The electronic whole blood aggregometer (WBA) has the advantage that it enables the study of platelet aggregation in whole blood shortly after blood collection. Using the WBA varying results have been obtained with respect to the anti-aggregating activity of dipyridamole. As dipyridamole is an efficient inhibitor of adenosine uptake, we tested whether the degree of red blood cell lysis (and thus availability of adenine nucleotides) affected its efficacy. Citrate (10.7 mM) blood was stored in sealed tubes and used between 20 and 100 min after venipuncture. One ml was placed in a Chronolog Model 540 WBA together with 10 μl 0.9 % NaCl, dipyridamole (final conc. 3, 10 or 30 μM) or its solvent (final conc. 0.03, 0.1 or 0.3 %). After reaching a stable baseline and WBA calibration, aggregation was induced by injection of 10 μM ADP dissolved in 10 μl 0.9 % NaCl (one channel) or 10 μl distilled water (other channel). Maximum impedance increase in 10 min was measured, red blood cells were removed by centrifugation, and from microhematocrit and absorbance at 416 nm the volume of lysed packed red blood cells was estimated. ADP caused aggregation (12.9 ± 1.9 and 11.1 ± 0.8, Ohm) and there was red blood cell lysis (2.8 ± 0.5 and 0.8 ± 0.2 μl red blood cells, ADP resp. in H20 and 0.9 % NaCF, n = 6). Dipyridamole (30 μM) suppressed aggregation when compared with solvent, but only when ADP was given in H20 (reduction resp. 4.4 ± 1.4 and 1.9 ± 1.6 ohm). Moreover, there was a negative correlation between the degree of haemolysis and the aggregation response at 10 as well as 30 μM dipyridamole. This reduced aggregation with increasing haemolysis was not observed in the presence of the corresponding solvent concentrations. The red blood cell lysis was proportional to the plasma ATP (luciferine-luciferase method) concentration as an index of adenine nucleotide leakage. In conclusion, a certain degree of haemolysis caused by stirring and injection with a microsyringe is inherent to the WBA, but use of hypotonic vehicles should be avoided. Release of red blood cell constituents may affect platelet aggregation as such, or interfere with the activity of adenosine uptake inhibitors and possibly other drugs. It may help to explain some of the variable results obtained with dipyridamole.
Inertial microfluidics combined with selective cell lysis for high throughput separation of nucleated cells from whole blood
