Platelet activation during hemodialysis measured through exposure of p-selectin: analysis by flow cytometric and ultrastructural techniques
Reverter, J.C.; Escolar, G.; Sanz, C.; Cases, A.; Villamor, N.; Nieuwenhuis, H.K.; López, J.; Ordinas, A.
Journal of Laboratory and Clinical Medicine 124(1): 79-85
1994
ISSN/ISBN: 0022-2143 PMID: 7518492 Document Number: 437499
P-selectin is a platelet protein in alpha-granules that is expressed on their membranes after platelet activation. Using a specific monoclonal antibody (RUU-2.17), we have studied platelet activation during hemodialysis with a cuprophan membrane in eight patients with uremia. Blood samples were obtained before hemodialysis from venipuncture (sample I), at the beginning of the hemodialysis from the arterial (sample II) and venous lines (sample (III), and after 2 hours of hemodialysis from the venous line (sample IV). Exposure of P-selectin on platelet surface was studied by flow cytometry, and morphologic and immunocytochemical studies were performed. No differences in P-selectin expression were observed by single-labeling flow cytometry in samples I, II, and IV. However, a significant increase in fluorescence occurred in sample III (percentage of positive platelets (% PP), 18.0% +- 6.1%) with respect to sample I (% PP, 6.9% +- 2.0%; p lt 0.01). An apparently decreased fluorescence was seen in sample IV (%PP, 8.8% +- 3.9%) with respect to sample III. Double-labeling flow cytometry showed statistically significant differences between sample I (%PP, 7.1% +- 2.6%) and sample III (%PP, 19.4% +- 9.4%; p lt 0.01) and IV (%PP, 19.0% +- 10.1%; p lt 0.01), but not between samples III and IV. The ultrastructural study revealed an increasing number of platelet morphologic signs of activation throughout the period of hemodialysis. The percentages of platelet with morphometrically detectable deviations from baseline discoid shapes were 8.3% +- 4.2% in sample I, 8.5% +- 4.0% in sample II, 26.1% +- 4.8% in sample III (p lt 0.01 versus sample I), and 49.8% +- 8.3% in sample IV (p lt 0.01 versus sample III). Immunocytochemical studies with colloidal gold markers confirmed an increased presence of P-selectin expression in sample IV. These results indicate that platelet activation occurs in vivo during the contact of blood with the hemodialysis membrane. Flow cytometry detects this activation but has certain limitations. Repeated platelet activation caused by current hemodialysis procedures might contribute to the platelet dysfunction observed in patients with uremia.