BackgroundPrevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia.MethodsSingle-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and β-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous β-thalassemia 3.5-kb gene deletion.ResultsHRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, β-thalassemia 3.5-kb gene deletion, and the wild-type β-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89℃, 85.66℃, 77.24℃, and 74.92℃, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR.ConclusionsSingle-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of α- and β-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.

Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)] is often missed by routine laboratory testing since its mRNA as well as gene product are unstable and presented at a low level in peripheral blood. This study aimed to analyze the efficacy of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) for detecting and quantifying Hb CS in 19 heterozygotes and 14 homozygotes with Hb CS as well as 10 Hb H-CS disease subjects who were detected by molecular analysis. In the CE electrophoregram, Hb CS was seen at zone 2 and was observed in all samples, while the chromatogram of Hb CS peaks was found in 26.32% heterozygotes, 42.86% homozygotes and 90% Hb H-CS disease subjects, respectively. In addition, the Hb CS levels in each group of subjects quantified by CE were significantly higher than those quantified by HPLC. Based on the CE method, the lowest Hb CS level was found in the heterozygotes, whereas the highest level was found in the Hb H-CS disease patients. Therefore, the CE method was superior to the HPLC method for detecting Hb CS. Furthermore, the level of Hb CS quantified by CE proved useful in screening heterozygotes and homozygotes with Hb CS as well as Hb H-CS disease.

CE is the preferable method for screening of heterozygote and homozygote of Hb CS. Moreover, in conjunction with a lower MCV (<70 fL), this approach provided a high resolution to identify Hb H-CS disease.

The β-chain hemoglobin (Hb) variants interfere with the diagnosis of β-thalassemia trait using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). We analyzed the effect of Hb Hope, a β-chain Hb variant frequently found in the Thai population, on β-thalassemia trait diagnosis. HPLC and CE were used to quantify the level of HbA(2) in 11 whole blood samples containing Hb Hope. The levels of Hb Hope detected by both methods were similar. An elevated HbA(2) level was found in all samples analyzed by the CE method, while 1 was increased when analyzed by HPLC, which was a compound heterozygous of Hb Hope and α-thalassemia-1 SEA-type deletion. Of 11 samples, 6 had mean corpuscular volumes within the reference range. All samples showed negative results for molecular analysis of β(0)-thalassemia codon 17, 41/42, and 71/72 mutations and β-thalassemia 3.5-kb deletion. Therefore, Hb Hope interfered with the diagnosis of β-thalassemia trait analyzed by CE but not by HPLC.

In patients receiving highly active antiretroviral therapy (HAART), increase of naive T-cell production, as measured by T-cell receptor rearrangement excision circles (TRECs), is an indicator of immune reconstitution. Our objective was to assess whether treating opportunistic infections (OIs) prior to HAART initiation affects CD4 T-cells recovery and TRECs in patients on HAART. HIV-infected patients presenting no OIs or treated OIs were prospectively enrolled prior to HAART initiation and followed-up over 12 months of HAART. CD4 T-cells and TRECs were measured at baseline, 6 and 12 months HAART and compared between patients presenting no OIs and those with treated OIs. Univariate and multivariate logistic regression models were used to identify potential factors associated with low TREC increase after 12 months HAART. Forty-four HIV-infected patients, 31 presenting no OIs and 13 with treated OIs at HAART initiation were enrolled. Patients presenting no OIs tended to have higher CD4 T-cell gain than those with treated OIs (151 vs 89 cells/μL; p = 0.05) after 6 months HAART but not after 12 months HAART (120 vs 149 cells/μL; p = 0.84). Among patients presenting no OIs, TREC levels significantly increased from baseline through 12 months HAART while among those with treated OIs, there was a trend for increase only after 12 months. Our study indicates that treatment of OIs prior to HAART does not lead to impaired CD4 T-cells recovery and thymic outputs.