The Evaluation of Leukocytes in Response to the In Vitro Testing of Ventricular Assist Devices

Chan, Chris Hoi Houng; Hilton, Andrew; Foster, Graham R.; Hawkins, Karl; Badiei, Nafiseh; Thornton, Catherine A.

doi:10.1111/aor.12161

Cited by 18 publications

(39 citation statements)

References 25 publications

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“…Interestingly, our previous study of bovine blood using the extracorporeal RotaFlow and the intracorporeal VentrAssist pumps observed an increase in platelet numbers . This increase was hypothesized due to LMP being falsely detected as platelets by the hematology analyzer due to the concomitant decrease in leukocytes and increase in CD45 + LMPs . Although the high speed condition in this current study caused a reduction in leukocytes and an increase in CD45 + LMPs, there was no increase in platelets.…”

Section: Discussioncontrasting

confidence: 58%

Ovine Leukocyte Microparticles Generated by the CentriMag Ventricular Assist Device In Vitro

et al. 2018

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Ventricular assist devices (VADs) are a life-saving form of mechanical circulatory support in heart failure patients. However, VADs have not yet reached their full potential due to the associated side effects (thrombosis, bleeding, infection) related to the activation and damage of blood cells and proteins caused by mechanical stress and foreign materials. Studies of the effects of VADs on leukocytes are limited, yet leukocyte activation and damage including microparticle generation can influence both thrombosis and infection rates. Therefore, the aim was to develop a multicolor flow cytometry assessment of leukocyte microparticles (LMPs) using ovine blood and the CentriMag VAD as a model for shear stress. Ovine blood was pumped for 6 h in the CentriMag and regular samples analyzed for hemolysis, complete blood counts and LMP by flow cytometry during three different pump operating conditions (low flow, standard, high speed). The high speed condition caused significant increases in plasma-free hemoglobin; decreases in total leukocytes, granulocytes, monocytes, and platelets; increases in CD45 LMPs as well as two novel LMP populations: CD11b /HLA-DR and CD11b /HLA-DR , both of which were CD14 /CD21 . CD11b /HLA-DR LMPs appeared to respond to an increase in shear magnitude whereas the CD11b /HLA-DR LMPs significantly increased in all pumping conditions. We propose that these two populations are released from granulocytes and T cells, respectively, but further research is needed to better characterize these two populations.

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Section: Discussioncontrasting

confidence: 58%

Ovine Leukocyte Microparticles Generated by the CentriMag Ventricular Assist Device In Vitro

et al. 2018

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“…Our laboratory has previously carried out hemolysis and leukocyte profile assays using the temporary RotaFlow CP and the now obsolete VentrAssist implantable rotary blood pump. These studies showed that hemolysis and leukocyte damage do not necessarily correlate closely, and that a pump with low hemolysis can still have a significant damaging effect on leukocytes as shown for the VentrAssist (9). With similar results using our total blood damage profiling approach, both the CentriMag and RotaFlow CP appear to be good comparators when undertaking in vitro testing of new implantable LVADs, although RotaFlow CP's effect on platelet activation would need to be established first.…”

Section: Discussionmentioning

confidence: 97%

The CentriMag Centrifugal Blood Pump as a Benchmark for In Vitro Testing of Hemocompatibility in Implantable Ventricular Assist Devices

et al. 2014

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Implantable ventricular assist devices (VADs) have proven efficient in advanced heart failure patients as a bridge-to-transplant or destination therapy. However, VAD usage often leads to infection, bleeding, and thrombosis, side effects attributable to the damage to blood cells and plasma proteins. Measuring hemolysis alone does not provide sufficient information to understand total blood damage, and research exploring the impact of currently available pumps on a wider range of blood cell types and plasma proteins such as von Willebrand factor (vWF) is required to further our understanding of safer pump design. The extracorporeal CentriMag (Thoratec Corporation, Pleasanton, CA, USA) has a hemolysis profile within published standards of normalized index of hemolysis levels of less than 0.01 g/100 L at 100 mm Hg but the effect on leukocytes, vWF multimers, and platelets is unknown. Here, the CentriMag was tested using bovine blood (n = 15) under constant hemodynamic conditions in comparison with a static control for total blood cell counts, hemolysis, leukocyte death, vWF multimers, microparticles, platelet activation, and apoptosis. The CentriMag decreased the levels of healthy leukocytes (P < 0.006), induced leukocyte microparticles (P < 10−5), and the level of high molecular weight of vWF multimers was significantly reduced in the CentriMag (P < 10−5) all compared with the static treatment after 6 h in vitro testing. Despite the leukocyte damage, microparticle formation, and cleavage of vWF multimers, these results show that the CentriMag is a hemocompatible pump which could be used as a standard in blood damage assays to inform the design of new implantable blood pumps.

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“…LVAD patients suffer from complications including infection, thrombosis, and bleeding which are linked to blood cell damage . The monitoring of blood cells during in vitro testing of new LVAD prototypes could help identify safer pump designs . Hematology analyzers can provide cell counts in this setting but cannot provide information about cell viability.…”

mentioning

confidence: 99%

“…The monitoring of blood cells during in vitro testing of new LVAD prototypes could help identify safer pump designs . Hematology analyzers can provide cell counts in this setting but cannot provide information about cell viability.…”

mentioning

confidence: 99%

“…LVAD patients suffer from complications including infection, thrombosis, and bleeding (3-5) which are linked to blood cell damage (6)(7)(8)(9)(10)(11). The monitoring of blood cells during in vitro testing of new LVAD prototypes could help identify safer pump designs (12,13). Hematology analyzers can provide cell counts in this setting (12,13) but cannot provide information about cell viability.Flow cytometry using absolute counting bead standards is considered the golden standard for enumerating cell subsets in clinical samples (14).…”

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confidence: 99%

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Quantification methods for human and large animal leukocytes using DNA dyes by flow cytometry

et al. 2016

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Ovine and bovine blood is used heavily within the development of blood-handling medical devices, such as heart pumps (left ventricular assist devices, LVADs), for which blood cell damage needs to be monitored during in vitro testing. Hematology analyzers provide cell counts but no information about cell viability. The anthraquinone DNA dyes CyTRAK Orange TM and DRAQ7 TM have practical and spectral properties rendering them suitable for multicolor assays. Compared to other DNA dyes such as Vybrant Dyecycle, CyTRAK Orange enables a faster staining protocol and does not require incubation at 1378C. Compared to traditional viability dyes such as propidium iodide and 7AAD, DRAQ7's unique spectral profile of excitation in both blue and red lasers and far-red emission enables identification of dual positive dead cell events and frees up detectors for use with other reagents. CyTRAK Orange and DRAQ7 could be used in combination with absolute counting bead standards to provide cell counts and viability but the combination of these dyes has previously only been used for microscopy on rodent cells. The purpose of this study was to evaluate the use of these dyes in combination in large animal blood samples for flow cytometry. A viability and cell counting protocol for bovine, ovine, and human leukocytes using CyTRAK Orange and DRAQ7 was prepared. Four different counting bead standards were evaluated using the Navios and FACSAria cytometers and compared to counts obtained from hematology analyzers. CyTRAK Orange successfully detected CD45 1 leukocytes in all species. The DRAQ7 single-stained dead cell counts correlated well with the CyTRAK Orange/ DRAQ7 double-stained dead cell counts in human and bovine blood, but not in ovine blood, which could be related to the blood source. In conclusion, for human and bovine blood, this method works well for viability counts using different flow cytometers and bead standards. V C 2016 International Society for Advancement of Cytometry Key terms flow cytometry; absolute counting; DNA dyes; hematology; bovine; ovine; viability; DRAQ7; CyTRAK Orange OVINE and bovine blood is used heavily within the research and development of blood-handling medical devices, such as heart pumps (left ventricular assist devices, LVADs) (1,2). LVAD patients suffer from complications including infection, thrombosis, and bleeding (3-5) which are linked to blood cell damage (6-11). The monitoring of blood cells during in vitro testing of new LVAD prototypes could help identify safer pump designs (12,13). Hematology analyzers can provide cell counts in this setting (12,13) but cannot provide information about cell viability.Flow cytometry using absolute counting bead standards is considered the golden standard for enumerating cell subsets in clinical samples (14). Multicolor flow cytometry using DNA dyes and absolute counts has been used for leukocyte viability counts in human blood (15), and has the benefit of enabling more detailed cell

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The Evaluation of Leukocytes in Response to the In Vitro Testing of Ventricular Assist Devices

Cited by 18 publications

References 25 publications

Ovine Leukocyte Microparticles Generated by the CentriMag Ventricular Assist Device In Vitro

Ovine Leukocyte Microparticles Generated by the CentriMag Ventricular Assist Device In Vitro

The CentriMag Centrifugal Blood Pump as a Benchmark for In Vitro Testing of Hemocompatibility in Implantable Ventricular Assist Devices

Quantification methods for human and large animal leukocytes using DNA dyes by flow cytometry

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