Hb Variants Research Articles

The Validation of Whole β-Globin Gene Sequencing for Detecting β-Thalassemia Mutations Found in Thailand Using Next-Generation Sequencing (NGS)

Beta-thalassemia is an inherited disorder prevalent in Thailand and Southeast Asia. Several molecular techniques for identifying β-thalassemia mutations have been reported. Next-generation sequencing (NGS) is a type of effective molecular testing with high throughput and accuracy. Hence, this study aims to evaluate a novel barcode-tagged NGS approach based on a short-read assay. A total of 258 samples with 54 different β-thalassemia genotypes related to 32 mutations were gathered and evaluated. A library was constructed with the BTSeqTM kit and sequencing was performed on the Illumina NGS machine. The validation results showed 98.45% concordance with conventional genotypes. Less discordant results (1.55%) were limited to insertional mutations and included one case of each of the following: HBB:c.27dupG, HBB:c.85dupC, HBB:c.216dupT, and HBB:c.440_441dupAC. Five single-nucleotide polymorphisms that derived from the NGS results were also analyzed in terms of allele frequency and revealed significant differences between the wild types and other β-genotypes. Furthermore, this paper is the first to describe rare single-nucleotide polymorphisms including IVS II-109 (C/T), IVS II-258 (G/A), IVS II-613 (T-C), and IVS II-806 (G/C). Interestingly, the C allele of IVS II-806 was found to have 100% linkage with two cases of Hb Tak. The haplotype and phylogenetic analysis was also constructed based on variants and revealed three clusters in the Hb variants, which represented their evolution and genetic background. Finally, NGS with the barcode-tagged method has a high throughput, which is suitable for large population screening. Its cost effectiveness and less complicated process promote its application in further works.

Upregulation of Interferon Gamma Gene Expression among Asymptomatic Malaria Adults in Bayelsa State, Nigeria

Despite concerted efforts, malaria remains a global public health problem. Treatment for asymptomatic malaria will reduce the health care burden of malaria. However evidence-based data for the presence of systemic pathology in asymptomatic malaria, which might inform treatment remains limited. The present study investigated the current prevalence of asymptomatic malaria in Bayelsa State, Nigeria and differences in interferon gamma (IFNγ) mRNA expression between asymptomatic malaria individuals (AMIs) and uninfected individuals (UIs), in relation to ABO blood groups and hemoglobin genotype (Hb) variants. A cross-sectional design was employed to randomly select 146 non-febrile participants from Sagbama LGA, Bayelsa State. Plasmodium falciparum infection was determined using high-resolution melting analysis and participants were grouped into UIs and AMIs. Hb, blood groups, and IFNγ mRNA expression were determined using electrophoresis, agglutination, and real-time PCR method respectively. Logistic regression and t-test were used to determine significant differences between groups at P < 0.05. The current prevalence of asymptomatic malaria in Bayelsa State was found to be 89.73%. Age and sex were associated with asymptomatic malaria (P < 0.05). Plasmodium falciparum DNA melt curves for AMIs aligned at 83.89±0.34°C, while the amplification cycle thresholds were below 30 cycles. Compared with UIs, the gene expression of IFNγ mRNA was significantly (P < 0.001) higher among AMIs. Also, among AMIs those with A, B, and AB blood groups expressed significantly higher levels of IFNγ mRNA compared with blood groups O AMIs. Furthermore, AMIs with HbAA expressed significantly higher levels of IFNγ mRNA compared with HbAS AMIs. The novelty of the present study is the demonstration of the existence of systemic pathology in asymptomatic malaria and the demonstration that the intensity of systemic pathology is dependent on blood types and haemoglobin genotype variants. Findings of the present study have implications for policy creation towards asymptomatic malaria treatment.

Carrier frequency and molecular basis of hemoglobinopathies among blood donors in eastern Morocco: Implications for blood donation and genetic diagnosis

BackgroundHemoglobinopathies represent the most commonly inherited autosomal recessive blood disorders in the world. The aim of this study was to determine the carrier frequency and molecular basis of hemoglobinopathies among blood donors in eastern Morocco. This is the first study of its kind for this country. MethodsHealthy blood donors of the BRO Biobank were included in this study. Blood samples were analyzed using an automatic blood cell analyzer for complete blood counts. Hemoglobin fractions were analyzed by capillary electrophoresis and serum ferritin was measured on a chemical and immunological analyzer. Suspected hemoglobinopathy carriers were further characterized by Sanger sequencing, Gap PCR and PCR-RFLP. ResultsThe study involved 2013 blood donors, of whom 1063 were male and 950 were female (sex ratio male-to-female of 1.1). The median age of these donors was 35 years. The overall carrier frequency of hemoglobinopathies was 1.84 %, with β-thalassemia carriers being the most prevalent (0.65 %) followed by HbAC (0.55 %), α-thalassemia carriers (0.30 %), HbAS (0.1 %), HbAG-Philadelphia (0.1 %), HbAD-Ouled Rabah (0.05 %) and HbAO-Arab (0.05 %). Additionally, novel β-thalassemia variants (C6(−G) and −83(A > G)) and a structural variant (Hb D-Ouled Rabah) were discovered for the first time in Morocco. ConclusionsThis study provided the first report on carrier frequency and molecular basis of hemoglobinopathies among healthy donors in Morocco. These findings are valuable for the implementation of carrier screening and genetic diagnosis for hemoglobinopathies. Furthermore, these results justify the need to introduce pre-donation screening for hemoglobinopathy carriers in Morocco, particularly in areas with a high prevalence of carriers to enhance the overall quality of the national blood supply.

A Rare Case of Homozygous Hemoglobin G-Philadelphia &amp; Alpha Thalassemia

Abstract Introduction/Objective Hemoglobin (Hb) G-Philadelphia (GPh) is an alpha chain gene variant caused by a missense mutation resulting in a change from Glu to Asp at position 68. This change leads to alterations in the Hb structure and oxygen affinity. The mutation is inherited in an autosomal codominant pattern allowing for an asymptomatic carrier state. While the most common alpha chain variant, GPh heterozygosity is still infrequent, estimated at 1:5000 in African Americans. In African Americans, GPh is frequently linked to an alpha thalassemia deletion. On its own it is considered a benign variant that does not result in significant health issues. Homozygous HbGPh frequency is estimated at 1 in 25 million. Methods/Case Report A postpartum woman in her 4th decade of life was found to have a Hb of 10.8 and a mean corpuscular volume of 74. Initial work up included Hb electrophoresis that quantified HbA at 0%, Hb F at 0%, HbA2 at 0%, and 2 Hb variants representing 96.1% and 3.8% of the total globin. The major Hb variant migrated in the D or G position by alkaline and acid gel Hb electrophoresis. The minor variant was consistent with HbG2. Due to this abnormal Hb electrophoresis, a sample was sent out for a Hb evaluation reflexive cascade (HERC). HERC analysis by high-performance liquid chromatography and capillary gel electrophoresis found 99.9% of Hb consistent with the alpha chain variant HbG. Analysis of the alpha globin gene region revealed 2 copies of an alpha globin gene deletion that was interpreted as alpha-thal trait (-a/-a). This result is consistent with homozygous alpha-thal-2 and homozygous HbGPh (alpha^G/thal + alpha^G/thal). Results (if a Case Study enter NA) NA Conclusion Alpha^G/thal + alpha^G/thal presents a unique and complex challenge for diagnosis due to its rarity and because HbG2 is not always visualized by alkaline gel electrophoresis. The presence of a Hb G or D variant, a HbG2 variant, and the lack of both HbA and HbA2 are key electrophoretic findings. Clinically, alpha^G/thal + alpha^G/thal behaves essentially like a homozygous alpha-thal-2 trait with persistent microcytosis and erythrocytosis. Thus, co- inheritance of the alpha-thal trait will be suggested by a thalassemic complete blood count pattern.

A Case for Co-oximetry: Methemoglobinemia secondary to a Rare Hemoglobin Variant

Abstract Introduction/Objective The presence of dyshemoglobins and some inherited hemoglobin (Hb) variants can cause over or underestimation of oxygen saturation (O2sat) when using a pulse oximeter (ox). These noninvasive medical devices rely on two wavelengths, a pulsing arterial bed, and light absorption properties of Hb to measure the difference between deoxy- and oxy-Hb. In contrast, a co-oximeter (co-ox) requires arterial blood. By using at least four wavelengths, co-ox measures the functional and fractional O2sat to detect all forms of Hb, including those that do not bind oxygen (O2), such as methemoglobin (metHb). Methemoglobinemia may be congenital or acquired, resulting from exposure to certain toxins, metabolic disorders, enzymatic deficiencies such as cytochrome b5 reductase deficiency, or hemoglobinopathies. Methods/Case Report A healthy, adolescent male undergoing a sports physical at his pediatrician’s office was hypoxic by pulse ox and sent to the emergency department (ED) for evaluation. On exam, he had cold, blue tinged fingertips and lips but was otherwise asymptomatic. His O2sat was 88-90% on room air with minimal improvement with 6L of supplemental O2. Co-ox revealed a metHb level &amp;gt; 20%. The father also mentioned that the mother had frequent ED visits for hypoxia. Capillary Hb electrophoresis showed peaks in the HbA, HbF, and HbA2 zones of 78.3%, 17.9%, and 3.6%, respectively. All CBC parameters were within the reference intervals. The large peak in the HbF zone would suggest hereditary persistence of fetal hemoglobin (HPFH); however, the clinical picture was not consistent. Further reference laboratory investigation determined that the patient was heterozygous for Hb Tübingen (HbTüb), a beta globin chain variant resulting from a point mutation at position 106. By mass spectrometry, the variant quantified as 38% of the total hemoglobin, indicating underestimation by electrophoretic techniques. Results (if a Case Study enter NA) NA Conclusion This case presents an essentially asymptomatic patient with hypoxia and elevated methemoglobin levels. Electrophoretic hemoglobinopathy and CBC workup were clinically inconsistent with HPFH and the presence of an aberrant hemoglobin migrating in the HbF must be considered in the differential diagnosis. The manifestation of HbTüb as methemoglobinemia in this context adds complexity to the diagnostic puzzle. Further laboratory exploration into the underlying mechanisms driving this unique presentation is crucial for a comprehensive understanding and tailored management approach.

Reemergence of Hemoglobin Yukuhashi

Abstract The abnormal hemoglobin, Hb Yukuhashi, identified in Japan in 1963, exhibits slower migration compared to Hb A and is associated with target cells on peripheral smear. A similar hemoglobin, Hb Dhofar, is found in an Omani tribesman and is defined as a substitution of arginine for proline at the 58th amino acid position of the beta-globin chain. Despite both Yukuhashi and Dhofar seemingly arising from the same exonic point mutation, variations in expression were clarified through beta-globin gene amplification. Hb Dhofar, unlike Yukuhashi, was linked to a splice-site mutation at codon 29, explaining its reduced expression. Despite these hemoglobins’ structural alterations, their functional properties on a molecular level, including oxygen affinity and heme-heme interaction, remained largely unaffected. While mutations at codon 58 have been observed alongside other mutations in Hb Dhofar and Hb C-Ziguinchor, this case represents the first sequencing and report of a beta-globin gene mutation solely at codon 58, as only Yukuhashi’s protein coding region had been sequenced, not the whole gene. “Hemoglobin D” was detected on newborn screening of a male child with no other significant medical history or physical abnormalities. Capillary Electrophoresis and Cation-Exchange HPLC showed abnormal hemoglobin fractions, with 20.1% in the S Zone and 25.7% in the D-window, respectively. This led to a presumptive diagnosis of Hemoglobin Dhofar trait; however, discrepancies in hematologic parameters and relative hemoglobin levels were noted. Genetic testing of the Beta Globin gene confirmed a C to G substitution at codon 58 but lacked the characteristic codon 29 mutations associated with Hemoglobin Dhofar. The patient, was discovered to possess a mutation at codon 58 on the beta globin gene, a mutation not previously reported in isolation. In contrast to previous studies on Hb Dhofar, where variant Hb levels were lower and accompanied by abnormal RBC indices indicative of beta thalassemia trait, this case exhibited higher variant Hb levels with normal RBC indices, suggestive of a typical beta globin variant. The mutation at codon 58 did not significantly alter intermolecular interactions of the globin which may mean the variant is clinically asymptomatic in nature. Continued follow-up is necessary to confirm this suspicion, although initial assessments at 3 and 6 months of age showed normal RBC parameters. This case represents the first instance of an isolated mutation at codon 58 of the beta globin gene, emphasizing the need to consider this mutation in interpreting hemoglobinopathy screening results.

False HbA1c value due to a rare variant of hemoglobin Petie Salpetriere coinherited with alpha thalassemia

Abstract Objectives To describe a variant hemoglobin that interferes with HbA1c analysis by cation exchange HPLC. Case presentation A 78 years-old Spanish male patient visited the Internal Medicine Clinic for a routine check-up, with HbA1c included to screen for diabetes. He had suffered hypertension and dyslipidemia, and the patient had no previous symptoms suggestive of diabetes such as hyperglycemia, weight loss, polydipsia, polyuria or tiredness. Diabetes screening by HbA1c measurement was assessed using cation exchange HPLC and an immunoassay point-of-care analyzer. Routine hemoglobinopathy screening was performed including CBC, HbF and HbA2 measurement by cation exchange HPLC and capillary electrophoresis (CE). Further variant characterization was undertaken by DNA sequencing. Discordant HbA1c results were obtained for our subject, with elevated HbA1c of 52 mmol/mol measured by cation exchange HPLC and a normal level of 34 mmol/mol by immunoassay. Abnormal HbA1c peak shape prompted hemoglobinopathy screening to investigate potential variant interference. A globin gene analysis was performed, and the results showed a variant hemoglobin named ‘Hb Petie Salpetriere’. This variant arises from a Val → Phe substitution due to a mutation of c.103G&gt;T of the beta-globin gene [BETA34 (B16) Val&gt;Phe; HBB:c.103G&gt;T]. Conclusions This is the first reported case involving the Hb Petie Salpetriere variant in a Spanish patient. The present results show that the Hb Petie Salpetriere variant can affect the results of HbA1c analysis through ion-exchange HPLC, but not that obtained from the latex agglutination immunoassay. Only ion-exchange HPLC suggested the presence of the Hb variant in this case, suggesting that a careful review of the resulting chromatogram might reveal a potential variant.

Presumptive Identification of Clinically Significant Hemoglobin Variants Hb E, Hb S, Hb D in Hb A1c Capillary Electrophoresis.

The quantitation of glycated hemoglobin (Hb A1c) represents an average blood glucose level for a period of 2 to 3 months for diagnosing, monitoring, and managing diabetes mellitus. Unreliable results are reported when hemoglobin (Hb) variants are present in the sample. Patients are advised to use an alternate method due to the presence of the variant Hb and a reflex test to Hb electrophoresis to obtain precise information about the Hb variant. The present study utilizes x axis values from Hb A1c capillary electrophoresis (CE) to identify clinically significant Hb variants Hb E, S, and D. Patient samples (n = 60) that showed a variant peak in the Hb A1c test with an x axis value of 190 to 240 were selected for the study. The migration position of the Hb variant (x axis value) and variant percent of the Hb A1c test were compared with the x axis value and variant percent in the Hb electrophoresis test to presumptively identify the variants. The identity of the variants was confirmed using mass spectrometry (MS). Out of 60 samples, 20 samples were identified as Hb E (x axis 225-227), 20 samples were identified as Hb S (x axis 210-214), and 18 samples were identified as Hb D-Punjab (x axis 200-201). Two variants with an x axis value of 194 were identified as an α variant Hb Q India using MS. There is an overall negative shift of the x axis (-1 to -13 units) and a lower variant percent (-0.2% to -8.7%) in Hb A1c CE when compared with Hb electrophoresis. The present study highlights the significance of the x axis value and variant percent to identify clinically significant Hb variants in the Hb A1c CE test.

A-052 Clinical usefulness of ADAMS A1c HA-8190V automatic switching mode for the HbA1c measurement in Hb variants

Abstract Background High-performance liquid chromatography (HPLC) is widely used as a HbA1c routine assay. However, quantification of glycated hemoglobin (HbA1c) is a challenge in patients with hemoglobin (Hb) variants. In this study, we evaluated the clinical utility and analytical performance of new developed ARKRAY ADMAS HA-8190V assay using autoswitch mode, that automatically switches between variant and fast mode depending on patient Hb characteristics, in patients with Hb variant suspected in Korea. Methods The measuring time for HbA1c assay was compared between the HA-8180V fast mode (existing model) and the autoswitch mode of HA-8190V (new model) in 60 normal Hb samples. And the HbA1c measured values was compared bewteen the HA-8180V fast mode and the HA-8190V autoswitch mode in 30 low Hb samples. In addition, flag detection for the abnormal Hb and measured values for 21 Hb variant samples were analyzed using the HA-8180V fast mode and the HA-8190V autoswitch mode. Results It took 50.8 minutes and 39.1 minute to measure 60 normal Hb samples by HA-8180V fast mode and HA-8190V autoswitching mode, respectively. The values could not be measured at the HA-8180V fast mode for 7 of 30 low Hb samples, whereas all samples could be measured at HA-8190V autoswitching mode. For 21 Hb variant samples, HA-8180V fast mode showed abnormal flag detections for only 14 samples, but HA-8190V autoswitch mode reported abnormal variant flags for 20 samples. In the result accuracy evaluation (% difference) based on capillary electrophoresis HbA1c assay (Sebia, Lisses, France) in the 21 Hb variant samples, the % difference of HA-8190 V autoswitch mode was 3.9%, which was superior to HA-8180V fast mode of -11.65%. Conclusions Autoswitch mode of The HA-8190V (automatically switch between variant and fast mode based on patient Hb characteristics) showed more accurate flag detection and HbA1c measurements in patients with Hb variants, along with shorter test time and manual reduction.

A-045 Analytical and Clinical Evaluation of A1c Test by HPLC - Lifotronic™ H100Plus Analyzer - in a Brazilian Clinical Laboratory

Abstract Background Over the years, the measurement of Glycated Hemoglobin (A1c) has solidified as an important risk marker for pre-diabetic patients and for the diagnosis and control of diabetic patients. The High-Performance Liquid Chromatography (CLAE) method has been adopted in prospective studies such as the DDCT, and international certification by the NGSP is available. In this study, analytical performance metrics including imprecision, correlation, linearity, carryover, and detection of hemoglobin variants were evaluated for the Lifotronic® H100 Plus analyzer from China. Methods The results were evaluated following CLSI guidelines on samples within stability, collected according to standardized procedures. For the comparative method, 40 patient samples ranging from 4 to 15.4% were analyzed, and two concentrations of internal quality control material were used for intra-assay (5 repetitions per day) and inter-assay (over five days) imprecision testing. Linearity was assessed using samples at two concentrations, one low (4.5%) and another high (17.2%). For carryover evaluation, low (4.98%), intermediate (9.97%), and high (14.96%) concentrations were assayed in an interleaved manner. Hemoglobin variants found in the studied population were determined using the same method employed in correlation tests (HbS, HbF, HbC, HbE, and HbD). Results The method comparison study revealed a Total Error for decision levels of 5.6%: 2.23% and 6.5%: 1.70%, which were lower than the maximum allowable Total Error of 3.3% (EFLM - EuBIVAS). The difference plot showed that 95% of the results fell within acceptable limits. Positive bias was observed in 7 out of 40 tested samples, predominantly in samples with concentrations above 6,5%, although the medical team assessed it as having no clinical impact. For intra-assay imprecision, the obtained result was 0.86%, and for inter-assay imprecision, it was 0.87%, both deemed acceptable according to the 3.0 % specification proposed by the supplier for the two tested levels.Linearity tests were approved for concentrations proposed by the manufacturer between 3 and 18%, and no carryover was found between samples. All hemoglobin variants previously detected in the comparative method were also identified in the test method without exception, although the specific type of Hb variant was not identified. Conclusions The Lifotronic™ H100Plus analyzer demonstrated approved analytical performance with some notable observations: positive bias was observed in samples with concentrations above 6,5%, though it was not considered clinically relevant, the throughput of 58 tests per hour could be a limitation for laboratories with high testing volumes and the analyzer detected the presence of Hb variants, although it did not identify their specific types.

B-248 Enhanced Detection of of Hemoglobin Variants in Clinical Research using Dried Blood Spot and HRAM MS

Abstract Background Hemoglobinopathies are one of the most prevalent genetic disorders, with more than 330,000 affected births occurring annually. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. However, there are challenges to differentiate isomers (i.e., between Hb C and Hb E), very similar masses, or new variants due to their lack of resolution. Mass spectrometry (MS) gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins. Here, we present the top-down analysis for enhanced detection of various hemoglobin variants using Orbitrap Exploris for clinical research. Methods Proteins were directly extracted from dried blood spots (DBS) and analyzed by Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option coupled to Vanquish HPLC system. A 3.2 mm disc was punched from the DBS cards to 96 well plate and 50 uL water was added to re-hydrate. For protein precipitation, 150 uL acetonitrile was added and incubated at -20 ⁰C for 15 minutes. After micro-centrifugation, 160 uL of supernatant was removed and 70 uL water was added to resuspend. For LC-MS analysis, 10 uL of supernatant was diluted 8 times with mobile phase A and transferred to another 96-well plate. LC separation was performed on MAbPac RP column 4 um, 1 x 100 mm, and data-dependent acquisition mode was operated. Results With direct protein extraction from DBS, the entire sample preparation time is less than 1 hour. The MAbPac column showed its capability of separating between different chains with minor overlap between normal beta and HbS beta chains. Data processing was performed by ProSightPD, resulting in ∼45% sequence coverage for both normal Hb and HbS beta chains. The mass difference between normal beta and Hb S beta chains is about 30 Da, which results in about 1.6 m/z difference at a charge state of 19. This mass difference can often be hindered by other proteins or interferences from the matrix if the mass analyzer doesn’t have sufficient resolving power. Here we successfully applied Orbitrap resolution at 120,000 to isolate these similar masses for accurate variants or subunits identification including normal alpha, normal beta, Hb S, and delta chains. The confirmation of the delta chain can be beneficial since its abundance is usually less than 0.5% in adults, which shows potential for detecting low abundant variants using this method. Also, the calibration curve was generated to demonstrate the method's feasibility which is not a typical quantitation method for hemoglobinopathies. For quantitation, three most abundant m/z values from 2 charge states were added to TraceFinder for peak area extraction and normal Hb beta chain was used as an alternative internal standard for data processing. The 3-day inter/intraday analysis generated great reproducibility with CV &amp;lt; 10% and LOQ was determined to be 2.5 mg/mL with R2 values &amp;gt;0.994. Conclusions Successful demonstration of a single-step protein extraction directly from DBS and top-down analysis of Hb variant for clinical research.

Interactions of electrophoretically silent hemoglobin Hekinan II [HBA1:c.84G>T] with various forms of α-thalassemias and other hemoglobinopathies: novel insights into the molecular and hematological characteristics and genetic origins

ABSTRACT 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, (ααHekinan/αCSα), (ααHekinan/αCSα,βA/βE), and (ααHekinan/αCSα,βE/βE), 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.

Analysis of the Haematological Phenotype and Molecular Characteristics of Rare Abnormal Haemoglobin.

Haemoglobinopathy refers to a group of common monogenic inherited conditions associated with variations in the haemoglobin molecule; however, there is relatively limited reporting on abnormal haemoglobinopathy in the Chinese population, especially rare abnormal haemoglobin (Hb). The aim of this study was to explore the clinical characteristics of haemoglobinopathy to supplement data for the epidemiological investigation of Hb variants in Guangdong province of China. Peripheral blood was collected from five patients (including a family) for Complete blood count, Hb electrophoresis, High-performance liquid chromatography analysis and degenerative globin body testing. Hb variants were further analysed by PCR and DNA sequencing. The research subjects were diagnosed with different types of abnormal Hb. The blood routine of the Hb Fukuyama (HBB:c.232C>T) diagnosed individual showed microcytic hypochromic anaemia, with a lower Hb level (64 g/L), mean corpuscular volume (MCV) of 71.5 fL and mean corpuscular haemoglobin (MCH) of 21.5 pg. Individuals diagnosed with Hb Port Phillip (HBA2:c.275T>C) exhibit a MCH level that is slightly below average, at 26.4 pg. The Hb Saint Etienne (HBB:c.279C>G) diagnosed individual showed macrocytic hypochromic anaemia, and the proband had a low Hb level (116 g/L), MCV of 102.2 fL and MCH of 29.4 pg. We confirmed the presence of Hb Fukuyama (HBB:c.232C>T) in China for the first time. Three rare patients with the Hb Saint Etienne (HBB:c.279C>G) phenotype and one patient with Hb Port Phillip (HBA2:c.275T>C) phenotype were included. Our research enriches the gene mutation map of haemoglobinopathy in Guangdong province and improves the detection system of haemoglobinopathy for population prevention and eugenics.

Clinical Risk Assessment and Comparison of Bias between Laboratory Methods for Estimation of HbA1c for Glycated Hemoglobin in Hyperglycemic Patients.

Hemoglobin A1c (HbA1c), also known as glycated hemoglobin, is a blood test used to evaluate and track a patient's blood sugar levels over the previous 2-3 months. We have compared the analytical performance of the D10 hemoglobin (HPLC) testing system to that of the immunoturbidimetric technique, which is a light-scattering immunoassay. To assess the clinical risk assessment between two methods (Compare the two Immunoturbidometric methods (AU680) vs. HPLC method (D10)) in hyperglycemic patients and assess the acceptability of the respective methods in the Clinical biochemistry laboratory. The charge of the globins in Hb was used as the basis for the HPLC method used to measure HbA1c. HPLC detects and quantifies even the tiniest Hb fractions and the full spectrum of Hb variants. HbA1c was measured using the immunoturbidimetric (AU 680 Beckmann coulter analyzer) and high-performance liquid chromatography (HPLC) techniques. Experiments also made use of immunoturbidimetric techniques (using an AU 680 Beckmann coulter analyzer equipment). There is no statistically significant difference in HbA1c readings between male and female patients, as measured by either the Immunoturbidimetric or HPLC techniques. The immunoturbidimetric and high-performance liquid chromatography techniques for estimating HbA1c yielded identical results. From the results of this study, we may deduce that both techniques are valid for estimating HbA1c. As a result, it may be suggested that both approaches can be used to estimate HbA1c in diabetic individuals.

Analytical performance evaluation of the GreenCare A1c and Cera-Stat HbA1c point-of-care testing assays.

The escalating prevalence of diabetes underscores the need for precise diagnostic tools to facilitate effective management. Hemoglobin A1c (HbA1c) is a crucial biomarker for long-term glycemic control in diabetic patients. Point-of-care testing (POCT) for HbA1c offers rapid, accessible alternatives to conventional laboratory methods, but uncertainties persist regarding the accuracy and reliability of POCT assays. This study evaluates the analytical performance of two boronate-affinity based HbA1c POCT assays, the GreenCare A1c and Cera-Stat HbA1c. Various analytical parameters including precision, linearity, comparison, and accuracy are assessed following guidelines from Clinical and Laboratory Standards Institute (CLSI), with results applied to certification criteria from the National Glycohemoglobin Standardization Program (NGSP) and International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Furthermore, 52 and 13 frozen EDTA whole blood samples were respectively used for additional evaluation of accuracy and interference due to Hb variants for the GreenCare A1c assay. Both GreenCare and Cera-Stat demonstrated good precision (repeatability CV% 1.5-1.9 and total imprecision CV% 1.6-2.2), linearity (R2 = 0.9996 & 0.9990), and correlation (r = 0.982 & 0.978) with an established HbA1c analyzer, the Bio-Rad D100. The GreenCare also exhibited good accuracy with frozen EDTA samples with known HbA1c values. Both assays met the certification criteria from NGSP and IFCC, classifying them as "standard" according to IFCC model for quality targets for HbA1c. This evaluation affirms the reliability of GreenCare and Cera-Stat POCT assays for HbA1c measurements, which can potentially reduce unnecessary referrals and enhance the overall quality of diabetes diagnosis and treatment.

Hemoglobin polymorphism and its association with morphometric and egg production traits in Ethiopian indigenous and Sasso chicken breeds.

The present study investigated the biochemical polymorphism of hemoglobin (Hb) and its relationship with performance traits of Ethiopian indigenous and Sasso chicken breeds. A total of 284 chickens reared in three agro-ecologies were examined for genetic diversity and associations with productive traits at Hb locus using agarose gel electrophoresis. The results showed that the HbA allele was dominant in both breeds, and a higher proportion of male chickens were HbAA genotypes, while females were predominantly HbBB types. In the highland agro-ecology, chickens with the HbAA genotype were the most dominant, whereas in mid- and low-land agro-ecologies, chickens with HbBB and HbAB genotypes were found to be more frequent. A moderate level of expected heterozygosity was obtained with 0.47 and 0.445 for indigenous and Sasso chickens, respectively, with an average effective number of alleles per locus of 1.89 and 1.80. Moreover, chickens with HbAA genotypes showed significantly (p ≤ 0.05) higher body weight and linear body measurements than those of HbAB and HbBB genotypes. However, for appendage body structures (comb and wattle dimensions), chickens with the HbAB and HbBB genotypes had higher mean values. Additionally, clutch size (14.2 ± 0.4), clutch length (21.8 ± 0.7), and eight-month egg production (84.1 ± 1.2) were significantly (p ≤ 0.05) higher for hens with HbBB genotypes, followed by those with HbAB-types. Therefore, the considerable hemoglobin variability and significant associations of Hb variants with the performance traits can be sought as guiding information for further genetic improvement interventions in the chicken breeds under investigation. Further microsatellite marker-based genotyping is recommended to validate the higher morphometric values for HbAA genotypes and the better egg production for HbBB and HbAB genotypes.

Coinherited Hemoglobin Lansing-Ramathibodi/Southeast Asian Deletional α0-thalassemia and Hemoglobin E Causing Falsely Low Oxygen Saturation on Pulse Oximetry

OBJECTIVE Hemoglobin (Hb) variants resulting from mutations in globin genes and leading to qualitative abnormalities of globin proteins can result in a spectrum of clinical presentations. Herein we describe a case of a 10-year-old boy with an Hb variant, Hb Lansing-Ramathibodi (HBA1:c.264C&gt;G, codon 87 His&gt;Gln), who was incidentally found to have low pulse oximetry oxygen saturation readings (low SpO2). Laboratory investigations revealed mild microcytic anemia. Arterial blood gas showed normal partial pressure of oxygen (PaO2) and arterial oxygen saturation (SpO2) values, indicating an oxygen saturation gap. Subsequent Hb analysis identified an abnormal peak, prompting further molecular studies which confirmed the coinheritance of Hb Lansing-Ramathibodi, Southeast Asian deletional α0-thalassemia and the Hb E trait. The patient’s mother, carrying the Hb Lansing-Ramathibodi and Hb E traits without Southeast Asian deletional α 0-thalassemia, was asymptomatic. This case underscores the importance of identifying and characterizing rare Hb variants presenting with an oxygen saturation gap for informed clinical management and genetic counseling. KEYWORDS alpha-thalassemia, coinheritance, hemoglobin Lansing- Ramathibodi, low SpO2, oxygen saturation

Analytical performance evaluation of the Mindray enzymatic assay for hemoglobin A1c measurement

Hemoglobin A1c (HbA1c) plays a crucial role in diabetes management. We aimed to evaluate the analytical performance of a new enzymatic method kit for HbA1c measurement. The performance of the enzymatic method, including precision, accuracy, and linearity, was evaluated. Moreover, the interference effect from conventional interferents, Hb derivatives, Hb variants, and common drugs were assessed. In addition, the agreement of HbA1c results was compared between enzymatic methods, cation-exchange high-performance liquid chromatography (HPLC), and immunoassays. The intra-assay, between-assay, and total precision of HbA1c were all lower than 2%. HbA1c showed good linearity within the range of 3.96–20.23%. The enzymatic assay yielded results consistent with the external quality control samples, with a bias of less than ± 6% from the target values. The enzymatic method showed no interference from bilirubin, intralipid, vitamin C, Hb derivatives, common Hb variants, as well as antipyretic analgesics and hypoglycemic drugs. The HbA1c results of the enzymatic assay showed good agreement and accuracy compared to those obtained from the HPLC method and the immunoassay. The enzymatic method kit performed on the BS-600M chemistry analyzer is a reliable and robust method for measuring HbA1c. It is suitable for routine practice in clinical chemistry laboratories.

Interference of hemoglobin variants with HbA1c measurements by six commonly used HbA1c methods.

Glycated hemoglobin, or hemoglobin A1c (HbA1c), serves as a crucial marker for diagnosing diabetes and monitoring its progression. We aimed to assess the interference posed by common Hb variants on popular HbA1c measurement systems. A total of 63 variant and nonvariant samples with target values assigned by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-calibrated methods were included. We assessed 6 methods for measuring HbA1c in the presence of HbS, HbC, HbD, HbE, and fetal hemoglobin (HbF): 2 cation-exchange high-performance liquid chromatography (HPLC) methods (Bio-Rad D-100 and HLC-723 G8), a capillary electrophoresis (CE) method (Sebia Capillarys 3 TERA), an immunoassay (Roche c501), an enzyme assay system (Mindray BS-600M), and a boronate affinity method (Primus Premier Hb9210). The HbA1c results for nonvariant samples from the 6 methods were in good agreement with the IFCC-calibrated method results. The Bio-Rad D-100, Capillarys 3, Mindray BS-600M, Premier Hb9210, and Roche c501 showed no interference from HbS, HbC, HbD, and HbE. Clinically significant interference was observed for the HLC-723 G8 standard mode. Elevated HbF levels caused significant negative biases for all 6 methods, which increased with increasing HbF concentration. Elevated levels of HbF can severely affect HbA1c measurements by borate affinity, immunoassays, and enzyme assays.

Exploring the Clinical and Hematological Characteristics of Beta-Thalassemia Trait: A Comprehensive Analysis in a Tertiary Care Hospital Setting.

Beta-thalassemia is one of the most common inherited hematological diseases caused by more than 350 mutations in the β-globingene (HBB). Beta-thalassemia carrier or trait is associated with defects in one allele of the HBB gene. The majority of beta-thalassemia trait cases remain concealed in society and remain unnoticed as they are mostly asymptomatic or present with mild symptoms of anemia. There is a 25% chance of having children with beta-thalassemia major and a 50% chance of having carrier babies when two people with beta-thalassemia trait are married. Hence, it is important to identify the individuals with beta-thalassemia trait and provide counseling to understand the risks of pregnancy and its outcome. Aim To study the identification of beta-thalassemia trait cases along with their clinical findings and hematological correlation. Materials and methods Study Design This was a retrospective study conducted at Saveetha Medical College and Hospitalfor a period of four years from January 2020 to December 2023. Inclusion Criteria Age group more than 18 years, antenatal mother, cases of anemia who were refractory to iron treatment, and screening of family members in the positive cases of beta-thalassemia trait. Exclusion Criteria History of blood transfusion within three months was excluded. Data Collection A total number of 837 cases were screened to rule out the presence of beta-thalassemia trait/hemoglobin (Hb) variants. A 2 mL of intravenous blood samples were collected in an ethylene diamine tetraacetic acid (EDTA) vacutainer tube and processed in a Sysmex XN 1000 (Hyogo, Japan: Sysmex Corporation)automated hematology analyzer. The hematological parameters were analyzed. Statistical Analysis The study included both descriptive and analytical characteristics. Mean and standard deviation (SD) were calculated for all the hematological parameters. Beta-thalassemia trait was diagnosed with an HbA2 level of more than 4.0% through high-performance liquid chromatography (HPLC) analysis. Results Among the 837 samples studied for HPLC screening, 74 (8.8%) cases were found to have beta-thalassemia trait. The age group included was from 18 years to 56 years. Of 74 cases studied, 32(43%) were females and 42(57%) were males. Among the 74 cases studied, the Mentzer index <13 was seen in 58 (78%) cases and the Mentzer index >13 was seen in 16 cases (22%). Thirty-four cases (46%) of beta-thalassemia traits presented to the hospital with a history of fever for evaluation and antenatal screening accounted for 19 cases (26%). The mean red blood cell (RBC) count was 5.5 million/cu.mm; mean corpuscular volume (MCV) was 63.8 fL; mean corpuscular hemoglobin (MCH) was 19.6 pg; red cell distribution width coefficient of variation (RDW-CV) was 17.8%. Among the 74 cases studied, 37(46%) cases had an Hb of more than 11 g/dL, 22 cases had mild anemia, 12 cases had moderate anemia, and three cases had severe anemia. Conclusion This study concluded that regular monitoring of the Mentzer index along with HPLC analysis is an effective approach in identifying beta-thalassemia trait cases and further providing genetic counseling among the couples that will help in reducing high-risk pregnancy and the birth of a child with thalassemia major.