In n = 9 independent experiments, we assessed the LVAD-related blood damage of the implantable continuous-flow LVAD HeartMate 3™ (HM3, Abbott, USA) and the extracorporeal BPX-80 Bio-Pump® (BPX-80, Medtronic, Ireland) with our recently described mini test loop.27 Analogous to the verification with porcine blood,27 the validation of the mini test loop with human blood was performed according to the ASTM F1841-97 (2017) standard.1
Test Loop Set-up
The mini test loops were set up as previously described.27 In brief, the mini test loops are downscaled to one-third (160 mL) of the ASTM F1841-97 (2017)1 priming volume, and include a throttle to yield the pressure head of 100 mmHg and mounted sensors for pressure (Xtrans, CODAN, Germany), temperature (Medos, Germany) and flow (Transonic, USA) (Fig. 1). To accommodate the size of the HM3 in- and outlet, 4 cm of tubing at each position are replaced with 1/2″-diameter PVC-tubing. To prevent bending of the tubing at the inlet and outlet of the mini test loop reservoir, connectors are fixed with a spacer and a clamp. Temperature of the mini test loops was controlled with heating-hoods.
Blood Collection and Preparation
The study was approved by the local ethics committee (Ethical Approval Number EK141/20) and informed written consent was obtained from all donors. For each of the 9 tests, a single donation of 450 mL human blood (15,000 international units L−1 sodium heparin (B. Braun, Germany), 0.09% (w/v) glucose, 2.0% (v/v) isotonic saline solution, 0.016 g L−1 gentamycin) was freshly collected (Compoflex Transferbag T2131, Fresenius Kabi, Germany) from healthy male volunteers free from antiplatelet or anticoagulant medication for at least 10 days prior to donation. The blood was inspected for hematocrit, red and white blood cell and platelet count (hematology analyzer Sysmex XT2000i vet, Sysmex, Germany) and hemoglobin and lactate concentration (blood gas analyzer ABL 825 Flex, Radiometer, Germany). Blood dilution to the set hematocrit of 30 ± 2% was performed during priming and filling of each test loop by adding pre-calculated volumes of isotonic saline solution and blood. Base excess was adjusted to 0 ± 5 mM with sodium hydrogen carbonate (8.4% (w/v), Fresenius, Germany).
Test Loop Handling
All test loops were primed with isotonic saline solution and de-aired before being filled with blood. To yield a hematocrit of 30 ± 2%, excess saline solution was drained and replaced with blood. The target volume was 160 ± 5 mL with 1% accepted deviation between the mini test loops and the static reference. After a second de-airing, all test loops were run for 5 minutes to mix completely before verification and adjustment of hematocrit and base excess. For similar pre-pumping conditions, the corresponding flow rate of 2.4 ± 0.0 L min−1 of the limiting pump’s minimal speed was set for both test loops. After pre-pumping sampling, flow rate and pressure head were adjusted to the ASTM operating point (5 ± 0.25 L min−1 and 100 ± 3 mmHg, respectively1) and timers were started for further sampling.
A 160-mL static reference reservoir was similarly prepared and kept at static conditions in a water bath.
Blood Sampling
After 0.5 mL discard, seven 2.5-mL samples were taken before starting the operating point (pre-pumping sample) and then every 60 minutes for 6 hours onwards. Samples were directly assessed for blood count, blood gas and activated clotting time and processed to platelet-poor plasma in 3.2%-tri-sodium-citrate tubes (Sarstedt, Germany). Plasma samples were stored at − 20 to − 80 °C according to standard clinical practice and manufacturer’s instructions until further analysis.
Cleaning Procedure
Re-usage of blood pumps requires thorough cleaning of all parts in contact with blood. To ensure removal of cells and proteins, all pumps were first rinsed thoroughly with tap water until effluent was visually clear. The pumps were then set up in a cleaning-in-place (CIP) loop27 and run with a pepsin/citrate solution for 1 hour followed by 15 minutes rinsing with de-ionized water. Pumps were then incubated in 1% (v/v) instrument disinfectant solution (Bomix plus, Bode Chemie, Germany) for 15 minutes and finally rinsed with de-ionized water for 30 minutes. Pumps were then dried overnight by filtered compressed air and boxed until the next experiment.
Analysis of Hemolysis
Analysis of hemolysis was performed according to DIN 58931:2010-086 by means of the cyanmethemoglobin (HiCN) method. In brief, plasma samples were thawed in a water bath at 37 °C for 8 minutes and diluted 1:5 (v/v) with HiCN conversion solution (fHb (HiCN), Bioanalytic GmbH, Germany) in duplicates in standard micro cuvettes (Brandt, Germany). After incubation, converted plasma free hemoglobin (pfHb) was photometrically detected at 540 nm with 680 nm reference wavelength. Duplicate results were accepted with a coefficient of variation (CV) ≤ 0.05. Hemolysis is presented as ΔpfHb (mg dL−1) and modified and normalized milligram index of hemolysis (MIH and mgNIH, respectively), with
$$ \Delta {\text{pfHb}}_{t} = {\text{pfHb}}_{t} - {\text{pfHb}}_{{{\text{pre}}}} , $$
(1)
$$ {\text{MIH}} = \frac{{\Delta {\text{pfHb}}_{t} \times \left( {100 - {\text{Hct}}_{t} } \right){\text{ }}/100}}{{{\text{Hb}}_{{{\text{pre}}}} }} \times \frac{{10^{6} }}{{\frac{{Q_{t} \times T}}{{V_{t} }}}}, $$
(2)
$$ {\text{mgNIH}} = \frac{{\Delta {\text{pfHb}}_{t} \times \left( {100 - {\text{Hct}}_{t} } \right)}}{100} \times \frac{100 }{{\frac{{Q_{t} \times T}}{{V_{t} }}}}, $$
(3)
$$ \frac{{Q_{t} \times T}}{{V_{t} }} = \# {\text{passages}}_{t } = 60 \times \mathop \sum \limits_{i = 60 }^{t} \frac{{Q_{i} }}{{V_{i} }}\quad {\text{with}}\quad \left( {t = 60, 120, 180, 240, 360} \right); $$
(4)
with ΔpfHbt: Increase of plasma free hemoglobin (mg L−1) in the sampling interval, Hbpre: Pre-pumping total hemoglobin (mg L−1), Hctt: Hematocrit (%), Qt: Flow rate (L min−1), Vt: Test loop volume (L), T: Elapsed time (min) and #passages: Absolute number of pump-passages
As previously recommended,27 the number of pump-passages in Eqs. (2) and (3) is corrected for the change of volume and flow rate over the sampling intervals by means of Eq. (4).
Analysis of vWF Degradation
vWF concentration (vWF:Ag) and collagen-binding functionality (vWF:CB) were analyzed by commercial enzyme-linked immunosorbent assay (ELISA) kits (vWF:Ag ELISA and vWF:CB ELISA Collagen Type I, both Technozym, Germany).Duplicate results were accepted with a CV ≤ 0.10 for vWF:Ag and ≤ 0.13 for vWF:CB.
Ratios of vWF:CB and vWF:Ag were calculated for each sample with a cut-off for clinically relevant vWF degradation of 0.8025 and were depicted as absolute and normalized (ΔvWF-) ratios with
$$ \Delta {\text{vWF-ratio}}_{t} = {\text{vWF-ratio}}_{t} - {\text{vWF-ratio}}_{{{\text{pre}}}} ; $$
(5)
with ΔvWF-ratiot: Decrease of vWF-ratio in the sampling interval.
Statistical Analysis
Statistical analysis was performed with Prism 9 (GraphPad, USA). Results were analyzed for each group, and normal distribution was verified with Shapiro Wilks test. Outliers were identified if below or above twice the interquartile range (IQR) or by means of the ROUT method15 with a maximum false discovery rate of 0.5%. Normally-distributed continuous variables are depicted as mean ± standard deviation (SD) or with 95% confidence interval (CI). Comparison within the groups were performed with mixed-effects analysis with Geisser-Greenhouse correction and Dunnett’s correction for multiple comparisons. For comparison between the groups, ANOVA or mixed-effects analysis with Geisser-Greenhouse correction and Tukey’s multiple comparison correction or t-test were used, as appropriate. An adjusted exact p-value was considered significant with p ≤ 0.05 (*).