Pattanapanyasat K, Yongvanitchit K, Heppner D G, Tongtawe P, Kyle D E, Webster H K
Thalassemia Center, Faculty of Medicine at Siriraj Hospital, Bangkok, Thailand.
Cytometry. 1996 Nov 1;25(3):287-94. doi: 10.1002/(SICI)1097-0320(19961101)25:3<287::AID-CYTO10>3.0.CO;2-S.
A novel culture system using biotin/streptavidin and flow cytometry was developed to compare maturation and growth rates in Plasmodium falciparum malaria parasites in two distinct red blood cell (RBC) populations. Biotin was used to label a selected RBC population which was then mixed with another distinct unbiotinylated RBC population. P. falciparum-infected RBCs were used to initiate co-cultures followed over 2-3 schizogonic growth cycles. Co-cultures were harvested and stained with streptavidin-fluorescein isothiocyanate (FITC) followed by fixation and staining of parasite DNA. The combination of biotin/streptavidin-FITC and DNA fluorochrome enabled simultaneous flow cytometric analysis of the two different RBC populations and of the parasitemias in each RBC population. We then used this system to study the in vitro susceptibility of RBCs from individuals with hemoglobin H (Hb H) disease to infection and growth of P. falciparum. Significant reduction in parasite multiplication was found in Hb H RBCs as compared with that in normal RBCs. This novel malaria culture system offers two major innovations: a method to compare directly the relative ability of any two red blood cell populations to support malaria parasite invasion and development under identical conditions, and a critical reduction in the volume of blood and reagents needed to assess parasite growth. The application of biotin-labeled RBCs in the flow cytometric analysis of parasite development may offer new insights in studies of the relationship between RBC defects and susceptibility to malaria parasites.
开发了一种使用生物素/链霉亲和素和流式细胞术的新型培养系统，以比较恶性疟原虫在两种不同红细胞（RBC）群体中的成熟和生长速率。生物素用于标记选定的红细胞群体，然后将其与另一个不同的未生物素化红细胞群体混合。使用感染恶性疟原虫的红细胞启动共培养，并在2-3个裂殖体生长周期内进行跟踪。收获共培养物，用链霉亲和素-异硫氰酸荧光素（FITC）染色，然后固定并染色寄生虫DNA。生物素/链霉亲和素-FITC与DNA荧光染料的组合能够同时对两种不同的红细胞群体以及每个红细胞群体中的疟原虫血症进行流式细胞术分析。然后，我们使用该系统研究了血红蛋白H（Hb H）病患者的红细胞对恶性疟原虫感染和生长的体外易感性。与正常红细胞相比，发现Hb H红细胞中的寄生虫增殖显著减少。这种新型疟疾培养系统有两个主要创新点：一种在相同条件下直接比较任何两个红细胞群体支持疟原虫入侵和发育的相对能力的方法，以及在评估寄生虫生长所需的血液和试剂体积方面的显著减少。生物素标记的红细胞在寄生虫发育的流式细胞术分析中的应用可能为研究红细胞缺陷与对疟原虫易感性之间的关系提供新的见解。
