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Published on: March 2, 2011
Determination of total interleukin-8 in whole blood after cell lysis
J Reinsberg1, J Dembinski, C Dorn
1Departments of Gynecological Endocrinology and Reproductive Medicine, and Neonatology, University of Bonn, Sigmund-Freud-Strasse 25, D-53127 Bonn, Germany.
Insights
This study validates a new method for measuring cell-associated interleukin-8 (IL-8) in whole blood. The method is reliable for assessing total IL-8 levels, with results influenced by sample handling and storage conditions.
Area of Science:
- Biochemistry
- Immunology
- Clinical Chemistry
Background:
- A significant portion of interleukin-8 (IL-8) in circulation is cell-associated.
- A novel method for quantifying cell-associated IL-8 in whole blood post-lysis has been recently developed.
Purpose of the Study:
- To evaluate the performance of a new method for determining cell-associated IL-8 in whole blood.
- To investigate the impact of pre-analytical sample handling on IL-8 measurements.
- To establish reference ranges for total IL-8 in healthy individuals and explore age and sex correlations.
Main Methods:
- Whole blood cells were lysed using Milenia((R)) cell lysis solution to determine total IL-8 content.
- Interleukin-8 (IL-8) concentrations in the resulting blood lysate were quantified using the IMMULITE((R)) IL-8 immunoassay.
Main Results:
- Storing whole blood on ice for up to 48 hours did not alter total IL-8 levels, while room temperature storage for 3 hours increased levels.
- Lysate stability was maintained for 48 hours at 4°C, but room temperature storage reduced recovery to 76%. Frozen lysate showed good recovery after 70 days.
- Median total IL-8 in healthy adults (22-61 years) was 83 ng/L, with levels increasing with age in females. Elevated IL-8 was observed in pregnant women with amniotic infection syndrome.
Conclusions:
- The evaluated method provides a reliable assessment of total IL-8 in blood when proper sample pretreatment is employed.
- The method's sensitivity allows for the detection of subtle changes in total IL-8.
- This technique is a valuable tool for investigating the diagnostic significance of total IL-8 in blood samples.
Background:
It has been shown that a high percentage of interleukin-8 (IL-8) in blood is cell associated. Recently, a simple method for determination of cell-associated IL-8 in whole blood after cell lysis has been described. The purpose of this study was to evaluate this method, to examine the influence of preanalytic sample handling, and to establish the concentration range of total IL-8 and its relation to age and sex in healthy subjects.
Methods:
Total IL-8 content of whole blood was determined after lysing blood cells with Milenia((R)) cell lysis solution. IL-8 in the resulting blood lysate was measured with the IMMULITE((R)) IL-8 immunoassay.
Results:
When freshly drawn blood was stored up to 48 h on ice, no significant changes in total IL-8 were measured in the subsequently prepared lysate, whereas with storage at room temperature, total IL-8 increased after 3 h from 94 +/- 13 ng/L to 114 +/- 16 ng/L (n = 10). In lysate stored for 48 h at 4 degrees C, marginal changes of the IL-8 concentration were noted, with storage at room temperature, only 76% +/- 5% (n = 12) of initial concentration was recovered. From lysate frozen at -20 and -80 degrees C, respectively, 84% +/- 4% and 93% +/- 2% of initial IL-8 was recovered after 70 days (n = 10). IL-8 was measured with comparable precision in plasma (CV, 3. 2-4.2%) and blood lysate (CV, 3.7-4.1%). When plasma was diluted with cell lysis solution, a slightly overestimated recovery (125% +/- 3%) was observed; for lysate specimens with a cell lysis solution content >/=75%, the recovery after dilution was 98% +/- 2%. In lysate prepared from 12 blood samples with exogenous IL-8 added, IL-8 recovery was 104% +/- 2% (recovery from plasma <35%). The median total IL-8 in blood lysates from 103 healthy subjects (22-61 years) was 83 ng/L of blood (2.5-97.5 percentile range, 49-202 ng/L of blood). In females but not in males, total IL-8 increased significantly with advancing age (P: <0.002). We found grossly increased total IL-8 in six pregnant women with amniotic infection syndrome.
Conclusions:
The evaluated method allows the assessment of total IL-8 in blood with good performance when appropriate conditions of sample pretreatment are considered. The values in healthy volunteers all were above the detection limit of the IL-8 assay; therefore, slight changes of total IL-8 could be noted. Thus, the present method is a suitable tool to study the diagnostic relevance of total IL-8 in blood.
