Protocol:HPLC method

Globin Chain Synthesis
Globin chain synthesis analysis was introduced in the study of thalassaemia syndromes more than 30 years ago (1). It has greatly contributed to the understanding of the pathophysiological mechanisms of the different thalassaemia syndromes. Moreover, even in the DNA era it still remains a very sensitive diagnostic tool, very useful to define some complex or atypical forms of thalassaemia.

There are two established methods for globin chain synthesis analysis described in this chapter: one is the first classical method of Weatherall and Clegg, based on carboxymethyl cellulose chromatography for globin chain separation, the other is based on reversed phase high performance liquid chromatography (HPLC) (2). Finally a rapid method using vertical isoelectric focussing is described. The three methods described below use some common methodologies including white cell removal, reticulocyte enrichment, incubation with amino acids and radioactive tritiated leucine. However the main difference in the three methods is the methodology of separating the globin chains. The protocols are described in full for each method despite the overlap.

HPLC Method
Globin chain synthesis can also be measured using reversed phase high performance liquid chromatography (HPLC) (6)

Reagents
 Resin  &alpha;-cellulose ( SIGMA C-8002 ) 10 g cellulose microcrystallin ( SIGMA S-5504 ) 10 g Dissolve in 300 ml 0.9% NaCl   Percoll ( SIGMA P-1644 )  Percoll gradient: 9 ml of Percoll is mixed with 1 ml KRP 10X; and the following concentrations are made up:   KRP <ol style="list-style-type:none"> 4000 ml NaCl 0.9%( 9 g / 1000 ml )</li> 160 ml KCl 1.15 %( 2.3 g / 200 ml )</li> 120 ml CaCl2 + 2 H2O 0.61% ( 1.22 g /200 ml)</li> 40 ml MgSO4 +H2O 3.82 %( 7.64/ 200 ml )</li> 800 ml Na2HPO4 0.1 M( 14.2 g /1000 ml )</li> Adjust to pH 7.4 with 4 M HCl or with 5 M NaOH.</li> </ol> </li> Prepare an amino acid mixture mixing 1ml of each solution see table 4.1 <ol style="list-style-type:none"> AB plasma (obtained from Blood Bank. Dyalise against +KRP)</li> Glucose</li> FeSO4</li> Tritiated 3H-Leucine (TRK 510 Amersham Life Science)</li> Incubation mix: <ol style="list-style-type:none"> 0.5 ml KRP 1x</li> 0.5 ml AB plasma</li> 20 ml of amino acid solution without leucine</li> <li>2 mg glucose</li> <li>20 ml FeSO4.7H2O (5 mg/ml)</li> </ol> </li> </ol> </li> <li>Elution Buffers: <ol style="list-style-type:none"> <li>Acetonitrile (CH3CN)</li> <li>Methanol (CH3OH)</li> <li>Sodium Chloride 0.155 M, pH 2.7 with HCl</li> </ol> <ol style="list-style-type:none"> <li>Buffer A:   <ol style="list-style-type:none"> <li>Acetonitrile 500 ml</li> <li>Methanol 200 ml</li> <li>NaCl 0.155 M300 ml</li> </ol> </li> <li>Buffer B:   <ol style="list-style-type:none"> <li>Acetonitrile 250 ml</li> <li>Methanol 400 ml</li> <li>NaCl 0.155 M350 ml</li> </ol> </li> </li> </ol>

Method

 * 1) WBC and platelets are removed from whole blood by filtering the specimen through a column of microcrystalline cellulose and alpha cellulose in 0.9% NaCl. Erythrocytes are washed three times with saline and resuspended in KRP buffer.
 * 2) The RBC pellet is stratified on a discontinuous Percoll gradient at the following concentrations: 75, 60, and 45%. After centrifugation in a variable angle rotor at 1,000 g for 20 minutes at +20°C, several cell rings are obtained, the top one being enriched in reticulocytes.
 * 3) The cells are washed in KRP and the pellet resuspended (vol/ vol) in the incubation mixture. Then 1 mCi 3H-Leucine /ml of cell suspension is added and tubes placed at 37 °C in a shaking water bath for 2 hours.
 * 4) The labelled reticulocytes and red cells are washed in cold saline and lysed by freezing and thawing.
 * 5) Blood lysates are centrifuged (1 minute at 13,000 rpm at +4oC) and then 10 &mu;l of supernatant are directly injected into the column.
 * 6) Globin chain separation is carried out at room temperature on the analytical System Gold (Beckham) using a LiChrospher RP8 column, 5 &mu;m beads, 250 x 4 mm (Bio-Rad) with Buffer A and Buffer B for elution. A linear gradient from 90% to 20% solvent B over 90 minutes is applied at a flow-rate of 0.7 ml/min.
 * 7) Peaks are identified by comparing their retention times with those of peaks obtained from known Hb solutions.
 * 8) 350 &mu;l aliquot of the fractions are mixed with 3 ml of scintillation liquid (Optiphase ‘Hisafe 2’ Wallac) and counted for 10 minutes in a liquid scintillation counter (LS 6000SE; Beckham).

Results
The &beta;/&alpha; ratio is 1.0 ± 0.08 in normal subjects, 1.43 ±0.15 in &alpha;-thalassaemia carriers and 0.65 ±0.05 in &beta;-thalassemia carriers.

''Comments: When not analysed immediately, samples should be stored at –20 °C. After lysis, and before column injection, the samples should be kept at +4oC. The control of temperature is critical. The method also allows the separation of the different g chains (G&gamma;, A&gamma;, and A&gamma;T), and since it only requires small quantities of globin, the method can also be used in the analysis of globin chain biosynthesis in BFU-e colonies derived cells.''

Table 4.1 Composition of single amino acid stock solutions used for the Weatherall and Clegg method.