High performance liquid chromatography(HPLC) in diagnosis of thalassemia and other hemoglobinopathies in India
Sachdeva, Annupam; Kumar, Shirish; Sood, S.K. and Gupta, Amrita (2002) High performance liquid chromatography(HPLC) in diagnosis of thalassemia and other hemoglobinopathies in India. In: The 44th American Society of Hematology Annual Meeting, 2002, Detroit, USA.
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Cation exchange HPLC is a convenient, efficient, reproducible & cost effective method for screening & diagnosis of Thalassemia & other hemoglobinopathies. We discuss the prevalence of usual & unusual hemoglobinopathies & the advantages, disadvantages & limitations of this method using a dedicated system (Bio Rad Variant). Practically, all antenatal women are screened for CBC & Hb A2 values to identify patients with b- Thalassemia trait at this hospital. Large number of samples is also received for diagnosis of hemoglobinopathies from other clinical departments. Since March 1999 till end of October 2000 we analyzed 3226 samples by HPLC (1210 samples from the antenatal clinic & rest from other units). Alkaline & acid hemoglobin electrophoresis on agarose gel was performed on all samples in which HPLC showed abnormal hemoglobin. Some abnormalities have been confirmed by DNA analysis. The prevalence of abnormal hemoglobin's in antenatal women is as follows: HemoglobinNumberPercent b-thal traitl03/12108.50 DPunjab014/1210* 1.16 Qlndia 005/1210*0.40 HbS004/12100.33 HbE004/1210*0.33 DIran003/12100.25 J002/12100.16 Some, in addition, were heterozygotes for b-thal trait. To the best of our knowledge Hb DIran has not been reported in India. Frequency of Hb Qlndia in Delhi is much higher than suspected earlier. Advantages: The biggest advantage of the HPLC system is excellent resolution, reproducibility & quantification of several normal & abnormal hemoglobin's -precisely measuring (Hb A2 and F) leading to early reporting in a b-thal screening programme. A larger number of abnormal hemoglobin's are identified than by electrophoresis, matching iso-electric focusing. System is very convenient, can be adapted for identification of other hemoglobin variants and for globin chain analysis. Automation and capability of interfacing with laboratory information system increase its usefulness. Limitations and disadvantages: Equipment is expensive and the cost per test is high. Calibration failures can cause errors in interpretation. Hb A2 cannot be measured in presence of co-eluting hemoglobin variants such as Hb A2 & Hb DIRAN. Interpretation: As several hemoglobin's elute within the same retention window, interpretation requires experience. Results should be interpreted keeping in mind the following: retention times of variant hemoglobin's, clinical data, history of blood transfusion, ethnic origin and family studies & whenever required, hemoglobin electrophoresis. We found a heterozygote for b-thal and Hb QINDIA had earlier undergone termination of pregnancy due to a mistaken diagnosis of heterozygote for b-thal and sickle cell trait. A combined approach using HPLC & alkali and acid electrophoresis can virtually identify most hemoglobin
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