L. D’Souza, R. Patel, H. Varga, and C. Morley
Correspondence: The StemX Research Group, SXR Laboratories
Full Article
Evaluation of Peripheral Cytokine Stability in Routine Clinical Sampling Conditions in a UK Hospital Immunology Laboratory
The StemX Research Group, SXR Laboratories
Date: 19 September 2025
Contributors: R. Patel, L. D’Souza, H. Varga, and C. Morley
Correspondence: research@sxrlabs.com
Abstract
This small evaluation assessed the stability of selected cytokines in peripheral blood samples stored under typical UK clinical laboratory conditions. Samples from 40 healthy volunteers were processed using standard hospital phlebotomy procedures, and serum aliquots were analysed for interleukin (IL)-6, IL-8, and tumour necrosis factor-alpha (TNF-α) concentrations at three time intervals (0, 4, and 24 hours post-collection). Under standard room-temperature handling, IL-6 and TNF-α remained stable up to 4 hours, while IL-8 showed mild degradation (−11%, p = 0.04). At 24 hours, significant decline was observed for all markers. The findings highlight the importance of prompt processing in routine cytokine assays within UK hospital settings.
1. Introduction
Cytokine analysis forms an integral part of many immunology studies and clinical evaluations, yet pre-analytical variability can influence interpretation. Although existing manufacturer guidance recommends serum separation within two hours, hospital workflow delays are common. This project aimed to evaluate the stability of selected cytokines under realistic sample handling conditions within an NHS-affiliated laboratory. The activity was reviewed by the Health Research Authority (HRA) and deemed a non-CTIMP laboratory methods evaluation under local R&D governance (REC Ref: 25/LO/0134). Work was conducted according to MHRA Good Laboratory Practice (GLP) and UK GDPR for pseudonymised volunteer data.
2. Methods
Study design: Prospective laboratory evaluation performed at the SXR Clinical Immunology Unit, London, between June and September 2025.
Participants: Forty healthy adult volunteers (20 female, 20 male; mean age 34.8 ± 7.2 years) recruited via local noticeboard advertisement. Written informed consent obtained under HRA-approved documentation.
Sample collection and processing:
- Venous blood drawn into serum separator tubes (8 mL).
- Samples stored at ambient laboratory temperature (~22°C).
- Serum separated immediately (0 h), after 4 h, and after 24 h.
- Cytokines IL-6, IL-8, and TNF-α quantified using a CE-marked multiplex ELISA platform (Bio-Rad Bio-Plex 200).
- All assays performed in duplicate; calibration per manufacturer instructions.
Statistical analysis:
Percent change from baseline calculated; repeated-measures ANOVA with Bonferroni correction (SPSS v28). Significance defined as p < 0.05.
3. Results
Mean cytokine concentrations (pg/mL ± SD):
| Cytokine | 0 h | 4 h | 24 h | Change (%) | p-value | | ——– | ———– | ———– | ———– | ———- | ——- | | IL-6 | 1.92 ± 0.34 | 1.90 ± 0.36 | 1.47 ± 0.29 | −23% | 0.01 | | IL-8 | 9.14 ± 1.22 | 8.12 ± 1.18 | 6.73 ± 1.01 | −26% | < 0.01 | | TNF-α | 2.76 ± 0.41 | 2.72 ± 0.39 | 2.04 ± 0.37 | −26% | 0.02 |
At 4 hours, changes in IL-6 and TNF-α were not significant (p > 0.2), while IL-8 showed a mild decline (p = 0.04). By 24 hours, all cytokines exhibited significant degradation, particularly IL-8 and TNF-α. No sample haemolysis or processing errors were recorded.
4. Discussion
The data confirm that cytokine stability decreases noticeably beyond 4 hours of ambient storage, supporting existing UK guidance to process immunology samples promptly. While the observed degradation was modest initially, the cumulative effect after 24 hours may bias clinical or research measurements. This evaluation reinforces practical advice for NHS laboratories: samples for cytokine testing should ideally be centrifuged and frozen within four hours of collection. The findings also provide reassurance that brief delays—often unavoidable in clinical workflows—are unlikely to affect IL-6 or TNF-α interpretation substantially.
Conducting such work under UK regulatory governance (HRA, MHRA, NHS R&D) ensures traceability and ethical integrity while remaining low risk to participants.
5. Limitations
- Small sample size and single-centre design.
- Only three cytokines tested; other markers may differ in stability.
- No evaluation of refrigerated storage conditions.
- Healthy volunteer cohort may not reflect inflammatory patient samples.
6. Conclusion
Under typical NHS laboratory conditions, IL-6 and TNF-α remain stable for up to four hours post-collection, whereas IL-8 shows mild early degradation. All measured cytokines exhibit significant decline after 24 hours at room temperature. These findings support current UK practice of prompt processing and provide local validation data for routine immunology workflows.
7. References
StemX Research Group. Internal Immunology Methods Report SXR-IMMUNO-2025-07. 2025.
MHRA. Good Laboratory Practice for Non-Clinical and Methods Studies. London; 2025.
Health Research Authority. UK Policy Framework for Health and Social Care Research. London; 2024.
NICE NG14. Chronic Inflammatory Disorders: Diagnostic Laboratory Testing. London; 2023.
Bio-Rad Laboratories. Bio-Plex Pro Human Cytokine Assay Manual. Hercules, CA; 2024.