Application Of High Performance Liquid Chromatography And Blood Cell Analysis Technique In Thalassemia And Abnormal Hemoglobin Disease

Objective:To evaluate the clinical diagnostic value on different types of thalassemia,rare thalassemia and abnormal hemoglobin disease by high performance liquid chromatography and blood cell analysis technology, in order to select the suitable screening program for diagnosis.Methods:1. 1782 patients visisting genetic counseling clinic and gynecology clinic in the Fujian Maternity and Children Health Hospital were selected with aged from 1 to 48 years old from January 2013 to December 2014;2. Blood samples were collected and detected with the blood cell analysis technology and the high performance liquid chromatography?HPLC? technology within the stipulated time. At the same time, the common sites of thalassemia gene mutation or deletion type were detected with polymerase chain reaction?PCR? and reverse dot bloting?RDB?. The subjects were divided into alpha thalassemia, beta thalassemia, alpha beta compound thalassemia and control groups based on the gene test results;.3. The negative specimens by routine gene detection which were defined as thalassemia by HPLC were futher sequenced for rare type of thalassemia as well as abnormal hemoglobin disease.4. Data from different screening methods were analyzed by SPSS17.0 statistical software with Chi-square test.Results:1. The sensitivity of erythrocyte mean corpuscular volume? MCV? in alpha thalassemia, beta thalassemia and alpha beta composite thalassemia were 87.8%, 98.5% and 100% respectively. The specificity, positive predictive value, negative predictive value and diagnostic accuracy of MCV were 65.2%, 67.3%, 90.8% and 76.8% respectively.The sensitivity of erythrocyte mean corpuscular hemoglobin?MCH? in alpha thalassemia, beta thalassemia and alpha beta composite thalassemia were 93.5%, 98.9%, 92.3% respectively.The specificity, positive predictive value, negative predictive value and diagnostic accuracy of MCH were 54.8%, 62.3%, 93.7% and 72.6% respectively; The sensitivity of and hemoglobin A₂?Hb A₂? in alpha thalassemia, beta thalassemia and alpha beta composite thalassemia were 28.1%, 96.6%, 100%.The specificity, positive predictive value, negative predictive value and diagnostic accuracy of Hb A₂ were 77.6%, 64.6%, 67.4% and 66.4% respectively. MCH was better than MCV for the sensitivity of thalassemia screening?95.3% vs 91.6%, P<0.01?and MCV was better than Hb A₂?91.6% vs 52.2%,P<0.01?;while Hb A₂ was better than MCV for the specificity of thalassemia screening?77.6% vs 65.2%,P<0.01?and MCV was better than MCH?65.2% vs 54.8%,P<0.01?.2. The sensitivity of MCV+MCH screening program in alpha thalassemia and beta thalassemia were 87.4% and 97.7%, the specificity,positive predictive value,negative predictive value and diagnostic accuracy in thalassemia were 66.4%, 67.9%, 90.3% and 77.2% respectively. The sensitivity of MCV+MCH +Hb A₂ screening program in alpha thalassemia and beta thalassemia were 26.8%,95.0%, the specificity, positive predictive value, negative predictive value and diagnostic accuracy in thalassemia were 84.3%, 71.7%, 68.6% and 69.5% respectively. The sensitivity of MCV+MCH screening program in alpha thalassemia was higher significantly than MCV+MCH +Hb A₂ screening program?87.4% vs 26.8%, P<0.001?, whlie there was no significant difference in beta thalassemia?97.7% vs 95.0%,P>0.05?. The specificity of MCV+MCH+Hb A₂ screening program in thalassemia was higher significantly than MCV+MCH screening program?84.3% vs 66.4%,P<0.001?.3. The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of MCV+MCH +Hb A₂ screening program for beta thalassemia group?were 95.0%, 99.3%, 97.3%, 98.7% and 98.4% respectively? was higher significantly than those?were 26.8%, 85.0%, 47.6%, 69.5% and 65.4% respectively? for alpha thalassemia group.4. The sensitivity and specificity of Hb A₂ for beta thalassemia were 96.6% and 99.5% which were higher than those?28.1% and 78.1% respectively? for alpha thalassemia. The most of gene type for alpha thalassemia with misdiagnosis was--SEA /???in 69.9%?, and the left was-?3.7/???in 19.5%?.5. One new type of beta gene mutation was first defined as codon 36?-C? beta thalassemia by gene sequencing with negative result by routine gene detection?the MCV, MCH and Hb A₂ were 72 fl, 25 pg and 4.9% respectively?.6. Two new types of beta gene mutation in Fujian Province was defined as codon 30?A?G? beta thalassemia and upstream +22?G? A? beta thalassemia by gene sequencing with negative result by routine gene detection, Which MCV, MCH and Hb A₂ in one case were 68 fl, 23 pg and 5.4% respectively, and the other were 71 fl,21pg and 5.2% respectively.7. Three cases of rare alpha thalassemia genotypes were detected including two cases of Thai alpha thalassemia and one case of Hk alpha thalassemia. The MCV, MCH and Hb A₂ of one Thai case were 78 fl, 26 pg, 2.2% and 76 fl, 25 pg, 2.2% in other respectively. The MCV, MCH and Hb A₂ in Hk alpha thalassemia were 75 fl, 22 pg and 2.4% respectively.8. Seven cases of abnormal hemoglobin disease were diagnosed by analyzing peak profile of HPLC, including 2 cases of Hb J-Bangkok, 2 cases of Hb E, 1 cases of sickle red blood cell anemia, 1 case of Hb D, and 1 case of Hb Abruzzo. Hb E was defined as codon26 mutation by routine gene detection. The left were verified by the gene sequenced instead of routine gene detection.Conclusion:1. MCV+MCH is the best for the screening of the thalassemia in different screening programs.2. MCV+MCH +Hb A₂ is the most suitable for screening beta thalassemia.3. HPLC is better for beta thalassemia screening than that for alpha thalassemia and could be applied for diagnosis of rare type of thalassemia and abnormal hemoglobin disease.