Laboratory diagnosis of haemoglobinopathies and reading the HPLC report

What it is

The laboratory investigations of hemoglobinopathies are a panel, not one test. The page lists six: the CBC (RBC, Hb, MCV, MCH and RDW); high performance liquid chromatography (HPLC); the Sickledex test, which is a screening test; alkaline (PH 8.4) and acid (PH 6.2) gel electrophoresis; isoelectric focusing; and molecular analysis.

How the HPLC separates the hemoglobin

HPLC here is cation exchange. The analytical cartridge contains negatively charged silica. Two acidic buffers containing different hydrogen ions are used. Hemolysates contain positively charged hemoglobin (Hb), and the Hb binds to the negatively charged silica at injection. The H+ concentration is then increased, and that separates the Hb from the silica.

What comes out is a Bio-Rad patient report. For every peak it gives the peak name, the calibrated area per cent, the area per cent, the retention time (min), and the peak area. Under the table it gives an F concentration and an A2 concentration, and it notes any value outside of expected ranges.