Center of Excellence for Flow Cytometry, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Ann Hematol. 2012 Nov;91(11):1685-94. doi: 10.1007/s00277-012-1522-2. Epub 2012 Jul 31.
Patients with thalassemia, an inherited hemolytic anemia, have increased risk of hypercoagulable complications. A whole blood flow cytometric (FCM) method has been used for studies of platelet activation and platelet-leukocyte aggregation in these patients. However, this FCM method presents technical difficulties because of the high proportion of immature red blood cells (RBCs) in these patients. A protocol for the simultaneous measurement of platelet activation and their aggregation with leukocyte populations in whole blood using four-color FCM which excluded immature RBC was devised, and evaluated for the evaluation of platelet function in patients with β-thalassemia/hemoglobin E (HbE). Whole blood from these patients and from healthy volunteers was stained for platelet activation and platelet-leukocyte aggregates using anti-CD42a, anti-CD62P, anti-CD45 and glycophorin A (GPA) conjugated with different fluorochromes. Our FCM method is simple, effective and based on the assumption that GPA is present on all immature RBCs, but is not expressed on CD45⁺ leukocytes. Results from the studies showed that blood samples from these patients contained a high frequency of circulating activated platelets (CD42a⁺/CD62P⁺) when compared to samples from healthy individuals. The percentage of platelet-neutrophil, platelet-monocyte-but not platelet-lymphocyte-aggregates were also elevated in both thalassemia genotypes with marked increase in patients who had undergone splenectomy. These findings suggest that platelets adhere to neutrophils and monocytes are activated which support the clinical observation that splenectomized thalassemia patients have an increased risk of arterial or venous thrombotic manifestations.
患有地中海贫血症（一种遗传性溶血性贫血症）的患者发生高凝并发症的风险增加。全血流式细胞术（FCM）方法已用于研究这些患者的血小板活化和血小板-白细胞聚集。然而，由于这些患者血液中未成熟红细胞（RBC）的比例较高，该 FCM 方法存在技术难题。本研究设计了一种排除未成熟 RBC 的四色 FCM 同时测量全血中血小板活化及其与白细胞群体聚集的方案，并评估其用于评估β-地中海贫血/血红蛋白 E（HbE）患者的血小板功能。使用不同荧光染料标记的抗 CD42a、抗 CD62P、抗 CD45 和糖蛋白 A（GPA）对这些患者和健康志愿者的全血进行血小板活化和血小板-白细胞聚集染色。我们的 FCM 方法简单、有效，基于 GPA 存在于所有未成熟 RBC 上但不存在于 CD45⁺白细胞上的假设。研究结果表明，与健康个体相比，这些患者的血液样本中循环活化血小板（CD42a⁺/CD62P⁺）的频率更高。两种地中海贫血基因型的血小板-中性粒细胞、血小板-单核细胞-但不是血小板-淋巴细胞聚集的百分比也升高，并且在接受脾切除术的患者中升高更为明显。这些发现表明，血小板黏附于中性粒细胞和单核细胞被激活，这支持了临床观察，即脾切除的地中海贫血患者发生动脉或静脉血栓形成表现的风险增加。
