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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 anaemia when bone marrow activity cannot compensate for the erythrocyte loss. The causes can be divided into three groups:
- 1.
Defects within red cells from dysfunction of enzyme- controlled metabolism, abnormal haemoglobins or thalassaemias
- 2.
Loss of structural integrity of red cell membrane and cytoskeleton in hereditary spherocytosis, hereditary elliptocytosis, paroxysmal nocturnal haemoglobinuria (PNH) and immune and drug-associated antibody damage
- 3.
Damage by extrinsic factors such as mechanical trauma, microangiopathic conditions (including thrombotic thrombocytopenic purpura) and chemical toxins.
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 is barely increased. In other disorders a major degree of haemolysis takes place within the bloodstream (intravascular haemolysis), the plasma haemoglobin concentration increases substantially and in some cases the amount of haemoglobin liberated is sufficient to lead to its being excreted in the urine (haemoglobinuria). However, there is often a combination of both mechanisms. The two pathways by which haemoglobin derived from effete red cells is metabolised are illustrated in Figure 11-1 .
Catabolic pathway of haemoglobin.
Investigation of haemolytic anaemia
The cardinal signs of haemolysis in adults (anaemia, jaundice and reticulocytosis) may also be seen in infants resulting from the shift from γ to β globin production, changes in glycolytic enzyme activities and reduction or absence of haptoglobins during the first month or so of life, and so it is essential to compare results with age-matched sample(s) or age-specific 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 resultant anaemia, namely erythroid hyperplasia and reticulocytosis.
The investigation of patients suspected of suffering from a haemolytic anaemia comprises several distinct stages: recognising the existence of increased haemolysis, determining the haemolytic mechanism and making a 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.
Is there evidence of increased haemolysis?
- 1.
Estimation of haemoglobin concentration (Hb); reticulocyte count; inspection of a stained blood film for the presence of spherocytes, elliptocytes, irregularly contracted cells, schistocytes or agglutination (see Chapters 3 and 5 )
- 2.
Tests for increased unconjugated serum bilirubin and urinary urobilinogen excretion; measurement of haptoglobin or haemopexin
- 3.
Detection of urinary haemoglobin or haemosiderin
What is the haemolytic mechanism?
- 1.
Direct antiglobulin test (DAT) with broad-spectrum antiserum
- 2.
Osmotic fragility and glycerol lysis test
- 3.
Measurement of haemoglobin concentration in urine and plasma; Schumm test
What is the precise diagnosis?
- 1.
If a hereditary haemolytic anaemia is suspected:
- a.
Eosin-5-maleimide (EMA) dye binding test or osmotic-fragility determination after 24 h of incubation at 37 °C; screening test for red cell glucose-6-phosphate dehydrogenase (G6PD) deficiency (or quantitative assay if reticulocytosis is present); red cell pyruvate kinase assay; assay of other red cell enzymes involved in glycolysis; estimation of red cell glutathione (see Chapter 12 )
- b.
Estimation of percentage of haemoglobins A 2 and F; high performance liquid chromatography or electrophoresis for an abnormal haemoglobin; tests for sickling; tests for an unstable haemoglobin; blood count parameters, especially mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC); gene analysis (see Chapter 8 )
- c.
Examination of the proteins of the red cell membrane and cytoskeleton (e.g. spectrin) by gel electrophoresis and by specific radioimmunoassay
- a.
- 2.
If an autoimmune acquired haemolytic anaemia is suspected:
- a.
Direct antiglobulin test using anti-immunoglobulin and anticomplement sera; tests for autoantibodies in the patient’s serum; titration of cold agglutinins; Donath–Landsteiner test; electrophoresis of serum proteins; demonstration of thermal range of autoantibodies; tests for agglutination and/or lysis of enzyme-treated cells by autoantibodies; tests for lysis of normal cells by autoantibodies
- a.
- 3.
If a drug-induced haemolytic anaemia is suspected:
- a.
Screening test for red cell G6PD; glutathione stability test; staining for Heinz bodies; identification of methaemoglobin (Hi) and sulphaemoglobin (SHb); tests for drug-dependent antibodies
- a.
- 4.
If mechanical stress is suspected:
- a.
Red cell morphology; platelet count; renal function tests; coagulation screen; fibrinogen assay; test for fibrinogen/fibrin degradation products (see Chapters 5 and 18 )
- a.
- 5.
In obscure cases:
- a.
Investigations for PNH, such as acidified serum test (Ham test), sucrose lysis test, flow cytometric immunophenotyping for erythrocyte and neutrophil glycosylphosphatidylinositol (GPI)-linked antigens (see Chapter 13 )
- b.
Measurement of lifespan of patient’s red cells (see Chapter 17 )
- c.
If splenectomy is contemplated, determination of sites of haemolysis by radionuclide imaging (see Chapter 17 )
- a.
Plasma haemoglobin
Methods for estimation of plasma haemoglobin concentration are based on (1) a peroxidase reaction and (2) direct measurement 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 concentration. Methaemoglobin and haemoglobin are measured together.
A pink tinge to the plasma is detectable by eye when the concentration is higher than 200 mg/l. When the concentration is > 50 mg/l, it can be measured as haemiglobincyanide (HiCN) or oxyhaemoglobin by a spectrometer at 540 nm (p. 21). 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, www.hemocue.com ), which can reliably measure plasma haemoglobin at a concentration of 100 mg/l or higher.
Sample collection
Every effort must be made to prevent haemolysis during the collection and manipulation of the blood. For this, it may be preferable to use a syringe rather than an evacuated tube system. A clean venepuncture is essential; a plastic syringe and relatively wide-bore needle should be used. When the required amount of blood has been withdrawn, the needle should be detached with care and 9 volumes of blood should be added to 1 volume of 32 g/l sodium citrate.
Peroxidase method
The test is now rarely performed and readers are referred to previous editions of this book.
Spectrophotometric method
Red cells from a normal ethylenediaminetetra-acetic acid (EDTA)-anticoagulated blood sample should be washed three times in isotonic saline (0.15 mol/l). Lyse 1 volume of washed packed red cells in 2 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 in 100 with phosphate buffer, pH 8, to obtain a 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 haemoglobin 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 haemoglobin 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 to 40 mg/l.
Significance of increased plasma haemoglobin
Haemoglobin liberated by the intravascular or extravascular breakdown of red cells interacts with plasma haptoglobin to form a haemoglobin–haptoglobin complex, which, because of its size, does not undergo glomerular filtration, but it is removed from the circulation by – and is degraded in – reticuloendothelial cells. Haemoglobin in excess of the capacity of haptoglobin 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 lost 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. 220); the globin competes with haemoglobin to form a complex with haptoglobin. Plasma haemoglobin concentration 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 is predominantly intravascular. 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 autoimmune haemolytic anaemia, sickle cell anaemia and severe β thalassaemia, the plasma haemoglobin concentration 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 and incorporated into ferritin and haemosiderin. When haemolysis is severe, the haemoglobin that appears in the glomerular filtrate leads to an accumulation of intracellular haemosiderin in the glomerular tubular cells; when these cells slough, haemosiderin appears in the urine (p. 221).
The presence of excess haemoglobin 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 on hard ground.
Serum haptoglobin
Haptoglobin is a glycoprotein that is synthesised in the liver. It consists of a pair of α chains and a pair of β chains. Following haemolysis, free haemoglobin 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 haemoglobin by column chromatographic separation or by its altered rate of migration in the α 2 position on electrophoresis.
Direct measurement of haptoglobin is also possible by turbidimetry or nephelometry and by radial immunodiffusion. The methods described below are cellulose acetate electrophoresis and radial immunodiffusion.
Electrophoretic method ,
Principle
Known amounts of haemoglobin are added to serum. The haemoglobin–haptoglobin complex is separated by electrophoresis on cellulose acetate; the presence of bound and free haemoglobin is identified in each sample and the amount of haptoglobin is estimated by noting where free haemoglobin appears.
Reagents
Buffer (pH 7.0, ionic strength 0.05)
Na 2 HPO 4 .H 2 O 7.1 g/l, 2 volumes; NaH 2 PO 4 .H 2 O 6.9 g/l, 1 volume. Store at 4 °C.
Haemolysates
Red cells from a normal EDTA-anticoagulated blood sample are washed three times in isotonic saline (0.15 mol/l). Lyse 1 volume of washed packed red cells in 3 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 Hb to 30 g/l with water and dilute this preparation further with water to obtain a batch of solutions with Hb of 2.5, 5, 10, 20 and 30 g/l. These solutions are stable at 4 °C for several weeks.
Stain
Dissolve 0.5 g of o -dianisidine (3,3′-dimethoxybenzidine) in 70 ml of 95% ethanol; prior to use, add together 10 ml of acetate buffer, pH 4.7 (sodium acetate 2.92 g, glacial acetic acid 1 ml, water to 1 litre), 2.5 ml of 3% (10 volumes) H 2 O 2 and water to 100 ml.
Clearing solution
Glacial acetic acid 25 ml, 95% ethanol 75 ml.
Acetic acid rinse
Glacial acetic acid 50 ml/l.
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 haemoglobin 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 haemoglobin and haemoglobin–haptoglobin complex migration are shown in Figure 11-2 . The complex appears in the α 2 globulin position. When there is more haemoglobin than can be bound to the haptoglobin, the free haemoglobin migrates in the β globulin position. The amount of haptoglobin present in the serum is determined semiquantitatively as between the lowest concentration of haemoglobin, which shows only a free haemoglobin band, and the adjacent strip, which shows a band of haemoglobin–haptoglobin complex. In the total absence of haptoglobin, a haemoglobin band alone will be seen, even at 2.5 g/l. In severe intravascular haemolysis with depleted haptoglobin, some of the haem may bind in the β globulin position to haemopexin (see below) and some to serum albumin to form methaemalbumin.
The concentration of haptoglobin can be determined quantitatively with a densitometer. The test is carried out as described earlier, but only one haemolysate is required with an Hb of 30–40 g/l. After the plate has cooled, the membranes are scanned by a densitometer at 450 nm with a 0.3-mm slit width. The density of the haptoglobin band is calculated as a fraction of the total Hb in the electrophoretic strip:
Radial immunodiffusion method
Principle
The test serum samples and reference samples of known haptoglobin concentration are dispensed into wells in a plate of agarose gel containing a monospecific antiserum to human haptoglobin. Precipitation rings form by the reaction of haptoglobin with the antibody; the diameter of each ring is proportional to the concentration of haptoglobin in the sample.
Reagents
Phosphate buffer, iso-osmotic, pH 7.4:
-
(A) NaH 2 PO 4 ·2H 2 O (150 mmol/l) − 23.4 g/l
-
(B) NaH 2 PO 4 (150 mmol/l) − 21.3 g/l.
Add 18 ml reagent A to 82 ml reagent B.
Single diffusion plates *
* Gel plates containing the antiserum are available commercially (e.g. The Binding Site Group Ltd, www.thebindingsite.com/radial-immunodiffusion .
Dissolve agarose (20 g/l) in boiling phosphate buffered water, pH 7.4. Allow to cool to 50 °C. Add 5% sheep or goat antihuman haptoglobin antiserum diluted in buffered water, pH 7.4. Mix well but without creating bubbles. Pour the gel onto thin plastic trays (plates) to a thickness of < 1 mm. After the gel has set, cut out a series of wells about 2 mm in diameter, about 2 cm apart. Extract the core by using a pipette tip with a negative pressure pump. Cover the plates with fitted lids and store in sealed packets at 4 °C until used.
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