A retrospective review of homozygous haemoglobin E patients.

Introduction

Haemoglobin E is a common [beta] chain haemoglobin (Hb) variant prevalent in South East Asia (SEA), resulting from a point mutation that creates an amino acid substitution of glutamic acid for lysine at position 26 ([[[alpha].sub.2][[beta].sub.2].sup.26Glu Lys]). The point mutation results in a false splicing site within an exon, leading to some mRNA transcripts being spliced abnormally, and a reduced rate of synthesis of [[beta].sup.E] chains. This reduced rate of synthesis is responsible for the thalassaemic red cell indices associated with the inheritance of Hb E.

Homozygous Hb E is usually an asymptomatic condition without anaemia, or any evidence of haemolysis (1). The blood film appearance is similar to beta thalassaemia trait, with hypochromia, microcytosis and the presence of target cells. On alkaline haemoglobin electrophoresis there is no Hb A, with the majority of the Hb migrating in the Hb [A.sub.2]/Hb E position (Figure 1). Haemoglobin F levels may be normal or elevated, with levels usually less than 5%. (1) On acid electrophoresis the Hb E band migrates in the Hb A position.

Hb E/[[beta].sup.0] thalassaemia demonstrates variable severity, ranging from a condition similar to [beta] thalassaemia minor to something approaching thalassaemia major. The blood count shows a hypochromic microcytic anaemia with the Hb varying from 25 to 130 g/L and an increased RDW (1). The blood film shows hypochromic microcytic red cells with varying degrees of anisocytosis and poikilocytosis, together with target cells and polychromasia. Basophilic stippling is often present and nucleated red blood cells are generally seen (2). On alkaline haemoglobin electrophoresis, there is an absence of Hb A with only haemoglobins E, [A.sub.2] and F present. The Hb F level is variably ranging from 5-87% (1).

Patients presenting with equivocal results may prove problematic in differentiating a diagnosis of homozygous Hb E from one of compound heterozygosity for Hb E and ?0 thalassaemia (Table 1).

Methods

For this review a computer search of the Canterbury Health Laboratory thalassaemia database was run using a diagnosis of homozygous Hb E as the search criteria. The database uses a Microsoft Access program, the haemoglobinopathy screen results, patient details and red cell indices together with a diagnosis are entered once investigation are complete. The search retrieved data on 43 patients with a diagnosis of homozygous Hb E.

Haemoglobin F quantitation was performed by either alkaline denaturation (3) or by high performance liquid chromatography (HPLC) on the Bio-Rad Variant II HPLC system using the Variant II-[beta] thalassaemia short program (Bio-Rad Laboratories, Hercules, CA, USA).

Results

On review of the data for each of the 43 patients diagnosed as homozygous Hb E, seven patients were found to have equivocal results (Table 2).

Patient 1 was reported as homozygous for Hb E with a comment that Hb E/[[beta].sup.0] thalassaemia could not be excluded, and family studies were suggested. Patient 2 was reported as homozygous Hb E with a comment that the abnormalities of the blood count and blood film seemed more severe than those normally associated with homozygous Hb E. On review, this patient appears to fit the criteria for Hb E/[[beta].sup.0] thalassaemia. Patient 3 was a 24 month old who was reported as homozygous Hb E with G6PD deficiency (confirmed by abnormal G6PD screening test and reduced assay level). At the time it was assumed that the Hb F of 15% was attributable to a combination of the patient's age and recovery from an oxidative haemolytic crisis. However, on review a diagnosis of Hb E/[[beta].sup.0] thalassaemia cannot be excluded. Patient 4 was only 11 months old at the time of testing, and the Hb F of 9% is quite possibly related to the age of the patient. This is probably a case of homozygous Hb E but Hb E/[[beta].sup.0] thalassaemia is not excluded. Patients 5,6 and 7 were all reported as homozygous Hb E but again a diagnosis of Hb E/[[beta].sup.0] thalassaemia cannot be excluded.

Discussion

In each of the seven cases the Hb F level is somewhat less than that usually associated with Hb E/[[beta].sup.0] thalassaemia, but is at the upper limit or slightly higher than that expected for homozygous Hb E. Atypical Hb F levels, together with an increased RDW, make a definitive diagnosis difficult. For some cases, blood film results or clinical findings were not available. No information was available on the iron status of these patients. Additional clinical information or the opportunity to investigate other family members, could have been helpful in establishing a definitive diagnosis.

Homozygous Hb E is a clinically benign disorder, the diagnosis of which is usually straightforward. However, patients may present with Hb F levels that overlap those seen in Hb E/[[beta].sup.0] thalassaemia. It now appears that although the Hb F level in homozygous Hb E usually less than 5% it may comprise up to 15% of the total Hb (1). In Hb E/[[beta].sup.0] thalassaemia the Hb F level is usually 30-60% but the range is extremely variable with levels from 5-87% being documented (1). The variability in the Hb F level highlights the importance of considering Hb E/[[beta].sup.0] thalassaemia in the differential diagnosis. Before making a diagnosis of homozygous Hb E the Hb F level needs careful consideration. In cases where the Hb F level approaches that seen in Hb E/[[beta].sup.0] thalassaemia, it may be necessary to consider other laboratory and clinical information in the differential diagnosis. Full blood count and blood film findings should be reviewed with particular emphasis on the RDW and red cell morphology. Clinical details may indicate other reasons for any anaemia, or may confirm a history of anaemia, jaundice or hepatosplenomegaly. In these cases investigation of other family members can provide important information regarding the inheritance and segregation of Hb E or beta thalassaemia alleles within the family. Sequencing of the globin gene may prove helpful in detecting a beta thalassaemia mutation.

It is important to recognise the difference between the clinically benign homozygous Hb E and the more severe condition of Hb E/[[beta].sup.0] thalassaemia. A correct diagnosis of Hb E/[[beta].sup.0] thalassaemia is important, not only for disease monitoring and treatment for the patient, but also in regard to reproductive choices, especially in the context of a partner who also carries a gene for either Hb E or [beta] thalassaemia.

References

(1.) Bain BJ. Haemoglobinopathy Diagnosis (2ndEd). Blackwell Science, Oxford, 2005: 204-8.

(2.) Weatherall D, Clegg JB. The Thalassaemia Syndromes. Blackwell Science, Oxford, 2001: 435.

(3.) Molden DP, Alexander NM, Neeley WE. Fetal hemoglobin: optimum conditions for its estimation by alkali denaturation. Am J Clin Path 1982; 77: 568-72.

Jaine M Duncan, Dip MLT, Medical Laboratory Scientist Haematology, Canterbury Health Laboratories, Christchurch

Address for correspondence: Jaine Duncan, Haematology, Canterbury Health Laboratories, P.O Box 151, Christchurch. Email: [e-mail]

Table 1. Differential diagnosis of homozygous Hb E and
Hb E/[beta][degrees] thalassaemia.

                                          Hb E/[beta][degrees]
                      Homozygous Hb E     thalassaemia

Hb (g/L)              normal              25-130

RDW                   normal              increased

Blood film            hypochromia         hypochromia
                      microcytosis        microcytosis
                      target cells        anisocytosis
                                          poikilocytosis
                                          target cells
                                          polychromasia
                                          often basophilic
                                          stippling
                                          usually NRBC

Hb F (%)              usually < 5         5-87

Reticulocytes (%)     usually normal      4-6

Table 2. Laboratory results for patients with an equivocal
diagnosis of homozygous Hb E.

              Hb (g/L)     MCV (fL)     MCH (pg)     RDW

Patient 1     113          72           24

Patient 2     94           56           17.4         25.7

Patient 3     101          63           20.4         20.7

Patient 4     87           57           18.3         21

Patient 5     127          67           20.7         18.3

Patient 6     107          65           21.1         16.7

Patient 7     89           65           21.3         16.3

              Film comment       Hb F (%)     Comment

Patient 1                        8

Patient 2     poikilocytosis     9.6
              anisocytosis
              target cells
              spherocytes
              fragments
              NRBC

Patient 3     target cells       15           24 month
              spherocytes                     old, G6PD
              polychromasia                   deficient
              blister cells

Patient 4                        9            11 month
                                              old

Patient 5                        5.7

Patient 6     target cells       4.8
              polychromasia
              spherocytes

Patient 7                        5            pregnant