Braune S, Zhou S, Groth B, Jung F
Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany.
Tianjin University, Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Teltow, Germany.
Clin Hemorheol Microcirc. 2015;61(2):225-36. doi: 10.3233/CH-151995.
Platelet adhesion to artificial surfaces is one of the most important indicators for the thrombogenicity of implant materials. Currently, a variety of enzyme activity-based colorimetric assays or microscopy-based techniques are commonly in use to assess this characteristic. Studies about how data of colorimetric assays correlate with the image-based quantification of adherent platelets are scarce. To address this question, the present study compared two colorimetric assays (lactate dehydrogenase (LDH) and acid phosphatase (ACP)) with an image-based quantification of the density of platelets adhering on polymer-based biomaterial surfaces.
Tri-sodium citrated whole blood was collected from apparently healthy subjects and platelet rich plasma (PRP) was prepared according to a standardized protocol. An in vitro static thrombogenicity test was applied to study platelet adhesion from PRP adjusted to 50,000 platelets per μL on three different polymers: medical grade polytetrafluoroethylene (PTFE), silicone and polyethylene terephthalate (PET). For the direct image-based approach, surface adherent platelets were fixed, fluorescently labelled and microscopically visualized. The image-based determination of platelet densities provided reference values for the comparison with data of the colorimetric assays. Correlation between standard platelet concentrations and ACP/LDH absorbance measurements were analysed to estimate accuracy and association of both parameters. ACP and LDH release from resting and ADP-stimulated platelets was studied to estimate how platelet activation influences colorimetric assay results.
The density of adherent platelets ranged from 15,693 ± 2,487 platelets·mm-2 (PTFE) to 423 ± 99 platelets·mm-2 (silicone) and 4,621 ± 1,427 platelets·mm-2 (PET) and differed significantly between the three polymers (ANOVA: p < 0.05). Correlation coefficients between microscopic and colorimetric determination of platelet densities ranged between r = 0.93 (LDH, p < 0.001) and r = 0.94 (ACP, p < 0.001). ACP absorbance measurements of platelet standards with different concentrations corresponded well to an ideal linear regression, while LDH data either deceeded or exceeded the expected values. The LDH release during ADP-induced platelet activation was significantly higher compared to the release of ACP.
For an adjusted platelet concentration of 50,000 platelets·μL-1, both colorimetric assays (ACP and LDH) allowed a similar accurate quantification of the mean platelet density compared to the microscopic evaluation. Better linearity of the assay standards, less variability of the results and a lower influence of platelet activation on the measurements mark the ACP assay as more suitable for the assessment of material surface adherent platelets compared to the LDH assay, particularly, if near physiological platelet concentrations are applied.
血小板在人工表面的黏附是植入材料血栓形成性的最重要指标之一。目前，多种基于酶活性的比色法或基于显微镜的技术常用于评估这一特性。关于比色法数据与基于图像的黏附血小板定量之间如何关联的研究很少。为解决这个问题，本研究将两种比色法（乳酸脱氢酶（LDH）和酸性磷酸酶（ACP））与基于图像的聚合物基生物材料表面黏附血小板密度定量进行了比较。
从表面健康的受试者采集枸橼酸钠全血，并按照标准化方案制备富血小板血浆（PRP）。应用体外静态血栓形成性试验研究将PRP调整至每微升50,000个血小板后在三种不同聚合物上的血小板黏附情况：医用级聚四氟乙烯（PTFE）、硅酮和聚对苯二甲酸乙二酯（PET）。对于基于直接图像的方法，将表面黏附的血小板固定、荧光标记并通过显微镜观察。基于图像的血小板密度测定为与比色法数据进行比较提供了参考值。分析标准血小板浓度与ACP/LDH吸光度测量值之间的相关性，以估计两个参数的准确性和关联性。研究静息和ADP刺激的血小板释放ACP和LDH的情况，以估计血小板活化如何影响比色法结果。
黏附血小板的密度范围为15,693±2,487个血小板·mm-2（PTFE）至423±99个血小板·mm-2（硅酮）和4,621±1,427个血小板·mm-2（PET），三种聚合物之间差异显著（方差分析：p<0.05）。血小板密度的显微镜测定与比色法测定之间的相关系数在r = 0.93（LDH，p<0.001）和r = 0.94（ACP，p<0.001）之间。不同浓度血小板标准品的ACP吸光度测量值与理想线性回归拟合良好，而LDH数据要么低于要么高于预期值。与ACP的释放相比，ADP诱导的血小板活化过程中LDH的释放显著更高。
对于调整后的血小板浓度为50,000个血小板·μL-1，与显微镜评估相比，两种比色法（ACP和LDH）对平均血小板密度的定量准确性相似。与LDH测定相比，ACP测定的标准品线性更好、结果变异性更小且血小板活化对测量的影响更低，这表明ACP测定更适合评估材料表面黏附的血小板，特别是在应用接近生理血小板浓度时。
