Enumeration of platelets by multiparameter flow cytometry using platelet-specific antibodies and fluorescent reference particles

Summary The correct enumeration of platelets is still an elusive matter. This is mainly due to the fact that commercial instruments which are used for platelet counting cannot discriminate platelets from other cellular particles and precipitates that cause similar signals. Visual (chamber counting) methods are still frequently used in routine laboratories to verify low automated platelet counts (< 50 × 10/l) despite obvious technical and statistical drawbacks. The following report shows how platelet counts can be measured by multiparameter flow cytometry with the help of reference particles (fluorescent latex beads) and platelet-specific antibodies i.e. anti-GPIIb/IIIa(CD41a), anti-GP Ib-α (CD42b) and anti-GP IIIa (CD61). The linearity of this method was highly satisfactory and the observed imprecision was within acceptable limits. At a platelet concentration of 10 × 10⁹/l the coefficient of variation (CV, n = 10) ranged from 5.3% (PCV = 0.456) to 5.6% (PCV = 0.148). Accuracy was evaluated by comparing results to the ICSH-selected method for platelet counting. The correlation of both methods was significant (P < 0.005) and Passing-Bablok's linear regression analysis showed no systematic differences between the two methods. Comparisons of this new platelet counting technique were also performed with routine visual methods, automated blood analysers (Technicon H-1, Sysmex E-5000) and a different flow cytometric method using only forward and side light scatter properties of platelets for their discrimination. The linear correlation of all methods was significant (P < 0.01) at platelet concentrations above 50 × 10⁹/l. At lower platelet concentrations, our new platelet counting technique correlated significantly only with the visual and the forward/side scatter methods. These findings stress the necessity to confirm low platelet counts by automated blood analysers and suggest that using multiparameter flow cytometry with platelet-specific antibodies may be a proficient way to do so. The possibility of using this technique as a reference method is discussed.