血小板计数参考方法的建立及应用研究

Reference method for platelet counting and its application

目的建立血小板(PLT)计数参考方法-PLT/RBC比值法。方法以CD41-FITC、CD61-FITC特异性标记血中的PLT,用流式细胞术(FCM)检测PLT/RBC比值,乘以血细胞自动分析所得RBC数,得PLT浓度。同时,将其计数83例PLT与电阻抗法、光散射法及目视计数法的检测结果进行比较。结果PLT/RBC比值法的批内重复性高值(760×109/L)、中值(130×109/L)、低值(8×109/L)标本的变异系数(CV)分别为1.5%、1.3%、13.3%;PLT在40×109/L-870×109/L范围内线性良好(r=0.9985),PLT计数在标本采集后6 h内稳定,携带污染率为0.19%;与其它血小板自动分析法—电阻抗法、光散射法计数PLT结果间相关性良好(r>0.98);受试者工作曲线(ROC)分析显示,电阻抗法和光散射法的各种检测性能较好,ROC曲线下面积(AUC)分别>0.99和0.98,其灵敏度均为100%,特异度分别为98%和93.5%;而目视计数法的检测性能欠佳。结论PLT/RBC比值法特异、准确、精密、线性和稳定性好、携带污染率低,可作为PLT计数的参考方法并用于评价其他PTL计数方法。

Objective To set up a reference method for platelet counting-the RBC/PLT ratio method. Methods Counting of specifically labeled platelets with CD41 and CD61 relative to the RBCs with a flow eytometer,together with an accurate RBC count determined with a semiautomated, single- channel aperture- impedance counter as a reference method for PLT counting; meanwhile,83 samples of daily whole blood of high,normal and low PLT values were collected and analyzed by the RBC/PLT ratio method as well as the methods of impedance, scatter and visual eounting. Results The within-replicate coefficient of variance （CV） of the RBC/PLT ratio method was 1.5 %, 1.3 %, 13.3 % for high count （760 × 10^9/L）, middle count （ 130 × 10^9/L） and low eount （8 × 10^9/L）, respectively. Linearity was demonstrated well （ r = 0.9985） when the PLT count were 40 × 10^9/L - 870 × 10^9/L. PLT eount was stable at room temperature within 6 h. The carry - over was 0.19%. The results between the RBC/PLT ratio method and other methods of automated PLT eounting including impedance or scatter methods were eomparable （ r 〉 0.98）. The analysis of receive operating characteristic （ROC） for impedance or scatter methods compared with the RBC/PLT ratio method showed the performance of them was good,the area under curve （AUC） of impedance or scatter methods 〉 0.99 and 0.98, respectively; and their sensitivity （Sen） was 100% and specificity （Spe） was 98% and 93.5% .However,the performance of visual PLT counting was not very good. Conclusion These findings confirm that the RBC/PLT ratio method for PLT eounfing is specific, accurate,precise, superior in linearity,lower in carry- over, could be as a reference method for PLT eounting and as a candidate for evaluating other PLT counting methods.