Protocol:Hb A2 measurement by column chromatography

Quantitative HbA2 determination is the most valuable test for &beta;-thalassaemia carrier identification. Several methods have been set up, but only a few are now recommended for their accuracy. It should be pointed out that the precision and accuracy of HbA2 determination using densitometry scanning after cellulose acetate electrophoresis is unsatisfactory and its use has to be avoided (1). Isoelectric focussing (IEF) has an excellent resolution allowing for an accurate quantification, but it is cumbersome and time-consuming. Capillary electrophoresis is an emerging method of haemoglobin analysis (2). One study showed an excellent correlation between capillary electrophoresis and high performance cation-exchange chromatography (HPLC) for the qualitative and quantitative haemoglobin analysis (3). However, this method is not widely used yet.

The most used methods for HbA2 measurement are the michrocromatography on DE-52 and the cation-exchange HPLC (4-7). Both methods are very accurate, fast and simple.

Principle
HbA2 is eluted from an ion-exchange resin (diethyl-amino-ethyl cellulose), using a pH gradient: at pH 8.3 HbA2 is eluted, while the other Hb components are eluted at pH 7.0.

Sample
Haemolysate with Hb concentration of 3-4 g/dl.

A past method to prepare the haemolysate is to add one drop of packed RBC to 12-14 drops of distilled water to

Reagents

 * 1) Resin: anion exchanger DE-52 (Whatman, Ltd, England)
 * 2) Glycine buffer (0.2 M)-KCN (0.01%):
 * Glycine 15 g + KCN 0.1 g in 1litre distilled water. Adjust pH to 7.1-7.2 with HCl 1 M (10 ml of 37% HCl to 100 ml with distilled water)
 * 1) 0.9% NaCl

Resin preparation
Place 100 g DE-52 in glycine-KCN buffer up to a total volume of 400 ml. Magnetic stirring for 15 min. Allow to settle for 20 min then pour off supernatant and discard. Repeat this washing procedure two times, then resuspend the ion exchanger in fresh 0.2 M glycine buffer. Adjust pH (7.3-7.4) with diluted HCl under magnetic stirring. Allow to settle again, then remove some of the supernatant (the volume of the settled resin should be about the same as the supernatant). Prepared resin can be stored at 4oC for up to two weeks, but the pH needs to be checked every 4 to 5 days.

Column preparation

 * 1) Put Pasteur pipettes vertically on stands.
 * 2) Position a small cotton plug at the tapered end. Do not pack this plug tightly.
 * 3) Fill the column with the prepared DE-52. The settled resin should be 6-7 cm in height. Prefilled columns are commercially available.

Application

 * 1) Apply the haemolysate to the top of the column with another Pasteur pipette.
 * 2) After the haemoglobin solution has settled into the top of the resin add the glycine buffer (5 ml) and collect this volume in a tube. (HbA2 will be eluted in the first 3 to 4 ml).
 * 3) Then add 10 ml of NaCl 0.9% to the top of the column. The other Hb components will be eluted.
 * 4) Collect the effluent and adjust the volume at 25 ml.
 * 5) Read the absorbance at 415 nm.

Interpretation
Calculate the HbA2 percentage with the following formula:

OD A2 x100

OD A2+ (5x OD R*)

* of the remaining haemoglobin

Results
Normal value of HbA2 in non carriers = 2-3.2%.

Increased values (3.8 to 7%) are found in &beta;-thalassaemia carriers.

''Comments: The proper adjustment of pH is critical and may vary between different laboratories and with different lots of DE-52. HbA2is increased slightly in carriers of unstable haemoglobins, while it is reduced in &delta;-thalassaemia carriers and sometimes in &alpha;-thalassaemia carriers.''