title: Quantification of adherent platelets on polymer-based biomaterials. Comparison of colorimetric and microscopic assessment.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Braune
authors.affiliation: 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.
authors.forename: S
authors.initials: S
authors.lastname: Zhou
authors.affiliation: Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Groth
authors.affiliation: Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Jung
mesh_terms: D000135:Acid Phosphatase / Q000097:blood; D001672:Biocompatible Materials; D001792:Blood Platelets / Q000166:cytology / Q000502:physiology*; D003124:Colorimetry; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000097:blood; D008853:Microscopy; D010973:Platelet Adhesiveness / Q000502:physiology*; D011108:Polymers
chemical_list: D001672:Biocompatible Materials; D011108:Polymers; D007770:L-Lactate Dehydrogenase; D000135:Acid Phosphatase
keywords: Platelet; acid phosphatase; biomaterial; colorimetric assay; lactate dehydrogenase; microscopy; thrombogenicity