Almost 300 beta (β)-globin gene mutations have now been characterized (http://globin.cse.psu.edu). Some mutations completely inactivate the β gene, resulting in the absence of β-globin production that leads to β0 thalassemia. Other types of mutations allow the production of some β globin and cause β+- or β++ (“silent”) thalassemia whereas β++ has more β globin production compared to β+. β0 / β+ /β++ thalassemia phenotype depends on the site and nature of the mutation.1 Therefore, the clinical and haematological spectrum of beta-thalassemia ranges from silent carrier to clinically manifested conditions, including severe transfusion dependent beta-thalassemia major and beta-thalassemia intermedia.2
The standard screening method for β-thalassemia includes full blood count (FBC), including the level of (MCV) < 80 fL and/or (MCH) <than 27 pg (being used as a cutoff level for a positive thalassemia screening result). The full blood picture (FBP) in thalassemia disease shows typical RBC morphology, consisting of microcytosis, hypochromia, and anisopoikilocytosis. Beta thalassemia major typically shows markedly elevated HbF (30–95%) levels with elevated HbA2. The proportion of HbA2 is dependent on the precise mutation of the β globin gene cluster.3,4 Therefore, the minimal deficit of β-globin production is not associated with any consistent haematological changes and are the limitation of standard screening method for β-thalassemia in carriers of very mild-or-silent types of β-thalassemia.3,4 Hence, the challenges faced during laboratory approaches and the importance of molecular genetic testing to confirm the diagnosis are discussed in this case report.
A 58-year-old male presented with symptomatic anaemia. Further history revealed that the patient has had anaemia since the age of 28 and on regular transfusion and follow-up at other hospital. However, bleeding did not manifest. The patient has no history of trauma or fever. The patient also has no family history of haematological disorders. The physical examination revealed pallor, jaundice and hepatosplenomegaly (liver 13 cm and spleen 16 cm below costal margin) but no lymphadenopathy.
Laboratory results at presentation showed haemoglobin (Hb) 5.9 g/dL, total leukocyte count 6.43 X 103/µL, MCV 48.9 fl, MCH15.4 pg MCHC31.5 g/dL and platelet count350X 109/L. Other investigations, including coagulation profile (activated partial thromboplastin time, prothrombin time and fibrinogen), liver and renal function tests were all within normal ranges. The peripheral blood smear showed hypochromic microcytic anaemia with anisopoikilocytosis, and many target cells. Occasional nucleated red blood cell. No eosinophilia or basophilia (Figure 1).
FBP stained with Wright Stain (X20 magnification)
Other investigations, including Hb analysis, high performance liquid chromatography (HPLC) showed A window (81.9%) with the presence of prominent peak at F (10.6%) and A2/E (7.5%) normal range is 2% to 3.2%. Alkaline gel electrophoresis showed prominent A2 band. The impression of Hb Analysis is a β thalassemia trait. The patient’s bone marrow aspirate (BMA) showed reactive changes and erythroid hyperplasia. There was no evidence of acute leukaemia and other haematological malignancy. Later, Multiplex Amplification Refractory Mutation System (M-ARMS) PCR revealed homozygous codon 19 mutation/Hb Malay.
Hb Malay was first described in 1989, being a β++ thalassemia phenotype with A → G mutation in codon 19, as detected in this case.5,6 The prevalence of Hb Malay in the Malaysian population was 5.5%.7 Homozygous Hb Malay usually presented with an average Hb of 7 to 8g/dL. Previously, it was reported that there was an increased production of Hb F between 9–25% in cases of homozygous Hb Malay and compound heterozygous Hb E/Malay.6 This was also seen in this case, where the Hb F level in homozygous Hb Malay was 10.6%. Hb Malay (5.5%) was detected in northeast Thailand8.
To date, many Hb variants have been discovered and can be detected by current screening methods for beta thalassemia; electrophoretic and HPLC methods. However, these techniques still have some limitations. It is because the available screening method is still unable to detect certain Hb variants with neutral substitutions.6,7 It is difficult to diagnose a variant causing silent β-thalassemia, especially heterozygous Hb Malay because the haematological parameters and Hb A2 levels remain within a normal range.9,10 Furthermore, as seen in this case, even though the Hb level is reduced, it is still challenging to confirm homozygous Hb Malay because both HPLC and capillary zone electrophoresis cannot differentiate between Hb A and Hb Malay. Hb Malay migrates as Hb A.6,7,8,9,10 Therefore, the definitive diagnosis of Hb Malay can only be made via molecular analysis; M-ARMS PCR. Based on this case, the presence of a variant causing silent β-thalassemia should be considered and emphasized in unexplained clinical presentation typical of thalassemia.6,7,8,9 Hence, it is a challenge or difficulty for the hospital or medical centre with no molecular technique facility to diagnose of Hb Malay. The hospital should therefore identify the nearest centre that has this service and send the sample to them for confirmation. Identification of this variant haemoglobin is important to prevent the birth of β-thalassemia major or intermedia children. Furthermore, for the couples at risk of conceiving a baby with β-thalassemia major or intermedia should be given genetic.6
In conclusion, well organized information, consisting of complete red cell indices and a Hb analysis result, together with a detailed history, including ethnic background, physical examination, and then followed up with molecular techniques such as M-ARMS PCR, can be used as a guideline for an effective tool in the investigation, detection, and confirmation of the diagnosis of Hb Malay. This is particularly important in the multi-ethnic populations of Malaysia as well as for proper clinical management of the patients.