2.5.1 Serum samples
2.5.1.1 Collection
Phlebotomy training was carried out with a UK accredited course (Phlebotomy Training Services, London UK) to carry out safe basic venepuncture on recruited adult subjects for this study (Certificate Level 2, Phlebotomy Training Services, London). Blood was collected once at day of surgery (between 7am and 5pm depending on surgery slot) prior to surgery and once again approximately six-months (±14 days) following microfracture surgery (between 10am and 1pm), prior to gait analysis.
Serum and plasma were collected via venepuncture using BD Vacutainer Safety-Lok Blood collection Sets (23g needle, 12” Tubing and Luer adaptor) (Fisher Scientific, UK) and Greiner Bio-one serum and plasma lithium heparin/EDTA collection tubes (Fisher Scientific, UK). The order of draw was conducted based on the Greiner Bio-one recommendations, which consisted of serum followed by plasma heparin and then EDTA tubes. Following successive collections, tubes were inverted five times and then kept at 4˚C before transportation to the Biosciences laboratory for processing. Serum tubes were left at room temperature to clot for 20 minutes before storing at 4˚C. Blood samples were kept no longer than 3 hours at 4˚C before transporting in a sample box with cool packs via courier to the Cardiff University School of Biosciences (BIOSI) Pathophysiology and Repair (PPR) laboratory. Care was taken not to agitate the samples during transport. Upon arrival, blood samples were processed immediately.
2.5.1.2 Processing
The protocol for processing of serum was identical to that used in the ARUK centre, permitting comparison of collected samples with pooled OA subject samples from the centre biobank. For both serum and plasma tubes, samples were first spun in their original tubes within a centrifuge at 2000G for 15 minutes, to separate serum/plasma from other blood constituents. Then, the clear fluid (plasma/serum) was carefully transferred to 1.5ml microcentrifuge tubes (Fisher-Scientific, UK) using a P1000 Gilson precision pipette, with care not to agitate the bottom layer of constituents. The clear fluids were then spun again at 3000G in a microcentrifuge for 15 minutes, to remove any
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additional cells and debris. Following centrifugation, supernatants were transferred to labelled 500µl cryovials in aliquots of 100µl, with care not to disturb the cell pellet. Final aliquoted samples were stored at -80˚C until day of immunoassay.
2.5.2 Synovial fluid samples
Synovial fluid samples were obtained from the affected knee by needle aspiration prior to introduction of the arthroscope by participating orthopaedic consultant Mr C Wilson. Aspirated fluids were transferred to a 5ml falcon tube and sent via courier to the BIOSI PPR laboratory in a cool packed container within 30 minutes. The protocol for processing of serum was identical to that used in the ARUK centre, permitting comparison of collected samples with pooled OA subject samples from the centre biobank. None of the collected fluids received contained blood staining, however this information was not recorded for pooled OA samples. Immediately upon arrival, synovial fluids underwent centrifugation at 5000G for 15 minutes to remove cells. Higher centrifugal forces were required for synovial fluid processing due to the increased viscosity of the fluid. Supernatants were then gently transferred to 500µl cryovials, in aliquots of 100µl. Finally, aliquoted samples were stored at -80˚C until day of immunoassay.
2.5.3 Immunoassays
Commercially available multiplex electrochemiluminescence (ECL) or single-plex enzyme-linked immunosorbent assays (ELISAs) were utilized to measure absolute concentrations of a total of seventeen biomarkers relating to bone and cartilage turnover and degradation, mechanical loading of bone and inflammation (Table 2.5-1). Both ECL assays and ELISAs were chosen as they have been extensively by clinical studies due to their high throughput, high protein specificity and low coefficient of variance of inter- and intra-assay signal readings (Watt et al., 2016, Struglics et al., 2015, Li et al., 2016, Lequin, 2005). ELISA assays were carried out at the BIOSI PPR Laboratory, whereas MSD multiplex assays were carried out at the Central Biotechnology Services centre, Cardiff, due to the availability of the specific MSD plate reader required for chemiluminescence measurements. Where possible, each sample aliquot was freeze-thawed the minimum number of times required to cover all tests and consistently with all other test samples to avoid concentration variability caused by repeated freeze-thawed cycles. The maximum number of freeze-thaw cycles any sample was exposed to was 2x.
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Table 2.5-1 – Biomarkers chosen for analysis and reference to their representation in known
homeostatic processes the joint
Biomarker Abbrev. Representation in the joint Reference
Cartilage Oligomeric Matrix
Protein
COMP
Cartilage matrix stabiliser, chondrocyte matrix synthesis activity (Recklies et al., 1998, Chen et al., 2007, Briggs et al., 1995, Attur et al., 2013) C-terminal telopeptide for collagen type I
CTX-I Bone resorption (osteoclast) activity (Garnero et al., 2003, Nikahval et al., 2016) Alkaline Phosphatase ALP
Bone formation (osteoblast) activity, bone and cartilage
mineralisation
(Poole et al., 1989, Burr and Gallant,
2012) Receptor activator
of nuclear factor κ-Β ligand
RANKL Canonical osteoclast pathway activation
(Odgren et al., 2003, Steeve et al.,
2004, Boyce et al., 2015) Osteoprotegerin OPG Canonical osteoclast
pathway inhibitor
(Odgren et al., 2003, Steeve et al.,
2004, Boyce et al., 2015)
RANKL:OPG ratio RANKL/OPG
Surrogate measure of net canonical osteoclast pathway activation (Odgren et al., 2003, Steeve et al., 2004, Boyce et al., 2015) Glutamate - Glutaminergic signalling agonist, Mechanical regulator of bone physiology, inflammatory regulator (Brakspear and Mason, 2012, Cowan et al., Wen et
al., 2015) Sclerostin - Wnt signalling inhibitor, mechanical regulator of bone physiology (Robling et al., 2008, Sebastian and Loots, 2017) Tumor necrosis factor alpha TNF-α Pro-inflammatory cytokine, osteoclastogenic signalling, osteoblast signalling (Kapoor et al., 2011, Lam et al., 2000, Osta et al., 2014) Interleukin-1β IL-1β Pro-inflammatory cytokine Molina-Holgado et (Attur et al., 1998,
al., 2000) Interleukin-2 IL-2 Pro-inflammatory cytokine (de Rham et al., 2007)
Interleukin-4 IL-4
Anti-inflammatory cytokine associated with IL-13
function
(Scanzello et al., 2008, Kapoor et al.,
2011, Onoe et al., 1996) Interleukin-6 IL-6 Pro-inflammatory cytokine,
96 Yoshitake et al., 2008, Tat et al., 2008a) Interleukin-8 IL-8 Pro-inflammatory chemokine, neutrophil chemotaxis (Merz et al., 2003, Wojdasiewicz et al., 2014)
Interleukin-10 IL-10 Anti-inflammatory cytokine, bone formation
(Wojdasiewicz et al., 2014, Jung et al.,
2013, Liu et al., 2006, Zhang et al.,
2016) Interleukin-12p70 IL-12p70 T cell stimulatory factor 2008, Scanzello and (Scanzello et al.,
Goldring, 2012) Interleukin-13 IL-13 Anti-inflammatory cytokine (Wojdasiewicz et al., 2014, Onoe et
al., 1996) Interferon-γ IFN-γ
Pro- and anti-inflammatory cytokine, neuropathic pain
pathway stimulant
(Mathieu et al., 2008, Zhang, 2007, Vikman et al., 2007)
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Table 2.5-2 – Immunoassay details for analytes selected for analysis.