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Published on: March 13, 2015
The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs
1Platform Technology & Science, Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Research & Development, Ware, Hertfordshire, SG12 0DP, UK. philip_denniff-1@gsk.com
Insights
Hematocrit levels significantly impact dried blood spot (DBS) sample area and analyte quantification. Variations outside normal ranges can cause unacceptable biases, necessitating method adjustments or specialized quality controls.
Area of Science:
- Biomedical Science
- Analytical Chemistry
- Clinical Diagnostics
Background:
- Hematocrit, a measure of red blood cell volume, naturally varies between individuals and can be altered by disease.
- Dried blood spot (DBS) sampling is a common method for collecting biological samples.
- Understanding factors affecting DBS integrity and accuracy is crucial for reliable testing.
Purpose of the Study:
- To investigate the impact of varying hematocrit levels on the physical properties of dried blood spot (DBS) samples.
- To assess the influence of hematocrit on the accurate quantification of analytes within DBS samples.
- To determine if hematocrit variations introduce significant biases in DBS-based measurements.
Main Methods:
- DBS samples were prepared on three different cellulose paper substrates.
- The area of DBS samples was measured.
- The concentrations of two specific analytes were quantified in DBS samples across a range of hematocrit levels.
- Observed biases in analyte concentrations were compared against acceptable validation limits.
Main Results:
- A linear inverse relationship was observed between DBS sample area and hematocrit levels.
- Significant biases in analyte concentrations were detected at varying hematocrit levels.
- These biases exceeded acceptable limits in certain cases, particularly with hematocrit values outside the normal physiological range.
Conclusions:
- The effect of hematocrit on DBS analyte quantification must be evaluated during method development and validation, especially when sample hematocrit is expected to deviate from normal.
- If significant biases are identified, analytical methods may require modification.
- Implementing quality control samples at different hematocrit levels can help manage and demonstrate assay control for variable hematocrit samples.
Background:
As hematocrit levels are known to vary between individuals and with disease state, its effect on the physical characteristics of dried blood spot (DBS) samples and on the accurate quantification of analytes within these samples is examined.
Results:
The area of DBS samples decreases with increasing hematocrit levels in a linear manner on the three cellulose paper substrates tested. Furthermore, a bias was observed in the concentrations of two analytes determined in DBS samples at different hematocrits, which in some cases exceeded acceptable values, particularly for hematocrits outside normal values.
Conclusion:
If it is expected that the hematocrit of study samples will vary from values considered normal, then its effect on the quantitative determination of an analyte in DBS samples should be investigated as part of the method development and validation. If an unacceptable effect is observed, then this will need to be addressed, by modification of the analytical method, or the inclusion of quality control samples at different hematocrit levels to show control of the assay.
