Cation-exchange High-performance Liquid Chromatography Research Articles

Novel Double Fusion (βδβ) Hemoglobin Variant with Comparison to Lepore Hemoglobins

Lepore hemoglobins (Hbs) such as Hb Lepore-Boston-Washington [δ(1-87) β(116-146)], Hb Lepore-Baltimore [δ(1-50) β(86-146)], Hb Lepore-Hollandia [δ(1-22) β(50-146)] are δβ gene fusion/deletion variants (ε- Gγ- Aγ-[δβ] v )(Figure 1). They are controlled by the weak δ-globin gene promoter and synthesized at approximately 10-15% of hemoglobin fractions. This results in a similar phenotype as β +-thalassemia (microcytosis and hypochromia ± mild anemia). Anti-Lepore hemoglobins such as Hb P-Nilotic (β(1-22) δ(50-146) are βδ gene fusion/duplication variants (ε- Gγ- Aγ-δ-[βδ] v-β) associated with normal CBC values. Herein we describe a novel βδβ double fusion variant (ε- Gγ- Aγ-δ-[βδβ] v). Due to high homology between the δ and β genes, this double fusion event encodes an identical protein to Hb Lepore-Hollandia, albeit linked to the β-promoter and therefore expressed at ~40% of hemoglobin fractions, which would be expected to enhance any intrinsic properties of the variant. This novel hemoglobin variant is named Hb Lepore Rochester-MN. Two cases have been identified (Table 1). Case 1 was a 10-month-old Caucasian/Native American infant referred for an abnormal newborn screen and a reported family history of Hb G-Coushatta (β22, GAA>GCA, Glu>Ala; HGVS: c.68A>C, p.E23A). Case 2 was a 3-month-old infant referred for an abnormal newborn screen. CBCs were normal for age including MCV. Cation-exchange high performance liquid chromatography (HPLC) showed a shouldered peak in the Hb A 2 window typical for Hb Lepore (elution time: 3.41-3.46 mins), although the variant percentage was higher than expected, mimicking Hb G-Coushatta (expected elution time: 3.33 mins). Capillary electrophoresis identified a peak in the D zone (Z(D) zone), which is expected for Hb Lepore or Hb G-Coushatta. Intact mass spectrometry was consistent with Hb Lepore-Hollandia at 15836 amu, differing from Hb G-Coushatta (Glu>Ala, 15809 amu). Multiplex ligand probe amplification (MLPA) was normal because the highly variable fusion regions are unsuitable for probe placement. Sanger sequencing of the β-globin gene identified multiple single base substitutions which were challenging to interpret but matched the sequence expected for a complex double crossover event (NM_000518.4:c.28_68delins41 (p.Ser10_Glu23delins14TAVNALWGKVNVDA). The first crossover is located between c.-29 and c.9, the second crossover is located between c.69 and c.92+17. Long-read sequencing was performed (Sequel, Pacific Biosciences) which detected the multiple missense mutations in cis conformation, as was corroborated by the mass spectrometry data. In summary, Hb Lepore Rochester-MN represents a novel double fusion [βδβ] variant class that will pose a diagnostic challenge for laboratories. Although it is likely a result of sequential crossover events (deletion and duplication) between Lepore and anti-Lepore variants, MLPA analysis is normal due to technical constraints and high homology. DNA sequencing (both Sanger and long read methods) yielded complex results that require sophisticated analysis and correlation with protein data including intact mass spectrometry. HPLC and CE data including variant percentage are very similar to Hb G-Coushatta. In addition, although resulting in an identical protein, the variant should not be mistaken for Hb Lepore-Hollandia, which is produced at lower levels and causes a thalassemic phenotype. Hb Lepore Rochester-MN is controlled by the β-promoter and therefore is associated with approximately 40% variant and apparently normal CBC values in the two infants.

A New Hemoglobin Variant: Hb Tangshan [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)] Detected by MALDI-TOF MS

In this report we decribed a new α-chain variant found during the measurement of hemoglobin A1c (Hb A1c) using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). MALDI-TOF MS analysis detected an α-chain variant with a mass of 15,155 Da. However, this Hb variant was not detected during Hb A1c measurement by cation-exchange high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) methods. Sanger sequencing validated the presence of a heterozygous missense mutation [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)]. The observed 28 Da mass difference exactly matches the theoretical mass difference (28 Da) resulting from the substitution of alanine (89.079) with valine (117.133). As this represents the initial documentation of the mutation, we named it Hb Tangshan after the proband’s residence.

A specific early eluting peak in Bio-Rad Variant II cation-exchange high-performance liquid chromatography (CE-HPLC) for the screening of alpha-thalassaemia in a Chinese population

The early eluting peaks in the first minute of cation-exchange high-performance liquid chromatography (CE-HPLC) are often not analysed in haemoglobin pattern studies, except for haemoglobin (Hb) Bart's andHb H peaks. In this study, we described the presence of a specific α-thalassaemia early eluting peak (αEEP) atthe retention time of 0.24 min generated by Variant II CE-HPLC (β-Thalassaemia Short Program; Bio-Rad Laboratories). We have evaluated the utility of αEEP for the screening of α-thalassaemia trait in our local Chinese population in comparison to the Hb H inclusion body test. A total of 303 blood samples presenting with microcytosis were sent for haemoglobin pattern study and were analysed for the presence or absence of αEEP and Hb H inclusions. Twenty cases with a normal mean corpuscular volume were assessed as a control. Discordant results between the αEEP and the Hb H inclusion test were reviewed with the α-globin genotyping test performed. The concordance rate of the αEEP and the Hb H inclusion body test was 96.0% (κ=0.921, p<0.001). Eight of 303 cases (2.6%) were initially negative for the Hb H inclusion test but positive for the αEEP. All eight cases were found to have occasional Hb H inclusion bodies upon review. Four of 303 cases (1.3%) were negative for the αEEP but positive for the Hb H inclusion test. Of these four cases, two (50%) showed heterozygous Southeast Asian (SEA) type deletion, one (25%) showed Hb Quong Sze mutation, and one (25%) showed no mutation detected upon molecular testing. All the Hb E trait cases with no Hb H inclusions and the negative control group showed the absence of the αEEP. The sensitivity and specificity of αEEP for detecting SEA deletion were 93.8% and 100% respectively, which is superior to the Hb H inclusion test (sensitivity 81.3%, specificity 95.2%). The αEEP is found to be a more sensitive method than the Hb H inclusion body test in the screening of α-thalassaemia trait in our Chinese population, in which SEA type deletion is prevalent. Further study is needed to explore the utility of the αEEP in the screening of α-thalassaemia traits in other populations. The exact nature of the αEEP is yet to be defined.

Thalassemia and Hemoglobinopathy Screening in Women Attending Antenatal Clinic at a Tertiary Care Center in Uttarakhand, India: A Re-look at the Laboratory Parameters Mandating High-Performance Liquid Chromatography Workup.

Thalassemia and hemoglobinopathies are the most common inherited hematological disorders. Of these, β thalassemia is the commonest disorder reported in India, followed by certain hemoglobinopathies encountered in different regions of the country. The data pertaining to the incidence of these disorders in the Uttarakhand region of India are sparse. To ascertain the prevalence and spectrum of thalassemia/hemoglobinopathies amongst antenatal women in Uttarakhand. The study also aimed to analyze the ability of red cell indices in differentiating beta thalassemia trait (BTT) from mild iron deficiency anemia (IDA). A total of 460 pregnant women in the first trimester of pregnancy were screened by cation exchange high-performance liquid chromatography. Retention time and proportions of normal/abnormal hemoglobin peaks were documented in all cases. Hemoglobin A2 (HbA2) values of ≥4% were taken as a cut-off for diagnosing BTT. Blood samples were also collected for complete blood counts, reticulocyte counts, and serum ferritin. The ability of the various discriminatory indices to differentiate between IDA and BTT was also assessed. The prevalence of BTT and hemoglobin D-Punjab trait amongst pregnant women was found to be 2.6% and 0.2%, respectively. RBC count, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were found to be moderately strong predictors of BTT, with an area under the curve of 0.860, 0.857, and 0.842, respectively, which were comparable to the discriminatory indices found to be most useful in this study. In view of the 2.6% prevalence of BTT in antenatal women in this region of Uttarakhand, a routine screening will be helpful in detecting carriers early in the antenatal period. Careful interpretation of red cell indices is crucial to the distinction between BTT and IDA. Discriminatory indices are reasonably accurate in differentiating BTT from mild iron deficiency, but for practical purposes, MCV and MCH provide equivalent information to identify cases that require further workup.

Comparison of HbA2 Using High Performance Liquid Chromatography Versus Haemoglobin Capillary Zone Electrophoresis.

Thalassemia is among the most common hereditary disorders in the world. Approximately 5% of the world's population are carriers of hemoglobinopathies, and 2.9% are carriers of beta thalassemia. Haemoglobin A2 (HbA2) constitutes less than 3% of the total hemoglobin (Hb) in adults, and the determination of Hb A2 levels is important to diagnose the beta thalassemia trait(BTT). In some cases, the level of HbA2 is not typically elevated, and some difficulties may arise in making the diagnosis. Cation exchange high-performance liquid chromatography (HPLC) and HbCZE (haemoglobin capillary zone electrophoresis) are considered acceptable methods to diagnose BTT, but these vary in their accuracy and cut-offs. In this study, we attempted to compare HbA2 values using two methods, HPLC and HbCZE, in 536 whole blood samples sent by physician-ordered hemoglobinopathy screening over two years. This included antenatal women, patients with anemia not responding to iron, and cases of familial screening where either a child or a sibling had been diagnosed with hemoglobinopathy or thalassemia. The performance characteristics of both machines were compared for the detection of the 5 most common hemoglobin variants: Hb A, HbF, HbS, Hb C, and HbE. On comparing the HbA2 values, the HPLC showed higher values for HbA2 as compared to HbCZE, while the HbF and HbS measurement agreement was good between both methods. Normal ranges and mean normal values of HbA2 differ between different methods and different manufacturers; hence, each institute using these machines should validate its cutoffs.

The Case Report of Hemoglobin Hamadan: HBB:c.169G&gt;C (B 56 (D7) Gly-Arg) in İstanbul

27-year-old case with hemoglobin (Hb) Hamadan was presented. During premarital thalassemia screening an unidentified Hb peak; retention time (RT): (5.55 min, 46.2%) was determined with cation-exchange high-performance liquid chromatography (CE-HPLC) at premier resolution (Trinity-Biotech) system. Variant Hb was eluted with Hb A2 (RT): 3.76 min, 45%) at Variant II Turbo (Bio-Rad), while it was 45.7% and detected at S zone at capillary zone electrophoresis system (CZE) Sebia (Lisses, France). She didn't show any clinical symptoms or hematological abnormality. Hemoglobin A1c (HbA1c) results by Boronate affinity, CE-HPLC and CZE methods were 5.7%, 4.7%, and 5.3% respectively. Beta globin gene sequencing revealed a heterozygote codon (c.169G>C (P.Gly57Arg) identical with Hb Hamadan. Hb Hamadan does not cause clinical complaints, but may be important in prenatal examination and cause interference in HbA1c measurements.

Hearing loss in Type 2 diabetes: Impact of duration, glycemic control, and oxidative stress

Background: Type 2 diabetes mellitus (T2DM) has been linked to many problems, including hearing loss, which research studies have not paid much attention to. This study looks into the possible links between hearing loss in people with T2DM, the duration of time they have had diabetes, how well they control their blood sugar, and measures of reactive stress. Understanding how these things work together can help doctors find problems faster, develop better ways to treat them, and improve their patients’ health. Aims and Objectives: This study aims to see if there are any links between hearing loss and duration of T2DM, levels of glycemic control, and measures of oxidative stress in people with T2DM. The results of this study will help come up with early identification and healing ways to improve patient care. Materials and Methods: In a cross-sectional study at Government Medical College, Kozhikode, 63 diabetic patients aged 30–60 were compared with 63 age and sex-matched controls. Blood sugar was assessed using the glucose oxidase peroxidase method, hemoglobin A1C (HbA1C) through cation exchange high-performance liquid chromatography, serum malondialdehyde (MDA) through the thiobarbituric acid test, and superoxide dismutase (SOD) using the technique by Marklund and Marklund, with the latter two assessed spectrophotometrically. Hearing abilities were evaluated using pure tone audiometry. Results: Diabetic participants exhibited a marked increase in hearing loss incidence compared to controls (P &lt; 0.001). Males with diabetes showed a higher prevalence of hearing loss than females (P = 0.043). A significant correlation was found between hearing loss and the duration of diabetes (P &lt; 0.001), elevated glycemic levels (fasting blood sugar, random blood sugar, and HbA1c, all P &lt; 0.001), and altered oxidative stress parameters. Specifically, MDA levels were higher in people with diabetes (P &lt; 0.001) and associated with hearing loss (P = 0.033), while SOD levels were reduced (P &lt; 0.001) and significantly linked to hearing loss (P = 0.047). Logistic regression identified age, sex, HbA1c, MDA, and SOD as independent risk factors for hearing impairment in Type 2 diabetes patients. Conclusion: Our findings emphasize the significant association between Type 2 diabetes and heightened risk of hearing loss, influenced by age, gender, glycemic levels, and oxidative stress markers. Regular audiological assessments are crucial for early detection and intervention in diabetic patients.

Evaluation of interference from 16 hemoglobin variants on hemoglobin A1c measurement by five methods

Hb variants prevalent in China are different from those in other countries. We aimed to assess the interference from Hb variants found in China on HbA1c measurement. All Hb variants were confirmed using Sanger sequencing. HbA1c was measured using a capillary electrophoresis method (Capillarys 3 OCTA), two cation-exchange high-performance liquid chromatography methods (ADAMS HA-8180V and HLC-723 G8 standard mode), an immunoassay (Cobas c501), and a boronate affinity chromatography method (Premier Hb9210). Premier Hb9210 was used as a comparative method. A total of 16 species of Hb variants were identified in 102 variant carriers. The most common variant was Hb E, followed by Hb Q-Thailand, Hb New York and Hb J-Bangkok. Clinically significant interference was observed for the Capillarys 3 OCTA (two Hb variants), ADAMS HA-8180V (seven Hb variants), HLC-723 G8 (14 Hb variants), and Cobas c501 (two Hb variants). The proportion of unacceptable HbA1c results was 13.7% for Capillarys 3 OCTA, 52.9% for HA-8180V, 83.3% for HLC-723 G8, and 3.9% for Cobas c501. Hb variants in China severely affect the accuracy of some commonly used HbA1c methods.

Quantification of lactoferrin in human milk using monolithic cation exchange HPLC

Human lactoferrin (hLF) is one of the most important whey proteins in human milk, known for its ability to modulate innate host immunity and multifunctional activities for neonatal growth. The objective of this study was to validate an efficient method for the detection and quantification of hLF using a unique technology of cation-exchange high-performance liquid chromatography (HPLC) on CIM® monolithic columns. Human milk samples were collected using manual expression or a breast pump, at different weeks of lactation. After sample preparation, hLF was detected and measured by HPLC method and further confirmed by SDS-PAGE. Selected fractions were analysed also by LC-MS/MS. Presumably, due to the high density of positive charge on the surface of the N-terminal domain, hLF binds strongly to the column and elutes last, enabling the high specificity of this method. The LC-MS/MS analysis indicated that hLF eluted in two clearly separated peaks, presumably representing two different molecular species of hLF. hLF concentration in the human milk samples ranged from 2.03 mg/mL to 5.79 mg/mL and was not significantly affected by the sample collection method whereas it was negatively correlated with the stage of lactation. These results suggest that cation exchange chromatography is an accurate, efficient, and robust method for the detection and quantification of hLF.

Hemoglobinopathies by HPLC: A 3 Year Study of 106,277 Cases

Introduction: Hemoglobinopathies and thalassemias are the most common single gene disorders in the world. World Health Organization figures estimate that 5% of the world populations are carriers of a potentially pathological hemoglobin (Hb) gene. The general incidence of thalassemia trait and sickle cell anaemia in India varies between 3-17% and 1-44% respectively3 but because of consanguinity, caste and area endogamy, some communities show a very high incidence, making the disease a major public health problem in our country. Cation exchange high-performance liquid chromatography (CE-HPLC) is one of the best methods for screening, detection, and identification of various hemoglobinopathies.&#x0D; Material &amp;Method: A retrospective study was carried out from period of 2017 to 2019 with 106277 cases evaluated with an aim to identify various hemoglobinopathies seen in Indian population by high-performance liquid chromatography. Cases outside Indian geographical location were excluded from the study&#x0D; Result: A total of 18,936(17.82%) cases with abnormal haemoglobin variants was reported in the study with 35 difference variants across India. Northeast India reported maximum abnormal hemoglobinopathies (50.16%). Beta Thalassemia Trait was the most common abnormal variant found.Such a high incidence emphasises premarital and prenatal screening for prevention of dangerous effects of hemoglobinopathies in the population.&#x0D; Conclusion: CE-HPLC should be used for early detection and proper management of these haemoglobinopathies. The most common hemoglobinopathy observed in our study was Beta thalassemia trait followed by Sickle cell trait and then sickle cell disease. It was also observed that Northeast India had maximum abnormal hemoglobinopathies.

Examination of the primary fraction of hemoglobin in individuals having sickle cell disease using high-performance liquid chromatography

Background: Hemoglobinopathies refer to a range of diseases, in which the hemoglobin (Hb) molecule’s synthesis or structure is aberrant. It is passed down through the generations (inherited). Hb C illness, Hb S-C disease, sickle cell anemia, and thalassemia are all part of this group. Hemoglobinopathies are the most frequent genetically inherited red blood cell condition on the planet. Hb variant screening and precise identification have become more crucial in the antenatal diagnosis and prevention of Hb diseases. Aims and Objectives: Hb analysis, cation exchange high-performance liquid chromatography (HPLC) has emerged as the method of choice. This research was brought up to diagnose numerous hemoglobinopathies and also to measure distinct Hb fractions. Materials and Methods: Four hundred and seventy participants were examined for hemoglobinopathies in the investigation. Different fractions of Hb were compared to patients with normal Hb after diagnosis. Results: Normal HPLC, SCA (Hb SS), sickle cell trait (SCT), and -thalassemia trait were discovered in 110 (23.4%), 130 (27.6%), 210 (44.6%), and 20 (4.2%) individuals, respectively, out of 470 patients. Rare hemoglobinopathies such as tenacious fetal Hb (PFH), Hb E, Hb D-Punjab, and Hb Q India accounted for 15 (3.1%) of the sufferers. Patients with SCA had a mean percent of Hb S, Hb F, Hb A, and Hb A2 of 81.6±74.6, 22.9±13.9, 3.1±6.1, and 2.5±1.02, respectively. Hb F was 22.9±13.9% on average, which was much higher than normal and SCT. A high HbA2 level (&gt;3.5%) indicated the existence of the β-thalassemia trait. Conclusion: In the included section of the population, SCD is the most common kind of hemoglobinopathy, both homozygous and heterozygous. Hb F concentrations are observed to be elevated in SCA patients, and it is thought to be a key element in protecting patients from problems. Raised Hb A2, which indicates the presence of the β-thalassemia trait, was also discovered in this area, and it is suspected when the value is &gt;3.7%. Cation exchange HPLC is ideal for routine hemoglobinopathies research due to its high resolution, short test time, and precise quantification.

Production and Conjugation of Truncated Recombinant Diphtheria Toxin to VEGFR-2 Specific Nanobody and Evaluation of its Cytotoxic Effect on PC-3 Cell Line.

Immunotoxins have represented a great potency in targeted therapeutics to encounter tumors. They consist of a protein toxin conjugated to a targeting moiety, which recognizes a specific antigen on surface of cancer cells and accordingly induces cell death by toxin segment. The targeting part could be a nanobody, which is a group of antibodies composed of an only functional single variable heavy chain (VHH).Therefore, this study was done to produce an immunotoxin (VGRNb-DT) by chemical conjugation of a truncated diphtheria toxin moiety to an anti-vascular endothelial growth factor receptor 2(VEGFR-2) nanobody, and to identify effectiveness of immunotoxin in recognizing the VEGFR-2- positive cancer cells and inhibiting cell growth and survival. Diphtheria toxin was expressed and purified by nickel affinity chromatography, and accordingly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis confirmed its expression. Function of heterobifunctional crosslinkers, Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), and SATP (N-succinimidyl-S- acetylthiopropionate) for bioconjugation purposes was acknowledged by cation exchange high-performance liquid chromatography (HPLC). Cytotoxicity of immunotoxin was evaluated on the VEGFR-2 positive PC-3 cell line by MTT assay.Overexpression of VEGFR-2 in the PC-3 cell line allowed immunotoxin to recognize them by anti-VEGFR-2 nanobodies. The concentrations above 5 μg/ml represented a significant decrease in cell survival rate in PC-3 cells compared to HEK293 cells (VEGFR-2 negative cells) as controls.VGRNb-DT demonstrated a successful bioconjugation; furthermore, variable concentrations were correlated with cell death in prostate cancer PC-3 cells.

Hb Kirikiriroa [α57(E6)Gly→Cys; HBA1: c.172G>T]: A Novel Unstable α-Globin Variant with Oxidized Derivatives Interfering with Hb A1c

We report the identification of a novel hemoglobin (Hb) variant [α57(E6)Gly→Cys; HBA1: c.172G>T], to be referred to as Hb Kirikiriroa. The variant was detected in five subjects from two families, with familial relationship established between the families following diagnosis. A persistently elevated Hb A1c over a 1-year period prompted hemoglobinopathy screening in an adolescent male of New Zealand (NZ) European descent (case 1). Capillary electrophoresis (CE) revealed the variant was negatively charged and susceptible to oxidation, with multiple abnormal peaks detected (0.4–5.1% total Hb). Hb A1c analysis by cation exchange high performance liquid chromatography (HPLC) was the first indication of the variant in a pregnant female of NZ European descent (case 2). Cases 1 and 2 had normal complete blood counts. Isopropanol stability testing provided evidence the variant was unstable. We herein describe the characterization of Hb Kirikiriroa and clinical significance of the variant for interference with Hb A1c analysis by CE and cation exchange HPLC.

Prevalence of Haemoglobin Variants and Haemoglobinopathies in a Single Paediatric Centre in Southern India: A Retrospective Cross-sectional Study

Introduction: Haemoglobinopathies are qualitative disorders of Haemoglobin (Hb) resulting from structural defects in the amino acid sequence of one of the globin chains, whereas, thalassaemia results from quantitative defects in the synthesis of one or more of the globin chain subunits of the Hb tetramer. Cation Exchange- High Performance Liquid Chromatography (CE-HPLC) is one of the methods for initial screening of Hb variants like HbS, HbD, HbE etc and for quantification of HbF, HbA and HbA2 levels. Aim: To find out the prevalence of haemoglobinopathies in patients of a Government paediatric tertiary care hospital in south India. Materials and Methods: In this retrospective, cross-sectional study, all laboratory requests, in the period from August 2019 to July 2021, for Hb variant analysis by HPLC were collected, irrespective of provisional diagnosis. The Hb variant analysis was carried out by CE-HPLC on the Bio-Rad D-10 analyser. This study was conducted in a Government Paediatric tertiary care hospital in Southern India for patients who had any clinical or familial suspicion of haemoglobinopathies. The Statistical analysis was performed using Microsoft Excel 2010. Results: Total data of 704 laboratory requests for Hb Variant analysis were obtained. Out of 704 laboratory request, 585 were from children younger than 12 years of age and 119 were parental screening. There were 164 abnormal chromatograms. Out of 164 patients, 91 were female patients and 73 were male patients. Out of 164 abnormal chromatograms, 97 (59.15%) were beta- thalassaemia trait, 18 (10.98%) were beta-thalassaemia major, 13 (7.93%) were sickle cell trait, 2 (1.22%) were sickle cell disease, 21 (12.80%) were HbE trait, 2 (1.22%) were homozygous HbE, 2 (1.22%) were HbD trait, 3 (1.83%) were Hereditary Persistence of Foetal Hb (HPFH)/delta beta-thalassaemia, 2 (1.22%) were HbJ trait, 1 (0.61%) was HbE beta-thalassaemia, 1 (0.61%) was sickle- beta-thalassaemia and 2 (1.22%) were alpha thalassaemia. Conclusion: From this study, beta-thalassaemia trait and beta- thalassaemia major were found to be the first and second most prevalent haemoglobinopathies in children below 12 years of age. This data suggests the importance of premarital and antenatal screening procedures that can help in reducing the possibility of such haemoglobinopathies in the future generation, suffering and burden of disease to the family and society.

The First Case of Hemoglobin Görwihl [&amp;#945;2&amp;#946;25(A2) Pro&amp;#8594;Ala] Identified in Türkiye

We present the first individual with hemoglobin (Hb) Görwihl in Türkiye and to our knowledge, this is the 3rd case reported worldwide. During premarital thalassemia screening an abnormal unidentified Hb peak [Retention Time (RT): 4.950 min., 42.4%] was determined by cation-exchange high-performance liquid chromatography (CE-HPLC) method at Premier Resolution (Trinity-Biotech) system. Variant Hb was eluted with HbA (RT: 2.47 min., 82%) at Variant II Turbo, Bio-Rad system. He was 22 years old and didn't show any clinical symptoms or haematological abnormality. HbA1c measured with Boronate affinity method at (Premier Hb9210, Trinity-Biotech) system was 5.2% and consistent with his blood glucose value (5.5 mmol/L) while it was 4.6% with CE-HPLC method at Variant II Turbo system. β-globulin gene sequencing revealed a heterozygote codon c16C>C variant which was identical to Hb Görwihl. Although clinically insignificant, Hb Görwihl may be important in hemoglobinopathy screening and HbA1c measurements.

Identification of Hemoglobin Variants Prevalent in China and Their Effects on Hemoglobin A1c Measurements.

We aimed to evaluate the effects of hemoglobin (Hb) variants prevalent in China on HbA1c measurements and to identify them during HbA1c measurements. We evaluated a cation-exchange high-performance liquid chromatography (HPLC) method (Bio-Rad D-100), a capillary electrophoresis (CE) method (Capillarys 3 TERA), an immunoassay (Cobas c501), and a boronate affinity method (Premier Hb9210, as a comparative method) for HbA1c measurements in the presence of Hb variants prevalent in China. The Bio-Rad D-100 and Capillarys 3 TERA gave specific retention times and numeric migration positions for each Hb variant, respectively, showing excellent interindividual reproducibility. All methods showed statistically significant differences (P < .01) for several variants. Clinically significant effects were observed for the Bio-Rad D-100 (Hb New York and Hb J-Bangkok), Capillarys 3 TERA (Hb New York and Hb J-Bangkok), and Cobas c501 (Hb New York). Among 297 samples with Hb variants, there were 75 (25.3%) unacceptable results for Bio-Rad D-100, 28 (9.4%) for Capillarys 3 TERA, and 19 (6.4%) for Cobas c501 compared with the results from Premier Hb9210. Some Hb variants prevalent in China affect HbA1c measurements. The HPLC retention time and CE migration position can aid in the presumptive identification of Hb variants.

Unexpected Discovery of Hemoglobin D Iran Families in Punjab, Pakistan by using two different Screening Methods

Background: Hemoglobin D Iran is frequently misdiagnosed as Hb E or Hb D Punjab if only one method of screening is used. The objective of our study was to highlight the importance of using two different screening techniques in diagnosis of a hemoglobin variant, Hb D Iran in our case. Hematological parameters of heterozygous Hb D Iran and compound heterozygous β/Hb D Iran were also compared. Methods: A descriptive study was carried out on results of 52,379 subjects which were part of thalassemia extended family cascade screening from 36 districts of Punjab from October 2019-March 2021. Cases of Hb D Punjab and Hb E were run on both CE-HPLC (cation exchange-high performance liquid chromatography) and CZE (capillary zone electrophoresis). Resulting Hb D Iran cases were confirmed by ARMS-PCR (Amplification refractory mutation system-polymerase chain reaction). Results: Forty cases of Hb D Iran were detected out of 160 initially suspected Hb D Punjab cases and 126 Hb E cases. Diagnosis was confirmed by molecular analysis. Statistical significance was found between RBC count, MCV, MCH, Hb F and diagnosis of “heterozygous Hb D Iran” and “compound heterozygous for β/ Hb D Iran”. Conclusion: Hb D Iran can be easily missed and misdiagnosed as Hb E or Hb D Punjab, if two screening methods are not used. This maybe a reason why Hb D Iran remains unreported in our region. CBC and HPLC indices can also be suggestive if a case is of heterozygous D Iran or compound heterozygous β/Hb D Iran. Keywords: Hb D Iran, Hb E, Hb D Punjab, Cation exchange High performance liquid chromatography, Capillary zone electrophoresis

[Speculation of hemoglobin A3 peak position in clinical cation exchange high performance liquid chromatogram of the diabetic blood sample with microarray isoelectric focusing].

血红蛋白A1c(HbA1c)是糖尿病诊断的关键生物标志物,目前其常用的分析方法为阳离子交换高效液相色谱法(CX-HPLC, 5/50 mm分离柱),此方法虽然具有稳定、快捷与自动化等众多优点,但临床CX-HPLC(VARIANT Ⅱ system)谱图中仍存在未知峰,尤其干扰HbA1c准确测定的谷胱甘肽化血红蛋白A3(HbA3)在色谱图中的相对位置仍不清楚。针对这一问题,该文以人新鲜血液为样本,首先利用低分辨CX-HPLC对血样进行分析,提示未知峰P3存在。然后通过微阵列等电聚焦(IEF)电泳和高分辨阳离子交换HPLC(Mono-S 5/50 mm分离柱)对血样的主要血红蛋白(Hb)成分进行分析,提示未知峰P3为HbA3峰。随后,通过血红蛋白谷胱甘肽化实验,利用HbA1c峰降低、HbA3峰明显增强这一信息,进一步推测未知峰P3即是HbA3峰,其在CX-HPLC中的保留时间在1.50 min左右。最后,结合前期研究讨论了体内应激情况下Hb谷胱甘肽化对HbA1c检测的影响。该研究丰富了CX-HPLC的未知峰P3的色谱信息,为更精准诊断糖尿病提供了有价值的参考。

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics.

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.

Role of cation-exchange high-performance liquid chromatography in detection of hemoglobinopathies- a study of 500 cases in a tertiary care hospital

Background: Abnormalities of hemoglobin synthesis are among the most common inherited disorders. Cation exchange high-performance liquid chromatography offers a reliable tool for early, accurate detection thereby aiding in the prevention and management of thalassemia major and various hemoglobinopathies. &#x0D; Materials and methods: This was a retrospective study carried out in the Department of Pathology, GCSMC Hospital and Research center, Ahmedabad over six years from August 2013 to August 2019. 500 cases were studied for the identification of various hemoglobin disorders in patients referred for screening and detection of hemoglobinopathies.Results: Abnormal hemoglobin fractions were seen in 104/500 (20.8%) cases. The β thalassemia trait was the predominant abnormality with a total of 69 cases (66.3%). β thalassemia major, β thalassemia intermedia, Hb D Punjab- β thalassemia, Acquired Hb F and Hereditary persistence of fetal hemoglobin/δβ thalassemia trait was found in 1 case (0.96%) each. Sickle cell heterozygous was found in 9 cases (8.6%), Sickle cell homozygous in 5 cases (4.8%), and Sickle-ß thalassemia in 6 cases (5.8%). Other variants detected included Hb Q India heterozygous and Hb D Punjab heterozygous in 3 cases (2.9%) each and 2 cases (1.9%) of Hb E heterozygous and Hb J each. &#x0D; Conclusions: Cation exchange high-performance liquid chromatography is an ideal and widely used methodology for routine clinical laboratory because of the simplicity of the automated system. The majority of the abnormal cases are diagnosed with it except a few inconclusive cases for which molecular and genetic studies are required.