Kisung Lee, A.V. Priezzhev, A.Yu. Maclygin, I.O. Obolenskii, M. Kinnunen, R. Myllylä

1. Russian-Chinese Workshop on Biophotonics and Biomedical Optics September 26-28, 2012 Investigation of red blood cells aggregation in plasma and in proteins solutions by optical trapping Kisung Lee, A.V. Priezzhev, A.Yu. Maclygin, I.O. Obolenskii, M. Kinnunen, R. Myllylä

2. Aggregation of RBC RBC aggregates. • Aggregation is a reversible process of RBCs forming so called “rouleaux” when they come into contact. • This process regulates the viscosity of blood at low shear rates and aims for achieving minimal energy dissipation for blood flow. • Aggregation parameters are major parameters affecting blood circulation.

3. Aggregation mechanism Crossbridge mechanism Depletion mechanism Aggregates are formed due to osmotic pressure from surrounding plasma proteins or other macromolecules. Aggregates are formed due to “crossbridges” made of plasma proteins or other macromolecules. Two aggregation mechanisms may coexist. Possibility of a single aggregation mechanism is not yet clear.

4. Content of work • Purpose: Investigation of RBC aggregation mechanism(s). • Methods: Measurement of aggregation forces and observation of the aggregation/disaggregation processes at single aggregate level using double channel optical tweezers. • Sample: Healthy RBCs in autologous plasma, dextran 500kDa (10, 20, 30 mg/ml) and fibrinogen (5, 10, 15 mg/ml) solutions. High molecular weight dextran and fibrinogen are known to induce the RBC aggregation.

5. Scheme of Optical Tweezers • Single cell trapping and manipulation. • Non-invasive, Non-contact measurement of forces when calibrated. • F~ 0.1…100pN Principle of optical trapping Tightly focused laser beam Trapping force Trapping force Microparticle

6. Measurement of RBC interaction forces in linear aggregates suspended in plasma and dextran solution. • The forces of RBC interaction were measured in linear aggregates containing 7~10 cells. • Two traps held the RBC aggregate at opposite ends and stretched it. The forces were measured by decreasing the power of one of the trapping beam until an RBC slips away from trap. • The interaction force was 8.4 ± 1.1 pN, about the same for both cases. Qualitatively RBC interaction force seemed to be the same also in the case of fibrinogen. Measurement procedure of the force of RBC interaction in an aggregate.

7. RBC disaggregation: three possible processes of disaggregation Small area of contact strongly keeps RBCs together 3 1 2 RBCs disaggregate easily without changing the shape of the membrane Strong thread-like strand keeps RBCs together

8. Measurement of disaggregation forces in dextran solution • The disaggregation forces were measured by similar method. One side of an aggregate was attached at the surface and the opposite side were trapped with optical tweezers. Trapped RBC was pulled away with increasing trapping force till disaggregation. The disaggregation forces were found to increase along with the concentration of macromolecules. • In the cases 1 and 2 the disaggregation was almost impossible at given maximum trapping foce (~ 50 pN). Instead the force was measured by decomposing the aggregate without breaking the final connection. The decomposing force increased with the concentration of dextran and was in the range from 18 to 38 pN.

9. Dependence of minimal aggregation force from dextran concentration Minimum disaggregation force, pN Trapped RBC Disaggregation of two RBC aggregate The differences in disaggregation forces between individual RBC aggregates were very wide. Those differences were observed to be very big even in the same type of aggregates. Dextran concentration. mg/ml

10. RBC aggregation in fibrinogen solution • In the case of fibrinogen increase of aggregate rate with increasing concentration of fibrinogen were observed. The aggregate was formed within several seconds at low concentration and almost instantly at higher concentration. • Visually, the number of RBC aggregates in the unit area of observation was higher in the presence of fibrinogen than in the presence of dextran at equal concentrations of macromolecules at given concentration of RBCs. • All three processes of disaggregation were also observed in the case of fibrinogen solution.

11. Conclusion • Dependence of the interaction forces between RBC on the concentration of macromolecules in solution was found. • Differences between individual RBC aggregates were confirmed. The difference appears to be wide including differences in aggregate types. • We assume that thread-like strands are acting like bridges made of macromolecules connected between themselves. Strands were found in both fibrinogen, dextran and in plasma. • In the case of dextran induced aggregation it can mean that bridge mechanism is working despite of widely known theory that only depletion mechanism is working for dextran induced aggregation.

12. Moscow State University, Department of Physics, Chair of General Physics and Wave Processes, Laboratory of Biomedical Photonics Research group This work was supported by Russian-Finnish agreement on cooperation A.Yu. Maclygin Diploma student A.V. Priezzhev Laboratory Head University of Oulu,Optoelectronics and Measurement Techniques Laboratory I.O. Obolenskii Researcher Lee Kisung Ph. D. Student M. Kinnunen Executive laboratory Head R. Myllylä Laboratory Head

13. Thank you!