Case presentation

A 20 year ago man presented to A&E department in July 2001 with repeated vomiting for 10 days. In 1996, he was diagnosed to have non A-C hepatitis, thalassaemic trait, mitral valve prolapse and polycythaemia rubra vera in Princess Margaret Hospital. He is a smoker of half a pack of cigarette per day.

Physical examination revealed no liver or spleen enlargement..

Investigations

Complete blood counts
Hb 18.4 g/dL, Hct 0.609, RBC 8.28 x 10¹²/L, MCV 73.4 fL, Plt 186 x 10⁹/L, WBC 8.4 x 10⁹/L

Arterial blood gas
PH 7.358, pCO₂ 5.38 kPa, pO₂ 14.95 kPa, HCO₃ actual 22.7 mmol/L, Base Excess -2.2 mmol/L, O₂ saturation 97.9

Red cell volume
62.1 ml/kg (Ref. M: 25 - 35 ml/kg)

Marrow examination showed mainly microcytic, hyperplastic erythropoiesis with normal granulopoiesis and megakaryocytopoiesis with normal iron store.. As the patient's age and other findings are not typical of polycythaemia vera, secondary causes of polycythaemia, especially those due to high affinity haemoglobin was sought. As we cannot perform P₅₀ and oxygen dissociation curve in our laboratory, haemoglobin electrophoresis was done to see if there is any haemoglobin variant with abnormal electrophoretic mobility.

Haemoglobin electrophoresis at alkaline pH showed absence of Hb A and an abnormal band with mobility between Hb F and Hb S.

Haemoglobin electrophoresis at acid pH showed an abnormal band moving towards the cathode.

Hb quantitation by HPLC showed absence of Hb A, Hb A2 6.4%, Hb F 18% and a haemoglobin variant in the D-window, which accounted for 75.4%.

DNA analysis of the patient showed that the patient is compound heterozygous for Hb Tak and IVSII-654 (C to T) beta-zero thalassaemia mutation

Family study results:

Discussion

The presence of a haemoglobin variant with high oxygen affinity would usually lead to polycythaemia, unless the high-affinity haemoglobin is also very unstable, e.g. Hb Koln. In fact, about one-third of unstable haemoglobins show an increased oxygen affinity. However, since the dominant feature is haemolysis rather than polycythaemia, they are categorized as unstable haemoglobin rather than as high-affinity haemoglobin.

High-affinity haemoglobin should be suspected in young patients with polycythaemia or patients with family history of polycythaemia. However, as people with high-affinity haemoglobin and polycythaemia are usually asymptomatic, they may be discovered later in life. In such cases, clinical and laboratory findings may give clues to the underlying cause of the polycythaemia. In patients with polycythaemia rubra vera (PRV), about 70% have palpable splenomegaly and 40% have hepatomegaly. Also in PRV, the peripheral blood usually shows mild leucocytosis due to neutrophila with absolute basophilia. The marrow usually shows marked erythroid hyperplasia often together with granulocytic and megakaryocytic hyperplasia and the megakaryocytes on average are large in size with deeply lobulated nuclei. Patients with absence of hepatosplenomegaly and the typical peripheral blood and marrow findings as described above should raise the suspicion that the polycythaemia may not be due to PRV.

More than 200 high-affinity haemoglobin variants have been reported since 1966. [1] Haemoglobin electrophoresis at alkaline pH sometimes, but not always, shows a haemoglobin variant with abnormal electrophoretic mobility. Electrophoresis at acid pH should be carried out even if electrophoresis at alkaline pH is normal as sometimes a haemoglobin variant is revealed only at acid pH. An oxygen dissociation curve and P50 measurement should be carried out whenever there is suspicion of a high-affinity haemoglobin, even if no electrophoretically abnormal variant or HPLC abnormality has been detected. However, these tests are not available in most clinical laboratories. There is an article that described the use of a simplified technique in the detection of haemoglobin variants with altered oxygen affinity.. Measurement of the pH, oxygen tension, and oxygen saturation of antecubital venous blood may permit an estimate of the strength of oxygen binding to hemoglobin. An equation can then be used to convert the venous oxygen tension (standardized to pH 7.4) and the oxygen saturation to the P50 of the oxygen-hemoglobin dissociation curve on which the observed point falls. [6]

Hb Tak results from an insertion of the dinucleotide CA into codon 147 [TAA® (AC)TAA], which abolishes the normal stop codon (TAA) at position 147, leading to an elongation of the beta chain by 11 amino acids - (147)THR-Lys-Leu-Ala-Phe-Leu-Leu-Ser-Asn-Phe-(157)Tyr-COOH.

The following DNA sequencing gel shows the molecular defect of Hb Tak.

The P50 of Hb Tak is very low and is not influenced by the addition of 2,3, diphosphoglycerate. [2]. Haemoglobin Tak has been described in patients of Malaysian [3], Thai [4], and Cambodian [5] descent.

It is interesting to note that the patient, compound heterozygous for Hb Tak and beta-zero thalassaemia, has higher haemoglobin level than his father, who is heterozygous for Hb Tak. This is understandable as the co-inheritance of the beta thalassaemia gene leads to absence of Hb A with high levels of Hb Tak and Hb F, both of which are high-affinity haemoglobins.

References

1. Wajcman H, Galacteros F. Abnormal hemoglobins with high oxygen affinity and erythrocytosis. Hematol Cell Ther 1996 Aug 38:305-12.

2. Imai K, Lehmann H. The oxygen affinity of haemoglobin Tak, a variant with an elongated beta chain. Biochim Biophys Acta 1975 Dec 412:288-94.

3. Lie-Injo LE, Randhawa ZI, Ganesan J, Kane J, Peterson D. Hemoglobin Tak in a newborn Malay. Hemoglobin 1977 1:747-57.

4. Fucharoen S, Fucharoen G, Sae-ung N, Sanchaisuriya K, Fukumaki Y. Molecular and haematological characterization of Hb Tak and Hb Pyros in Thailand. Southeast Asian J Trop Med Public Health 1997 28 Suppl 3:110-4.

5. Hoyer JD, Wick MJ, Thibodeau SN, Viker KA, Conner R, Fairbanks VF. Hb Tak confirmed by DNA analysis: not expressed as thalassemia in a Hb Tak/Hb E compound heterozygote. Hemoglobin 1998 Jan 22:45-52.

6. Lichtman MA, Murphy MS, Adamson JW. Detection of mutant hemoglobins with altered affinity for oxygen. A simplified technique. Ann Intern Med 1976 May 84:517-20.