Hemolysis Causes Falsely Increased Serum Total Protein Results with the Biuret Method
Serum total protein results obtained using the biuret reaction can be falsely increased if the specimen is hemolyzed (option C).
Why Hemolysis Affects Biuret Reaction Results
Hemolysis introduces hemoglobin into the serum sample, which directly interferes with the biuret reaction through several mechanisms:
Direct Protein Contribution: Hemoglobin is a protein that contributes additional protein content to the sample, artificially increasing the total protein measurement 1.
Spectrophotometric Interference: The biuret method measures protein by detecting the purple complex formed when peptide bonds react with copper in an alkaline solution, measured at 540-560 nm. Hemoglobin has intrinsic absorbance in this wavelength range, causing spectrophotometric interference 1.
Quantifiable Impact: Studies have shown that hemoglobin concentrations of 3 g/L can increase apparent serum protein by approximately 0.4 g/L 1.
Analysis of Other Options
Option A (lipids are removed prior to analysis): Removing lipids would actually improve accuracy, not cause false elevation. Lipids can cause turbidity that interferes with spectrophotometric measurements, potentially causing a positive bias in protein measurement 2.
Option B (arterial blood is used): The source of blood (arterial vs. venous) does not significantly affect total protein measurements using the biuret method. The reaction depends on protein content, not blood source.
Option D (a fasting specimen is used): Fasting status does not significantly affect total protein measurement with the biuret method. While glucose at very high concentrations could potentially interfere, studies have shown that glucose up to 10 g/L does not interfere with the biuret reaction 1.
Other Potential Interferences with Biuret Method
Beyond hemolysis, other factors can affect serum total protein measurements:
Leukocyte and Platelet Contamination: Incomplete removal of white blood cells and platelets can lead to falsely elevated protein measurements due to their protein content 3.
Bilirubin: At very high concentrations (>300 mg/L), bilirubin may interfere with the biuret reaction 1.
Lipemia: Triglycerides can cause turbidity that interferes with spectrophotometric measurements, potentially causing a positive bias 2.
Cross-reactive Compounds: Certain amino acids, dipeptides, and organic compounds capable of forming chelation complexes with copper can cross-react in the biuret assay, potentially leading to falsely elevated results 4.
Best Practices for Accurate Measurement
To ensure accurate serum total protein measurement:
- Avoid hemolyzed samples whenever possible
- Use proper sample processing techniques to minimize platelet and leukocyte contamination
- Consider using serum-based calibrators rather than pure albumin calibrators to compensate for matrix effects 2
- Follow standardized protocols for reaction time and temperature
- Use appropriate blanking procedures to minimize interference from colored or turbid substances
The biuret method remains a reference measurement procedure for serum/plasma total protein when performed correctly, but understanding these potential interferences is critical for accurate clinical interpretation.