Sebia Capillarys Research Articles

A-037 A1c by separation and enzymatic methods: what's the difference?

Abstract Background Accurate values for hemoglobin A1c (HbA1c) are crucial for diagnosing and monitoring diabetes mellitus. Two common methods to measure HbA1c are capillary electrophoresis (CE) and immunoassay (IA). Many methods used to measure HbA1c are prone to interferences from hemoglobin variants and abnormalities affecting the red blood cell’s (RBC) turnover. Consequently, the average estimated glucose levels using the HbA1c value can be unreliable in diabetics with hemoglobinopathies and other RBC lifetime disorders. Separation methods such as CE can capture hemoglobinopathy information, while IA methods may not. We performed this study in a population with relatively high levels of hemoglobin disorders to evaluate the performance of HbA1c testing on the Alinity platform (IA) against the Sebia Capillarys 3 Tera instrument (CE). Methods Samples were identified retrospectively based on a search of the Electronic Medical Record for all patients with an ordered and resulted HbA1c and complete blood count in the Montefiore Medical Center the day prior to sampling. Routine HbA1c testing was performed by an Abbott Immunoassay method on the Abbott Alinity Instrument and CBC results were obtained from a Sysmex XN2100 instrument. Records were compared by patient name, medical record number, and sample collection time to identify all patients with CBC and HbA1c drawn within one hour of each other. Those samples were retrieved from storage (a 4° automated refrigerator) within 48 hours of draw and tested via the Sebia Capillarys 3 Tera instrument using HBA1c kits. Results In this large representative population study (N=2075), there was overall excellent agreement between IA and CE. The correlation coefficient (R2) of the whole dataset was 0.989, with an average bias of 2.7% between the two methods. Additional analysis was performed on those with possible RBC turnover disorder (patients with RBC counts below 4x106/uL or above 6x106/uL, MCVs above 96 fL or below 80 fL, RDW above 17%, or abnormal A2 value: total of 638 samples), those with a hemoglobin variant as detected by the Capillarys 3 instrument (162 samples), or with a measurable hemoglobin F value detected by the Capillarys 3 instrument (37 samples). Samples were sorted into these groups independently and thus there was significant overlap between groups, notably with 23 of 37 samples with elevated HbF also having a hemoglobin variant. In both the RBC disorder and hemoglobinopathies group, similar low bias and high correlations were seen. The elevated F samples showed a clear negative bias (5.6%) for all patients on the IA method as compared with CE. The IA method states that samples with HbF above 5% should not be tested. However, as this is not a separation method, there is no way to test samples for HbF before analysis, and bias was seen for samples below 5%. Conclusions The IA and CE methods showed similar performance across patients regardless of RBC lifetime or hemoglobin disorder status, though presence of HbF is a potential interference and should be considered. Further questions remain for these instruments in terms of correlation with average glucose levels as calculated through CGM instruments.

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

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

Evaluation of effects from hemoglobin variants on HbA1c measurements by different methods.

The impact of seven hemoglobin variants (Hb Q-Thailand, Hb G-Honolulu, Hb Ube-2, Hb New York, Hb J-Bangkok, Hb G-Coushatta, and Hb E) on the outcome of HbA1c was investigated for six methods by comparing withliquid chromatography-tandem mass spectrometry (LC/MS/MS) reference method. Twenty-nine normal and 112 variant samples were measured by LC/MS/MS, Sebia Capillarys 3 TERA, Intelligene Biosystems QuanTOF, Premier Hb9210, Arkray HA-8190V, Bio-Rad D-100, and Tosoh G11, then evaluated for correlation, consistency, and mean relative bias among six methods. The lowest biological variation bias of ±2.8 % was an acceptable standard. All methods showed poor correlation and consistency with LC/MS/MS for Hb E. The unacceptable biases were observed for Capillarys 3 TERA (-14.4 to-3.7 % for Hb Q-Thailand, Hb Ube-2, Hb New York, Hb J-Bangkok and Hb E), QuanTOF (-8.3 to-2.9 % for Hb Ube-2, Hb New York and Hb G-Coushatta), Premier Hb9210 (-18.3 to-3.6 % for Hb Q-Thailand, Hb Ube-2, Hb New York, Hb J-Bangkok and Hb E), HA-8190V variant mode (-17.3 to 6.6 % for Hb G-Honolulu, Hb Ube-2, Hb New York, Hb G-Coushatta and Hb E). All variant samples showed larger biases than ±2.8 % comparing HA-8190V fast mode, D-100, and G11 with LC/MS/MS. The accuracy of different HbA1c methods was influenced by some Hb variants, especially Hb Ube-2 and Hb New York. Thus, laboratories need to choose appropriate methods to measure HbA1c with different Hb variants.

Effects of lipemia on capillary serum protein electrophoresis in native ultra-lipemic material and intravenous lipid emulsion added sera.

This study aims to investigate the effect of natural ultralipemic material (NULM) and intravenous lipid emulsion (IVLE) on capillary serum protein electrophoresis (SPEP). NULM material was prepared from leftover patients' lipemic serum sample (triglyceride concentration >2,000mg/dL) pool by a refrigerated high-speed centrifuge, and IVLE Omegaven lipid emulsion (30%) was used. Serum pools for interference study were prepared from patient samples for which serum protein electrophoresis was studied as Normal SPEP and M Peak SPEP. For both types of lipemia (DULM and IVLE), five pools with triglyceride concentrations of ∼4.52 mmol/L, ∼7.91 mmol/L, ∼14.69 mmol/L, ∼21.47 mmol/L, and ∼28.25mmol/L were prepared. SPEP was studied in each pool with Sebia Capillarys Minicap. A repeated measure ANOVA test was used to determine the difference between the pools, and interferograms were used to evaluate the interference effect. Interference was not detected in IVLE added Normal SPEP and M Peak SPEP pools, either % or concentrations of fractions. In NULM-added Normal SPEP and M Peak SPEP pools, significant positive interference in albumin % (p=0.002 and p<0.001 respectively) and significant negative interference in gamma% (p<0.001 and p=0.005 respectively) and M protein peak (p=0.002) fractions were detected. However, significant positive interference was seen only for albumin concentration fractions (p<0.001 for both pools). It is vital to use NULM instead of IVLE solutions in lipemia interference studies for all laboratory tests, including CZE SPEP. The fractions concentration values calculated with the total protein concentration should be used for evaluating SPEP results.

Differential Effects on HbA1c Detection by HPLC and Capillary Electrophoresis in Five Types of Hb Variants in China.

Hemoglobin A1c (HbA1c) can be used to evaluate blood glucose control. Its measurement will be affected by many factors, but Hb variation is the most critical factor. This study aimed to explore the types of variants found in routine work and their impact on test results. Samples with abnormal HbA1c chromatograms found in routine testing were tested with high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) and then further tested with polymerase chain reaction (PCR) and sequencing. Five recessive heterozygous mutations were identified after PCR and sequencing. Hb Riccarton-II (a mutation in the HBA2 gene), Hb E, Hb G-Coushatta, Hb G-Taipei, and Hb North Manchester (a mutation in the HbB gene) were identified. All HbA1c values of these variants detected by HLC-723 G8 (HPLC method) were lower than those of Sebia Capillarys 2 FP (C2FP, CE method) with P < .0001. Five Hb mutations were identified in our routine HbA1c test, and their HPLC detection values were significantly lower than those obtained with the CE method.

Does size matter? Two new deletions in the HBB gene cause β0-thalassemia.

Most β-thalassemias are caused by mutations involving one or a limited number of nucleotides within the gene or its adjacent regions. They can be substitutions or deletions; in these cases, the loss ranges from a single nucleotide to even the entire HBB gene, so we wonder if the phenotype is due to the size of the deletion or the location of the mutation. To clarify this, we present two new deletions in the β-globin gene that cause β0-thalassemia. The hematological parameters were determined with an automated cell counter; the Hb A2 and Hb F levels were measured by performance liquid chromatography. Hemoglobins were analyzed by capillary zone electrophoresis (Sebia Capillarys Flex system) and ion-exchange HPLC (BioRad Variant II β-thalassemia Short Program). Molecular characterization was performed by automatic Sanger sequencing. The screening of common α-thalassemia point mutations and deletions in the world (21 in total) were carried out using multiplex PCR followed by reverse-hybridization with a commercial Alpha-Globin StripAssay kit. We have characterized two new mutations-(1) 1-bp deletion [CD61/62(-G)] [HBB:c.186_187delG], (2) 105-bp deletion [IVS-2-nt767-CD111] [HBB:c.316-84_333del]-and we have described, for first time in Spain, the 25-bp deletion [β nts 252 - 276 deleted] [HBB:c.93-22_95del] mutation. These mutations were classified as pathogenic by UniProt Variants confirmed according to the American College of Medical Genetics and Genomics guidelines. These mutations present a phenotype compatible with β0-thalassemia, supported by hematological parameters that correlate the degree of reduction in the synthesis of the β-globin chain. Identification of this type of mutation is important for genetic counselling of partners where both are carriers, so that they are aware of the genetic risk of having affected children, allowing them to take an informed decision about their reproductive choices.

High-resolution capillary electrophoresis for the determination of carbamylated albumin

Abstract Objectives Carbamylation is a non-enzymatic post-translational reaction of a primary amino group of a protein with isocyanate. The albumin carbamylation is a negative prognostic factor in chronic kidney disease (CKD) patients and induce charge difference implying an observed shift in electrophoretic mobility that can be measured through a symmetry factor (SF). Methods The Helena V8 and the Sebia Capillarys 2 systems were used for all experiments. The effect of in vitro carbamylation on the SF by spiking increasing concentrations of potassium isocyanate (KCNO) in serum of three healthy volunteers was investigated. Theoretical plate numbers (N) as a surrogate of separation efficiency were also calculated and correlations between SF and renal function biomarkers were performed on 284 patients. Results A dose-dependent impact of KCNO on the SF was observed for both methods with the Helena V8 being more sensitive. The mean N was significantly higher on the Helena V8 as compared to the Sebia Capillarys 2 (2,972 vs. 444.1, p&lt;0.0001). The SF correlated significantly with eGFR (r=0.50, p&lt;0.0001), creatinine (r=−0.31, p&lt;0.0001) and urea (r=−0.34, p&lt;0.0001) on the Helena V8. On the Sebia Capillarys 2, a significant correlation was only observed with eGFR (r=0.17, p=0.004). A better discrimination between CKD stages was also observed using the Helena V8. Conclusions Thanks to a higher mean N, the Helena V8 might offer new possibilities, including detection of carbamylated albumin through SF calculation. Further studies are still needed to confirm the interest of using this type of assays in clinical routine.

Immunoglobulin D Multiple Myeloma with a “Hidden” Lambda Light Chain

Abstract Introduction/Objective In rare cases, the conventional immunofixation gel electrophoresis technique fails to detect the light chain of an M-protein. We report a case of immunoglobulin (Ig) D multiple myeloma with a hidden lambda (λ) light chain. Methods/Case Report Capillary electrophoresis (CE) (Sebia CAPILLARYS 2) was used to detect and quantify M- proteins in serum specimens. Immunosubtraction (IS) on the CAPILLARYS 2 systems was used to identify the classes of M-proteins. Conventional gel immunofixation electrophoresis (IFE) was performed, using monospecific antisera for IgD, IgE, kappa (κ) or λ in the Sebia HYDRASYS system, and IgG, IgA, IgM, κ or λ in the Helena SPIFE3000 system. Beta-mercaptoethanol (BME) with Fluidil were used as reduction agents. Results (if a Case Study enter NA) Results of serum CE showed two abnormal peaks in beta 2 and gamma regions, suspected to be positive for M-proteins. IS results showed subtraction for λ light chain only in both peaks, suggesting two monoclonal λ light chains. In contrary, no monoclonal λ light chain was detected in gamma region by IFE (Sebia). Epitope masking in the folded monoclonal protein was suspected to cause the “hidden λ light chain” and was further investigated by two laboratory approaches. IFE performed on the Helena SPIFE3000 system found two λ bands in beta 2 and gamma regions, which was consistent with the results from IS. The treatment of BME with Fluidil helped unmasking the hidden epitope and revealed the λ band in gamma region on IFE (Sebia). Conclusion The medical laboratories should be aware of the described scenario. The failure to detect light chains of certain intact M-proteins is most likely due to the structurally inaccessibility of light chains. It is recommended that treatment with reduction agents or use of an alternative methodology or IS might be helpful for investigating suspected heavy chain only cases, due to the limitation of conventional methodology.

Evaluation of a Capillary Electrophoresis System for the Separation of Proteins.

Serum protein electrophoresis is one of the core investigations for screening for monoclonal proteins. Among the available capillary systems, the Helena V8 system has been evaluated in a limited number of studies. In total, 310 sera samples were assessed on the Helena V8 system and compared with the Sebia Capillarys instrument. Abnormalities suggestive of monoclonal proteins were confirmed by immunofixation. Imprecision studies and reference intervals were determined. The imprecision of the Helena V8 was inferior or equal to 5.8%. The mean bias of Helena V8 vs Sebia Capillarys was about -0.9 g/L for albumin; -0.2 g/L for alpha-1; 1.1 g/L for alpha-2; -0.2 g/L for beta; 0.3 g/L for gamma; -0.5 g/L for monoclonal protein in beta; and 0.3 g/L for monoclonal protein in gamma. Among the 56 samples with monoclonal proteins confirmed by immunofixation, all were seen on both methods, with only 1 discordant result at a cutoff of 5.0 g/L. Reference intervals were statistically different between the 2 analyzers, except for the beta fraction. Our evaluation confirms the good analytical performance of the Helena V8 analyzer as a suitable alternative to the Sebia Capillarys instrument.

Hemoglobin fractionation by Sebia Capillarys 2 Flex Piercing System as primary method for evaluation of hemoglobinopathies

BackgroundHemoglobin fractionation by capillary zone electrophoresis (CE) is becoming a popular method for the identification of hemoglobin variants that can cause hemoglobinopathies. The goal of this study was to compare the performance of capillary electrophoresis using Sebia Capillarys 2 Flex Piercing system (CE-S) with high-pressure liquid chromatography (HPLC) using Primus Ultra2 Resolution Variants System (HPLC-P) as a primary method in hemoglobinopathy work-up. MethodsA total of 306 blood specimens submitted for evaluation of hemoglobinopathies were studied using HPLC-P and CE-S. ResultsThe reference ranges for Hb A, A2 and F agreed well between methods. All common variants containing Hb S and Hb C were detected by both methods. Quantification of Hb A2 with HPLC-P required a correction in the presence of Hb S, while quantification of Hb A2 was slightly overestimated by CE-S in the presence of Hb C. Of 41 samples containing other variants, 2 were not identified by HPLC-P and 3 were not identified by CE-S. ConclusionCE-S provides comparable information to that obtained by HPLC-P and it is a reliable primary method for the evaluation of hemoglobin variants.

Identification of Paraproteins via Serum Immunofixation or Serum Immunosubtraction and Immunoturbidimetric Quantitation of Serum Immunoglobulins in the Laboratory Testing for Monoclonal Gammopathies.

In laboratory testing for monoclonal gammopathies, paraproteins are identified via serum immunofixation or serum immunosubtraction, and immunoturbidimetric quantitation of serum immunoglobulins is often used. To evaluate methodologic differences between serum immunofixation and serum immunosubtraction, as well as in the quantitation of serum immunoglobulins on different clinical chemical platforms. Three hundred twenty-two unique routine patient samples were blinded and used for comparison between serum immunofixation on Sebia's HYDRASIS 2 and serum immunosubtraction on Sebia's CAPILLARYS 2, as well as between quantitation results of immunoglobulin A, G, and M on Abbott's ARCHITECT c16000PLUS and Roche's Cobas c 502 module. Microsoft Excel 2019 with the add-on Abacus 2.0 and MedCalc were used for statistical analysis and graphic depiction via bubble diagram, Passing-Bablok regressions, and Bland-Altman plots. The median age of patients was 75 years, and samples with paraproteinemia were nearly evenly split between sexes. Paraprotein identification differed remarkably between immunofixation and immunosubtraction. Quantitation of serum immunoglobulins showed higher values on Abbott's ARCHITECT c16000PLUS when compared with Roche's Cobas c 502 module. Identification of paraproteins via serum immunosubtraction is inferior to serum immunofixation, which can have implications on the diagnosis and monitoring of patients with monoclonal gammopathy. If immunoturbidimetric quantitation of immunoglobulins is used for follow-up, the same clinical-chemical platform should be used consistently.

Reference intervals for commonly requested biochemical and haematological parameters in a healthy Irish adult Caucasian population.

In laboratory medicine, reference intervals (RIs) are key decision support tools used to guide the clinical interpretation of numerical test results. Best practice suggests each laboratory establishes RIs in the local population prior to introducing an assay into routine clinical practice. The aim of this study was to define RIs for frequently requested biochemical/haematological parameters in a healthy adult Irish Caucasian population. A cross-sectional study of non-pregnant apparently healthy volunteers was conducted. Baseline demographics, anthropometric and laboratory measurements were recorded. In total, 37 commonly requested biochemical (serum, n=26) and haematological (venous blood, n=11) ISO15189:2012 accredited tests were analysed, using the Roche Cobas® Sebia Capillarys 3 Tera and Siemens Advia® 2120i platforms following standard operating procedures. RIs were defined according to the International Federation of Clinical Chemistry (IFCC) recommended method. Of 208 apparently healthy volunteers, 76 failed to meet the study inclusion criteria. The reference population comprised of132 participants (males: n=65, 49.2%) with a median age of 29.7 (18.1-62.2) years. RIs for the majority of biochemical/haematological parameters were broadly in accord with those provided by Pathology Harmony (UK)/Irish RI Harmonisation Project and the manufacturer Roche Diagnostics. However, the established RI defined for HbA1c: 27-37mmol/mol was markedly different from that quoted nationally, HbA1c: 20-42mmol/mol. Normative biological intervals established in a healthy adult Irish population for 37 commonly requested biochemical/haematological parameters will be a valuable aid to result interpretation in clinical laboratories after appropriate verification in accordance with ISO 15189: 2012.

Unexpected HbA1c results in the presence of three rare hemoglobin variants

Hemoglobin (Hb) variants, characterized by structural abnormalities in the globin chains, are among the most common inherited disorders. It has been shown that Hb variant remains an important cause of erroneous HbA1c results. Thus, it is important to be aware of the extent of the interference of each Hb variant encountered to avoid reporting unreliable results. However, the effects of many types of Hb variants on the measurement of HbA1c remain unclear. Here, we describe three rare Hb variants, Hb J-Tashikuergan [HBA2: c.59 C > A], Hb Pyrgos [HBB: c.251G > A], and Hb Hope [HBB: c.410 G > A], which lead to extremely high values (>25%) determined by Variant II Turbo 2.0. We further investigated their effects on HbA1c measurement by an HPLC system (Bio-Rad D100), a CE system (Sebia Capillarys 3 TERA), a boronate affinity chromatography system (Premier Hb9210), and an immunoassay method (Roche Diagnostics), and found that these Hb variants severely interfered with HbA1c measurement by Variant II Turbo 2.0 and Bio-Rad D100. This study demonstrates that patients with abnormally high HbA1c levels should be highly suspected of carrying Hb variants. When the HbA1c results are considered unreliable, other indicators such as glycated albumin may be a possible alternative to HbA1c in diabetic patients.

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.

Relevant aspects of identification and interpretation of the glycated hemoglobin research findings.

The article contains the literature review on laboratory criteria of detection and monitoring of the progression of the disease in patients with the diagnosis of diabetes mellitus. It also covers the issues of methodical approaches to the identification of glycated hemoglobin (HbA1c). The findings of author's researches of glycated hemoglobin in 149 patients have been given within the framework of comparison of two methodical approaches and comparison of the results with the subsequent classification of the received data. A random laboratory finding of qualitative hemoglobinopathy has been demonstrated, and the results recognized as unqualifiable and the approach to classification of such values have been discussed.Comparison of the results of glycated hemoglobin identification performed by different methods. 149 patients underwent a one-stage identification of glycated hemoglobin from plasma stabilized with K2-EDTA on Bio-Rad D10 and Sebia Capillarys Flex Piercing 2. Comparative study of the results of glycated hemoglobin identification has shown a difference in absolute values. However, a statistically reliable (p < 0.05) correlation between the values of glycated hemoglobin, expressed as a percentage obtained by different methods, has been revealed. In this case, the choice of a method for identifying glycated hemoglobin is not a matter of principal but it is important to adhere to the same method in treatment and long-term monitoring.

Capillary hemoglobin electrophoresis of healthy and anemic dogs: Quantification, validation, and reference intervals of hemoglobin fractions.

Despite the advances in canine medicine and the rapid gaining of attention of canine models in biomedical field and particularly in hemoglobin genes research, the studies on canine hemoglobin composition are sparse with ambiguous findings. Our aim was: i) to investigate the electrophoretic pattern of canine hemoglobin and the possible effects of age, sex, and anemia using a capillary electrophoresis assay, and ii) to validate this assay and calculate reference intervals (RIs) for canine hemoglobin fractions. Blood samples were collected from 53 healthy and 42 dogs with regenerative and non-regenerative anemias. The Sebia Capillarys 2 flex-piercing was used for hemoglobin analysis and it was validated using canine blood samples. R statistical language was employed for the statistical analyses. A major hemoglobin fraction (named HbA0) and a minor one (named HbA2) were identified in 100% and 47.4% of samples, respectively. The within-run and between-run CV was 0.1% for HbA0, and 9.1% and 11.2% for HbA2, respectively. The extremely narrow range of HbA0 and HbA2 values hampered a linearity study using canine blood samples. The RIs for HbA0 and HbA2 were 98.9–100% and 0–1.1%, respectively. HbA0 and HbA2 values were not significantly correlated with age (P = 0.866) or reticulocyte count (P = 0.731). No differences were observed in the median HbA0 and HbA2 between the two sexes (P = 0.887), and healthy and anemic dogs (P = 0.805). In conclusion, the capillary electrophoresis revealed a major hemoglobin fraction and an inconsistently present minor fraction. No effect of age, sex, anemia, or regenerative status of anemia was detected. The assay used was validated and RIs were generated, so as to be suitable for use in future investigations.

Hb Palencia: a novel δβδ-type two-way fusion variant with β-globin-like expression levels

AimsFusion proteins of unequal recombination events at the β-globin locus have pathological effect. The haemoglobin (Hb) variants of type Lepore are fusion proteins characterised by β-like globin chains with a...

Utility of Reflexing Suspicious Beta Region Findings on Capillary Serum Protein Electrophoresis to Immunofixation for Detecting Monoclonal Gammopathies

Serum protein electrophoresis (SPEP) detects alterations suspicious for monoclonal immunoglobulins (M-protein/MP). Immunofixation (IFE) confirms/characterizes MPs. Beta region (BR) proteins, transferrin, and C3 can obscure beta-migrating MPs. There is no standardization of how to handle BR abnormalities. This study evaluates the effectiveness of our laboratory’s strategy of reflexing BR abnormalities to IFE and of separate measurement of beta1 and beta2 proteins for MP detection. In total, 2,134 SPEPs, performed on Sebia Capillarys, containing the comment “possible restriction in the beta region” (PRB) were identified by retrospective review (Michigan Medicine, 6/2013–6/2017). PRB was used in three situations: suspicious BR spike, beta1-beta2 bridge, or increased total beta region (TB) without obvious explanation (beta-gamma bridge), resulting in reflexed IFE. Inclusion of only the initial sample for each patient and exclusion of patients with MP history resulted in 1,904 cases. TB, MP, isotype, and increased polyclonal immunoglobulins were recorded. Separate beta1 and beta2 measurements were performed. Construction of references ranges (RRs) (mean ± 2 SD) using 31 normal patients showed RR 0.31 to 0.61 g/dL (beta1) and 0.24 to 0.57 g/dL (beta2). Normal TB RR, 0.65 to 1.03 g/dL. A total of 272 cases had MP detected by IFE (14.3%); 928 cases had increased TB (48.7%), while 660 had increased beta1/beta2 (34.7%). MP was present in 169 cases with increased TB (8.88%) and 183 with increased beta1/beta2 (9.61%). Low/normal TB, beta1, beta2, and beta1/beta2 were present in 103 (5.41%), 234 (12.3%), 119 (6.25%), and 88 (4.62%) cases with MP, respectively. Most common isotypes were IgA kappa and IgA lambda. Most common polyclonal increase was IgA (316). This technique had a true-positive rate of 14.3% with many negative IFEs, similar to another study reporting a 10% detection rate for elevated beta-fraction. Separate beta1 and beta2 measurements modestly increased MP detection. Further improvement of true-positive rates will require additional exclusion criteria.

New HPLC instrument performance evaluation in HbA1c determination and comparison with capillary electrophoresis

Glycated hemoglobin (HbA1c) measurement provides the most important medium to long-term marker of time-averaged glycemic status. Its relationship to clinical outcome in diabetes has been convincingly demonstrated for both type 1 and type 2 diabetes. The main HbA1c measurement methods for clinical routine are as follows: ion-exchange chromatography; affinity chromatography, capillary electrophoresis, immunoassay and enzymatic methods. In this study, we evaluated the analytical performances of a new HPLC instrument (Tosoh HLC-723 G11 in the VAR mode) in HbA1c analysis and compared it with a capillary electrophoresis instrument (Sebia Capillarys 2 Flex Piercing). HbA1c analysis was performed in parallel by both methods for 250 samples randomly chosen from healthy and diabetic subjects at ‘Tor Vergata’ University Hospital of Rome. Tosoh HLC-723 G11 showed good reproducibility for 10 days both in quality controls and in samples analyzed (%CV < 2%). We found good linearity for HbA1c values ranging from 15 mmol/mol (3.5%) to 178 mmol/mol (18.5%), with a correlation coefficient R2 = 1. In a comparison between Tosoh HLC-723 G11 and Capillarys 2FP a good correlation (r = 0.99) was found; however, Tosoh HLC-723 G11 showed higher values in the low range of HbA1c and lower in the high range (Tosoh HLC-723 G11 = 4.3043 + 0.913 Capillarys 2FP; p < 0.001). Tosoh HLC-723 G11 showed good repeatability, reproducibility, accuracy and automated simplicity, and it seemed suitable for routine use in clinical chemistry laboratories.

Effects of common hemoglobin variants on HbA1c measurements in China: results for α- and β-globin variants measured by six methods.

HbA1c is a widely used biomarker for diabetes mellitus management. Here, we evaluated the accuracy of six methods for determining HbA1c values in Chinese patients with common α- and β-globin chains variants in China. Blood samples from normal subjects and individuals exhibiting hemoglobin variants were analyzed for HbA1c, using Sebia Capillarys 2 Flex Piercing (C2FP), Bio-Rad Variant II Turbo 2.0, Tosoh HLC-723 G8 (ver. 5.24), Arkray ADAMS A1c HA-8180V fast mode, Cobas c501 and Trinity Ultra2 systems. DNA sequencing revealed five common β-globin chain variants and three common α-globin chain variants. The most common variant was Hb E, followed by Hb New York, Hb J-Bangkok, Hb G-Coushatta, Hb Q-Thailand, Hb G-Honolulu, Hb Ube-2 and Hb G-Taipei. Variant II Turbo 2.0, Ultra2 and Cobas c501 showed good agreement with C2FP for most samples with variants. HLC-723 G8 yielded no HbA1c values for Hb J-Bangkok, Hb Q-Thailand and Hb G-Honolulu. Samples with Hb E, Hb G-Coushatta, Hb G-Taipei and Hb Ube-2 produced significant negative biases for HLC-723 G8. HA-8180V showed statistically significant differences for Hb E, Hb G-Coushatta, Hb G-Taipei, Hb Q-Thailand and Hb G-Honolulu. HA-8180V yielded no HbA1c values for Hb J-Bangkok. All methods showed good agreement for samples with Hb New York. Some common hemoglobin variants can interfere with HbA1c determination by the most popular methods in China.