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Updated: Aug 7, 2026
Methodology for Sputum Induction and Laboratory Processing
Published on: December 17, 2017
Validation of assays for inflammatory mediators in sputum
1Dept of Medicine, Queen Elizabeth Hospital, Birmingham, UK.
Insights
Commercial immune assays for lung inflammation studies are reproducible but require validation in complex samples like sputum. Reliability varies, necessitating pre-assessment for accurate interpretation of results.
Area of Science:
- Pulmonary Medicine
- Immunology
- Biochemistry
Background:
- Immune assays are increasingly used for lung inflammation research.
- Performance assessment of these assays in complex biological fluids like sputum is often lacking.
Purpose of the Study:
- To evaluate the reliability and interpretability of commercially available immune assays in sputum samples.
- To determine the reproducibility and recovery accuracy of specific assays in complex lung fluid matrices.
Main Methods:
- Sputum sol phase from chronic lung disease patients was used.
- Spiking and dilutional experiments were performed with pure reagents.
- Reproducibility and recovery rates of myeloperoxidase, interleukin-8, leukotriene B4, and secretory leukoproteinase inhibitor assays were assessed.
Main Results:
- Assays for myeloperoxidase (enzyme), interleukin-8, leukotriene B4, and secretory leukoproteinase inhibitor were reproducible (CV 3.8-7.7%) with >85% recovery.
- The immune assay for myeloperoxidase showed poor recovery, dependent on sample dilution and elastase content, despite being reproducible.
Conclusions:
- While some immune assays demonstrate reliability in sputum, others, like the immune assay for myeloperoxidase, exhibit limitations.
- Thorough assessment of assay reliability in complex biological samples is crucial before widespread application in lung inflammation studies.
Abstract:
Commercially available immune assays are being used with increasing frequency in the study of lung inflammation. However, their performance in complex biological fluids is rarely assessed. The authors wished to assess their reliability to determine whether the results obtained in sputum samples can be easily interpreted. The reproducibility of several such assays was therefore determined together with their ability to recover known amounts of pure reagent. Sputum sol phase was obtained from several patients with chronic lung disease and used together with the reagents in a series of "spiking" and dilutional experiments. Results confirmed that the enzyme assay for myeloperoxidase and the immune assays for interleukin-8, leukotriene B4 and secretory leukoproteinase inhibitor were all reproducible (intra-assay coefficient of variation 3.8-7.7%). Furthermore, each of these assays gave >85% recovery of a "spike" with pure reagent. However, the immune assay for myeloperoxidase (although reproducible) gave poor recovery and was dependent on the degree of sample dilution and elastase content. These studies confirm that the reliabilities of fluid phase measurements should be assessed before being widely applied to complex biological samples.
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