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Determination of Total Interleukin-8 in Whole Blood after Cell Lysis

Departments of
¹ Gynecological Endocrinology and Reproductive Medicine, and
² Neonatology, University of Bonn, Sigmund-Freud-Strasse 25, D-53127 Bonn, Germany.
^a Address correspondence to this author at: Zentrum für Frauenheilkunde und Geburtshilfe, Universität Bonn, Sigmund-Freud-Strasse 25, D-53127 Bonn, Germany. Fax 49-228-2874651.

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^® cell lysis solution. IL-8 in the resulting blood lysate was measured with the IMMULITE^® 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 °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 °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.

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