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Published on: December 23, 2014
Fresh haemolysis interferes with blocked p-nitrophenylmaltoheptaoside amylase methods
Abstract:
Two commercial amylase methods which employ p-nitrophenol derivatives of blocked maltoheptaosides suffer from negative interference due to fresh haemolysate. Specimen storage at room temperature, or pre-incubation at 37 degrees C or 57 degrees C removes the effect. Incubation of amylase reagent at 37 degrees C with fresh haemolysate where the final haemoglobin concentration was 0.011 g/L, showed a rapid fall in absorption around 414 nm which became stable after 150 min. Since p-nitrophenol, the product of the amylase reaction, is measured at 405 nm it is concluded that the negative interference from fresh haemolysis is due to the transitory fall in absorption around 405 nm. It is recommended that amylase measurements using this technique, particularly those performed as an emergency, should not be done on haemolysed specimens.
Insights
Fresh haemolysis negatively impacts amylase testing using p-nitrophenol methods. Storing samples or pre-incubating the reagent prevents interference, which is caused by a temporary dip in absorption around 405 nm.
Area of Science:
- Clinical Chemistry
- Biochemical Assays
Background:
- Commercial amylase assays utilizing p-nitrophenol derivatives of blocked maltoheptaosides are susceptible to interference.
- Fresh haemolysate is identified as a source of negative interference in these amylase measurement methods.
Purpose of the Study:
- To investigate the cause of negative interference in amylase assays due to fresh haemolysate.
- To determine optimal conditions for amylase testing to mitigate interference from haemolysis.
Main Methods:
- Two commercial amylase methods employing p-nitrophenol derivatives were tested.
- Specimens were stored at room temperature and pre-incubated at various temperatures (37°C, 57°C).
- Absorption changes at 414 nm and 405 nm were monitored during incubation of amylase reagent with haemolysate.
Main Results:
- Specimen storage at room temperature or pre-incubation effectively removed the negative interference.
- A rapid, temporary fall in absorption around 414 nm was observed upon incubation of amylase reagent with fresh haemolysate.
- This absorption decrease stabilized after 150 minutes, correlating with the measurement wavelength of the p-nitrophenol product (405 nm).
Conclusions:
- The negative interference observed in amylase assays with fresh haemolysate is attributed to a transient decrease in absorption around 405 nm.
- It is recommended to avoid testing haemolysed specimens for amylase using these p-nitrophenol-based methods, especially in emergency situations.
- Proper sample handling and pre-incubation protocols are crucial for accurate amylase determination.
