The Laboratory Diagnosis of Haemoglobinopathies*,†

British Journal of Haematology, 1998, 101, 783–792 Guideline THE LABORATORY DIAGNOSIS OF HAEMOGLOBINOPATHIES*,† SUMMARY (ii) to explain a haematological abnormality such as anaemia or microcytosis; (iii) to identify an abnormality in The laboratory diagnosis of haemoglobinopathies, including the presymptomatic phase, as in neonatal screening; (iv) to the thalassaemias, is of growing importance, particularly predict serious disorders of globin chain synthesis in the fetus because of an increasing requirement for antenatal diagnosis and offer the option of termination of pregnancy; (v) to of significant disorders of globin chain synthesis. This permit genetic counselling of prospective parents; (vi) as guideline discusses the laboratory tests which are most preoperative screening for the presence of sickle cell useful in the diagnosis of haemoglobinopathies and describes haemoglobin. Improved fully automated systems and their role in specific clinical circumstances. Of the newer reagents for techniques such as high-performance liquid technical methods, high-performance liquid chromatogra- chromatography (HPLC) and isoelectric focusing (IEF) have phy (HPLC) is of considerable importance whereas isoelectric led to their introduction in many laboratories. Immunolo- focusing (IEF) and immunoassay for variant haemoglobins gical methods for the identification of variant haemoglobins have a more minor role. have also become available. There is therefore a need for an Specific recommendations have been formulated for updated guideline defining the role of new techniques in testing in relation to genetic counselling and for neonatal relation to traditional techniques. To save repetition, diagnosis. Methods used in specialized laboratories for fetal previous guidelines (Globin Gene Disorder Working Party diagnosis have been tabulated. Genetic counselling requires: of the BCSH General Haematology Task Force, 1994; (i) identification of haemoglobins S, C, D-Punjab, O-Arab, E, The Thalassaemia Working Party of the BCSH General Lepore and H, and (ii) the detection of carriers of aЊ and b Haematology Task Force, 1994) should be consulted. This thalassaemia. It is recommended that subjects of all ethnic guideline discusses techniques and defines their place in groups be screened for b-thalassaemia trait, all except specific diagnostic settings. The detection of unstable Northern European Caucasians for variant haemoglobins, haemoglobins, methaemoglobins and high oxygen affinity and selected ethnic groups for aЊ-thalassaemia trait. Testing haemoglobins is not discussed but laboratories should either for b-thalassaemia trait should be carried out when the have methods for detecting these variant haemoglobins or mean cellular haemoglobin (MCH) is < 27 pg and testing for should refer such samples to a reference laboratory. aЊ-thalassaemia trait should be considered when the MCH is It should be noted that the identification of haemoglobins < 25 pg. Appropriate methods include HPLC or haemoglobin is often presumptive, based on a characteristic electro- electrophoresis for identification of variant haemoglobins phoretic mobility or other characteristics in an individual of and HPLC or microcolumn chromatography for appropriate ethnic origin. Definite identification usually quantification of haemoglobin A2. requires DNA analysis or amino acid sequencing. Family studies are also of considerable importance in elucidating the INTRODUCTION nature of disorders of haemoglobin synthesis. Disorders of globin chain synthesis, both thalassaemias and structurally abnormal haemoglobins, are common in the TECHNIQUES U.K. and constitute a significant public health problem. Diagnosis may be required: (i) to confirm a provisional Full blood count diagnosis such as sickle cell disease or b-thalassaemia major; A full blood count (FBC) is usually indicated in all individuals being investigated for a suspected disorder of globin chain * Prepared by a Working Party of the General Haematology Task synthesis. The exception is in neonatal screening. Red cell Force of the British Committee for Standards in Haematology. indices are of particular importance in screening for b- Members of the Working Party: Dr B. J. Bain, Dr R. J. Amos, Dr D. and aЊ-thalassaemia trait and in distinguishing between Bareford, Dr C. Chapman, Professor S. C. Davies, Dr J. M. Old and db-thalassaemia and hereditary persistence of fetal Dr B. J. Wild. Members of the General Haematology Task Force: haemoglobin. Dr R. J. Amos, Dr B. J. Bain (Secretary), Dr I. Cavill, Dr C. Chapman, Dr K. Hyde, Dr E. Matutes, Dr J. Reilly (Chairman), Dr J. K. Wood. When are further tests indicated? A blood film is useful, in † Although the advice and information contained in these addition to a blood count, when an unstable haemoglobin, guidelines is believed to be sound and accurate at the time of sickle cell disease or thalassaemia is suspected. A reticulocyte going to press, neither the authors nor publishers can accept liability count is indicated if haemolysis is likely, e.g. if an unstable for any errors or omissions. haemoglobin, haemoglobin H disease or sickle cell disease is Correspondence: The Secretary, British Society for Haematology, suspected. In addition, definitive tests to identify normal and 2 Carlton House Terrace, London SW1Y 5AF. variant haemoglobins are indicated in suspected ᭧ 1998 Blackwell Science Ltd 783 784 Guideline haemoglobinopathies, regardless of the results of the full haemoglobin A2 is not elevated) and also when hereditary blood count and film. persistence of fetal haemoglobin (HPFH) is suspected. Haemoglobin electrophoresis on cellulose acetate at alkaline pH Haemoglobin electrophoresis on citrate agar or agarose gel (pH 8·2–8·6) at pH 6·0–6·2 Cellulose acetate electrophoresis enables the provisional Electrophoresis at acid pH on citrate agar or appropriate identification of haemoglobins A, F, S/G/D, C/E/O-Arab, H agarose gel is usually used as a supplement to cellulose and a number of less common variant haemoglobins. With acetate electrophoresis at alkaline pH. There are differences good electrophoretic techniques, haemoglobin F levels >2% in the relative mobilities of variant haemoglobins between can be recognized visually; when an increased level is citrate agar and agarose gel. Both techniques distinguish detected, quantification is required. Good techniques also haemoglobin S from D/G but do not distinguish between enable a split A2 band to be recognized. This is useful in most types of D and G. They will distinguish haemoglobin C helping to distinguish a-chain variants, e.g. haemoglobin G from E, C-Harlem and O-Arab. Philadelphia, from b-chain variants, e.g. haemoglobin D Electrophoresis at acid pH is indicated in the investigation Punjab. It is also essential if b-thalassaemia trait is to be of suspected high-affinity haemoglobins even when electro- diagnosed in individuals who also have a d-chain variant (see phoresis at alkaline pH is normal, since some high-affinity below). haemoglobins have abnormal mobility at acid pH but normal Haemoglobin A2 can be quantified by cellulose acetate mobility at alkaline pH. electrophoresis followed by elution and spectrometry, but this When are further tests indicated? Further tests are needed to is a labour-intensive technique if large numbers of samples distinguish haemoglobins D and G from each other (see require testing. Quantification of haemoglobin A2 by above). A sickle solubility test is indicated when mobility at scanning densitometry is not recommended as the precision acid pH suggests haemoglobin C-Harlem, a variant haemo- is not good enough for the diagnosis of b-thalassaemia trait globin in which the sickle mutation is one of two mutations, (ICSH, 1978). rather than haemoglobin C. A sickle cell solubility test is Variant haemoglobins, including haemoglobin S, can be similarly indicated if electrophoretic mobility suggests the quantitated by scanning densitometry (Legg et al, 1995). presence of one of the other variant haemoglobins in which However, it is necessary to have good separation of bands the sickle mutation is one of two mutations. Performing a and it should be noted that this method is imprecise if the sickle solubility test when any variant haemoglobin is concentration is low, e.g. haemoglobin F can be quantified detected (World Health Organization, 1994) will avoid with reasonable precision only when it is appreciably missing any such cases. elevated. Quantification of variant haemoglobins can be diag- nostically useful, e.g. in helping to distinguish haemoglobin Quantification of haemoglobin A2 by microcolumn Lepore from heterozygosity for haemoglobins S/D/G and in chromatography the differential diagnosis of sickle cell/bþ thalassaemia (Sbþ This is a satisfactory method for quantification of haemo- thal) and sickle cell anaemia (SS). globin A2 for the diagnosis of b-thalassaemia trait. Special When are further tests needed? Patients who show a band columns are required for haemoglobin A2 quantification in with the mobility of haemoglobin S require a sickle solubility the presence of haemoglobin S, but it should be noted that test (or alternative) to confirm the presence of haemoglobin this test is not essential for diagnosis since Sbþ thal can be S; if this is negative an alternative technique to identify distinguished from sickle cell trait (AS) on the basis of haemoglobins D and G is needed. Those with a single band haemoglobin S comprising a higher proportion of total with the mobility of haemoglobin S require not only a sickle haemoglobin

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