Protocol:Weatherall and Clegg 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.

Weatherall and Clegg method
This method has been used for prenatal diagnosis and the first prenatal diagnosis for haemoglobin disorders used this technique (3). It is now rarely used in prenatal diagnosis except in situations where the parental mutations are not known, if a couple present late for prenatal diagnosis, or if the partner is unavailable for testing. The diagnosis is based on the relative synthesis of &beta;-globin (representing HbA) and &gamma;-globin (representing HbF). A &beta;/&gamma; chain synthesis ratio above 0.02 indicates an unaffected fetus. However the results should be interpreted with great care as mild &beta;+-thalassaemia mutations can produce higher levels of &beta;-globin, which may lead an incorrect diagnosis of an unaffected fetus (4).

This method is also useful in risk assessment when the full blood count and haemoglobin electrophoresis do not give a conclusive diagnosis, an example is when an individual has microcytic hypochromic indices and a normal HbA2 level. If globin chain biosynthesis gives a biosynthesis ratio indicating beta thalassaemia trait the diagnosis is made of normal HbA2 &beta;-thalassaemia trait. Globin chain synthesis is also useful for patients undergoing bone marrow transplant to monitor the &beta;/&alpha; activity during the transplant and follow up of the patient.

In vitro labelling of haemoglobin
When using fetal blood for prenatal diagnosis, the white cell removal and reticulocyte enrichment steps are not required.

Method

 * 1) Blood samples are collected in Li-heparin anticoagulant and kept at 4oC.
 * 2) The blood is sedimented by centrifugation at 4000 rpm for 15 minutes at 4oC and the plasma and buffy coat removed.
 * 3) The top 0.5 cm, slightly reticulocyte-enriched portion of cells are resuspended in about 5 ml cold Krebs-Ringer phosphate solution and loaded onto a column, 2 cm long x 1cm diameter, containing a mixture of &alpha; cellulose (Sigma C 8002) and Sigmacell Type 50 microcrystalline cellulose (Sigma S 5504) (20 g of each in 1 litre of normal saline), in order to remove white cells.
 * 4) The red cells are eluted from the column with another 5ml of cold KRP, and washed twice in cold KRP.
 * 5) The red cell pellet is labelled using 0.5 mCi of 3H Leucine and 0.1 ml of incubation mix in a 2 hour incubation at 37oC in a shaking water bath.
 * 6) The samples are then washed twice with cold KRP to remove free 3H Leucine. The red cell pellet is either used directly for globin extraction or stored at –80oC.

Reagents

 * 1) Krebs-Ringer phosphate (KRP) buffer
 * KRP is a balanced salt solution buffered at a physiological pH, made up from:
 * 1) NaCl 1.5 M
 * 87.66 g NaCl in 1 Litre ion-free water.
 * 1) Phosphate buffer pH 7.4:
 * 0.1 M NaH2PO4. 2H2O (MW 156) = 15.6 g in 1 Litre.
 * 0.1 M Na2HPO4. 2H2O (MW 178) = 17.8 g in 1 Litre.
 * Add 19 parts of A to 81 parts of B, adjust pH to 7.4.
 * 1) KCl 0.15 M
 * 5.6 g KCl (MW 74.56) in 500 ml distilled water.
 * 1) MgSO4. 0.15 M
 * 3.7 g MgSO4.7H2O (MW 246.48) in 100 ml distilled water.
 * 1) CaCl2 0.11 M
 * 1.62 g CaCl2.2H2O (MW 147.02) in 100 ml distilled water.

To make KRP for use:
 * 1) Make up 100 ml of 1.5 M NaCl to 1 litre.
 * Add: 120 ml of phosphate buffer pH 7.4
 * 40 ml of 0.15 M KCl
 * 10 ml of 0.15 M MgSO4
 * 10 ml of 0.11 M CaCl2 – stir carefully to avoid precipitation.
 * 1) Adjust pH to 7.4 with NaOH (1 M)

Check osmolarity and adjust with 1.5 M NaCl stock solution or distilled water if necessary, to 280 – 300 m OsM.

Preparation of incubation medium
One pack of AB Rhesus negative blood is obtained from the blood bank. The plasma transferrin is saturated with iron by adding 3.0 ml ferrous ammonium sulphate solution (10.5 mg/10 ml H2O) and leaving it to stand for 30 minutes at room temperature. Free amino acids are removed by dialysis at 4oC for 48 hours against KRP using boiled dialysis tubing. The KRP is changed at 1 hour, 6 hours and 24 hours. The calcium in the KRP causes clotting and the fibrin is removed by filtering the dialysed plasma through a nylon mesh. Glucose (6 mg/ml plasma) and leucine free amino acid mix (0.133 ml/ml plasma, details below) are added to the plasma and the pH adjusted to 7.4 with 1 M NaOH. The resulting incubation medium is stored in 0.5 ml aliquots at –40oC until required.

Preparation of the leucine-free amino acid mix
20 mM (10 ml) of stock solutions of each amino acid are made in water, and stored at –40oC until required. Table 4.1 shows the composition of the amino acid stock solutions. To prepare the leucine-free amino acid mix, 1 ml of each stock solution, except leucine are mixed to give 20 ml of a 1 mM stock solution of all amino acids except leucine.

Preparation of 3H Leucine
The 3H Leucine (TRK 170) obtained from Amersham Biosciences at a concentration of 5 mCi/5 ml aqueous solution containing 2% ethanol is too dilute particularly for the active labelling required for prenatal diagnosis samples. It is concentrated by using a freeze dryer. The 3H Leucine is then taken up in a small measured amount of KRP – usually 0.5 ml/5 mCi 3H Leucine, to give a final concentration of 10 mCi/ml.

Carrier Haemoglobin
In some circumstances it is necessary to add carrier haemolysate before globin extraction, in order to localise the ratioactive globin chains that may be present in very small quantities. This is particularly important for fetal samples, since the fetal beta globin chains are present in small quantities and do not register on the optical density monitor during carboxymethyl cellulose column chromatography. Carrier haemolysate is a non-radioactive lysate made from an adult blood sample diluted to a Hb concentration of 2 g/dl and stored in aliquots at –40oC. Approximately 0.5 ml of this carrier solution is added to the radioactive test sample prior to globin extraction.

Preparation of Globin
Globin is prepared by acid/acetone precipitation (5). The incubated cells are lysed by freezing and/or by the addition of distilled water to make up a concentration of 2-4 g/100ml. The lysate is then added dropwise with stirring to acetone containing 1.5% (v/v) 11.3 M HCl previously cooled to –20oC. 20 ml of acid/acetone is used for 1 ml of haemoglobin solution. During this procedure the haemoglobin is denatured and the protein moiety precipitates while the haem remains in solution. The globin is then centrifuged at 3000 rpm at –20oC for about 1 minute. The acid/acetone containing the haem is removed by suction and the globin washed twice for 3 minutes with cold acetone that has been cooled to –20oC. A third wash is carried out in diethyl ether, the supernatant discarded and the tubes placed horizontally or slightly inverted to allow the ether to evaporate and drain out. Before the pellet is quite dry it is broken up with a Pasteur pipette so that by the time it dries completely it is clean, white and powdery. If the globin is not to be used immediately it is stored at –40oC.

Carboxymethyl Cellulose Chromatography
The method used is that of Clegg et al (1) with some modifications. The globin chains are separated on carboxymethy cellulose (CM23) (Amersham Biosciences) column chromatography using a urea-phosphate buffer gradient. The phosphate buffer pH 6-7 is optimal for the separations of globin chains, but at this pH globins are relatively insoluble, so urea is needed as a solvent. Furthermore a reducing agent (dithiothreitol) must be added to prevent formation of mixed disulphides by the free sulphhydryl groups of the globins. Since the ability to bind to the CM- cellulose depends on the net positive charge of the globins, the affinity of each globin for cellulose at pH 6-7 is &alpha;>&beta;>&gamma;. Thus &gamma;-chain is eluted out of the column first, then &beta;, &delta; and &gamma; chains respectively. The method described is for the preparations of 6 columns.

Preparation of Urea Buffers
1440 g of urea is dissolved in distilled water and made up to 3 Litres with distilled water to make an 8 M solution. The urea solution is filtered through a column of mixed-bed resin deionizier. The filtered solution is divided into two lots as follows:


 * 1) Weak Buffer:

1800 ml 8 M urea solution plus 1.05 g of disodium hydrogen orthophosphate (Na2HPO4), adjusted to pH 6.4 with 20% orthophosphoric acid and 180 mg dithiothreitol added.


 * 1) Strong Buffer:

1200 ml 8 M urea solution plus 5.1 g of Na2HPO4, adjusted to pH 6.4, and 120 mg dithiothreitol added. The Na2HPO4 gives a gradient of 0.004 – 0.028 M.

Column Chromatography
Figure 4.1 shows the chromatography set up. 15 g Carboxymethyl cellulose (CM23) is suspended in about 200 ml weak buffer for a few minutes and the suspension poured into 6 columns (0.5 cm x 15 cm). The columns are allowed to settle partially and then refilled to a level of 12 cm. Each column is then connected via polythene tubing to a Uvicord S11 spectrophotometer (Amersham Biosciences) and a fraction collector. The optical density of the effluent is monitored continuously at 280 nm, and recorded on a six-channel recorder. One channel is used for recording the number of fractions each time the fraction collector moves, thus the peak optical densities of the &alpha;, &beta; & &gamma; fractions can be localised. After the columns are packed they are washed with weak buffer at a flow rate of 0.5 ml/min for 10-15 minutes. The globin samples are then dissolved, 5-10 mg of globin in 2-3 ml weak buffer is applied carefully to the columns, allowed to pass through the resin, and washed with 10 ml weak buffer. The columns are stoppered and joined to 6 peristaltic pumps. Elution with weak buffer is started immediately at a flow rate of 5 ml/12 min.

A sodium phosphate gradient is made with a two-chamber linear gradient maker (each chamber is 9 cm in diameter connected to each other by polythene tubing- (this can be made in house). In this set-up, the principle compartment that flows out to the columns starts with 1200 ml of a weak buffer, the other compartment starts with 1200 ml of strong buffer. Prior to starting the run, the compartments are separated by a clip in the connecting tubing. The weaker buffer is first added, this is used to rinse out the tubing between the gradient maker and the fraction collectors. Once the columns are joined to the peristaltic pumps, the level of the weak buffer compartment is adjusted to the same level as the strong buffer compartment, and the two compartments are then joined by releasing the clip on the connecting polythene tubing.

The flow rate of the peristaltic pumps is regulated to 5 ml/12 min, and the fraction collectors pre-set to collect 12 minute fractions. All the equipment is connected to a timer pre-set for 17 hours, ensuring that at least 80 x 12-minute fractions are collected by the end of the run.

Radioactivity Counting
At the end of the run, the optical density profile from the recorder is examined and aliquots for counting are taken beginning from the point where the protein starts to be eluted, to the end of the run. 0.5 ml aliquots are taken from the selected fractions into minivials and 4.5 ml scintillation fluid is added. The tubes are shaken vigorously and once the gel is homogeneous the vials are placed in a scintillation counter. Each sample is usually counted for 5 minutes.

Processing of Results
The cpm (counts per minute) for each column is plotted against the fraction numbers and the radioactivity profile compared with the optical density profile, to locate the &alpha;, &beta; and &gamma; peaks. The number of counts under each peak is calculated as shown in the example in figure 4.2 and table 4.3, and the biosynthetic ratios of the different chains obtained.

Pre peaks are included in the calculations. The background is calculated by drawing a line across the two lowest points of the peak. Some centres calculate this by drawing a parallel line from the lowest point of the peak, and drop a perpendicular line to it, from the lowest point. The method chosen here is the previous method, although minimum values are calculated for the globin chains. This is particularly important for prenatal diagnosis samples.

Standardization of Columns and Results
Columns should be standardized using normal adult and normal cord blood samples. Conditions (pH, gradient, temperature) should be adjusted until a balanced non &alpha;/&alpha; ratio is obtained in these normal samples. Figs 4.3 and 4.4 show elution patterns obtained with standard normal adult and beta thalassaemia trait samples. During a column run, a control sample should be run in parallel.

In the UCLH centre a biosynthesis ratio of &beta;/&gamma; above 0.02 indicates an unaffected fetus. Non-fetal samples: Normal &beta;/&alpha; 0.96 (range 0.78-1.14); &beta; thalassaemia trait &beta;/&alpha; 0.50 (range 0.38-0.62); &alpha;0-thalassaemia trait &beta;/&alpha; 1.44 (range 1.22-1.82); &alpha;+-thalassamia trait &beta;/&alpha; 1.20 (range 0.95-1.39).

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

Table 4.2 Recipe for 100 ml of glutamine and leucine free amino acid stock solution cocktail to be used with the vertical IEF method . Due to variable solubility it is convenient to weight the following mg. in separate tubes and to dissolve each amino acid separately in the indicated volumes. The separate volumes can than be thoroughly collected in single cylinder and the final volume adjusted to 100 ml with RTBS

Table 4.3 The calculation of the &beta;/&alpha;, &gamma;/&alpha; and non &alpha;/&alpha; globin chain synthesis ratios for the example shown in Fig. 4.2 (which is from a thalassaemia intermedia patient).