Measurement of fluorescent-labeled LMM-heparin in biological fluids using protamine-linked microbeads

A quantitative assay for fluorescent heparin in a purified system and in plasma was developed (Piazolo et al: Semin Thromb Hemostas 20:227-235, 1994). The protamine microbeads (1.6 microns) showed a broad size distribution and a large standard variation in low concentrations. Our aim was to optimize these protamine microbeads for the measurement of fluorescent heparin. The following results were obtained: Paramagnetic protamine microbeads of different average diameters (0.8, 1.6, 2.8, and 4.5 microns) were synthesized by cyclocarbodiimide and tosyl activation. These microbeads bind heparin and are assayed using flow cytometry. The protamine concentration on the surface of the beads ranged between 2.0 and 61 mg/mL. The protamine microbeads bound fluorescent heparin and were analyzed by flow cytometry. The protamine microbeads bound LMM-heparin-tyramine-FITC dose dependently in saline solution, plasma, and blood. There are substantial differences between the microbeads of different origins with regard to the amount of protamine bound, the sensitivity of the detection, and the reliability for the determination of heparins in plasma and blood. The minimal sensitivity of the final method was 0.001 U/mL LMMH-tyramine-FITC in saline solution and in plasma. Human blood cells were not bound to protamine microbeads. The half-maximal binding of LMMH-tyramine-FITC of the different protamine-coated microbeads ranged from 1.7 to 8.0 micrograms/mL in saline solution, 2.3 to 8.7 micrograms/mL in plasma, and 3.1 to 6.4 micrograms/mL in blood. We conclude that all protamine microbeads can be used to quantify the concentration of LMMH-tyramine. Protamine Dynabeads M-450 (diameter 4.5 microns) have advantages over other microbeads because of their more homogeneous size distribution, a higher selectivity, and they can be measured together with leukocytes. They are currently used to develop a competitive binding assay for heparin in plasma.