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A new sensitive method for determining endotoxin in whole blood
H Tamura1, S Tanaka, T Obayashi
1Tokyo Research Institute, Seikagaku Corporation, Japan.
Summary
A new microplate method accurately measures endotoxin in whole blood using Endospecy. This whole blood assay offers advantages over traditional methods, requiring less time, sample, and reagent for endotoxin detection.
Area of Science:
- Biochemistry
- Clinical Chemistry
- Microbiology
Background:
- Endotoxins are potent pyrogens that can cause severe inflammatory responses.
- Accurate endotoxin detection in whole blood is crucial for diagnosing and managing sepsis and other infections.
- Existing methods for endotoxin determination often require processing of platelet-rich plasma (PRP), which can be time-consuming and increase contamination risk.
Purpose of the Study:
- To develop and validate a novel microplate method for direct endotoxin determination in whole blood.
- To assess the efficiency of sample preparation for removing interfering factors in whole blood.
- To compare the performance of the new whole blood method with the conventional PRP method.
Main Methods:
- A microplate assay was developed using Endospecy, an endotoxin-specific chromogenic limulus test reagent.
- Whole blood samples were treated with a solution of 0.66 mol/l nitric acid and 0.25% Triton X-100 to remove interfering substances.
- Endotoxin recovery was evaluated using spiked samples from humans and experimental animals.
- Measurements were performed on paired whole blood and PRP samples from normal and diseased subjects.
Main Results:
- The developed method successfully removed interfering factors in whole blood samples.
- High recoveries of various endotoxins spiked into whole blood were achieved, with no enhancement from sample dilution.
- Normal endotoxin concentration in human whole blood was determined to be less than 10 pg/ml (using Escherichia coli 0111:B4 endotoxin as reference).
- A strong correlation (r = 0.901, P < 0.01) was observed between whole blood and PRP measurements.
Conclusions:
- The microplate whole blood method provides a reliable and efficient means for endotoxin determination.
- This new method offers significant advantages over the conventional PRP method, including reduced time, sample volume, reagent usage, and risk of contamination.
- The findings support the clinical utility of this direct whole blood assay for endotoxin detection.