Li N, Goodall A H, Hjemdahl P
Department of Laboratory Medicine, Karolinska Hospital, Stockholm, Sweden.
Cytometry. 1999 Feb 1;35(2):154-61. doi: 10.1002/(sici)1097-0320(19990201)35:2<154::aid-cyto7>3.0.co;2-y.
Platelet-leukocyte aggregates (PLAs) may be important in thrombotic and inflammatory disease states, but accurate assessment of PLA formation in vivo is hampered by the propensity for in vitro artefacts caused by sample manipulation. A whole blood flow cytometric assay for circulating PLAs, based on minimal sample manipulation, was thus developed.
Citrated whole blood was labeled with a RPE-CD45 MAb (leukocyte marker) and an FITC-CD42a (GPIX) MAb (platelet marker). The latter was used to avoid possible influences of platelet glycoprotein proteolysis by neutrophil-derived proteases. The samples were mildly fixed with 0.5% formaldehyde saline. The cytometer was triggered by RPE-CD45 fluorescence. Leukocyte subpopulations were separated according to their typical light scattering and CD45 expression.
Minimal sample manipulation and mild sample fixation resulted in minor in vitro artefacts and good sample stability. Fluorescence triggering increased the efficiency of the flow cytometric analysis approximately 5-fold compared with triggering with light scatter, and allowed discrimination of leukocyte subpopulations. The majority of PLAs involved monocytes and neutrophils, rather than lymphocytes, both without and with in vitro stimulation by ADP or thrombin. A cocktail of blocking MAbs to CD62P, CD15, GPIIb/IIIa and the CD11b/CD18 complex had no effect on unstimulated samples, whilst totally inhibiting aggregation induced by 10(-5) M ADP, suggesting that the PLAs in unstimulated blood were preformed in vivo.
This whole blood flow cytometric assay for PLAs is simple and efficient, and appears to reflect closely platelet-leukocyte aggregates in circulating blood in vivo.
血小板 - 白细胞聚集体（PLAs）在血栓形成和炎症性疾病状态中可能起重要作用，但由于样本处理易导致体外假象，使得体内PLA形成的准确评估受到阻碍。因此，开发了一种基于最小样本处理的循环PLAs全血流式细胞术检测方法。
用RPE - CD45单克隆抗体（白细胞标志物）和FITC - CD42a（GPIX）单克隆抗体（血小板标志物）标记枸橼酸化全血。使用后者以避免中性粒细胞衍生蛋白酶对血小板糖蛋白的蛋白水解可能产生的影响。样本用0.5%甲醛盐水轻度固定。流式细胞仪由RPE - CD45荧光触发。根据白细胞亚群典型的光散射和CD45表达进行分离。
最小样本处理和轻度样本固定导致体外假象较少且样本稳定性良好。与光散射触发相比，荧光触发使流式细胞术分析效率提高约5倍，并能区分白细胞亚群。无论有无ADP或凝血酶体外刺激，大多数PLAs涉及单核细胞和中性粒细胞，而非淋巴细胞。针对CD62P、CD15、GPIIb/IIIa和CD11b/CD18复合物的封闭单克隆抗体混合物对未刺激样本无影响，而完全抑制10(-5) M ADP诱导的聚集，这表明未刺激血液中的PLAs在体内已预先形成。
这种用于PLAs的全血流式细胞术检测方法简单有效，似乎能密切反映体内循环血液中的血小板 - 白细胞聚集体。
