Sitthichai Panyasai

Non-deletional hemoglobin (Hb) H and Hb H-like diseases are clinically significant forms of α-thalassemia with variable severity. In this study, we aimed to update the molecular spectrum of these conditions in southern Thailand and revise the allele proportions of α-thalassemia mutations among screened carriers from different regions in Thailand. A total of 118 patients with non-deletional Hb H or Hb H-like disease were characterized using DNA sequencing and multiplex ligation-dependent probe amplification analysis, and 679 possible α-thalassemia carriers were analyzed to determine allele proportion. Ten distinct genotypes were identified. Hb H-Constant Spring (CS) disease was the most frequent (79.7%), followed by non-deletional Hb H with Hb E heterozygosity (13.6%). Among patients with non-deletional Hb H disease, 76.5% of those with Hb H-CS disease and 68.8% of those with non-deletional Hb H disease and Hb E heterozygosity were classified as NTDT. Three cases of Hb H-like disease with a TDT phenotype caused by compound heterozygous Hb CS/Hb Adana were identified for the first time in Thailand. Homozygous Hb Chumphae was also reported for the first time to exhibit a mild clinical phenotype. Hb Adana was detected at an allele proportion of 1.8% among screened carriers from Southern Thailand, and a multiplex AS-PCR for simultaneous detection of Hb CS, Hb Paksé, and Hb Adana was developed.

Monthon Mayoon, B.S. and Sitthichai Panyasai, Ph.D.. Ann Lab Med -0001;0:. https://doi.org/10.3343/alm.2026.0183

Introduction: No evidence-based preanalytical guideline exists for EDTA blood storage before hemoglobin H (HbH) inclusion body detection. We evaluated the stability of rapid (30-minute) brilliant cresyl blue staining across α-globin and β-globin genotypes in blood stored at 4 °C. Methods: Residual EDTA blood from 322 patients with microcytic hypochromic anemia was stored at 4 °C and tested on days 0, 2, 4, and 7 by 3 blinded medical technologists. Hemoglobin typing and genotyping followed inclusion body counting. Wilcoxon and Mann-Whitney U tests as well as intraclass correlation coefficient(2,1) were applied. Results: Six α-globin genotype groups were identified, including HbH disease (n = 103), by β-globin genotype background. Non-HbH groups showed no meaningful inclusion bodies, confirming stable specificity. In HbH disease with a normal β-globin gene, 100% qualitative concordance was maintained at day 7, with no statistically significant quantitative change from day 0 (P = .28); the intraclass correlation coefficient(2,1) was 0.858. A transient increase at day 2 (P < .001) may have reflected in vitro HbH autoxidation during cold storage. Hemoglobin H disease co-inheriting HbE showed stable but markedly reduced counts throughout storage; a consistent pattern was observed in isolated cases co-inheriting β-thalassemia. Discussion: EDTA blood stored at 4 °C maintains qualitative stability for HbH inclusion body detection for up to 7 days; HbE co-inheritance substantially reduces diagnostic sensitivity, with a similar pattern observed in isolated β-thalassemia co-inheritance.

Background: Hemoglobin Constant Spring (HbCS) and Hb Paksé are clinically significant α‐globin variants prevalent in Southeast Asia that cause severe non‐deletional HbH disease when combined with α ⁰ ‐thalassemia deletions. Their inherent instability and low circulating levels (0.1%–1.0%) present analytical challenges, often yielding false negatives with conventional screening methods. This study aimed to evaluate Premier Resolution HPLC performance in detecting HbCS and Hb Paksé vs. capillary electrophoresis (CE), and to characterize chromatographic patterns across α‐ and β‐globin genotype combinations. Methods: A two‐phase validation study was conducted. Phase 1 analyzed 616 samples (307 DNA‐confirmed HbCS/Hb Paksé cases) to characterize chromatographic patterns and compare Premier Resolution with CE. Phase 2 prospectively validated the system in 2032 consecutive individuals undergoing routine health screening. Molecular analysis served as the reference standard. Results: Premier Resolution HPLC demonstrated distinctive multi‐peak patterns spanning the S and C chromatographic windows for HbCS/Hb Paksé, with quantification limited to homozygous and α ⁰ ‐thalassemia co‐inherited cases. It outperformed CE with 96.4% sensitivity, 97.1% specificity, and 96.8% accuracy vs. CE (92.8% sensitivity, 93.5% specificity, 93.2% accuracy). False negatives occurred mainly in heterozygotes (Premier Resolution: 5/73 [6.8%] vs. CE: 14/73 [19.2%]); heterozygous Hb Paksé remained undetectable by both techniques (3/3, 100%). Clinical validation confirmed excellent performance with 94.9% sensitivity, 99.6% specificity, and 99.36% accuracy. Conclusions: Premier Resolution HPLC provides a reliable and cost‐effective method for HbCS detection in α‐thalassemia–risk populations. Its enhanced resolution and characteristic chromatographic patterns support routine thalassemia screening in high‐prevalence regions, though molecular confirmation remains necessary in heterozygous cases for genetic counseling.

Wibhasiri Srisuwan, M.S., Surada Satthakarn, Ph.D., and Sitthichai Panyasai, Ph.D.. Ann Lab Med -0001;0:. https://doi.org/10.3343/alm.2025.0552

To determine the molecular basis, genotype - phenotype relationship, and genetic origin of Hemoglobin (Hb) Hekinan associated with several forms of α-thalassemia and other hemoglobinopathies for a better understanding of its diverse clinical phenotypes. Seventeen participants with suspected abnormal Hb were studied. Hb analysis was performed using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Mutational and α-haplotypic and structural analyses were conducted, and the effects of mutations on globin-chain stability were determined. All participants harbored Hb Hekinan II (HBA1:c.84 G>T) co-inherited with another α-globin gene anomaly. Three novel genotypes, (αα^Hekinan/α^CSα), (αα^Hekinan/α^CSα,β^A/β^E), and (αα^Hekinan/α^CSα,β^E/β^E), were characterized. Despite being co-inherited with both α- and β-Hb variants Hb Hekinan II led to minimal changes in erythrocyte parameters, suggesting a non-pathological nature. HPLC but not CE revealed a distinct small shoulder-like Hb pattern. Thai Hb Hekinan II was strongly associated with haplotype [+ - S + - - -] and the possibility of four different haplotypes, while two Burmese Hb Hekinan II were associated with haplotypes [± - S + - + -] and [± - S + - - -]. The novel genotypes identified provide a fresh perspective on Hb Hekinan II diversity. HPLC has superior identification capabilities for samples of Hb Hekinan II co-inherited with α-thalassemia. Thai and Burmese Hb Hekinan II have diverse origins.

Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype–phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and β-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a β-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with β⁰-thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.