Laboratory methods used in the investigation of the haemolytic anaemias

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Chapter 11 Laboratory methods used in the investigation of the haemolytic anaemias

Red cells are typically removed from the circulation at the end of their lifespan of about 120 days. A shortened lifespan due to premature destruction may lead to haemolytic anemia when bone marrow activity cannot compensate for the erythrocyte loss. The causes can be divided into three groups:

At the end of a normal lifespan, red cells are destroyed within the reticuloendothelial system in the spleen, liver and bone marrow. In some haemolytic anaemias, the haemolysis occurs predominantly in the reticuloendothelial system (extravascular) and the plasma haemoglobin concentration (Hb) is barely increased. In other disorders, a major degree of haemolysis takes place within the bloodstream (intravascular haemolysis), the plasma Hb increases substantially and in some cases, the amount of Hb so liberated may be sufficient to lead to Hb being excreted in the urine (haemoglobinuria). However, there is often a combination of both mechanisms. The two pathways by which Hb derived from effete red cells is metabolized are illustrated in Figure 11.1.

Investigation of haemolytic anaemia

The cardinal signs of haemolysis in adults – anaemia, jaundice and reticulocytosis – may be mimicked by non-haemolytic conditions unique to the newborn. Because of changes associated with Hb F and Hb A concentrations as a result of the shift from γ- to β-globin production, differences in glycolytic enzyme activities and reduction or absence of haptoglobins during the first month or so of life, it is essential to compare results with age-matched sample(s) or age-adjusted reference values.

The clinical and laboratory associations of increased haemolysis reflect the nature of the haemolytic mechanism, where the haemolysis is taking place and the response of the bone marrow to the anaemia resulting from the haemolysis, namely, erythroid hyperplasia and reticulocytosis.

The investigation of patients suspected of suffering from a haemolytic anaemia comprises several distinct stages: recognizing the existence of increased haemolysis; determining the type of haemolytic mechanism; and making the precise diagnosis. In practice, the procedures are often telescoped because the diagnosis in some instances may be obvious to the experienced observer from a glance down the microscope at the patient’s blood film.

The following practical scheme of investigation is recommended. In each group, tests are listed in order of importance and practicability.

What is the Precise Diagnosis?

Plasma haemoglobin

Methods for estimation of plasma Hb are based on (1) peroxidase reaction and (2) direct measurement of Hb by spectrometry. In the peroxidase method, the catalytic action of haem-containing proteins brings about the oxidation of tetramethylbenzidine* by hydrogen peroxide to give a green colour, which changes to blue and finally to reddish violet. The intensity of reaction may be compared using a spectrometer with that produced by solutions of known Hb. Hi and Hb are measured together.

A pink tinge to the plasma is detectable by eye when the Hb is higher than 200 mg/l. When the plasma Hb is >50 mg/l, it can be measured as haemiglobincyanide (HiCN) or oxyhaemoglobin by a spectrometer at 540 nm1 (p. 26). Lower concentrations can also be measured reliably provided that the spectrometer plots of concentration/absorbance give a linear slope passing through the origin. This facility is provided by the Low Hb HemoCue (Hemocue Ltd, Dronfield, Derbyshire, UK), which can reliably measure plasma Hb at or higher than 100 mg/l.2

Spectrophotometric Method

A normal EDTA anticoagulated blood sample should be washed three times in isotonic saline (0.15 mol/l). Lyse one volume of washed packed red cells in two volumes of water. Alternatively, lyse by freezing and thawing. Centrifuge the haemolysate at 3000 rpm (1200 g) for 30 min and transfer the clear solution to a clean tube. Adjust the haemoglobin concentration to 80 g/l.

Dilute 1:100 with phosphate buffer, pH 8, to obtain an Hb concentration of 800 mg/l. By six consecutive double dilutions with phosphate buffer, make a set of seven lysate standards with values from 800 to 12.5 mg/l.

Read the absorbance of each solution at 540 nm, with water as a blank. Prepare a calibration graph by plotting the readings of absorbance (on y axis) against Hb concentration (on x axis) on arithmetic graph paper and draw the slope. Check that the slope is linear.

Read the absorbance of the plasma directly at 540 nm with a water blank and read the Hb concentration from the calibration graph. If absorbance is greater than the maximum value plotted on the graph, repeat the reading with a sample diluted with buffer.

When using the Low Hb HemoCue haemoglobinometer, fill the special cuvette with plasma and carry out the test in accordance with the instructions that are provided.

Normal Range

The normal range is 10–40 mg/l.

Significance of increased plasma haemoglobin

Hb liberated from the intravascular or extravascular breakdown of red cells interacts with the plasma haptoglobins to form an Hb–haptoglobin complex,4 which, because of its size, does not undergo glomerular filtration, but it is removed from the circulation by and is degraded in, reticuloendothelial cells. Hb in excess of the capacity of the haptoglobins to bind it passes into the glomerular filtrate; it is then partly excreted in the urine in an uncomplexed form, resulting in haemoglobinuria, and partly reabsorbed by the proximal glomerular tubules where it is broken down into haem, iron and globin. The iron is retained in the cells and eventually excreted in the urine (as haemosiderin). The haem and globin are reabsorbed into the plasma.

The haem complexes with albumin forming methaemalbumin and with haemopexin (p. 235); the globin competes with Hb to form a complex with haptoglobin. In effect, the plasma Hb level is further increased in haemolytic anaemias when haemolysis is sufficiently severe for the available haptoglobin to be fully bound. The highest levels are found when haemolysis takes place predominantly in the bloodstream (intravascular haemolysis). Thus, marked haemoglobinaemia, with or without haemoglobinuria, may be found in PNH, paroxysmal cold haemoglobinuria, cold-haemagglutinin syndromes, blackwater fever, march haemoglobinuria and other mechanical haemolytic anaemias (e.g. that after cardiac surgery). In warm-type autoimmune haemolytic anaemias, sickle cell anaemia and severe β thalassaemia, the plasma Hb level may be slightly or moderately increased, but in hereditary spherocytosis, in which haemolysis occurs predominantly in the spleen, the levels are normal or only very slightly increased.

Haem within the proximal tubular epithelium undergoes further degradation to bilirubin with liberation of iron, some of which is retained intracellularly incorporated into ferritin and haemosiderin. When haemolysis is severe, the excess of Hb that occurs in the glomerular filtrate will lead to an accumulation of intracellular haemosiderin in the glomerular tubular cells; when these cells slough, haemosiderin will appear in the urine (p. 236).

The presence of excess Hb in the plasma is a reliable sign of intravascular haemolysis only if the observer can be sure that the lysis has not been caused during or after the withdrawal of the blood. It is also necessary to exclude colouring of the plasma from certain foods and food additives.

Increased levels may occur as a result of violent exercise, as well as occurring in runners and joggers as a result of mechanical trauma caused by continuous impact of the soles of the feet with hard ground.4

Serum haptoglobin

Haptoglobin is a glycoprotein that is synthesized in the liver. It consists of two pairs of α chains and two pairs of β chains. With haemolysis, free Hb readily dissociates into dimers of α and β chains; the α chains bind avidly with the β chains of haptoglobin in plasma or serum to form a complex that can be differentiated from free Hb by column chromatographic separation or by its altered rate of migration in the α2 position on electrophoresis.

Direct measurement of haptoglobin is also possible by turbidimetry or nephelometry and by radial immunodiffusion.5 The methods described below are cellulose acetate electrophoresis and radial immunodiffusion.

Electrophoresis Method6,7

Method

Serum is obtained from blood allowed to clot undisturbed at 37°C. As soon as the clot starts to retract, remove the serum with a pipette and centrifuge it to rid it of suspended red cells. The serum may be stored at –20°C until used.

Mix well 1 volume of each of the diluted haemolysates with 9 volumes of serum. Allow to stand for 10 min at room temperature.

Impregnate cellulose acetate membrane filter strips (12 × 2.5 cm) in buffer solution and blot to remove all obvious surface fluid. Apply 0.75 ml samples of the serum–haemolysate mixtures across the strips as thin transverse lines. As controls, include strips with serum alone and Hb lysate alone. Electrophorese at 0.5 mA/cm width. Good separation patterns about 5–7 cm in length should be obtained in 30 min (see Fig. 11.2).

After electrophoresis is completed, immerse the membranes in freshly prepared o-dianisidine stain for 10 min. Then rinse with water and immerse in 50 ml/l acetic acid for 5 min. Remove the membranes and place in 95% ethanol for exactly 1 min. Transfer the membranes to a tray containing freshly prepared clearing solution and immerse for exactly 30 s. While they are still in the solution, position the membranes over a glass plate placed in the tray. Remove the glass plate with the membranes on it, drain the excess solution from the membranes, transfer the glass plate to a ventilated oven preheated to 100°C and allow the membranes to dry for 10 min.

Interpretation

The patterns of free Hb and Hb–haptoglobin complex migration are shown in Figure 11.2. Hb–haptoglobin complex appears in the α2

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