Measuring red blood cell aggregation forces using double optical tweezers.

Classic immunohematology approaches, based on agglutination techniques, have been used in manual and automated immunohematology laboratory routines. Red blood cell (RBC) agglutination depends on intermolecular attractive forces (hydrophobic bonds, Van der Walls, electrostatic forces and hydrogen bonds) and repulsive interactions (zeta potential). The aim of this study was to measure the force involved in RBC aggregation using double optical tweezers, in normal serum, in the presence of erythrocyte antibodies and associated to agglutination potentiator solutions (Dextran, low ionic strength solution [LISS] and enzymes). The optical tweezers consisted of a neodymium:yattrium aluminium garnet (Nd:YAG) laser beam focused through a microscope equipped with a minicam, which registered the trapped cell image in a computer where they could be analyzed using a software. For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities, was determined. The median of the RBC aggregation force measured in normal serum (control) was 1 × 10(-3) (0.1-2.5) poise.cm. The samples analyzed with anti-D showed 2 × 10(-3) (1.0-4.0) poise.cm (p < 0.001). RBC diluted in potentiator solutions (Dextran 0.15%, Bromelain and LISS) in the absence of erythrocyte antibodies, did not present agglutination. High adherence was observed when RBCs were treated with papain. Results are in agreement with the imunohematological routine, in which non-specific results are not observed when using LISS, Dextran and Bromelain. Nevertheless, false positive results are frequently observed in manual and automated microplate analyzer using papain enzyme. The methodology proposed is simple and could provide specific information with the possibility of meansuration regarding RBC interaction.

Towards absolute calibration of optical tweezers.

Aiming at absolute force calibration of optical tweezers, following a critical review of proposed theoretical models, we present and test the results of Mie-Debye-spherical aberration (MDSA) theory, an extension of a previous (MD) model, taking account of spherical aberration at the glass-water interface. This first-principles theory is formulated entirely in terms of experimentally accessible parameters (none adjustable). Careful experimental tests of the MDSA theory, undertaken at two laboratories, with very different setups, are described. A detailed description is given of the procedures employed to measure laser beam waist, local beam power at the transparent microspheres trapped by the tweezers, microsphere radius, and the trap transverse stiffness, as a function of radius and height in the (inverted microscope) sample chamber. We find generally very good agreement with MDSA theory predictions, for a wide size range, from the Rayleigh domain to large radii, including the values most often employed in practice, and at different chamber heights, both with objective overfilling and underfilling. The results asymptotically approach geometrical optics in the mean over size intervals, as they should, and this already happens for size parameters not much larger than unity. MDSA predictions for the trapping threshold, position of stiffness peak, stiffness variation with height, multiple equilibrium points, and "hopping" effects among them are verified. Remaining discrepancies are ascribed to focus degradation, possibly arising from objective aberrations in the infrared, not yet included in MDSA theory.