Interference by haemoglobin with the cresolphthalein complexone method for serum calcium measurement

Area of Science:

• Clinical Chemistry
• Biochemistry
• Analytical Chemistry

Background:

• The calcium-cresolphthalein complexone (CCPC) reaction is a common method for determining calcium levels.
• Spectral interference from haemoglobin (Hb) can significantly affect the accuracy of CCPC assays, particularly in haemolysed samples.
• Understanding Hb derivative formation is crucial for mitigating assay interference.

Purpose of the Study:

• To investigate the spectral interference of haemoglobin in the CCPC reaction.
• To determine how the order of sample and reagent addition influences Hb derivative formation and subsequent interference.
• To identify optimal methods for minimizing haemoglobin-related spectral interference in calcium determination.

Main Methods:

• Investigated spectral interference of haemoglobin in the calcium-cresolphthalein complexone reaction.
• Examined the influence of different orders of sample and reagent addition on Hb derivative formation.
• Assessed the impact of haemolysis on assay results under various procedural conditions.

Main Results:

• The nature of the haemoglobin derivative formed is dependent on the order of sample and reagent addition.
• Alkaline CCPC methods are highly susceptible to interference from persistent oxyhaemoglobin in haemolysed samples.
• Adding sample to an acid CCPC reagent, followed by alkali, results in acid haematin formation with reduced interference.
• Adding sample to a buffer before CCPC addition can correct for moderate haemolysis.

Conclusions:

• The order of addition in CCPC assays critically impacts haemoglobin derivative formation and spectral interference.
• Procedural modifications, such as sample-to-buffer addition, can mitigate haemoglobin interference in calcium assays.
• Optimizing reagent addition sequences is essential for accurate calcium measurements in the presence of haemolysis.