Flow Cytometric Analysis of Coronary Stent-Induced Alterations of Platelet Antigens in an in Vitro Model

Background: Coronary artery stents can induce platelet activation by shear forces and contact to the biomaterial. This activation is one important trigger for thrombosis. Coating of stents is a possible approach to prevent this side effect. The purpose of this study was to evaluate in vitro the biocompatibility of stents coated with diamond-like carbon (DLC). Materials and Methods: For in vitro testing, DLC-coated stents were compared with noncoated 316L stainless steel stents. For this purpose, cell culture assays, videomorphometric, electron microscopic and flow-cytometric techniques were applied. Results: Growth assays with smooth muscle cells and endothelial cells revealed that DLC did not affect proliferation rates and did not have cytotoxic effects. Video-based morphometry and scanning electron microscopy showed an ultrasmooth surface and homogenous expansion patterns of the DLC stents. For analysis of platelet antigens in a circulating loop model, flow cytometry was applied. Our experiments showed no significant changes in mean channel fluorescence intensity for the structural antigens CD41a (p = 0.6) and CD42b (p = 0.1). In contrast, the expression of the activation-dependent antigens CD62p and CD63 increased significantly in noncoated stents compared to DLC-coated stents (p < 0.05). Conclusion: Coating of intracoronary stents with DLC significantly reduces platelet activation. Hereby, biocompatibility is improved.

Section: Flow Cytometry and Light Microscopysupporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

In vitro Biocompatibility Analyses of Stents Coated with Diamond-Like Carbon by Flow Cytometry, Cell Growth Assays and Electron Microscopy

Gutensohn¹,

Beythien²,

Koester³

et al. 2000

“…Stents in general are foreign biomaterial [32] and consist of a thrombogeneic wire mesh that has been proven in vitro to induce significant platelet activation [33]. Stent length as well as coating have influences on platelet activation in vitro as measured by flow cytometry [33]. In our study, stent implantation led to higher platelet activation, measured as higher MCFIs for expressions of activation-dependent antigens CD62p, CD63 and fibrinogen binding, than PTCA alone.…”

Section: Discussionmentioning

confidence: 56%

“…Stents are well established in interventional cardiology [1]. Stents in general are foreign biomaterial [32] and consist of a thrombogeneic wire mesh that has been proven in vitro to induce significant platelet activation [33]. Stent length as well as coating have influences on platelet activation in vitro as measured by flow cytometry [33].…”

Section: Discussionmentioning

confidence: 99%

Flow-Cytometric Analysis Comparing Platelet Activation during Percutaneous Transluminal Coronary Angioplasty, Stent Placement and Rotablation

Bau¹,

Beythien²,

Brockmann³

et al. 2001

Background: Acute occlusion and subacute restenosis of the coronary artery remain the limiting factors of the otherwise successful techniques of interventional cardiology. Platelets and especially activated platelet subpopulations play a key role in these sometimes fatal complications. In this study, we used flow cytometry to compare the influences of percutaneous transluminal coronary angioplasty (PTCA, n = 12 patients), stenting (n = 8) and rotablation (n = 10) on platelet antigens and their possible alteration, indicating platelet activation. Material and Methods: Samples for flow cytometry were taken directly from the arterial sheath in free flow before the procedures, directly after the procedures, and 30 min after finishing either PTCA, stenting or rotablation. One aliquot of the sample was immediately fixed and then stabilized. Aliquots were labeled with saturating amounts of antibodies against CD41a (GPIIb-IIIa), CD42b (GPIb-V-IX), CD62p (P-selectin), CD63 (GP53), and antihuman fibrinogen, and measured within 2 h. For flow-cytometric analyses we used a FACScan cytometer. Results: CD41a and CD42b did not show significant alterations in mean channel fluorescence intensity (MCFI) before, directly after, and 30 min after finishing PTCA, stenting, or rotablation (PTCA: CD41a p = 0.8 directly after and p = 0.9 30 min after finishing; CD42b p = 0.5 and p = 0.2; stenting: CD41a p = 0.3 and p = 0.2, CD42b p = 0.5 and p = 0.7; rotablation: CD41a p = 0.2 and p = 0.2; CD42b p = 0.4 and p = 0.1). However, measuring the MCFI of CD62p, CD63, and fibrinogen binding (all p < 0.05), platelet activation could be detected directly after PTCA, stenting, or rotablation. Compared with values obtained directly after the procedures, there were further significant changes 30 min after finishing the procedures in MCFI after PTCA and stenting (CD62p, CD63, fibrinogen binding, all p < 0.05), but not after rotablation (CD62p p = 0.1; CD63 p = 0.9; fibrinogen binding p = 0.5). Conclusions: The results of our study show that all three techniques induce significant platelet activation. Activation differs between the procedures. Detecting platelet activation may help to determine patients at risk for thrombophilic adverse effects.

“…the determination of platelet activation markers in donors, patients or blood components, were investigated by flow cytometry, [106][107][108][109][110][111][112][113][114]. Finally, platelet activation markers were investigated to determine the biocompatibility of different biomaterials using in vitro models [115][116][117][118][119][120][121].…”

Section: Platelet Integrity and Immunologymentioning

confidence: 99%

Flow Cytometry in Transfusion Medicine: Development, Strategies and Applications

Greve¹,

Valet²,

Humpe³

et al. 2004

Nowadays, flow cytometry represents an essential tool for the daily work in laboratories for transfusion medicine. After cytophotometry of immobilized cells, flow cytometry was developed since the 1960s using moving fluorescence-stained cells. This technique enabled the determination of several thousand cells per second, e.g. from blood, bone marrow, or organ-associated cell suspensions. The introduction of monoclonal fluorescence-labeled antibodies in diagnostics has further accelerated the development of flow cytometry. In the meantime, numerous cellular and nuclear properties can simultaneously be measured on single cell level in automated devices at high speed and precision. Internal standards and quality control systems further increased the accuracy of flow cytometric analyses. Flow cytometry is therefore likewise suitable for routine diagnostics in patients, quality control of blood components and scientific purposes in transfusion medicine. Nevertheless, many flow cytometric applications are related to counting of rare events or detection of cells with low antigen expression. Therefore, several pre-analytic and analytic factors (e.g. erythrocyte lysing procedures, cell gating strategies, etc.) may essentially affect the accurate determination of cell numbers and functions. In this issue, TRANSFUSION MEDICINE AND HEMOTHERAPY starts a recurrent sequence of reviews focused on ‘Flow Cytometry in Transfusion Medicine’. In order to introduce the flow cytometry series, this review summarizes the development, principles, and strategies of flow cytometry as an increasing diagnostic tool in medical laboratories. Also, current and possibly future flow cytometric applications in transfusion medicine are epitomized, e.g. quality control of blood products, immunohematologic diagnostics, hematopoietic progenitor cell transplantation, adoptive immunotherapy, and further therapeutic applications. In following issues of this journal, reviews of notable authors will then focus on special applications and give more detailed insights into single diagnostic fields.