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

Chapter contents

Investigation of haemolytic anaemia 230

Plasma haemoglobin 231

Sample collection 231

Peroxidase method 232

Spectrophotometric method 232

Serum haptoglobin 233

Electrophoresis method 233

Radial immunodiffusion (RID) method 234

Normal ranges 234

Significance 235

Serum haemopexin 235

Examination of plasma (or serum) for methaemalbumin 235

Schumm’s test 236

Quantitative estimation by spectrometry 236

Demonstration of haemosiderin in urine 236

Chemical tests of haemoglobin catabolism 237

Serum bilirubin 237

Urobilin and urobilinogen 237

Qualitative test for urobilinogen and urobilin in urine 238

Porphyrins 238

Demonstration of porphobilinogen in urine 238

Aminolaevulinic acid 239

Demonstration of porphyrins in urine 239

Spectroscopic examination of urine for porphyrins 239

Abnormal haemoglobin pigments 240

Spectroscopic examination of blood for methaemoglobin and sulphaemoglobin 240

Measurement of methaemoglobin 241

Screening method for sulphaemoglobin 242

Demonstration of carboxyhaemoglobin 242

Identification of myoglobin in urine 243

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:

1. Defects within red cells from dysfunction of enzyme-controlled metabolism, abnormal haemoglobins and 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 outside 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 (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.

Figure 11.1 Catabolic pathway of haemoglobin.

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.

Is There Evidence of Increased Haemolysis?

1. Hb estimation; 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. Test for increased unconjugated serum bilirubin and urinary urobilinogen excretion; measurement of haptoglobin or haemopexin

3. Detection of urinary Hb or haemosiderin.

What is the Type of Haemolytic Mechanism?

1. Direct antiglobulin test (DAT) with broad-spectrum antiserum

2. Osmotic fragility and glycerol lysis test

3. Measurement of Hb in urine; plasma Hb; Schumm’s test.

What is the Precise Diagnosis?

1. If a hereditary haemolytic anaemia is suspected:

a. Osmotic-fragility determination after 24 h of incubation at 37°C or eosin-5-maleimide (EMA) dye binding test; screening test for red cell glucose-6-phosphate dehydrogenase (G6PD) deficiency (formal assay if reticulocytosis 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 % Hb A₂ and % Hb F; electrophoresis or high-performance liquid chromatography for abnormal Hb; tests for sickling; tests for unstable Hb; blood count parameters, especially mean cell volume (MCV) and mean cell Hb (MCH); 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.

2. If an autoimmune acquired haemolytic anaemia is suspected:

a. Direct antiglobulin test using anti-immunoglobulin and anti-complement 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.

3. If the haemolytic anaemia is suspected of being drug induced:

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.

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).

5. In obscure cases:

a. Investigations for paroxysmal nocturnal haemoglobinuria (PNH) (e.g. acidified serum test [Ham’s test], sucrose lysis test, flow cytometric immunophenotyping for erythrocyte and neutrophil 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).

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 nm ¹ (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

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 venipuncture 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 ³

Reagents

Benzidine compound

Dissolve 1 g of 3,3′,5,5′-tetramethylbenzidine in 90 ml of glacial acetic acid and make up to 100 ml with water. The solution will keep for several weeks in a dark bottle at 4°C.

Hydrogen peroxide

Dilute 1 volume of 3% (‘10 vols’) H₂O₂ with 2 volumes of water before use.

Acetic acid

Use 100 g/l glacial acetic acid.

Standard

A blood sample of known Hb content is diluted with water to a final concentration of 200 mg/l. It is convenient to use an HiCN standard solution as the source of Hb.

Method

Add 20 μl of plasma to 1 ml of the benzidine reagent in a large glass tube. At the same time, set up a control tube, in which 20 μl of water are substituted for the plasma and a standard tube, containing 20 μl of the Hb standard. Add 1 ml of the H₂O₂ solution to each tube and mix the contents well.

Allow the mixture to stand at about 20°C for 20 min and then add 10 ml of the acetic acid solution to each tube; after mixing, allow the tubes to stand for a further 10 min. Compare the coloured solutions at 600 nm, using the colour developed by the control tube as a blank. If the Hb of the plasma to be tested is unusually high, it can be measured by the method used with whole blood (described later).

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 Method ⁶^,⁷

Principle

Known amounts of Hb are added to serum. The Hb–haptoglobin complex is separated by electrophoresis on cellulose acetate; the presence of bound and free Hb is identified in each sample and the amount of haptoglobin is estimated by noting where free Hb appears.

Reagents

Buffer (pH 7.0, ionic strength 0.05)

Na₂HPO₄.H₂O 7.1 g/l, 2 volumes; NaH₂PO₄.H₂O 6.9 g/l, 1 volume. Store at 4°C.

Haemolysates

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 three 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 concentrations 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) H₂O₂ 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 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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