Anandani Garima, Motiani Anita, Goswami Parth, Sonagra Amit
Department of Pathology, AIIMS, Rajkot, Gujarat, India.
Department of Biochemistry, AIIMS, Rajkot, Gujarat, India.
Int J Appl Basic Med Res. 2025 Jul-Sep;15(3):197-205. doi: 10.4103/ijabmr.ijabmr_70_25. Epub 2025 Aug 20.
Cation exchange high-performance liquid chromatography (HPLC) serves as a rapid, reproducible, and accurate method for diagnosing hemoglobinopathies. This study outlines the diagnostic approach and the challenges faced in the routine diagnosis of hemoglobinopathies through HPLC, particularly in laboratories with limited resources.
The aim of the study was to identify the challenges encountered in identifying abnormal hemoglobin (Hb) variants and to determine the significance of HbA2 and/or fetal Hb (HbF) analysis in the HPLC methodology for hemoglobinopathies.
A total of 1900 samples were analyzed using the ARKRAY ADAMS HA-8180T HPLC automated analyzer for the purpose of hemoglobinopathy testing. The samples were classified into normal or abnormal hemoglobin variants based on the percentage levels of HbA2, HbF, HbA, and the identification of any abnormal peaks. Among these, 113 cases were diagnosed to have thalassemia or hemoglobinopathy. The clinical presentations and red blood cell (RBC) indices were compared with the HPLC findings for each case, thereby contributing to the accuracy of the diagnosis.
The study examined the distribution of Hb variants, revealing that β-thalassemia trait was the most prevalent at 44.2%, followed by sickle cell trait at 13.3% and HbD Punjab trait at 10.6%. There were many challenging cases with elevated HbA2, like HbE thalassemia and Hb Lepore. Furthermore, there was identification of some abnormal peaks which were not exactly in the instrument's predetermined HbA2, HbF, HbA, or sickle windows, like HbJ Meerut. There were a few cases with abnormally elevated HbF, which can be seen in homozygous β-thalassemia, sickle cell disease, compound double heterozygous sickle cell β-thalassemia, δβ-thalassemia, and hereditary persistence of HbF. Carriers of β-thalassemia were generally identified by an HbA2 level of 4% or higher; however, there were nine cases which exhibited borderline HbA2 levels ranging from 3.5% to 3.9%, which might turn out to be β-thalassemia trait, especially in high-prevalence areas like Gujarat.
Any case scenario with abnormally elevated HbA2 is not always β-thalassemia trait. Nor abnormally elevated HbF may always indicate β-thalassemia major. Furthermore, some clinico-pathologically relevant hemoglobinopathies might show an abnormal peak on HPLC at any retention time, which may not be necessarily determined by the machine to be in some specific window. We need to correlate the clinical context, RBC indices, HPLC findings, and family studies to effectively detect most Hb variants.
阳离子交换高效液相色谱法（HPLC）是诊断血红蛋白病的一种快速、可重复且准确的方法。本研究概述了通过HPLC进行血红蛋白病常规诊断的方法及面临的挑战，尤其是在资源有限的实验室中。
本研究的目的是确定在鉴定异常血红蛋白（Hb）变体时遇到的挑战，并确定HbA2和/或胎儿血红蛋白（HbF）分析在血红蛋白病HPLC方法中的意义。
总共1900份样本使用ARKRAY ADAMS HA - 8180T HPLC自动分析仪进行血红蛋白病检测分析。根据HbA2、HbF、HbA的百分比水平以及是否存在异常峰，将样本分为正常或异常血红蛋白变体。其中，113例被诊断为地中海贫血或血红蛋白病。将每个病例的临床表现和红细胞（RBC）指标与HPLC结果进行比较，从而提高诊断的准确性。
该研究检测了Hb变体的分布情况，发现β地中海贫血特征最为常见，占44.2%，其次是镰状细胞特征，占13.3%，HbD旁遮普特征占10.6%。有许多具有挑战性的病例，其HbA2升高，如HbE地中海贫血和Hb Lepore。此外，还鉴定出一些异常峰，这些峰并不完全在仪器预先设定的HbA2、HbF、HbA或镰状窗内，如HbJ密拉特。有少数病例HbF异常升高，这在纯合β地中海贫血、镰状细胞病、复合双杂合镰状细胞β地中海贫血、δβ地中海贫血和HbF遗传性持续存在中可见。β地中海贫血携带者通常通过HbA2水平4%或更高来鉴定；然而，有9例病例的HbA2水平处于临界范围，为3.5%至3.9%，这些病例可能是β地中海贫血特征，尤其是在古吉拉特等高发地区。
任何HbA2异常升高的病例并不总是β地中海贫血特征。HbF异常升高也不一定总是表明是重型β地中海贫血。此外，一些临床病理相关的血红蛋白病在HPLC上的任何保留时间都可能出现异常峰，而仪器不一定能确定其处于某些特定窗口。我们需要将临床背景、RBC指标、HPLC结果和家族研究相关联，以有效检测大多数Hb变体。
