Cation-exchange High-performance Liquid Chromatography Research Articles

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. 
 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. 
 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.

Rapid and Efficient Enrichment of Snake Venoms from Human Plasma Using a Strong Cation Exchange Tip Column to Improve Snakebite Diagnosis.

Snake envenomation is a serious public health issue in many tropical and subtropical countries. Accurate diagnosis and immediate antivenom treatment are critical for effective management. However, the venom concentration in the victims’ plasma is usually low, representing one of the bottlenecks in developing clinically applicable assays for venom detection and snakebite diagnosis. In this study, we attempted to develop a simple method for rapid enrichment of venom proteins from human plasma to facilitate detection. Our experiments showed that several major protein components of both Naja atra (N. atra) and Bungarus multicinctus (B. multicinctus) venoms have higher isoelectric point (pI) values relative to high-abundance human plasma proteins and could be separated via strong cation exchange–high-performance liquid chromatography (SCX-HPLC). Based on this principle, we developed an SCX tip column-based protocol for rapid enrichment of N. atra and B. multicinctus venom proteins from human plasma. Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the identification of cytotoxin and beta-bungarotoxin as the major proteins enriched by the SCX tip column in each venom sample. The entire process of venom enrichment could be completed within 10–15 min. Combination of this method with our previously developed lateral flow strip assays (rapid test) significantly enhanced the sensitivity of the rapid test, mainly via depletion of the plasma protein background, as well as increase in venom protein concentration. Notably, the SCX tip column-based enrichment method has the potential to efficiently enrich other Elapidae snake venoms containing proteins with higher pI values, thereby facilitating venom detection with other assays. This simple and rapid sample preparation method should aid in improving the clinical utility of diagnostic assays for snakebite.

Incidental Detection of Hemoglobin Variants During Evaluation of HbA1c.

HemoglobinA1c (HbA1c) is used to diagnose Diabetes mellitus and monitor glycemic control over the previous eight to twelve weeks in diabetic patients. Detection of HbA1c by cation exchange-high performance liquid chromatography (CE-HPLC) gives a chromatogram by which abnormal hemoglobin variants are also picked up. Some of these may interfere with HbA1c values affecting clinical management. Due to increased inter-state migration as well as medical tourism, there is a high possibility of finding various hemoglobin variants in any part of India. We did a prospective analysis over 1.5years, of the hemoglobin variants detected during all the HbA1c runs. The HbA1c was tested on Bio-Rad D10 dual HbA2/F/A1c platform, which uses the CE-HPLC method. Every chromatogram was carefully studied to look for unknown peaks. The samples showing unknown peaks >6% were re-run in extended HbA2/F mode to categorize the hemoglobin variants. We had 9595 HbA1c samples, of which 70 cases showed a variant window. There were 40 males and 30 females, age ranging from 28 to 76years. The different hemoglobin variants detected were HbD, HbE and HbS in heterozygous state, high HbF (with a differential diagnosis of HPFH heterozygous and delta-beta thalassemia heterozygous), HbE homozygous, HbQ heterozygous and HbJ heterozygous. We conclude that in the process of monitoring glycemic control using HbA1c, we can also pick-up hemoglobin variants. Hence, it is essential to review HbA1c graphs, so that the diagnosis of hemoglobin variants is not missed and the HbA1c reported is reliable.

Association of hemoglobin H (HbH) disease with hemoglobin A1c and glycated albumin in diabetic and non-diabetic patients.

Hemoglobin A1c (HbA1c) and glycated albumin (GA) are glycemic control status indicators in patients with diabetes mellitus. Hemoglobin H (HbH) disease is a moderately severe form of α-thalassemia. Here we examine the usefulness of HbA1c and GA in monitoring glycemic control in patients with HbH disease. HbA1c, GA, and an oral glucose tolerance test were performed in 85 patients with HbH disease and 130 healthy adults. HbA1c was measured using five methods, including two systems based on cation-exchange high-performance liquid chromatography (Variant II Turbo 2.0 and Bio-Rad D100), a capillary zone electrophoresis method (Capillarys 3 TERA), a boronate affinity HPLC method (Premier Hb9210), and an immunoassay (Cobas c501). Significant lower levels of HbA1c were observed in patients with HbH disease than in healthy adults. In contrast, GA showed no statistically significant differences between participants with and without HbH disease. A considerable number of diabetic patients with HbH disease would be missed if using HbA1c as a diagnostic criterion for diabetes mellitus. GA but not HbA1c is suitable for monitoring glycemic control in patients with HbH disease that can modify the discriminative ability of HbA1c for diagnosing diabetes.

Physicochemical stability of the bevacizumab biosimilar, ABP 215, after preparation and storage in intravenous bags

Study Objectives: To evaluate extended in-use stability of bevacizumab biosimilar, ABP 215, after dilution into intravenous bags, extended storage, and simulated infusion to enable advanced preparation and storage. Methods: Two lots of ABP 215 were diluted to high- (16.5 mg/mL) and low- (1.4 mg/mL) dose concentrations in two types of intravenous bag under ambient light conditions. Dosed intravenous bags were stored at 2°C–8°C for 35 days, followed by 30°C for 2 days, and each bag was infused on Day 37. Analysis of purity and physicochemical stability was performed using size-exclusion high-performance liquid chromatography (SE-HPLC), cation-exchange high-performance liquid chromatography (CEX-HPLC), reduced capillary electrophoresis-sodium dodecyl sulphate (rCE-SDS), subvisible particle detection assays, visual inspection, and by measuring protein concentration and potency. Results: No meaningful changes were seen in ABP 215 purity when analysed by SE-HPLC, CEX-HPLC and rCE-SDS following dilution, storage and infusion of two lots, bags, and doses. Protein concentration remained consistent throughout the study for all samples and no significant loss in potency was detected. No potentially proteinaceous particles or increases in subvisible particles were observed. Discussion: This study investigated the in-use stability of ABP 215 following dilution, extended storage, and infusion, that represent worst-case handling conditions. ABP 215 exhibited consistent product quality and activity, with no significant degradation observed under the conditions tested. Conclusion: ABP 215 retains physicochemical stability after dilution over the recommended dosing concentrations, extended storage, and simulated infusion. This supports the advance preparation and storage of ABP 215 in intravenous bags for infusion.

Spectrum of Haemoglobinopathies in Sikh population of Bangalore: A pilot study

Objectives: To study the burden of different Hemoglobinopathies in the Sikh community of Bangalore. To study their patterns on gel electrophoresis (EPH) and compare the results with hematological parameters and peripheral smear. To confirm the results by cation exchange- High Performance Liquid Chromatography (CE-HPLC) wherever necessary. Materials and Methods: A prospective pilot study was conducted on one hundred and fifty one cases who visited Gurudwara for a regular visit every Sunday. About 2-3 ml. intravenous blood samples were collected after obtaining informed consent from each individual. Background data of each individual were recorded like age, sex, caste, place of origin, consanguinity, etc. Hematological indices were measured using MS4 Cell Counter. Hemoglobin electrophoresis was carried out for the quantification of A2 fraction of hemoglobin by elution method. The use of high performance liquid chromatography is used to separate and quantify abnormal haemoglobin (Hb) fractions that were inseparable on Gel electrophoresis. Results: Most common hemoglobinopathies observed were β?thalassemia trait (5.3%), thalassemia major (0.6%), Hb D trait (1.9%), HbD-β?thalassemia (0.6%), sickle cell trait (0.6%), HbQ disease (0.6%). Conclusions: Proper screening camps and counselling needs to be done on the population at risk about awareness and detection of heterozygous carriers; to prevent the birth of homozygous Beta Thalassemia Major Child. Keywords: Sikh population, Hemoglobinopathies, Electrophoresis, High Power Liquid Chromatography.

Hb broomhill [α1 or α2 114(GH2) pro > ala;HBA1 or HBA2:c.343C > G]: a rare Hb variant found in a diabetic chinese individual

We report a clinically silent hemoglobin (Hb) variant, Hb Broomhill [α1 or α2 114(GH2) Pro > Ala;HBA1 or HBA2:c.343C > G] in a diabetic Chinese man. The Hb fractions of the subject were analyzed using various chromatographic and electrophoretic techniques. The glycated hemoglobin (HbA1c) levels measured using cation-exchange high-performance liquid chromatography (CE-HPLC) and the boronate affinity method showed nearly identical results. Analysis of the chromatogram of the CE-HPLC revealed an abnormal shoulder peak that appeared towards the end of the elution profile. Though the capillary electrophoresis method did not interpret the results, a manual examination revealed an abnormal shoulder on the HbA0 peak. Similarly, the electropherogram of the capillary zone electrophoresis also had an abnormal shoulder on the HbA peak. A missense mutation specific to the Hb Broomhill variant was found using Sanger sequencing.

Evaluation of four different HPLC devices for hemoglobinopathy screening

Abstract Objective Hemoglobinopathies are a common public health problem in Turkey. In the screening of these disorders in population, cation-exchange high performance liquid chromatography (HPLC) is accepted as the gold standard method. In this study, the aim was to assess four different HPLC devices used in hemoglobinopathy screening. Materials and methods A total of 58 blood samples were analyzed with four different HPLC methods (Bio-Rad variant II, Agilent 1100, Tosoh G8 and Trinity Ultra2 trademarks). Results The comparison study demonstrated a good correlation between the results of each HPLC analyzer and the reference value obtained by averaging all the HbA2 results belonging to the methods tested in the study [ (Tosoh G8 (r=0.988), Bio-Rad variant II (r=0.993), Agilent 1100 (r=0.98) and Trinity Ultra2 (r=0.992) ]. HbA2 determination in the presence of HbE was interfered in both Bio-Rad variant II and Tosoh G8. Conclusion The analyzers were found to have compatible HbA2 results but with accompanying different degrees of proportional and systematic biases. HPLC analyzers may be affected by different hemoglobin variants at different HbA2 concentrations, which is an important point to take into consideration during the evaluation of HbA2 results in thalassemia screening.

Hemoglobin variants in southern China: results obtained during the measurement of glycated hemoglobin in a large population.

Hemoglobin (Hb) variant is one of the most common monogenic inherited disorders. We aimed to explore the prevalence and hematological and molecular characteristics of Hb variants in southern China. We collected blood samples from all patients with suspected variants found during HbA1c measurement via a cation-exchange high-performance liquid chromatography system (Bio-Rad Variant II Turbo 2.0) or a capillary electrophoresis method (Sebia Capillarys). Hematological analysis, Sanger sequencing, and gap-PCR were performed for these samples. Among the 311,024 patients tested, we found 1,074 Hb variant carriers, including 823 identified using Capillarys and 251 using Variant II Turbo 2.0, with a total carrier rate of 0.35%. We discovered 117 types of Hb variants (52 HBB, 47 HBA, and 18 HBD mutations) containing 18 new mutations. The most common variant found was Hb E, followed by Hb New York, Hb J-Bangkok, Hb Q-Thailand, Hb G-Coushatta, Hb G-Honolulu, Hb G-Taipei, and Hb Broomhill. Most heterozygotes for the Hb variant exhibited normal hematological parameters. However, most patients with compound heterozygotes for the Hb variant and thalassemia showed varied degrees of microcytic hypochromic anemia. The prevalence of hemoglobin variants remains high and exhibits genetic diversity and widespread distribution in the population of southern China.

TRACE LEVEL DETERMINATION OF SODIUM CHLORIDE AND SODIUM SULFATE CONTENT IN SODIUM LAURETH SULFATE RAW MATERIAL USING COUNTER CATION-EXCHANGE HPLC WITH INDIRECT ULTRAVIOLET DETECTION

Objective: A novel study on a new liquid chromatographic approach has been developed and validated for simultaneous determination of trace level determination of sodium chloride and sodium sulfate measures its impurities using counter cation-exchange high-performance liquid chromatography with indirect ultraviolet (UV) detection.
 Methods: Chromatographic separation is developed and validated on a Hamilton PRP-X100 column with a mobile phase contained a mixture of the para-hydroxybenzoic acid buffer with a pH of 9.0 and methanol. Chromatography is developed at a flow rate of 2.0 mL/min with an indirect UV determination at 310 nm at a sensitivity level of 0.5%. The optimized method was validated as per the ICH Q2 guidelines.
 Results: The retention times of chloride and sulfate were about 2.8 and 7.6 min, respectively. The resolution between chloride and sulfate peaks is >4. Regression analysis confers a correlation coefficient for the stated compounds that are found to be >0.999.
 Conclusion: A novel analytical method was validated as per the ICH method validation guidelines and found to be selective. Hence, the validated analytical method was precise, specific, and accurate, and it is more economic and simple for the determination of inorganic impurities.

Effects of Voxelotor (GBT440) Therapy on Laboratory Testing of Sickle Cell Patients

Background: Voxelotor (GBT440) is a small molecule allosteric effector with high specificity for hemoglobin (Hb) that inhibits Hb S polymerization by increasing hemoglobin oxygen affinity. Developed as an oral, once-daily medication, it is undergoing phase 3 randomized clinical trials in sickle cell disease patients. Therapeutic monitoring of Hb quantitative fractions is routine in sickle cell disease patients to assess medication and transfusion effects and future treatment requirements. In the clinical laboratory, some therapies can alter the Hb fraction percentages or characteristic protein migration/elution patterns and can mimic complex hemoglobinopathy disorders causing misinterpretation or unnecessary additional testing. We present the effects of voxelotor on hemoglobin identification/monitoring testing in multiple testing platforms. Methods: A whole blood anticoagulated sample from a male patient who was undergoing treatment for sickle cell disease was received for routine hemoglobin testing. Five additional samples with similar features were also received from three male patients (age range of all patients was 18 to 22 years) with sickle cell disease. Hemoglobin protein analysis was performed using standard methods including cation-exchange high performance liquid chromatography (HPLC), capillary electrophoresis (CE), isoelectric focusing (IEF), intact globin chain mass spectrometry (MS), sickle solubility and hemoglobin stability studies. DNA sequencing of the alpha (HBA1/HBA2) and beta globin (HBB) genes was performed on one sample. Results: CE (initial and 50:50 mix, figures 1A and 1B, respectively) revealed a Hb S peak with a shoulder and additional small peaks at position 203 (D zone) and 234 (cusp of zones E/A2). In some cases the 234 peak interfered with instrument designation of the A2 peak (50:50 mix with normal) and shifted the Hb S peak into the E zone which risks variant misidentification (figure 1C). Our laboratory overcame this limitation by substituting the normal sample with beta thalassemia trait to enhance the Hb A2 peak after mixing. HPLC tracings (figure 1D) showed a duplex peak pattern in the D and S windows with separate peak quantification percentages (index case showed elution at 4.18 and 4.37 minutes with 14.9% and 66.4%, respectively). The dual peaks detected by HPLC partially mimicked compound Hb S/D-Punjab. IEF (figure 1E) showed additional bands at -4.7, and -11.5 (Schneider-Barwick ratios). The additional peaks identified by CE and IEF mimicked an alpha chain hemoglobin variant; however, the expected hybrid patterns were atypical prompting further investigation. Sickle solubility was positive, heat stability studies were negative and mass spectrometry did not definitively detect abnormal peaks other than βS. DNA sequencing of the alpha and beta globin genes did not reveal abnormalities other than homozygous Hb S. Similar hemoglobin patterns were identified in all samples tested. Conclusion: Voxelotor treatment in sickle cell patients results in a distinctive pattern alteration in hemoglobin testing. Methods may not give accurate variant percentage estimation and the treatment may result in shifts in variant mobility causing erroneous laboratory results. Novel therapies are increasing in the treatment of hemoglobin disorders and medication effect should be a consideration when atypical patterns are encountered in the laboratory. Knowledge of these artifacts by clinicians and laboratorians is paramount to avoid erroneous diagnosis, unnecessary additional testing and inaccurate Hb variant quantitation for monitoring purposes. Notification of current treatment regimens to testing facilities is highly recommended in these patients. Figure 1 Disclosures No relevant conflicts of interest to declare.

Effect of hemoglobin F and A2 on hemoglobin A1c determined by cation exchange high-performance liquid chromatography

AbstractBackgroundThe potential effect of increase in hemoglobin (Hb) A2and HbF on glycated hemoglobin (HbA1c) measurements were investigated using a high-performance liquid chromatography (HPLC) method compared with an immunoturbimetric assay.MethodsSamples producing abnormal chromatograms during the measurement of routine HbA1ctesting with HPLC were further analyzed to characterize abnormal Hb variants. Patients were divided into three groups that had only high HbF (group 1), only high HbA2(group 2), and both high HbA2and HbF (group 3). HbA1cvalues of patients were re-assayed using the immunoturbidimetric method (Advia, Siemens Healthcare, Germany).ResultsHbA1clevels were significantly higher in all groups measured by immunoassay than in HPLC. We found a positive correlation between HPLC and immunoturbidimetry in the group 2 and a slight correlation in the group 1. There was no correlation between the two methods in group 3.ConclusionsHbA1cmeasurement by HPLC method interfering with elevated HbA2and HbF, especially HbF, should be verified by an immunoturbidimetric method.

Analytical Evaluation of BioRad D-100 HPLC Analyzer and Workflow Comparison to BioRad TurboLink Variant II HPLC With Reflex to Boronate Affinity

Abstract Introduction Measurement of hemoglobin A1c (HbA1C) is used for the diagnosis and management of patients with diabetes. Methods for measuring HbA1C are classified on the basis of charge differences (cation exchange chromatography) or structural differences (boronate affinity chromatography). Some cation exchange high-performance liquid chromatography (HPLC) analyzers may be prone to interferences from hemoglobin variants. Historically, our lab used two methods to report HbA1C results: cation exchange HPLC (VariantII) with reflex to boronate affinity HPLC (Ultra2) methods. A new analyzer (BioRad D-100) with improved interference detection and thresholds for interference was evaluated. The objectives of this study were (1) assess the comparability of HbA1c results between D-100, VariantII, and Ultra2; (2) evaluate the need for maintaining a reflex method; and (3) calculate cancellation rates before and after implementing D-100. Methods HbA1c was measured by cation exchange methods using VariantII Turbolink, D-100 (BioRad), and boronate affinity using Ultra2Affinity (Trinity Biotech) according to the manufacturer’s recommendations with the following analytical measuring ranges (AMRs), 4.0% to 17.6%, 4.0% to 18.0%, and 4.0% to 17.6%, respectively. D-100 was compared to VariantII (n = 26) and Ultra2 (n = 31) using residual patient samples from provider-ordered HbA1c having no chromatography flags and with the following chromatography flags on VariantII (n = 113): variant window (n = 41), HbA1c <4.0% (n = 24), HbA1a + HbA1b >5% (n = 19), HbF >5% (n = 9), P3 >10% (n = 6), HbA1c >17.6% (n = 4), unknown peak (n = 3), labile 5% to 15% (n = 3), labile >15% (n = 3), and HbS >60% (n = 1). The cancelation rates were calculated at baseline (January 1-31, 2018) and postimplementation of D-100 (September 20, 2018, to January 22, 2019). Results HbA1c results from D-100 were within ±0.3 or 5% with the following frequencies: Ultra2 in 96.2% (25/26) with VariantII in 96.8% (30/31) using samples with no chromatography flags. A total of 113 samples with abnormal VariantII flags were tested using D-100 with 82% (n = 92) not exceeding interference limits for result reporting. These D-100 results were within ±0.3 or 5% of Ultra2 results with the following frequencies: 38.5% (10/26) of samples below (n = 23) or above (n = 3) the AMR, 85.3% (29/34) of results with Hb variant flags, 68.0% (17/25) with unknown/minor peaks, and 71.4% (5/7) with HbF <15%. The clinical concordance was also assessed according to the following decision limits: normal <5.6%, prediabetic = 5.7% to 6.4%, and diabetic >6.5%. Results were concordant in 88% with variant peaks (12/13 w/HbA1c <5.6%, 8/11 w/HbA1c = 5.7%-6.4%, 10/10 w/HbA1c >6.5%), 84% with minor peaks (5/6 w/HbA1c <5.6%, 0/2 w/HbA1c = 5.7%-6.4%, 16/17 w/HbA1c >6.5%), and 86% with HbF (3/3 w/HbA1c <5.6%, 2/3 w/HbA1c = 5.7%-6.4%, 1/1 w/HbA1c >6.5%). Results above/below AMR had 100% concordance (>16.0% or <4.4%). The frequency of VariantII rule violation was 2.1% in January 2018, with 53 of 117 having results confirmed as <4.0% or >16.0% by Ultra2, 2 of 117 reported by Ultra2, and 62 being cancelled due to HbF >15% (n = 23) or variant Hb >60% (n = 39). The frequency of rule violation using only D-100 since implementation was 2.2%. Conclusion Implementation of D-100 has removed the need for boronate affinity reflex testing and maintained similar cancellation rates.

Hemoglobin Phenotypes in Nigeria: Data From a National Reference Laboratory

Abstract On a global scale, 5% to 7% of the population carries an abnormal hemoglobin. With a sickle hemoglobin (HbS) carrier prevalence of 25% to 40%, Nigeria bears the greatest burden of sickle cell disorder worldwide. Until recent times, detection of other clinically significant hemoglobin variants associated with HbS has been unavailable, resulting in missed/wrong diagnosis, weak national data, and suboptimal control of hemoglobin disorders. This study aimed to review hemoglobin assays over a 2-year period at a national reference laboratory in Nigeria, in order to describe the prevalent hemoglobin phenotypes for health planning. The study was a retrospective, laboratory data-based survey. Sociodemographics and hemoglobin assay results of 6,851 specimens between January 2016 and December 2017 were analyzed. Study protocol was approved by the Institutional Review Board. Participants aged less than 1 year and who had recent transfusions and a history of hydroxyurea therapy were excluded. At the hemoglobin laboratory, specimens were analyzed using cation exchange high-performance liquid chromatography (CE-HPLC) (Bio Rad D10, California). When indicated, further analysis of suspected abnormal hemoglobins was confirmed using a second method, capillary electrophoresis (SEBIA Capiflex II, France). The distribution of the identified hemoglobin phenotypes was determined as a proportion of the total number of participants. The mean (SD) age of the participants was 31.8 (14.9) years. The most frequent hemoglobin phenotypes were AA (59.4%), AS (23.3%), and SS (13.3%). Hemoglobin phenotypes AC (2.1%), SC (0.9%), AGPhiladelphia (0.2%), and CC (0.2%) were less frequent. Least occurring phenotypes were ADIbadan, AE, AGPhiladelphia, AOArab, DD, and hereditary persistence of fetal hemoglobin (HPFH). In the Nigerian context, accurate diagnosis of hemoglobin variants using quantitative, high-resolution hemoglobin assays, compared to zone (cellulose acetate) electrophoresis at alkaline pH will provide data for health planning, better access to genetic counseling, informed reproductive health choices, and secondary prevention of hemoglobinopathies.

Genetic and Biochemical Evidence That Enterococcus faecalis Gr17 Produces a Novel and Sec-Dependent Bacteriocin, Enterocin Gr17.

Bacteriocins are ribosomally synthesized antibacterial peptides or proteins from microorganisms. We report a novel bacteriocin producing strain, Enterococcus faecalis Gr17, that was isolated from the Chinese traditional low-salt fermented whole fish product Suan yu. E. faecalis Gr17 displayed potent antibacterial activity against foodborne pathogenic and spoilage bacteria. The complete genome of E. faecalis Gr17 contained one circular chromosome and plasmid. The gene cluster of a novel bacteriocin designated enterocin Gr17 was identified. The enterocin Gr17 structural gene encodes a precursor of the bacteriocin. Two other transporter genes and an immunity gene within two divergent operons were identified as being associated with enterocin Gr17 secretion and protection. The novel enterocin Gr17 was purified by ammonium sulfate precipitation, cation exchange, gel filtration, and reverse-phase high-performance liquid chromatography. The molecular weight of enterocin Gr17 was 4,531.01 Da as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and its mature amino acid sequence of enterocin Gr17 was RSYGNGVYCNNSKCWVNWGEAKENIIGIVISGWATGLAGMGR. Sequence alignment revealed that enterocin Gr17 is a class IIa bacteriocin with similarities to enterocin P. The merits of bactericidal activity, sensitivity to enzymes, and pronounced stability to chemicals, temperature (60°C, 30 min and 121°C, 15 min), and pH (2–10) indicated practicality and safety of enterocin Gr17 in the food industry. The complete genome information of E. faecalis Gr17 will improve the understanding of the biosynthetic mechanism of enterocin Gr17, which has potential value as a food biopreservative.

Case Report: Rare Hemoglobin Variant Causes Elevated A1c, False Diagnosis of Pre-gestational Diabetes in Pregnancy [33H

INTRODUCTION: Hemoglobin variants result from missense mutations that lead to substitution of amino acids within globin proteins, altering the structure of the hemoglobin molecule. Hemoglobin A1c (HbA1c) is commonly used to diagnosis and assess control of diabetes mellitus and can be falsely affected by hemoglobinopathies, hyperbilirubinemia, and hypertriglyceridemia. METHODS: We present a case of an obese Caucasian 27 year-old Gravid 3 Para 0020 with early HbA1c of 10.0 percent at 9-weeks gestation. The patient was diagnosed with pre-gestational diabetes, and referred for nutrition counseling with initiation of four times daily home blood sugar monitoring notable for consistently normal fasting and postprandial values. Due to continued discordant results in HbA1c level with her glucometer log and random glucose testing, hemoglobin electrophoresis was performed at 22-weeks gestation with findings suggestive of Hemoglobin Wayne, a rare hemoglobin variant that is clinically silent. Hemoglobin Wayne results from a frameshift mutation within the alpha chain resulting in a net charge that is similar to HbA1c. This common charge manifests as similar retention times on cation-exchange high-performance liquid chromatography, leading to an overestimation of HbA1c concentration. Genetic sequencing was pursued and the patient completed appropriately timed routine screening for gestational diabetes with normal results. CONCLUSION: Our case, the first reported in a pregnant patient, should remind clinicians of the few conditions that can lead to misrepresentation of HbA1c levels. Remaining vigilant in clarifying such an atypical patient presentation will decrease the risk of mismanaging, particularly over-treating, patients believed to be high-risk for diabetes.

Characterization of Subtilin L-Q11, a Novel Class I Bacteriocin Synthesized by Bacillus subtilis L-Q11 Isolated From Orchard Soil.

Bacteriocins are peptides or proteins synthesized by bacterial ribosomes that show killing or inhibitory activities against different groups of bacteria. Bacteriocins are considered potential alternatives to traditional antibiotics, preservatives in pharmaceutical and food industries. A strain L-Q11 isolated from orchard soil was phylogenetically characterized as Bacillus subtilis based on 16S rRNA gene sequencing analysis. A novel class I bacteriocin (Subtilin L-Q11), was identified and purified from L-Q11 cell-free supernatant in a four-step procedure, including salt precipitation, cation exchange, gel filtration, and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass (3,552.9 Da) of this novel bacteriocin was determined by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The purified Subtilin L-Q11 exhibited optimal features in pH tolerance, thermostability, and sensitivity to proteases. Further, Subtilin L-Q11 showed inhibitory activities against a number of bacteria including some human pathogens and food spoilage bacteria, in particular Staphylococcus aureus. All these important features make this novel bacteriocin a potential candidate for the development of a new antibacterial drug or food preservative in the future.