ENUMERATION OF PLATELETS BY MULTIPARAMETER FLOW-CYTOMETRY USING PLATELET-SPECIFIC ANTIBODIES AND FLUORESCENT REFERENCE PARTICLES

The correct enumeration of platelets is still an elusive matter. This is mainly due to the fact that commercial instruments which are used f or platelet counting cannot discriminate platelets from other cellular particles and precipit;ates that cause similar signals. Visual (chamb er counting) methods are still frequently use:d in routine laboratorie s to verify low automated platelet counts (< 50 x 10/1) despite obviou s technical and statistical drawbacks. The following report shows how platelet counts call be measured by multiparameter now cytometry with the help of reference particles (fluorescent latex beads) and platelet -specific antibodies i,e. anti-GPIIb/IIIa(CD41a), anti-GP Ib-alpha (CD 42b) and anti-GP IIIa (CD61), The linearity of this method was highly satisfactory anti the observed imprecision was within acceptable limit s. At a platelet concentration of 10 x 10(9)/1 the coefficient of vari ation (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 me thod for platelet counting. The correlation of both methods was signif icant (P < 0.005) and Passing-Bablok's linear regression analysis show ed no systematic differences between the two methods, Comparisons of t his new platelet counting technique were also performed with routine v isual methods, automated blood analysers (Technicon H-l, Sysmex E-5000 ) and a different flow cytometric method using only forward and side l ight scatter properties of platelets for their discrimination, The lin ear correlation of all methods was significant (P < 0.01) at platelet concentrations above 50 x 10(9)/1. At lower platelet concentrations, o ur 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 analy sers and suggest that using multiparameter now 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.