应用高效液相色谱技术检测小儿β地中海贫血的研究

Study on high performance liquid chromatography for rapid diagnosis of children β-thalassemia

目的探讨应用高效液相色谱技术(HPLC)快速诊断小儿β地中海贫血(β地贫)的实际应用价值。方法应用HPLC技术,对150例临床疑似珠蛋白生成障碍性贫血患儿进行血红蛋白分析,检测其血红蛋白A2(Hb A2)与血红蛋白F(Hb F)的含量。同时对上述患儿采用缺口聚合酶链反应技术(GAP-PCR)检测α地中海贫血(α地贫)缺失型突变,反向点杂交法(RDB)检测α地贫基因点突变与17种β珠蛋白基因突变。结果 1.以Hb A2>4.0%或Hb F>10.0%作为HPLC方法诊断β地贫的诊断标准,共检测出90例β地贫。与基因分析方法比较,应用HPLC方法检测小儿β地贫的敏感性为98.9%,特异性为100.0%,准确性为99.3%,阳性预测值为100.0%,阴性预测值为98.3%。2.β地贫纯合子或双重杂合子与β地贫杂合子的Hb F含量有显著性差异。结论应用HPLC方法检测小儿β地贫敏感性高,特异性强,与基因分析方法有很高的符合率,且其操作简便,快速,适用于小儿β地贫的诊断分型。

Objective： To evaluate the usefulness of analysis of hemoglobin by high performance liquid chromatography （HPLC） as a rapid diagnosis method for children β-thalassemia. Methods： The content of adult hemoglobin 2 （HbAz） and fetal hemoglobin （HbF） were detected by HPLC on 150 suspected β-thalassemia children. At the same time, after DNA extraction, amplification by PCR, the common deletional mutation types of α-thalassemia were assayed by electrophoresis.Reverse dot blot （RDB） were performed to detected the non-deletional mutation types of α-thalassemia and β gene mutations. Results： 1.Among the 150 samples, 90 cases were diagnosed as β-thalassemia by HPLC according to the principle that the cases with HbA2〉4.0% or HbF〉10.0% were considered to be β-thalassemia.The sensitivity is 98.9%, the specificity is 100.0%, the accuracy is 99.3%, the positive predictive value is 100.0% and the negative predictive vaiue is 98.3%. 2.There was apparent differrentce between homozygotes （double heterozygotes） and heterozygotes on HbF concentration. Conclusion. The usage of HPLC technique in identifying β-thalassemia has the advantages of high sensitivity, specificity and accuracy, as well as simplicity, rapid and the high agreement rate with gene analysis method.This technique is an alternative method for β-thalassemia screen.