Study cohort
Children with heart disease undergoing cardiac and non-cardiac surgery were recruited into the Heart Centre Biobank Registry [11]. The study was approved by the Research Ethics Board of the Hospital for Sick Children. Written informed consent to participate and publish results was obtained from all parents/legal guardians/participants. Serum samples were collected prior to cardiac surgery and on post-operative days 1 and 2. Serum samples were stored at 4 °C for 24–48 h and then frozen at -80 °C until analysis. Demographic data, diagnosis, surgery type, and cardiopulmonary bypass duration were collected from medical records. Patients were categorized by presence or absence of a left ventricular (LV) ventriculotomy (incision in LV), or myectomy (resection of myocardium from the LV). Patients undergoing non-cardiac surgery i.e. liver or renal transplant were included as controls.
ELISA for SIRPA
All serum samples were analyzed for SIRPA concentration using commercially available Sandwich-SIRPA ELISA kits purchased from Cusabio Biotech Co, Ltd, Wuhan, China (Catalog # CSB-EL021334HU) and Elabscience- Biotech Co.Ltd, Wuhan, China (Catalog # E-EL-H1573). ELISA assays on pre- and post-operative serum were performed according to manufacturer’s instructions. After assay, Optical Density (OD) was measured using Gemini EM microplate reader (Molecular Devices, USA) set at 450 nm and change in SIRPA concentrations after surgery were compared between the different surgical groups.
Western blot analysis of SIRPA ELISA kit calibrators
To verify the identity of the protein detected, Western blot was performed on the calibrators from Cusabio and Elabscience ELISA kits and compared to recombinant human SIRPA (rhSIRPA) which was purchased from Elabscience (Catalog # PN201773) to use as positive control. Briefly, equal amount of protein from each sample was separated by gel electrophoresis using Novex 10 % Tris-Glycine Gel (Life technologies, catalog no# EC6078Box) following manufacturer’s instructions. Protein was transferred to PVDF membrane and blocked with 5 % milk in TBST for 1 h. at room temperature followed by overnight incubation with anti-human SIRPA antibody (Biolegend, catalog # 323805) followed by 1 h incubation with horseradish peroxidase-conjugated secondary antibody at room temperature.
Mass spectrometry analysis of ELISA kit calibrators
SIRPA ELISA kit calibrators from Cusabio and Elabscience were also analyzed on an Orbitrap analyzer (Q-Exactive, ThermoFisher, San Jose, CA) outfitted with a nanospray source and EASY-nLC nano-LC system (ThermoFisher, San Jose, CA) in order to determine if SIRPA was the target antigen for both calibrators. This was performed at the SickKids SPARC Biocentre (Toronto, ON). The samples were trypsin digested and lyophilized peptide mixtures were dissolved in 0.1 % formic acid and loaded onto a 75 μm x 50 cm PepMax RSLC EASY-Spray column filled with 2 μM C18 beads (ThermoFisher San, Jose CA) at a pressure of 800 Bar. Peptides were eluted over 60 mins at a rate of 250 nl/min using a 0 to 35 % acetonitrile gradient in 0.1 % formic acid. Peptides were introduced by nano-electrospray into the Q-Exactive mass spectrometer (Thermo-Fisher). The instrument method consisted of one MS full scan (400–1500 m/z) in the Orbitrap mass analyzer with an automatic gain control (AGC) target of 1e6, maximum ion injection time of 120 ms and a resolution of 70,000 followed by 10 data-dependent MS/MS scans with a resolution of 17,500, an AGC target of 1e6, maximum ion time of 120 ms, and one microscan. The intensity threshold to trigger a MS/MS scan was set to 1.7e4. Fragmentation occurred in the Higher –energy C-trap dissociation with normalized collision energy set to 27. The dynamic exclusion was applied using a setting of 5 s.
Statistical analysis
Serum SIRPA concentrations were expressed as mean ± standard deviation. Samples were grouped by the type of cardiac surgery i.e. no-ventriculotomy (n = 27), myectomy (n = 14), and ventriculotomy (n = 7). Samples from non-cardiac surgery i.e. liver and renal transplant (n = 6) were used as a controls. Statistical analysis was performed by Student t-test to compare SIRPA concentrations in cases and controls and to compare change in SIRPA concentrations in paired samples from pre- to post-operative time-points. Differences were considered statistically significant if p < 0.05. GraphPad PRISM 6.05 (GraphPad software, USA) was used for statistical analysis.