流式细胞术诊断X连锁高IgM血症的临床应用

Application of flow cytometry in the diagnosis of X-linked hyper-IgM syndrome CAO

目的建立流式细胞术诊断x连锁高IgM血症（X-linked hyper-IgM syndrome，XHIM）的方法。方法取患者、患者母亲和健康对照者外周血，肝素抗凝，用RPMI-1640培养基稀释，以15μl的佛波醇酯（1ns／μl）和6μl的离子霉素（50ns／斗1）作刺激剂，从而将外周血标本分为刺激组和非刺激组，运用荧光标记的抗细胞表面抗原单抗检测CD3＋CD4-T淋巴细胞表面CD40配体的表达情况，同时检测CD3＋淋巴细胞表面的CD69分子作为激活对照，以确定细胞活化的程度。结果刺激组细胞表面CD69的表达率均在96％以上；患者母亲及健康对照者CD3＋CD8＋T淋巴细胞表面CD40L的表达率分别为60．04％和62．87％，而患者仅为0．8％。非刺激组CD69的表达率均小于3％；患者、患者母亲和健康对照者CD40L的表达率分别为0．88％、4．15％和5．51％。结论流式细胞术可以快速诊断X连锁高IgM血症，有可能成为该类疾病的初筛方法。

Objective To develop a flow cytometric assay to for diagnosis of X-Linked Hyper-IgM Syndrome （XHIM）. Methods Heparinized peripheral blood obtained from patient, mother and a healthy control was diluted with RPMl-1640 （unstimulated control） or with RPMI-1640 containing 15μl PMA （1 ng/μl）and 6μl ionomycin（50 ng/μl）（ stimulated cell）. Using directly labeled antibodies, we have examined CD40 ligand levels on CD3＋CD8- T lymphocyte surface, and CD69 levels on CD3＋ lymphocyte surface to determine whether the cells were activated. Results CD69 levels on CD3 lymphocyte surface from stimulated group and from unstimulated group were above 96% and below 3%, respectively. CD40L levels on CD3＋CD8-T lymphocyte surface from stimulated group were 0.8% （patient）, 60.04% （mother） and 62. 87% （healthy control）. CD40L levels on CD3＋ CD8- T lymphocyte surface from unstimulated group were 0. 88% （patient） ,4. 15% （mother） and 5.51% （ healthy control）. Conclusion This flow cytometric assay is accurate and convenient, which can be used in neonatal screening.