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Evaluating the long-term biological stability of cytokine biomarkers in ocular fluid samples.

Author(s): Felfeli T, Park J, Nestor B, Altomare F, Rai AS, Mandelcorn ED, Chow DR, Wong DT

Publication: BMJ Open Ophthalmol, 2023, Vol. 8, Page

PubMed ID: 38081779 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared levels of cytokines in aqueous humor and vitreous fluid specimens that were stored frozen for 1 week with those stored for 3 months, 9 months and 15 months and among specimens subjected to 1 versus 2 and 3 freeze-thaw cycles. Aqueous humor was collected by anterior chamber paracentesis from fifteen patients undergoing cataract surgery, pars plana vitrectomy (PPV), or scleral buckle surgery.  Vitreous fluid was obtained by standard scleral incision and aspiration using the vitrector during PPV from 20 patients. Specimens were aliquoted and stored at 4°C for 5 h and then frozen at -80°C. Aliquots were thawed after 1 week, 3 months, 9 months, and 15 months. To investigate the effect of freeze-thaw cycling, specimens were freeze-thawed (details not provided) 1 to 3 times. Cytokines were quantified using the Bioplex Pro-Human Cytokine Grp I Panel 27-Plex 96-well Assay Kit.  

   Summary of Findings:

   Significant declines in interleukin (IL)-2, IL-10, IL-12, and platelet-derived growth factor (PDGF)-bb occurred after prolonged (3-15 months) frozen storage at -80°C relative to levels in specimens that were frozen for 1 week (P<0.001, all);  the duration of frozen storage did not affect levels of fibroblast growth factor (FGF), eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interferon gamma (IFN-ɣ), IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6,  IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17A, inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory proteins (MIPs)-1α, MIP-1β, PDGF-bb, regulated on activation, normal T cell expressed and presumably secreted (RANTES), tumor necrosis factor alpha (TNF)-α, and VEGF. Only the decrease in PDGF-bb was progressive (−30.8% at 3 months, −33.8% at 9 months, and −37.6% at 15 months), as a larger decline (relative to the 1 week control) was observed among specimens stored for 9 months than 15 months for IL-2 (-27.2% versus -13.7%), IL-10 (-17.2% versus -9.0%), and IL-12 (-22.6% versus -12.5%).  None of the analytes investigated were significantly affected by 2 or 3 freeze-thaw cycles compared to 1 freeze-thaw cycle. Importantly, the effects of frozen storage were similar in aqueous humor and vitreous fluid specimens.  

    

   Biospecimens

   • Bodily Fluid - Vitreous Humor
   • Bodily Fluid - Aqueous Humor

   Preservative Types

   • Frozen

   Diagnoses:

   • Cataracts
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   Protein	Immunoassay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Aqueous humor Vitreous fluid
   Storage	Storage duration	1 week 3 months 9 months 15 months
   Storage	Freeze/thaw cycling	1 cycle 2 cycles 3 cycles

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