haemoglobinopathies. The latter included 3 beta thalassaemia trait, 3 Hb. S trait, one each for Hb E trait and Hb C trait, and 2 heterozygous for delta globin chain ...
Alpha thalassaemia screening using an immunochromatographic method to detect Hb Bart’s Carolyn Bunkall1, Nikhil Ghallyan2, Catherine Elliott1, Neil van de Water2, George Chan1 Departments of Haematology1 and Diagnostic Genetics2, Laboratory Services, Auckland City Hospital, Auckland, New Zealand.
Department of Pathology and Laboratory Medicine, Auckland City Hospital
Results
Introduction Alpha thalassaemia due to mutation, usually deletion, of the alpha globin gene/s on Chromosome 16 is ubiquitous, with global incidence of 26.5% for alpha-plus thalassaemia mutations and 0.3% for alpha-zero thalassaemia carriers. Alpha-zero thalassaemia however is found mainly in people from the WHO South East Asia and Western Pacific Regions, with incidence of around 4% to 5% in Mainland Southeast Asia (Indochinese peninsula) and southern China1. The clinical severity ranges from asymptomatic carrier state (single-gene alpha thalassaemia carrier) through non-transfusion dependent thalassaemia (two-gene defect alpha thalassaemia in cis- or trans-, and HbH Disease) to severe anaemia incompatible with survival (Hb Bart’s hydrops foetalis). Diagnosis of individuals with alpha thalassaemia mutation is important for proper management of affected individuals and genetic counselling and diagnosis of severe cases. In alpha thalassaemia the reduced production of alpha globin chain results in an excess of non-alpha globin chains, with beta globin chains being the predominant excess globin chain and only very small amounts of excess gamma and delta chains in post-natal life. The excess beta globin chains tetramerise to form HbH inclusions. Scanning red cells for HbH inclusion bodies (HbH-i) after supravital staining is the conventional and most common way to screen for alpha thalassaemia problems. The HbH-i staining however is not standardised across laboratories with different dyes, blood:dye ratio and incubation time used2. Detection of small numbers of HbH inclusions in alpha thalassaemia traits requires operator skill and time, hence relatively expensive. The excess gamma chains tetramerise to form Hb Bart’s, but in postperinatal life the minute amount of Hb Bart’s is difficult to detect. Recently an immunochromatographic test (ICT) to identify the small amount of Hb Bart’s for alpha thalassaemia diagnosis became available. This study assessed the suitability of ICT for alpha thalassaemia screening.
Material and Methods The ICT is a strip test for alpha thalassaemia screening (iLAB alpha-thal, i+Med Laboratories, Bangkok, Thailand). The whole blood haemolysate applied to a test strip diffuses through the absorbent area containing anti-Hb Bart’s-colloidal gold conjugate which forms an antibody-antigen complex with Hb Bart’s in the specimen. This complex binds to the anti-Hb Bart’s antibody in the test area to form a coloured band. The unbound antiHb Bart’s conjugate is captured by the goat-anti-mouse antibody further down the reaction tract to give a band in the control area. The absence of a band in the test region indicates a negative result (Figure 1). Residual specimens from 58 cases for haemoglobinopathy study were tested with the ICT. These included 8 with normal haemoglobin concentration and MCV/MCH, 40 with thalassaemia-like red cell indices but no beta haemoglobinopathy and normal iron store, and 10 cases with other haemoglobinopathies. The latter included 3 beta thalassaemia trait, 3 Hb S trait, one each for Hb E trait and Hb C trait, and 2 heterozygous for delta globin chain variants. Alpha thalassaemia multiplex PCR were done on 40 cases using the method of Tan ASC et al3 and the 3.7 type III deletion by a modification of the method of Dode C et al4. The molecular study was done on 2 of the 8 cases with normal red cell indices, 34 of the 40 with abnormal indices and no beta haemoglobinopathy, and 4 of the 10 with other haemoglobinopathies. The ICT results were compared with the HbH-i and molecular findings. Figure 1. Principle of the ICT for Hb Bart’s. Insets: Photograph of the actual strips.
Acknowledgement: The iLab alpha-thal kits for this study were provided free of charge by Biospecifix Pty Ltd for routine reagent kit assessment.
The results of the ICT and HbH-i tests and the alpha thalassaemia genotyping are summarised in Table 1. The ICT is more sensitive than HbH-i in detecting an alpha thalassaemia condition, and neither test gave positive result on cases with normal red cell indices but without an alpha thalassaemia mutation. There were 36 cases with alpha thalassaemia mutations. Their detail genotypes and the sensitivity of ICT and HbH-i in detecting these mutations are shown in Table 2. Table 1. Overall findings by haemoglobinopathy status N = α-thal genotyping done/positive ICT+ HbH-i + 8 2/0 0 0 34 34/34 25 11 6 0 2 0
Normal indices Genotype confirmed α-thal Thal-like indices, no other haemoglobinopathies Other haemoglobinopathies
10
4/2*
0
0
* Heterozygous alpha-plus thal
Table 2: Performance of ICT and HbH-i by α-thal genotypes Genotype Alpha-plus thal: Heterozygous -α3.7 (III) -α3.7 (I or II) -α4.2 Homozygous -α3.7 (III) -α3.7 (I or II) Alpha-zero thal Heterozygous: --SEA --FIL HbH Disease TOTAL
N= 14
14
ICT+ 5 8 1
11 3
7 6 1 1 36
HbH-i +
6 (43%) 1 (20%) 4 (50%) 1 (100%) 11 (79%) 9 (82%) 2 (67%)
1 (6%)
7 (100%)
7 (100%)
6 (100%) 1 (100%) 1 25 (69%)
0 (0) 1 (17%) 0 (0) 2 (14%) 1 (9%) 1 (33%)
6 (100%) 1 (100%) 1 11 (31%)
Conclusion This study showed that in confirmed alpha thalassaemia mutation carriers with or without other haemoglobinopathies, the ICT has a higher overall detection sensitivity of 69% compared to 31% using HbH-i. Further analysis of the 36 genotypically confirmed alpha thalassaemia mutations showed ICT has a detection sensitivity of 43% for heterozygous and 79% for homozygous alpha-plus thalassaemia. These are much higher than the 6% and 14% by HbH-i in our laboratory. The HbH-i sensitivity for alpha-plus thalassaemia detection in our laboratory is low compared to the reported sensitivities of 40-60 % for alpha-plus thalassaemia carrier whether in heterozygous or homozygous state5,6. Using the reported HbH-i sensitivities as the reference ICT still has a comparable, if not higher sensitivity in detecting alphaplus homozygosity and the sensitivity for alpha-plus heterozygosity at the lower end of the range. The alpha-plus thalassaemia mutation is considered clinically and genetically harmless, particularly when present in heterozygous state, and not detecting them is therefore not a disadvantage. Both the ICT and HbH-i can reliably detect alpha-zero thalassaemia mutations with sensitivity of 100%. There were two ICT+ cases with hypochromic microcytic red cells and HbH-i negative. The alpha thalassaemia status of these two cases has not been confirmed by molecular study. These subjects however had normal iron store. It is therefore most likely that these positive ICT results reflected true alpha thalassaemia carrier state than being false positive. The ICT for alpha thalassemia detection is objective, requiring less operator time and skill and no special equipment, and has higher sensitivity and specific than HbH-i. It is therefore suitable for alpha thalassaemia screening in most clinical laboratories. References 1. UCL Centre for Health Informatics & Multiprofessional Education (CHIME). Modell’s Haemoglobinopathologist’s Almanac 2008. < http://www.modell-almanac.net/world.pdf >. Accessed 12 April 2016. 2. Kwan CS, Todd D. The clinical significance of occasional red-cells with Haemoglobin H inclusions. Journal of the Hong Kong Medical Technologist Association, 1977; 2:7-12. 3. Tan ASC, Quah TC, Low PS, Chong SS. A rapid and reliable 7-deletion multiplex polymerase chain reaction assay for α-thalassemia. Blood 2001;98:250-251. 4. Dode C, Krishnamoorthy R, Lamb J, Rochette J. Rapid analysis of -α3.7 thalassaemia and α α α 3.7 triplication by enzymatic amplification analysis. Br J Haematol 1992;82:105-111. 5. Galanello R, Paglietti E, Melis MA, Giagu L, Cao A. Haemoglobin inclusions in heterozygous alpha-thalassaemia according to their alpha-globin genotype. Acta Haemat 1984; 72:34-36. 6. Skogerboe KJ, West SF, Smith C, Terashita T, LeCrone CN, et al. Correlation of Hemoglobin H inclusion bodies with DNA-determined genotype. Arch Pathol Lab Med 1992; 116: 1012-1018.
