Methods
Pre-HD and post-HD serum samples
Renal patients attending the HD were invited to participate and informed consent was obtained. Whole blood was obtained before (pre-HD) and immediately post-HD therapy in Vacutainer serum separator gel (SST) tubes (Becton-Dickinson, Oxford, UK). Following clotting, centrifugation at 3000 rpm for 10 min and routine biochemical analysis for clinical patient management, residual serum samples were aliquoted and stored at −70°C until further analysis of cTn.
Immunohistochemical staining of cellulose acetate membranes
The experimental design occurred in two phases. In phase 1, passive diffusion of cTn from a commercial source in cut dialyser membranes was examined. This was followed by phase 2, in which the counter-current flow of dialyser fluid and fluid in the blood compartment was used to replicate the process of dialysis to demonstrate the location of cTnI binding to the cuprophan membrane. Capillary middle-flux polysulfone Helixone, FX80 (Fresenius Medical Care, Nottinghamshire, UK) HD membranes were used throughout.
Phase 1: passive diffusion in dialyser membrane
A sterile cuprophan dialyser was flushed with reagent grade distilled water. The plastic casing was cut with a hacksaw to remove the inlet and outlet caps. Twenty-millimetre long sections of membrane tubing were cut using a scalpel and the bundle was loosely placed in a 0.5 mL Eppendorf tube. Each bundle was washed twice with 0.1 M phosphate buffered saline (PBS), pH 7.2. The membrane sections were then incubated overnight in 1000 μL of serum spiked with free cTnT, free cTnI and cTn I-T-C complex (HyTest Ltd, Finland). The concentrations were 78 000 and 29 150 μg/L for free cTnI and cTnT, respectively. For the human cTn complex, the cTnT and cTnI concentrations were 27 420 and 30 370 μg/L, respectively.
A 1:100 concentration of M7 anti-cTnT MAb (Roche Diagnostics, Tutzing, Germany) was incubated in one Eppendorf tube, the other was incubated with a 1:100 dilution of 19C7 anti-cTnI MAb (HyTest Ltd, Finland) for 1 h. The membranes were rinsed three times with 0.1 M PBS, pH 7.2, to remove any excess unbound primary antibody. The membranes were then incubated with biotin-labelled mouse anti-IgG (1:1000) for a further hour, washing unbound secondary antibody again with PBS. The membranes were then incubated with the fluorescent marker streptavidin-labelled fluorescein isothiocyanate (FITC). Membranes were transferred to cork tissue boards and orientated either longitudinally (LS) or transversely (TS). The entire block was covered in cryo-embedding media (OCT, Tissue-Tek, Lamb Ltd, East Sussex, UK) and snap frozen by submerging in liquid nitrogen. The frozen tissue board was transferred to a cryotome cryostat (−70°C) and allowed to equilibrate to environmental temperature for 3 min prior to sectioning. LS and TS sections, 5 μm thick, were cut and mounted on aminoalkylsilane-coated (Silane-Prep, Sigma Diagnostics) slides. The slides were allowed to air dry before being visualised using fluorescent microscopy (Olympus BX-40-FLA, Olympus Optical Ltd, London, UK) and video images were captured using Image Grabber PCI V.2.05, (Neotech Ltd, London, UK) for Microsoft Windows.
Phase 2: replication of counter-current flow of dialysis
In order to ascertain if cTnI crosses the dialyser membrane during the dialysis process or if it remains within the vascular compartment of the dialyser, dialysis membranes were examined after simulating the process of dialysis. Dialysate fluid and a volume of serum spiked with cTn (representing the blood compartment) were passed through the cuprophan membrane in counter-current flow, similar to that of dialysis. A water-driven vacuum pump was attached via the dialysate outlet and the tubing cannulated for effluent dialysate sample collection. The inlet was connected to a 5 L reservoir containing dialysate fluid (figure 1). Renalyte acid concentrate bicarbonate dialysate fluid (Fresenius Medical Care, Nottinghamshire, UK) was reconstituted 1 to 1.26 to 32.78 in 8.4% sodium hydrogen carbonate and reagent grade water, respectively, as per the recommended protocol of the manufacturers. The final solution had the following composition: Na+ 103 mmol/L, K+ 2 mmol/L, Ca++ 1.25 mmol/L, Mg++ 0.5 mmol/L, Cl− 108.5 mmol/L, CH3COO− 3 mmol/L and glucose 5.6 mmol/L. An aliquot of the renalyte dialysate fluid working solution was tested for possible interference in the cTnT and cTnI assays. The counter-current flow rate of dialysis fluid (Qd) was controlled at 200 mL/min. The membranes were primed with 200 mL dialysate and an effluent sample was collected for cTnT and cTnI assay testing before introduction of the blood component.
Figure 1In vitro setup of simulated haemodialysis. (A) Blood compartment inlet; (B) blood compartment outlet; (C) dialysate outlet; (D) dialysate inlet; (E) dialysate reservoir; (F) water-driven vacuum pump; (G) efferent dialysate collection syringe.
Five serum pools of 50 mL volumes were constructed from healthy volunteers free from a history of AMI and who did not demonstrate serum cTn positivity. The serum pools were filtered and further centrifuged at 3000 rpm for 10 min to remove any particulate matter. The pools were prepared as follows: pool A: troponin free serum pool (no spiking); pool B: unbound free cTnI; pool C: unbound free cTnT; pool D: troponin I-C; pool E: troponin T-I-C complex.
For each pool constructed, a fresh sterile membrane filter was used in the simulation. The serum pools were injected into the blood compartment of the HD membrane using a 20 mL syringe. Two-millilitre aliquots were drawn from the blood compartment outlet repeated at 2 min intervals. In addition, 2 mL aliquot fractions of efferent dialysate were also collected at 2 min intervals for a period of 15 min. Samples from the blood compartment outlet and the efferent dialysate were assayed for cTnT and cTnI.
Following simulated HD procedures, all membranes were disconnected and the outer plastic shell was cut using a hacksaw at the collar of the housing to remove the inlet and outlet caps. Two to 4 cm long bundles of fibres were cut from the mid-section of the dialyser with a sterile scalpel and placed into 0.5 mL Eppendorf tubes and incubated with anti-cTn antibodies and visualised with fluorescent microscopy as described above.
cTnT assay
cTnT was determined using the fourth generation Troponin T STAT assay on an Elecsys 2010 (Roche Diagnostics, Haywards Heath, UK). The assay total imprecision was 5.4–9.3% in the range 0.47–11.5 μg/L. The measuring range was 0.01–25 μg/L. The 10% CV was at 0.03 μg/L with a 99th centile of <0.01 μg/L.
cTnI assay
cTnI was determined using the TnI-Ultra assay for the ADVIA Centaur (Siemens Healthcare Diagnostics, Frimley, UK). The detection limit of the instrument was 0.006 μg/L, upper limit 50 μg/L. The manufacturers claim was 10% CV at 0.03 μg/L with a 99th centile of 0.04 μg/L.
Data handling and statistics
All data were exported to Microsoft Excel (Microsoft Corporation). All statistical analyses will be performed using the Analyse-it, add-in software for Excel. Data were examined for normal distribution. Box and whisker plots were constructed to demonstrate the distribution of pre-HD and post-HD cTnT and cTnI concentrations and formally tested for statistical significance with non-parametric Wilcoxon signed-ranks testing. A p≤0.05 was deemed significant. All biomarker concentrations were measured in triplicate and are reported as mean±SD.