret reaction. When red blood cells rupture:
- Hemoglobin is released into the serum, creating a falsely elevated protein measurement
- According to research, hemoglobin at a concentration of 3 g/L can increase apparent serum protein by 0.4 g/L 1
- Hemoglobin forms complexes with copper in the biuret reagent, contributing to the measured absorbance
The Biuret Reaction Principle
The biuret method measures total protein through a colorimetric reaction:
- Peptide bonds in proteins react with copper ions in an alkaline solution
- This forms a purple-colored complex that can be measured spectrophotometrically
- The intensity of the color is proportional to the protein concentration
- The reaction is named after biuret, a compound formed by heating urea that gives a similar color reaction
Other Potential Interferences
While hemolysis is the correct answer, other factors can also affect serum total protein measurements:
- Lipemia: Lipids can cause falsely elevated results due to light scattering effects 2
- Bilirubin: High bilirubin levels can interfere with the biuret reaction 2
- Incomplete removal of interferents: Short-term blanking procedures used in automated analyzers may not adequately eliminate all interfering substances 2
- Certain medications: Some drugs may potentially interfere with the reaction
Why the Other Options Are Incorrect
A. Lipids removed prior to analysis: Removing lipids would actually decrease interference and improve accuracy, not cause falsely increased results.
B. Arterial blood used: The source 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 protein results; if anything, non-fasting specimens might have more potential interferents.
Clinical Implications
Understanding these interferences is important because:
- Falsely elevated total protein results can lead to misdiagnosis of conditions like multiple myeloma or chronic inflammatory disorders
- Laboratory professionals should inspect specimens for hemolysis before analysis
- Hemolyzed specimens should be rejected or results should be reported with appropriate comments noting potential interference
- Alternative methods may be needed when hemolysis cannot be avoided
Best Practices for Accurate Total Protein Measurement
To minimize interference in the biuret reaction:
- Proper specimen collection to avoid hemolysis
- Use of serum-based calibrators rather than pure albumin calibrators 2
- Appropriate blanking procedures to account for sample color
- Consideration of alternative methodologies when significant hemolysis is present
The biuret method remains a reference procedure for total protein measurement when performed correctly 3, but understanding its limitations and potential interferences is essential for accurate clinical interpretation.