Protocol:Hb A2 variant analysis by HPLC

Cation – exchange high performance liquid chromatography (HPLC) has emerged as the method of choice for quantification of HbA2, Hb F and for detection and quantitation of the Hb variants, particularly those which may interact with &beta;-thalassaemia such as Hb S, E, C, O-Arab, D and Lepore.

Principle
In this method phosphate buffers at different concentrations (mobile phase), pass under pressure through an ionic exchange column (stationary phase). The stationary phase consists of a temperature controlled analytical cartridge containing a resin of anionic or cationic particles (3 – 5 &mu;m). The chromatographic station delivers a programmed buffer gradient of increasing ionic strength and pH to the cartridge by two dual-piston pumps, and the haemoglobins are separated according to their ionic interaction with the stationary phase.

The separated haemoglobins then pass through the flow cell of the filter photometer, where changes in the absorbance (415 nm) are measured; background variations are corrected by an additional filter at 690 nm. Each haemoglobin is characterised by a specific retention time, which is the elapsed time from the sample injection to the apex of a haemoglobin peak.

The calibration factors for HbA2, F, A1c are automatically calculated by processing a calibration sample at the beginning of each run. A specific software turns the raw data collected from each analysis into a report showing the chromatogram, with all the haemoglobin fractions eluted, the retention times, the areas of the peaks and the values (%) of the different haemoglobin components. The report presents the percentages of haemoglobins F, A1C, A and A2 and provides qualitative and quantitative determination of abnormal haemoglobins.

Sample
Venous blood in any anticoagulant.

Methods
A commonly used apparatus is the Variant (Bio-Rad Laboratories). However, reliable results have been recently obtained also with other instruments, such as HA816O (Menarini Arkay) and G7 (Tosoh- Bioscience).

Results and interpretation
The expected normal range for HbA2 is between 1.7 and 3.2% in normal subjects, while in &beta;-thalassaemia carriers is between 4.0 and 7%. HbA2 values are considered borderline when between 3.2 and 3.8%. Samples with these levels need further investigation (see Volume 1). The normal range for HbF is usually less than 1.5% of total haemoglobin.

HPLC machines have analyte identification windows that help in the interpretation of normal and abnormal haemoglobins detected in the blood sample (8). The windows are defined retention time intervals in which the common haemoglobin variants are eluted (eg Hb S, C and D). However, it should be pointed out that since other Hb variants may have a similar retention time to the common variants, (table 3.1), the Hb variant identification is only presumptive (Fig. 3.1) and DNA or globin amino acid analysis is necessary for definitive Hb variant identification (9). HPLC is also being used for neonatal haemoglobinopathy screening programmes (10).

Table 3.1