Proposed Topic (Most preferred): :
HA Young Investigators Session (Projects to be presented by HA staff who had joined HA for 10 years or less)
Proposed Topic (Second preferred): :
Research and Innovations (new projects / technology / innovations / service models)
Authors (including presenting author) :
Lam WK(1), Law YFW(1), Wong TF(1), Woo HKV(1), Wu HHA(2), Chow YDE(2), Lau KN(1), Wong CCA(1), Yip SF(1)
Affiliation :
(1)Department of Clinical Pathology, Tuen Mun Hospital, (2)Department of Pathology, United Christian Hospital
Introduction :
The α- and β-thalassaemias are the most common inherited single-gene disorders in the world. The prevalence of a-thalassaemia is 5% in Hong Kong, in which 90% of the patients carry SEA type of deletion. The screening of α-thalassaemia is commonly based on the haemoglobin (Hb) H inclusion test as a screening test, which is a labour-intensive and time-consuming test. Detection of β-thalassaemia can be shown by an elevated Hb A2 level on cation-exchange high-performance liquid chromatography (HPLC). However, the use of HPLC for the detection of α-thalassaemia has not been described before. We described the novel finding of an early eluting peak at about 0.24 minutes of the chromatogram in Variant II® HPLC, which is commonly observed in our local α-thalassaemia trait patients but is not present in normal individuals.
Objectives :
We aim to investigate the use of the novel α-thalassaemia early eluting peak (αEEP) observed before the first minute of elution in high-performance liquid chromatography as a potential alternative for α-thalassaemia screening compared with HbH inclusion test in our population.
Methodology :
303 archived blood samples for haemoglobin pattern study were analysed for the presence or absence of αEEP and HbH inclusions with blinded observers. The concordance of the Hb H inclusion test and the αEEP was reviewed in each case. Whenever the results of the αEEP and the Hb H inclusion body test were discrepant, additional molecular analysis would be performed to determine the patient’s α-globin genotype if the blood sample was sufficient for testing. In cases where the αEEP was detected but the initial Hb H inclusion body test result was negative, a second search for the Hb H inclusion bodies was done by a trained observer on the original slides prepared.
In addition, all the patients with the haemoglobin pattern study conclusion of “α-thalassaemia trait cannot be excluded” were tested for the α-globin genotyping if the remaining blood sample was sufficient for testing. 20 random samples of α-thalassaemia trait were also tested for the α-globin genotyping. Moreover, genotyping study results previously performed for special clinical need (e.g. antenatal testing) were also reviewed. The performance characteristics of αEEP and HbH inclusion test in detecting the α-thalassaemia trait with SEA type deletion were compared.
Result & Outcome :
The concordance rate of αEEP and HbH inclusion body test was 96.0% (κ=0.921, p< 0.001). 6 cases with negative HbH inclusion test but positive αEEP showed heterozygous Southeast Asian (SEA) type deletion by genotyping study. Among the 4 cases with negative αEEP but positive HbH inclusion test, 2 cases showed heterozygous SEA type deletion,1 case showed nondeletional HbQS mutation and 1 case showed no mutation. The performance of αEEP for detecting SEA deletion (sensitivity 93.8%, specificity 100%) was superior to the HbH inclusion test (sensitivity 81.3%, specificity 95.2%). Inter-observer assessment of the αEEP showed perfect agreement. Staff operation time of the αEEP (10 minutes per 100 cases) was significantly shorter than the HbH inclusion test (1800 minutes per 100 case).
The novelly-described αEEP was found to be a more sensitive and specific method than the HbH inclusion test in detecting the reproductively significant α-thalassaemia trait with SEA type deletion. The αEEP could be a potential alternative of HbH inclusion test for α-thalassaemia screening, with over 98% reduction in reagent and labour cost.
