Hemoglobin A Research Articles

Preclusion of methemoglobinemia caused by nitrate drugs in diabetics and nondiabetics: Possible role of Vitamin C

The drugs containing nitrates like isosorbide dinitrate, isosorbide mononitrate and glyceryl trinitrate, etc., trigger the oxidation of hemoglobin which is manifested in the pathological disorder named methemoglobinemia. It was considered interesting to investigate the preventive roles of vitamin C towards the toxic effects of nitrate containing drugs used for the treatment of angina. The aim is to find whether these drugs need to be administered with special care to diabetic patients who are more prone to develop methemoglobinemia. Vitamin C (500 mg/day) was administered orally to reduce the methemoglobin (metHb) level in both the diabetic and nondiabetic patients consuming nitrate containing drugs regularly, keeping diabetic and nondiabetic patients not on nitrate drugs as control. Concentration of metHb and hemoglobin A (HbA) was estimated spectrophotometrically assuming the molar extinction coefficient values of metHb as 3.78 mM‐−1 cm‐−1 at 630 nm and HbA as 125,000 M ‐−1 cm ‐−1 at 415 nm. MetHb level was found to be lower after the treatment with vitamin C for 30 consecutive days than that before the trial with statistically significant two tailed p value. Additionally, fasting insulin level was also found to decrease after 4 weeks of consumption of vitamin C with moderate lowering of fasting serum glucose level as well, indicating a higher insulin sensitivity for the treated patients.

Serum Visfatin Level in Sickle/β Thalassemia in Correlation with Frequency of Vaso Occlusion Crises

Background: Hemoglobinopathies are the most common heritable disorders of erythrocytes, with sickle cell diseases (SCDs) and thalassemia being the most common. SCDs are characterized by the presence of sickle hemoglobin within red blood cell (RBC) as a result of point mutation (β6glu→val). SCDs include sickle cell anemia, hemoglobin C disease (HbSC), and sickle/β thalassemia. The latter is caused by coinheritance of two different mutations in β globin gene, one from each parent: one for sickle hemoglobin and the other for β thalassemia. Vaso occlusion is a key feature of this disease that occurs due to a series of interactions leading to painful crisis. Visfatin is a pro-inflammatory adipocytokine that contributes to vaso occlusive crises (VOC) through its role in the inflammatory process. Visfatin can directly promote endothelial dysfunction and stimulates vascular smooth muscle cells proliferation. Objectives: The primary aim of this study was to assess serum visfatin level in sickle/β thalassemia patients and evaluate the possible association between serum visfatin level in sickle/β thalassemia patients and the frequency of VOC, serum ferritin level, complete blood count (CBC) and high-performance liquid chromatography (HPLC) parameters. The secondary aim of this study was to compare the findings in two centers (Al-Karama Teaching Hospital, in Baghdad and Babylon Teaching Hospital for Maternity and Children, in Babylon). Materials and Methods: This was a cross-sectional study conducted from December 2018 until the end of August 2019 and included 77 individuals. Among them, 57 were sickle/β thalassemia patients (Group I): 28 from Al-Karama Teaching Hospital (Group IA) and 29 from Babylon Teaching Hospital for Maternity and Children (Group IB). The remaining 20 individuals act as a healthy control group (Group II). Clinical data were gathered, with collection of 5 mL of peripheral blood in order to examine CBC, C-reactive protein (CRP), serum ferritin, and serum visfatin. Results: Mean age was (13.14 ± 5.40) years. Males formed (59.74%), whereas females formed the remaining (40.26%). The mean number of annual frequency of VOC events was (3.05 ± 0.95) with no significant difference between the two subgroups. White blood cells count was significantly higher among cases compared to controls (P = 0.004). Hemoglobin was significantly lower among cases compared to controls (P < 0.001). Also, hemoglobin was significantly higher in cases of group IA compared to group IB with P = 0.022. Similarly, hemoglobin A2 (HbA2) was significantly higher in cases of group IA compared to group IB with P = 0.013. Serum ferritin was markedly higher among cases compared to controls with P < 0.001. Serum visfatin was significantly higher among cases compared to controls with P < 0.001. No significant difference was observed between the two subgroups regarding serum ferritin and visfatin.There was positive correlation between visfatin and annual frequency of VOC (r = 0.821, P < 0.001), moderate negative correlation between visfatin and HbA (r = –0.46, P < 0.001), moderate positive correlation between visfatin and HbS (r = 0.54 and P < 0.001), and strong positive correlation between visfatin and ferritin among cases group (r = 0.60 and P < 0.001) but not in control group. Conclusions: Serum visfatin level is significantly higher among patients with sickle/β thalassemia compared to healthy individuals with positive correlation exists between visfatin level and the annual frequency of VOC, ferritin level, and HbS; negative correlation with HbA, among those patients with sickle/β thalassemia. Hemoglobin level and HbA2 percentage significantly higher among Al-Karama hospital patients compared with those in Babylon hospital.

Genetic research and clinical analysis of β-globin gene cluster deletions in the Chinese population of Fujian province: A 14-year single-center experience.

BackgroundHeterozygotes of HPFH and δβ thalassemia are clinically asymptomatic or have mild hemoglobin (Hb) values. However, when both HPFH and δβ‐thalassemia are coinherited with heterozygous β‐thalassemia, patients may progress to a clinical phenotype of thalassemia intermedia or thalassemia major. The purpose of this study was to characterize the genotypes and analyze the phenotypes of these disorders in Fujian Province, to offer advice for genetic counseling and accurate prenatal diagnosis in this region. A total of 55 001 subjects were participated in thalassemia screening. 142 subjects with HbF levels ≥10%, before the blood transfusion, were selected for further investigation.MethodsMultiplex ligation‐dependent probe amplification (MLPA) and Gap‐PCR were used to screen for three β‐globin gene cluster deletions: Chinese Gγ(Aγδβ)0 thalassemia and Southeast Asia HPFH (SEA‐HPFH) deletion and 1357 bp deletion (NG‐000007.3:g.69997‐71353 del 1357).ResultsA total of 142 patients with HbF (≥10%) were enrolled to characterize the molecular basis of β‐globin gene cluster deletions in our study; 22 cases 0.04% (22/55 001) were definitively diagnosed with β‐globin gene cluster deletions. Ten cases were heterozygous for the Chinese Gγ(Aγδβ)0‐thal mutations, 10 cases were heterozygous for SEA‐HPFH, and one case was compound heterozygous for SEA‐HPFH and the α‐thal mutation. The 1357 bp deletion (NG‐000007.3:g.69997‐71353 del 1357) was detected in one case. Moreover, the hemoglobin A2 levels in patients who were heterozygous for Chinese Gγ(Aγδβ)0‐thal were statistically lower than in cases with SEA‐HPFH deletion(p < 0.05).ConclusionIn Fujian Province, the prevalence of common β‐globin gene cluster deletions was 0.04%. What's more, the most common β‐globin cluster deletions are the Chinese Gγ(Aγδβ)0 and SEA‐HPFH.

Hemoglobin A2 and Heterogeneous Diagnostic Relevance Observed in Eight New Variants of the Delta Globin Gene.

Background: Hemoglobin A (Hb A) (α2β2) in the normal adult subject constitutes 96–98% of hemoglobin, and Hb F is normally less than 1%, while for hemoglobin A2 (Hb A2) (α2δ2), the normal reference values are between 2.0 and 3.3%. It is important to evaluate the presence of possible delta gene mutations in a population at high risk for globin gene defects in order to correctly diagnose the β-thalassemia carrier. Methods: The most used methods for the quantification of Hb A2 are based on automated high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). In particular Hb analyses were performed by HPLC on three dedicated devices. DNA analyses were performed according to local standard protocols. Results: Here, we described eight new δ-globin gene variants discovered and characterized in some laboratories in Northern Italy in recent years. These new variants were added to the many already known Hb A2 variants that were found with an estimated frequency of about 1–2% during the screening tests in our laboratories. Conclusions: The knowledge recognition of the delta variant on Hb analysis and accurate molecular characterization is crucial to provide an accurate definitive thalassemia diagnosis, particularly in young subjects who would like to ask for a prenatal diagnosis or preimplantation genetic diagnosis.

A Method to Measure Voxelotor Exposure in People with Sickle Cell Disease Using Capillary Zone Electrophoresis

Background:Voxelotor (Oxbryta®) is a small molecule that binds to the alpha chain of hemoglobin (Hb) and increases the affinity of Hb for oxygen which reduces sickle Hb polymerization. It was approved by the FDA in 2019 for the treatment of sickle cell disease (SCD). Currently, the only method available to estimate the concentration of voxelotor in the blood is to obtain exposure measurements which are only available in select research laboratories. A method to measure voxelotor at a standard laboratory would allow clinicians to assess compliance and may be useful in determining optimal dosing. Case studies have reported that voxelotor binding to Hb interferes with capillary zone electrophoresis (CZE). As previously reported, in CZE the characteristic peaks of hemoglobin A2 (HbA2) and hemoglobin F (HbF) split in the presence of voxelotor. Interestingly, it does not cause the hemoglobin S peak to split. We posited that we could use this split to estimate the presence of voxelotor and whole blood concentration. Biophysical measurements of voxelotor binding to Hb were also measured in these samples.Methods:Patients were enrolled prospectively in an IRB approved protocol. Voxelotor was initiated at 1500 mg daily on day 0 and samples were taken at day 0 (pre-dose), 14, 30, and 60. Samples had Hb variants quantified by CZE using the Capillarys 2 FlexPiercing Instrument (Sebia, Georgia). Hematology parameters were measured with the Sysmex XN-1000 automated analyzer (Sysmex, Illinois).To determine whole blood concentration of voxelotor, samples were sent to Worldwide Clinical Trials where a validated liquid chromatography-tandem mass spectrometry method was used.Voxelotor's interference with HbA2 on CZE is dependent on the HbF percentage, therefore samples from patients with SCD were combined into three pooled samples (5-10 samples per pool) of HbF percentages spanning 5-30%. Three hundred µL aliquots of each pool were spiked with voxelotor in DMSO in triplicate to different concentrations between 0 and 600 µMol/L and were incubated at room temperature for 1 hour before being tested with CZE. Samples were then analyzed for voxelotor interference resulting in split peaks of HbF and HbA2. HbA2 interference percent (%VarA2) was calculated as the reported value of the HbA2 split peak over the total HbA2 value (both split and parent peak) times 100. F% was used as directly reported by the instrument without consideration of voxelotor interference. Results were then analyzed in Excel (Data Analytics package) using a multiparameter regression to generate a line of best fit. To allow for logarithmic fit when examining the correlation of calculated concentration with increase in Hb due to voxelotor, samples with negative Hb rises were excluded and concentrations which resulted as negative values were changed to 0.01 µM.Results:Of 20 patients which have been enrolled to date, 9 patients have completed the study and their data was used for these analyses. Using the CZE method described above the concentration of voxelotor was quantifiable using the following equation.Equation 1: uM voxelotor = -99.13 + 7.10*%HbF +12.52*%VarA2The calculated concentrations of voxelotor based on CZE results had a strong correlation with whole blood concentration (R 2 = 0.85, p <0.001). (Figure 1)When calculated concentration was compared to change in Hb at days 14, 30, and 60 there was a significant positive logarithmic correlation between concentration and change in Hb (R 2=.56, p<0.01). (Figure 2)Conclusions:Using equation 1, CZE can be used to detect the presence of voxelotor and estimate its whole blood concentration. This will allow clinicians to have a better understanding of how their patients are using voxelotor. Additionally, higher calculated whole blood concentrations correlated with higher increases in Hb. It was previously shown that patients who receive higher doses of voxelotor have on average larger increases in Hb. If it could be shown that increasing concentration in an individual on voxelotor is associated with an increased Hb for that individual, then our method could also be used to help clinicians select and adjust doses of voxelotor in a similar manner to how HbF is used in hydroxyurea dosing. [Display omitted] DisclosuresCurtis: GBT: Consultancy. Minniti: CSL Behring: Other: Endpoint adjudicator; Bluebird Bio: Other: Endpoint adjudicator; F. Hoffmann-La Roche: Consultancy; Chiesi: Consultancy; Novo Nordisk: Consultancy; Forma: Consultancy; Novartis: Consultancy; GBT: Consultancy. Ngyuen Dang: GBT: Current Employment. Pochron: GBT: Current Employment. Campbell: GBT: Research Funding; Sebia: Research Funding.

Hemoglobin Variants Influence Plasmodium Falciparum Sexual Differentiation

It has long been recognized that individuals who express variations of the hemoglobin-A (HbA) protein experience less severe malaria disease. As malaria remains to be one of the most significant infectious diseases in history, this human adaptation has led to the persistence of HbA variants (HbVARs) in the population. The intricate lifecycle of the parasite which causes the most cases of clinical malaria, Plasmodium falciparum, relies on both asexual and sexual reproductive cycles, with host to vector transmission reliant on sexual stage gametocyte formation. Multiple epidemiological studies have shown that HbVARs may influence gametocyte production during P. falciparum infection, with greater gametocyte numbers reported in individuals with hemoglobin variant containing erythrocytes (Hb VAR-Ery) when compared to hemoglobin A containing erythrocytes (Hb A-Ery). Here we provide experimental support for these studies by showing significantly higher sexual differentiation rates among parasites grown in Hb S containing erythrocytes (Hb S-Ery) obtained from sickle cell patients than those differentiated in Hb A-Ery (p=0.038). Because the digestion of hemoglobin is such an integral part of the intraerythrocytic cycle, we then sought to determine whether there was a difference between the hydrolysis efficiencies of HbA and other hemoglobin variants (HbVAR). By using a prominent recombinant P. falciparum hemoglobinase we found the hydrolysis efficiency of HbA to be significantly (p=0.0058) more efficient after 24 hours compared to a HbVAR sample containing mixed amounts of HbA, HbF, and HbS. To further determine whether there is a link between hemoglobin digestion efficiency and sexual differentiation, we therapeutically inhibited the hemoglobin digestion and hemozoin formation process in a culture of P. falciparum using sub-optimal doses of chloroquine diphosphate. We found a significant difference (p<0.001) among gametocyte conversion rates between treated and non-treated cultures, as well as a moderate negative correlation between hemozoin formation and gametocyte conversion rate (Pearson r=0.72, p=0.008). Gene expression analysis also revealed patterns of expression that were consistent with increased gametocytogenesis. We conclude that hemoglobin type plays a significant role in the process of sexual conversion in P. falciparum. Though further studies should be completed in order to confirm these results, these findings may suggest hemoglobin digestion efficiency as a causative factor for sexual differentiation. As individuals with hemoglobinopathies make up approximately 7% of the global population, and malaria infection rates have been shown to differ depending on these genetic dynamics, these findings may support the creation of targeted initiatives to reduce transmission specifically in areas where there is a high percentage of hemoglobinopathy carriage. DisclosuresNo relevant conflicts of interest to declare.

LIN28B and ZBTB8B Genes Are Highly Expressed in Vitro in a CD34⁺ Cells Subpopulation of β-Thalassemia Major Patients and May be Involved in Increased HbF Production

Patients with homozygous beta thalassemia and the β⁰β⁰ genotype produce almost only fetal hemoglobin and a small proportion of hemoglobin A2. However, in the bone marrow of these patients there are descriptions of two different subpopulations of erythroblasts, one with high production of HbF (High HbF cells) and another with low production of HbF (Low HbF cells). The High F cells are probably those that survive through erythropoiesis and originate the viable red blood cells. The molecular mechanisms that are responsible for this high production of HbF are not clear. Therefore, the central aim of our work was to study, in cultures of CD34⁺ cells from β⁰β⁰ patients, differentiated to erythrocytes in vitro, the molecular characteristics of cells related to high production of HbF (High HbF), compared to those with low production of HbF (Low HbF). For this purpose, peripheral blood was collected from patients and CD34⁺ cells were identified, and further differentiated in vitro and then isolated by flow cytometry (FACS Aria). After separating the High HbF and Low HbF groups, the cells were morphologically evaluated by optical and confocal microscopy and image cytometry. Next RNA was extracted from the pool of isolated cells and the RNA sequencing assay (RNAseq) was performed, so that it was possible to analyze the transcripts that were differentially expressed between each of the groups. Finally, we validated the sequencing data by real-time PCR. The sorting was performed on the 10th day of cell culture, when the cells presented a double labeling profile for anti-transferrin (CD71) and anti-glycophorin (CD235) antibodies, the mean intensity and fluorescence (MIF) of the markers was 1058 and 1365 for CD71 in healthy control and β-thalassemia groups, respectively, and for CD235 was 430 in controls and 516 for patients. The cells were in an intermediate stage of maturation. After the isolation of sub-populations of High HbF and Low HbF cells, confocal microscopy indicated a clear difference in the intracellular HbF levels of the analyzed cells, confirming that the sorting by cytometry had occurred as desired. Imaging cytometry analysis revealed a MIF of 7.8 x 10⁵ for High HbF cells and 1.7 x 10⁵ for Low HbF cells, when we assessed the cell pools and when single cells were studied, these values ranged from 2.2 x 10⁵ for Low HbF cells to 1.9 x 10⁶ of mean intensity and fluorescence for High cells. After all data generated by RNAseq were filtered, we obtained a list of 16 enriched and differentially-expressed genes between High HbF and Low HbF cells. From these genes, it was possible to identify two that were apparently associated with the increased production of fetal hemoglobin, the genes ZBTB8B and LIN28B. ZBTB8B is a protein-coding gene with a zinc finger domain and may be involved in transcriptional regulation, it was included in our analyses since this gene is part of the same family as ZBTB7A, an important transcription factor that represses several genes involved in cell differentiation and proliferation and that has already been described as a regulator of HbF production. The LIN28B gene encodes an RNA-binding protein, which is described as a possible regulator of HbF levels during fetal development, by increasing the expression of gamma globin, culminating in the increase of intracellular levels of fetal hemoglobin through a direct action on the let-7 microRNA family and BCL11A gene.These results not only contribute to a better understanding of the mechanisms of gene regulation involved in the production of HbF, but indicate a potential new therapeutic approach to increase the production of HbF in hemoglobinopathies. DisclosuresCosta: Novartis: Consultancy.

Jaundice in a Preterm Infant Twelve Hours After Birth.

Jaundice in a Preterm Infant Twelve Hours After Birth.

The Roles of Liver Inflammation and the Insulin Signaling Pathway in PM2.5 Instillation-Induced Insulin Resistance in Wistar Rats.

To elucidate the mechanism of how the liver participates in PM2.5-caused insulin resistance. A novel Wistar rat model was developed in this study by instilling a suspension of lyophilized PM2.5 sample (2.5 mg/kg, 5 mg/kg, or 10 mg/kg) collected from the atmosphere. Systemic insulin resistance indicators, including serum fasting blood glucose (FBG), fasting insulin (FINS), Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), and hemoglobin A1 (HbA1), were upregulated by the PM2.5 instillation. The area under the curve (AUCglu) calculated by intraperitoneal glucose tolerance testing (IPGTT) was also significantly greater in the PM2.5 instillation groups. Additionally, PM2.5 instillation was found to cause liver damage and inflammation. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were significantly elevated by PM2.5 instillation. PM2.5 also triggered IL-6 and TNF-α transcription but inhibited mRNA synthesis and suppressed signaling activation of the insulin-phosphoinositide 3-kinase- (PI3K-) Akt-glucose transporter 2 (GLUT2) pathway in the rat liver by reducing the ratio of phosphorylated Akt to phosphorylated insulin receptor substrate 1 (IRS-1). Thus, PM2.5-induced inflammation activation and insulin signaling inhibition in the rat liver contribute to the development of systemic insulin resistance.

Screening for hemoglobinopathy with capillary electrophoresis in adult patients

Abstract Hemoglobinopathy screening is frequently needed in adult patients, including prenatal carrier screen, workup of unexplained anemia, and bone marrow donor and recipient screening. However, the preferred test method for screening of hemoglobinopathy is not well established due to limited guidance from professional societies. American College of Obstetricians and Gynecologists’ Committee on Genetics recommended hemoglobin electrophoresis as the screening method of hemoglobinopathy in pregnancy; nevertheless, electrophoresis employs various methodologies, including acid gel electrophoresis, alkaline gel electrophoresis, and capillary electrophoresis (CE) with alkaline buffer. For other adult patient populations who need hemoglobinopathy screening, no clear guidelines dictate the method of choice. A previous study has shown that CE captures major hemoglobinopathies with comparable performance to high-performance liquid chromatography (HPLC) in pediatric patients but no study has investigated using CE alone in adult patient screening. In this retrospective study, we evaluated the utility of CE as a screening method to rule out clinically significant hemoglobin variants. During eight months, 312 adult patients without previously identified hemoglobin variants had hemoglobinopathy screening performed using a comprehensive testing algorithm. This cascade algorithm screens for hemoglobinopathy using both CE (Capillarys, Sebia, Paris, France) and HPLC (laboratory-developed test) with reflex to more advanced variant identification such as mass spectrometry and genetic analyses. Categories of abnormal findings were reviewed to determine if hemoglobinopathy can be identified by using CE only. The patient population mainly consists of pregnant women and anemic patients with hematologic malignancies with an average age of 42. Out of the 312 screened patients, 47 had abnormal results. The most frequent condition was elevated hemoglobin F (N=25) ranging 2-5% seen in leukemia patients on chemotherapy attributed to bone marrow stress. Eight cases of beta plus thalassemia (featuring hemoglobin A2 &amp;gt;4%) and 3 cases of hemoglobin C trait were identified in patients with little to mild clinical manifestations (red blood cell indices suggesting anemia). Decreased hemoglobin A2 fraction was observed in 7 patients, and potential causes were alpha thalassemia or iron deficiency. Other less common hemoglobinopathies included heterozygote A2 prime (N=3, a benign delta chain variant that migrates separately from hemoglobin A2 on CE) and hemoglobin G-Philadelphia (N=1). All of the abnormal results are identifiable by CE alone, although HPLC and more advanced methods help confirm the diagnosis. Our study shows that CE as the first line of screening method would rule out major hemoglobinopathies in adults. There have been reports that rare but clinically significant hemoglobin variants like hemoglobin Malmo may not be detected by CE, and therefore, certain pre-test probability factors need to be considered when testing for hemoglobinopathies, such as race/ethnicity background, family history, red blood cell indices, and iron deficiency status.

Gastrointestinal Hormones in Healthy Adults: Reliability of Repeated Assessments and Interrelations with Eating Habits and Physical Activity.

Background: Gastrointestinal hormones (GIHs) are crucial for the regulation of a variety of physiological functions and have been linked to hunger, satiety, and appetite control. Thus, they might constitute meaningful biomarkers in longitudinal and interventional studies on eating behavior and body weight control. However, little is known about the physiological levels of GIHs, their intra-individual stability over time, and their interaction with other metabolic and lifestyle-related parameters. Therefore, the aim of this pilot study is to investigate the intra-individual stability of GIHs in normal-weight adults over time. Methods: Plasma concentrations of ghrelin, leptin, GLP-1 (glucagon-like-peptide), and PP (pancreatic polypeptide) were assessed by enzyme-linked immunosorbent assay (ELISA) in 17 normal-weight, healthy adults in a longitudinal design at baseline and at follow-up six months later. The reliability of the measurements was estimated using intra-class correlation (ICC). In a second step, we considered the stability of GIH levels after controlling for changes in blood glucose and hemoglobin A1 (HbA1c) as well as self-reported physical activity and dietary habits. Results: We found excellent reliability for ghrelin, good reliability for GLP1 and PP, and moderate reliability for leptin. After considering glucose, HbA1c, physical activity, and dietary habits as co-variates, the reliability of ghrelin, GLP1, and PP did not change significantly; the reliability of leptin changed to poor reliability. Conclusions: The GIHs ghrelin, GLP1, and PP demonstrated good to excellent test–retest reliability in healthy individuals, a finding that was not modified after adjusting for glucose control, physical activity, or dietary habits. Leptin showed only moderate to poor reliability, which might be linked to weight fluctuations, albeit small, between baseline and follow-up assessment in our study sample. Together, these findings support that ghrelin, GLP1, and PP might be further examined as biomarkers in studies on weight control, with GLP1 and PP serving as anorexic markers and ghrelin as an orexigenic marker. Additional reliability studies in obese individuals are necessary to verify or refute our findings for this cohort.

Anti-interference hemoglobin analysis system by high performance liquid chromatography

High performance liquid chromatography (HPLC) is currently the mainstream technology for detecting hemoglobin. Glycated hemoglobin (HbA1c) is a gold indicator for diagnosing diabetes, however, the accuracy of HbA1c test is affected by thalassemia factor hemoglobin F (HbF)/hemoglobin A2 (HbA2) and variant hemoglobin during HPLC analysis. In this study, a new anti-interference hemoglobin analysis system of HPLC is proposed. In this system, the high-pressure three-gradient elution method was improved, and the particle size and sieve plate aperture in the high-pressure chromatography column and the structure of the double-plunger reciprocating series high-pressure pump were optimized. The system could diagnose both HbA1c and thalassemia factor HbF/HbA2 and variant hemoglobin, and the performance of the system was anti-interference and stable. It is expected to achieve industrialization. In this study, the HbA1c and thalassemia factor HbF/HbA2 detection performance was compared between this system and the world's first-line brand products such as Tosoh G8, Bio-Rad Ⅶ and D10 glycosylated hemoglobin analysis system. The results showed that the linear correlation between this system and the world-class system was good. The system is the first domestic hemoglobin analysis system by HPLC for screening of HbA1c and thalassemia factor HbF/HbA2 rapidly and accurately.

THE ROLE OF COMPLETE BLOOD COUNT IN PATIENTS WITH HETEROZYGOUS BETA THALASSEMIA

OBJECTIVE: The goal of this study was to compare the Mean Platelet Volume (MPV), Platelet Lymphocyte ratio, and Neutrophil Lymphocyte ratio by evaluating the parameters of Complete Blood Count in healthy children and patients with heterozygous Beta-thalassemia (HBT).MATERIAL AND METHODS: Between July 2016 and September 2019, 60 patients admitted to the pediatric hematology-oncology outpatient clinic diagnosed with HBT and 60 healthy control subjects of similar age and sex were analyzed retrospectively. Children over one year old, whose hemoglobin A2 levels were above 3.5% were included in the study population.RESULTS: A total of 120 subjects were enrolled in this study. The HBT group included 60 subjects (32 males and 28 females) and the control group included 60 (36 males and 24 females). There were no significant differences between the two groups in terms of age and gender (p=0.29 and p=0.27). Platelet count, MPV, lymphocyte, and ferritin were significantly higher in the HBT group, whereas hemoglobin was significantly lower in the HBT group as compared with the control group.CONCLUSIONS: Increased MPV and platelets in healthy HBT patients may cause hypercoagulability and may play a role with other risk factors in the predisposition of thrombosis in adult cases. There are very few studies on the incidence of thromboembolic events in cases with HBT. It is necessary to monitor adult HBT cases especially in terms of thromboembolic events, and to study with more cases.

Screening of Some Indicators for Alpha-Thalassemia in Fujian Province of Southern China.

BackgroundCarrier screening is the most effective means of controlling the prevalence of alpha-thalassemia. However, due to the differences in ethnic populations and genotypes, the distribution of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and hemoglobin A2 (HbA2) varies in different regions. This study aimed to examine screening efficiency of these indicators in different genotypes of alpha-thalassemia in Fujian Province, China.MethodsThe data of 13,294 subjects collected from May 2016 to December 2019 were reviewed. The participants were categorized as alpha-thalassemia group and negative-for-alpha-thalassemia group based on the results of the genetic analysis. The distribution of MCV, MCH, and HbA2 in different groups was analysed statistically. And the screening efficiency of different indicators and schemes was compared in different genotypes. The positive criteria of MCV < 80fL, MCH < 27pg, and Hb A2< 2.5% were applied.ResultsAmong the 13,294 subjects, 2658 were alpha-thalassemia carriers. The genotypes of –SEA/αα and -α3.7/αα are the most prevalent with 63.9% and 21.9% in Fujian Province, China. There were significant differences in the distribution of the three indicators in different groups. The detection rate of the three indicators combined screening was 92.6%.ConclusionThe distribution of the three indicators overlapped partly between alpha-thalassemia group and negative-for-alpha-thalassemia group. They showed significant differences in the median comparison of seven common genotypes. Combined screening with MCV, MCH and HbA2 improved the detection rate of alpha-thalassemia. The results of this study provide a data basis for clinical laboratories and a reliable reference for clinical consultation.

Differences in insulin dosing in women with type 1 diabetes before and after the menopause.

The menstrual cycle increases insulin requirements in a subset of women with type 1 diabetes as a result of reduced insulin action through sexual hormones. If exposure to sexual hormones declines during the menopause, adaptions of insulin dosing may be required. However, there are no validated recommendations available on how to adapt insulin treatment in postmenopausal women with type 1 diabetes. The present study compared insulin dosing profiles of 630 premenopausal and 548 postmenopausal women, who had long-term type 1 diabetes and used continuous subcutaneous insulin infusion. Data were extracted from the German "Diabetes-Patienten- Verlaufsdokumentation". It was found that total daily insulin (p <0.0001), daily insulin per kilogram bodyweight (p <0.0001), total daily basal insulin (p <0.0001), daily basal insulin per kilogram bodyweight (p <0.0001) and estimated glomerular filtration rate (eGFR) (p <0.0001) were lower in postmenopausal women. Total daily bolus insulin, daily bolus insulin per kilogram, glycated haemoglobin A1, body mass index and the incidence of severe hypoglycaemic events were similar in both cohorts.Postmenopausal women with type 1 diabetes used lower insulin doses as compared with premenopausal women, whereas glycaemic control and body mass index were comparable. This observation might be explained by lower exposure to sexual hormones and lower eGFR, even though the contribution of other factors such as body composition and eating habits requires further investigation.

English

BACKGROUND Sickle delta beta thalassemia is a rare genetic disorder, with varied symptoms, signs, requiring careful monitoring for potential complications. It is due to sickle mutation and thalassemia mutation occurring together, with sparse data available worldwide. The purpose of this study was to assess the clinical and laboratory profile of possible sickle delta beta thalassemia. METHODS The study design is retrospective analysis of clinical information of those selected patients done in our multi-specialty tertiary care referral hospital situated in Telangana state in south India. The case material was collected from December 2017 to December 2019 (2 years duration). All haemoglobin electrophoresis reports were collected with no prior blood transfusions in preceding 4 months. The information collected was analysed and presented. RESULTS Total 9 patients were diagnosed as possible sickle delta beta thalassemia, with male to female ratio of 5 : 4 and age ranging from 12 years to 45 years of age. The commonest symptoms were joint pain and jaundice in 5 patients and sign was splenomegaly in 2 patients. Ultrasonogram of abdomen showed that 3 patients had gall stones, 1 patient had gall bladder sludge, 1 patient had autosplenectomy and 3 patients had splenomegaly. Mild to moderate anaemia was seen with reticulocytosis, sickling test positive in all patients, with haemoglobin in the range of 5.5 g/dl to 12.7 g/dl. 3 patients had iron over load, 2 patients had hepatopathy, 5 patients had unconjugated hyperbilirubinemia, Acute chest syndrome, hepatic necrosis, and nephropathy was seen in 1 patient each. Haemoglobin electrophoresis showed Hb S was from 46.1 % to 76.4, Hb A from 5.3 % to 34.7 %, Hb F from 4.8 % to 22.7 %, Hb A2 from 1.5 % to 3.3 %. 2 patients were treated with hydroxyurea. 2 patients had mutation analysis elsewhere that was reported as compound heterozygous for β-globulin gene for Hb S (GAG-GTG) and IVS 1 - 5 (G-C). CONCLUSIONS Sickle delta beta thalassemia presents as mild to moderate anaemia haemolysis, splenomegaly with vaso-occlusive crises. Hydroxyurea may help in the treatment. Genetic analysis helps in diagnosis and future therapies. KEYWORDS Haemoglobin S, Haemoglobin A2, Haemoglobin A, Haemoglobin F

Microcytic Anaemia as Susceptibility Factors in Bipolar Spectrum Disorders: Review of the Literature, Replication Survey, and Co-Segregation within Families.

Background:Potential interactions between mood disorders and microcytic anaemias have been suggested by case reports, surveys of haematological parameters in psychiatric populations, and surveys of psychiatric morbidity in thalassaemic carriers.Objectives:a) To review published studies.b) To study the prevalence of microcytic anaemia in a sample of Sardinian outpatients with recurrent mood disorders.c) To check whether mood disorders and microcytic anaemia co-segregate within families.Methods:We extracted data on blood count and serum iron concentrations from the records of patients admitted between January 1st, 2001 and December 31st, 2016, to our clinic for mood disorders. Moreover, we studied siblings of subjects with both major mood disorders (according to Research Diagnostic Criteria) and heterozygous thalassaemia (according to Mean Corpuscular Volume, serum iron, and haemoglobin A2 concentrations). Siblings affected with a major mood disorder were examined for haematological concordance with the proband (reduced MCV and/or increased HbA2 in case of heterozygous β-thalassaemia, or presence of gene deletions in case of α-thalassaemia).Results:Microcytic anaemia was highly prevalent (81/337 = 24.0%) among outpatients with mood disorders. Starting from 30 probands with heterozygous ß-thalassaemia, concordance for reduced MCV and/or increased HbA2 was found in 78% (35/45) of affected siblings. Starting from 3 probands with heterozygous α-thalassaemia, only one of the 5 affected siblings carried four α-globin functional genes.Conclusion:Based on the review of the literature, the high prevalence of microcytic anaemia in outpatients, and the concordance between affected siblings, we can conclude that a role of heterozygous thalassaemias is highly probable. Future studies are required to establish the relevance of heterozygous thalassaemias and evaluate the magnitude of the effect, possibly using a molecular diagnosis also in the case of heterozygous β-thalassaemia.

DIAGNOSTIC EFFICACY OF RED BLOOD CELL INDICES IN DIAGNOSIS OF BETA THALASSEMIA TRAIT TAKING HAEMOGLOBIN ELECTROPHORESIS AS GOLD STANDARD

Objective: To determine the diagnostic efficacy of haematological indices for the diagnosis of beta thalassemia trait taking haemoglobin electrophoresis as a gold standard.&#x0D; Study Design: Cross sectional study.&#x0D; Place and Duration of Study: Department of Haematology Combined Military Hospital Lahore, from Aug to Dec 2019.&#x0D; Methodology: Two hundred and six anaemic patients 20-70 years of age of both genders were included in the study. Blood was collected from all the patients and complete blood count were generated through automated haematology analyser Sysmex KX-21. Based on the complete blood count parameters like mean cell volume, red blood cell count, red cell distribution width and mean cell haemoglobinthe haematological indices such as Mentzer, Ricerca, mean density of Hb/litre of blood, mean cell Hb density and red cell distributon width indices were calculated. The haemoglobin A2 band of &gt;3.5% on haemoglobin electrophoresis was taken as cut off for Beta thalassemia trait.&#x0D; Results: Out of the 206 blood samples analysed, 120 (58%) were labelled as Beta thalassemia trait after Hb electrophoresis and 86 cases had anemia due to other causes. Mentzer index established the highest sensitivity of 92.5% whereas RICERCA index established the highest specificity of 94.1%. Mentzer and red cell distribution width indexhad highest diagnostic efficacy value up to 90.3%.&#x0D; Conclusion: We conclude that Mentzer and red cell distribution width index showed the highest diagnostic significance. The Mentzer index has the highest sensitivity whereas Ricerca index has the highest specificity. The red blood cell count, haemoglobin and mean cell volume.............

Response to: Acute Liver Failure in a Sickle Cell Patient.

Reply: We thank Maji et al for their interest in our case report (1). Our patient had considerable evidence of hemolysis on initial presentation, evident by anemia of 7.2 g/dL, with an increase reticulocyte percentage of 11.94 (0.50–2.20%), and an elevated absolute reticulocyte number of 386.9 (19.0–124.0 × 109). Additionally, he was noted to have elevated lactate dehydrogenase with the value of 910 (140–271 U/L) with low haptoglobin of <5.8 mg/dL (36.0–195.0 mg/dL). Upon initial presentation, infectious work up including Epstein–Barr virus, parvovirus, cytomegalovirus, human herpesvirus 6, human immunodeficiency virus, hepatitis B, and hepatitis C were all negative. Liver biopsy was done to rule out etiologies including autoimmune and metabolic rather than making a diagnosis of sickle cell intrahepatic cholestasis (SCIC). SCIC can present with renal impairment, but it is not the defining feature. Impressive hyperbilirubinemia, mostly conjugated, and transaminase levels in the thousands are the hallmark of this condition (2). Fortunately, our patient did not have any renal impairment Table 1. TABLE 1 - Hemoglobin electrophoresis pre- and post-exchange transfusion Electrophoresis Hemoglobin A1 (92–99%) Hemoglobin A2 (1.0–5.0%) Hemoglobin F (1.0–4.0%) Hemoglobin S Sickle cell solubility Pre 61 3.0 6.0 30.0 Positive Post 87 3.0 2.0 8.0 Positive Presentation of SCIC can be variable resulting in an impediment to know an exact number of cases. Ahn et al retrospective chart review study is limited due to incomplete data and lacks a clear diagnosis of intrahepatic cholestasis (3). Additionally, transaminase levels are usually in the thousands in patients with SCIC (2), and half of the reported patients had a mild elevation of transaminases, mostly <500 U/L (3). There may be more reported cases of SCIC, but our aim was timely recognition of rare presentation of sickle cell hepatopathies and consider exchange transfusion with aggressive hydration for SCIC to reverse the hepatic sinusoidal sickling, and reverse liver failure.

Retrospective Analysis of the HB A2 Value in Sickle Cell Positive Population in Central Gujarat, India

Introduction:Sickle cell disease (SCD) is one of the commonest autosomal inherited diseases globally. India has high Sickle cell disease burden, presented with very wide clinical phenotypes. Gujarat state particularly central and south region of state has a high prevalence of the sickle cell anemia. Though being commonly encountered in this region, there are hardly few large scale studies done on sickle cell anemia and its clinical impact on health. With this background in mind, we did this retrospective analysis of laboratory parameters to understand the correlation of HbA2 values in sickle cell population.Materials and Methods:This retrospective analysis of Capillary electrophoresis (CE) or High performance liquid chromatography (HPLC) results of total 30845 sample size was done. Study period June 2013 to June 2017. Investigation carried out at 4 different laboratories was reviewed for analysis of Hemoglobin A2 (HbA2) values in Sickle cell population in the central Gujarat region of India. Hematological indices, Solubility tests, were also taken into considerations. Initially, all the data were reviewed and analyzed separately and then they were compiled together and analyzed. Out of 30845, 28495 results were reviewed. Rests were omitted either due to incomplete data. On capillary electrophoresis 19383 tests were conducted, whereas, 9112 tests were conducted on HPLC.ResultOut of total 28495 samples, 2327 were sickle positive, 846 had HBS >50% (SCD), 1471 had HBS<50% Sickle cell trait (SCT), 26168 no sickle window. With respect to HbA2 value, we divided them further into 3 groups. Group 1 : HbA2 between 2 - 3.5%, Group 2 : HbA2 more than 3.5% and Group 3:HbA2 less than 2%. Further sub analysis of all the 3 groups was done on the basis of MCV. As we assume that high HbF with HbA2<2% may be associated with δ-β thalassemias and low HbF with HBA2<2% may be associated with α-thalassemia, in group 3, Hb F was also considered in sub analysis. The results of the analysis are as depicted in table.ConclusionIn this retrospective analytic study we found that SCD can be categorized into 3 groups based on HB A2 value, further sub analysis with respect to MCV and HB F value shows, 644 (75.2%) have probability of having co inheritance of Thalassemia (MCV< 80 FL), remaining 212(24.8%)may be representing homozygous SCD with no co inheritance of α or β or δ-β Thalassemia (MCV>80 FL).As we could not find single sample having HBA2 <2% with HBF(<10%) with low MCV in SCD category, raises possibility are we dealing with δ-β Thalassemia in this study population? These patients require further family studies and molecular study to confirm co-association of Thalassemia.There is high possibility that heterogeneity in clinical presentation of SCD patients found in Gujarat may have correlation with low MCV and variability in A2 value (indirect evidence of underlying co-inheritance of Thalassemia). Prospective large cohort study is required to document clinico-pathological correlation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.