Automated Systems Detect Variant Hemoglobins

The HPLC systems have become the method of choice for the evaluation of patients suspected with hemoglobinopathies, but they vary in their performance and ability to discriminate variant hemoglobins.

Scientists at the University of California Davis Health System, (Sacramento, CA, USA), evaluated the performance of two HPLC methods used in the detection of common hemoglobin (Hb) variants. They tested 377 samples, of which 99 were from patents with HbS, 32 with HbC, 78 with other hemoglobin variants or thalassemia, and 11 with increased hemoglobin A1c. The interpretations of each chromatograph produced by the HPLC systems were compared.

The two systems were the Variant, (Bio-Rad Laboratories, Hercules, CA, USA), and the Ultra2, (Trinity Biotech, Kansas City, MO, USA). Both the Variant and Ultra2 methods use a relatively short program that measures the separation process over approximately 4–6 minutes. The Ultra2 has a secondary feature that will reflex repeat test samples with abnormal peaks or unusual findings using a high-resolution method that observes the separation process over 6-8 minutes, allowing for better separation of the hemoglobin peaks.

There were no differences noted for hemoglobins A₀, S, or C. There were significant differences between the HPLC methods for hemoglobins F, A2, and A1c. However, there was good concordance between normal and abnormal interpretations (97.9% and 96.2%, respectively). The authors concluded that both the Variant and Ultra2 HPLC methods were able to detect most common hemoglobin variants. There was better discrimination for fast hemoglobins, between hemoglobins E and A2, and between hemoglobins S and F using the Ultra2 HPLC method.

Hemoglobin variants are a result of genetic changes resulting in abnormal or dys-synchronous hemoglobin chain production, as in thalassemia or the generation of hemoglobin chain variants such as hemoglobin S, as seen in sickle cell anemia.

Automated high-pressure liquid chromatography (HPLC) systems have become the method of choice for the evaluation of hemoglobinopathies. With this method, samples are mixed with buffers and salts and injected through an ion exchange column where physical properties of the hemoglobin such as surface charge and hydrophilic group, are separated during migration. HPLC methods allow for better isolation of hemoglobin variants and are less subjective than electrophoretic methods.

The study was published in the April 2011 edition of the International Journal of Laboratory Hematology.

Related Links:
Bio-Rad Laboratories
Trinity Biotech
UC Davis