# Impact of Delayed Centrifugation on Interleukin 6 Determination in Human Blood

## TL;DR

Delaying centrifugation of blood samples affects IL-6 measurements, leading to false high or low results depending on the collection tube type.

## Contribution

The study reveals that delayed centrifugation causes significant IL-6 changes in heparin plasma and serum, but not in EDTA blood.

## Key findings

- Delayed centrifugation increased IL-6 levels in heparin plasma and serum by up to 50-fold and 12-fold, respectively.
- Preincubation of EDTA blood decreased IL-6 levels in most samples from both healthy individuals and critically ill patients.
- False high IL-6 results increased by over 50% in heparin plasma and serum due to delayed centrifugation.

## Abstract

Background/Objectives: Clinical experience indicates that the determination of interleukin 6 (IL-6) in human blood can vary depending on time span between sample collection and centrifugation. Here, we evaluated confounding effects in various blood specimens. Methods: The blood of healthy individuals and critically ill patients was collected in EDTA-, heparin-, and serum collection tubes. Tubes were facultatively incubated (20 °C, 24–48 h) before centrifugation, and IL-6 was measured in the supernatant. Results: The preincubation of the blood collection tubes increased the IL-6 values in heparin plasma (in 17/20 samples up to 50-fold) and serum (in 17/20 samples up to 12-fold). These changes were relevant since the normal values were thereby lifted above the upper confidence limit in 12/20 heparin plasma samples and 4/20 serum samples. These IL-6 increases were probably due to in vitro synthesis as opposed to the release of preformed IL-6 from blood cells because subjecting uncentrifuged collection tubes to mechanical cell lyses had negligible effects on IL-6, while incubation with microbial stimulators dramatically increased these values. In the case of EDTA blood, collection tube preincubation induced IL-6 decreases in 17/20 samples from healthy individuals and 20/23 samples from critically ill patients. Conclusions: IL-6 determination in heparin plasma and serum is compromised by delayed centrifugation. This effect is relevant for normal values. It increased the number of false high results by >50%. The delayed centrifugation of EDTA blood decreased the IL-6 values, which caused a single false-negative result in 1/43 healthy and critically ill people. The false-negative rate is possibly higher in EDTA blood from non-critically ill out-patients, exhibiting moderately increased IL-6 levels.