Hemoglobinopathies: Utilizing HPLC

Oct. 1, 2011

Due to the increase in immigration of different ethnic groups, it is becoming more important to diagnose disorders of hemoglobin chain synthesis, or hemoglobinopathies. Adequate presumptive identification usually requires a combination of at least two techniques, one qualitative and one quantitative.

In today's laboratories, automated High Performance Liquid Chromatography (HPLC) is the methodology of choice because both qualitative and quantitative information can be obtained at the same time. HPLC is used for rapid, accurate, and precise separation of the hemoglobin fractions. Analysis is carried out without any off-line specimen pretreatment. Separation is achieved by utilizing differences in ionic interactions between the cation exchange group on the column resin surface and the various hemoglobins and hemoglobin components.

Alternative methodologies such as electrophoresis are time, labor and cost intensive. Utilizing HPLC is a faster, easier and less expensive way for presumptive identification of hemoglobinopathies; laboratories can reduce turnaround time from seven days to 24 hours.

HPLC is used to confirm the identity of a hemoglobin variant in an HbA1c analysis and to screen for Beta-thalassemia and other hemoglobinopathies. For example, people with thalassemia can develop diabetes, and a hemoglobin variant may interfere with their HbA1c result. Transfusion patients need to see a relative decrease in a specific hemoglobin variant after treatment. Hereditary Persistent Fetal Hemoglobin (HPFH) patients have high HbF, above 20%, and severe blood disorders such as E Beta-thalassemia or Sickle Beta-thalassemia need to be properly diagnosed and treated. Therefore, HPLC instruments are no longer solely dedicated for HbA1c testing; they are also used for HbF and HbA2 measurement and hemoglobinopathy screening.

In summary, presumptive identification of hemoglobin variants that cause hemoglobinopathies and the identification of hemoglobinopathy carriers is crucial because children born to parents who are carriers have a 25% chance of acquiring the disease. Furthermore, hemoglobinopathies may interfere with an HbA1c result and merit further investigation. As a service to customers, many analyzers come with a chromatogram interpretation guide to help identify a particular hemoglobin variant based on the specific pattern and retention time.

Ranka Milojkovic is senior HPLC product manager for Tosoh Bioscience, Inc.