Another Exotic Cause of Haemolytic Anemia
Dr Wai-Chiu Tsoi
Haematology Laboratory and Blood Bank,
Prince of Wales Hospital
A Chinese boy initially presented at 3 months old because of symptoms of anaemia. His parents and two siblings were normal. Physical examination showed jaundice and hepatosplenomegaly. Complete blood counts are depicted in Table 1. Red cell morphology revealed moderate polychromasia, anisocytosis, a few target cells and moderate number of irregularly contracted cells (Fig. 1). He received blood transfusion on three occasions. Splenectomy was performed on account of hypersplenism and thrombocytopenia at four years of age. His haemoglobin level was stable at around 10-11 g/dl and platelet count returned to normal in the years postsplenectomy. Later, this child had two episodes of transient left side weakness attributable to transient ischemic attack of the central nervous system. Findings of a MRI angiography of the brain were in keeping with moyamoya disease. The patient was put on aspirin for prophylaxis of thrombosis.
Table 1 Hematological data: the complete blood count of the patient was taken at 9 years old when the hemoglobin level was stable.
Further laboratory investigations:
A screening test for unstable haemoglobin on the patient's haemolysate by n-butanol test was positive (Fig. 2), and 25% of the hemoglobin precipitated after 10 minutes at 56 oC (normal control: 5%) by heat stability test. Heinz bodies were observed when the red cells were treated with brilliant cresyl blue (Fig. 3). Globin chain synthesis experiment showed that the relative synthetic rate for a-chain to b-chain (a/b) was 1.22. Further evaluation by starch gel electrophoresis revealed Hb A, Hb A2 and Hb X; the latter appeared as a diffuse band between Hb A and Hb S that accounted for approximately 5% of the haemoglobin. The use of globin chain electrophoresis in polyacrylamide gel in the presence of urea, acid and Triton X showed a pattern identical with that of Hb A, i.e., aA + bA. Separation of haemoglobin by a high performance liquid chromatography (HPLC) system which employed a weak cation exchanger (PolyCATA column), Bis-Tris buffer and sodium acetate gradients revealed Hb A1c (2.1%), Hb A1d (4.7%), Hb A0 (88%), Hb A2 (4.6%) and no abnormal haemoglobin variants were detected. However, the bX chain could be separated from the bA chain by reversed phase HPLC using Vydac C4 column and the recovery ratio of bX : bA was 22.4 : 77.6.
Molecular studies by PCR showed a GCT (for alanine)®CCT (for proline) point mutation at codon 138 (Fig. 4).
Hb Brockton [a2b2138(H16)ala®pro], an unstable haemoglobin
Unstable haemoglobins are a group of structurally abnormal haemoglobin variants showing molecular instability. The extent of the instability may vary from subclinical in-vitro findings to severe congenital Heinz body haemolytic anaemias; and if the proteolysis of the extremely unstable globin chains takes place in the bone marrow, this may produce a thalassaemic phenotype. There are many mutations associated with unstable haemoglobins, mostly by single amino acid replacements affecting certain key areas of the tertiary haemoglobin structure, such as the haem pocket, intersubunit contacts and the globin interior.
Unstable haemoglobin follows an autosomal dominant pattern of inheritance. A significant proportion of cases are due to de novo mutations without evidence of unstable haemoglobin in the parents and other family members. The unstable haemoglobins have a greater tendency to spontaneous oxidation to methaemoglobin and subsequent haemichromes formation and precipitation. The latter can be recognized as Heinz bodies attaching to the interior red cell membrane by hydrophobic interactions, after supervital stains with brilliant cresyl blue (Fig. 3). These Heinz body-containing red cells are less deformable, pliable and filterable, and are easily entrapped in the spleen microcirculation. Moreover, autologous antibodies, that recognize modified membrane proteins, can bind to red cells containing Heinz bodies, and these cells are then partially or completely removed from the circulation by macrophages in the liver and the spleen. These processes are significantly accelerated by increased temperature and exposure to oxidative substances, such as superoxide, hydrogen peroxide and hydroxyl radicals, which may apply during infections.
The heat and isopropanol precipitation tests are simple qualitative screening methods for the detection of unstable haemoglobin. They may also be applied to quantitate the amount of labile haemoglobin present, by spinning down the precipitated haemoglobin and measuring the concentration of the supernatant haemoglobin. The more sophisticated method of DNA analysis for the identification of the globin mutations by selective amplification and direct sequencing of globin genes now provides a useful and precise approach to the characterization of these mutations.
Of interest, HbA2 levels are increased in many cases of unstable haemoglobin, that may lead to a misdiagnosis of heterozygous b-thalassaemia, as in this patient. Therefore, remember to include unstable haemoglobin as a differential diagnosis when an elevated HbA2 concentration is encountered during interpretation of haemoglobinopathy studies.
In this patient, Hb Brockton is an unstable haemoglobin variant, arising from a single amino acid replacement, the substitution of proline for the alanine residual in the H-helix, rendered it unable to form a buried hydrogen bond between Pro 138b and Val 134b, and resulted in considerable instability. However, Hb Brockton exhibits normal oxygen-binding property.
Tsoi WC, Li CK, Plaseska-Karanfilska D, Efremov GD. Hb Brockton [a2b2138(H16)Ala®Pro] observed in a Chinese boy. Hemoglobin 1998; 22(4): 397-400.
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