Multispectral Imaging for MicroChip Electrophoresis Enables Point-of-Care Newborn Hemoglobin Variant Screening

Hemoglobin (Hb) disorders affect nearly 7% of the world's population. Globally, around 400,000 babies are born annually with sickle cell disease (SCD), primarily in sub-Saharan Africa where morbidity and mortality rates are high. Although treatments are available for Hb disorders, screening, early diagnosis, and monitoring are not widely accessible due to technical challenges and cost, especially in low-and-middle-income countries. We hypothesized that multispectral imaging will allow sensitive hemoglobin variant identification in existing affordable paper-based Hb electrophoresis, which is a clinical standard test for Hb variant screening. To test this hypothesis, we developed the first integrated point-of-care multispectral Hb variant test: Gazelle-Multispectral. Here, we evaluated the accuracy of Gazelle-Multispectral for Hb variant newborn screening in 321 completed tests in subjects younger than 6 months with known hemoglobin variants including hemoglobin A (Hb A), hemoglobin F (Hb F), hemoglobin S (Hb S) and hemoglobin C (Hb C). Gazelle-multispectral detected levels of Hb A, Hb F, Hb S, and Hb C, demonstrated high correlations with the results reported by laboratory gold standard high performance liquid chromatography (HPLC) at Pearson Correlation Coefficient = 0.97, 0.97, 0.89, and 0.94. Gazelle-multispectral demonstrated 100% sensitivity and 100% specificity in both disease vs normal and disease vs trait, 98.1% sensitivity and 97.0% specificity in trait vs normal in comparison to HPLC in newborns. The ability to obtain rapid and accurate results on newborn samples suggest that Gazelle-Multispectral is suitable for large-scale newborn screening and potentially for accurate diagnosis of SCD in low resource settings.

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

ObjectivesHemoglobin (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.MethodsWe 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.ResultsAmong 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.ConclusionsThe prevalence of hemoglobin variants remains high and exhibits genetic diversity and widespread distribution in the population of southern China.

EXPRESS: Assessment of the analytical performance of point-of-care faecal immunochemical tests for haemoglobin

Background: The faecal immunochemical test (FIT) detects the presence of haemoglobin (Hb) in faeces. It is used as a screening tool for colorectal cancer (CRC) and increasingly to triage patients presenting with symptoms of CRC. A number of quantitative point-of-care (POC) FIT systems marketed for professional use and intended for use in a clinical setting are available. Here we reviewed the POC FIT systems available; three (Eurolyser Cube, OC-Sensor iO and QuikRead go) were evaluated to assess their performance against manufacturers’ claims and suitability for use in a clinical setting. Methods The analytical evaluation of the POC FIT systems was undertaken using Hb lysates, patient samples and an external quality assessment sample. The evaluation focused on linearity, recovery, imprecision, prozone effect, Hb variant detection and suitability for use in a clinical setting. Results: All three POC FIT systems performed to their manufacturer’s claims and demonstrated good analytical performance with acceptable linearity, recovery, within- and between-run imprecision. The QuikRead go and OC-Sensor iO were able to accurately detect samples with results above their measuring range. However, because of a prozone effect the Eurolyser Cube gave falsely low results when using high concentrations of Hb. The QuikRead go performed best in the usability assessment due to portability and timeliness of result. Conclusion: Each system performed according to their manufacturers’ claims. The QuikRead go and OC-Sensor iO are suitable for use. The Eurolyser Cube is not recommended because of the risk of falsely low results.

Computer Vision and Deep Learning Assisted Microchip Electrophoresis for Integrated Anemia and Sickle Cell Disease Screening

Introduction: Anemia affects a third of the world's population with the heaviest burden borne by women and children. Anemia leads to preventable impaired development in children, as well as high morbidity and early mortality among sufferers. Inherited hemoglobin (Hb) disorders, such as sickle cell disease (SCD), are associated with chronic hemolytic anemia causing high morbidity and mortality. Anemia and SCD are inherently associated and are both prevalent in the same regions of the world including sub-Saharan Africa, India, and south-east Asia. Anemia and SCD-related complications can be mitigated by screening, early diagnosis followed by timely intervention. Anemia treatment depends on the accurate characterization of the cause, such as inherited Hb disorders. Meanwhile, Hb disorders or SCD treatments, such as hydroxyurea therapy, requires close monitoring of blood Hb level and the patient's anemia status over time. As a result, it is crucially important to perform integrated detection and monitoring of blood Hb level, anemia status, and Hb variants, especially in areas where anemia and inherited Hb disorders are the most prevalent. Blood Hb level (in g/dL) is used as the main indicator of anemia, while the presence of Hb variants (e.g., sickle Hb or HbS) in blood is the primary indicator of an inherited disorder. The current clinical standards for anemia testing and Hb variant identification are complete blood count (CBC) and High-Performance Liquid Chromatography (HPLC), respectively. State-of-the-art laboratory infrastructure and trained personnel are required for these laboratory tests. However, these resources are typically scarce in low- and middle-income countries, where anemia and Hb disorders are the most prevalent. As a result, there is a dire need for high accuracy portable point-of-care (POC) devices to perform integrated anemia and Hb variant tests with affordable cost and high throughput. Methods: In 2019, the World Health Organization (WHO) listed Hb electrophoresis as an essential in vitro diagnostic (IVD) technology for diagnosing SCD and sickle cell trait. We have leveraged the common Hb electrophoresis method and developed a POC microchip electrophoresis test, Hemoglobin Variant/Anemia (HbVA). This technology is being commercialized under the product name "Gazelle" by Hemex Health Inc. for Hb variant identification with integrated anemia detection (Fig. 1A&B). We hypothesized that computer vision and deep learning will enhance the accuracy and reproducibility of blood Hb level prediction and anemia detection in cellulose acetate based Hb electrophoresis, which is a clinical standard test for Hb variant screening and diagnosis worldwide (Fig. 1C). To test this hypothesis, we integrated, for the first time, a new, computer vision and artificial neural network (ANN) based deep learning imaging and data analysis algorithm, to Hb electrophoresis. Here, we show the feasibility of this new, computer vision and deep learning enabled diagnostic approach via testing of 46 subjects, including individuals with anemia and homozygous (HbSS) or heterozygous (HbSC or Sβ-thalassemia) SCD. Results and Discussion: HbVA computer vision tracked the electrophoresis process real-time and the deep learning neural network algorithm determined Hb levels which demonstrated significant correlation with a Pearson Correlation Coefficient of 0.95 compared to the results of reference standard CBC (Fig.1D). Furthermore, HbVA demonstrated high reproducibly with a mean absolute error of 0.55 g/dL and a bias of -0.10 g/dL (95% limits of agreement: 1.5 g/dL) according to Bland-Altman analysis (Fig. 1E). Anemia determination was achieved with 100% sensitivity and 92.3% specificity with a receiver operating characteristic area under the curve (AUC) of 0.99 (Fig. 1F). Within the same test, subjects with SCD were identified with 100% sensitivity and specificity (Fig. 1G). Overall, the results suggested that computer vision and deep learning methods can be used to extract new information from Hb electrophoresis, enabling, for the first time, reproducible, accurate, and integrated blood Hb level prediction, anemia detection, and Hb variant identification in a single affordable test at the POC. Disclosures An: Hemex Health, Inc.: Patents & Royalties. Hasan:Hemex Health, Inc.: Patents & Royalties. Ahuja:Genentech: Consultancy; Sanofi-Genzyme: Consultancy; XaTec Inc.: Consultancy; XaTec Inc.: Research Funding; XaTec Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Genentech: Honoraria; Sanofi-Genzyme: Honoraria. Little:GBT: Research Funding; Bluebird Bio: Research Funding; BioChip Labs: Patents & Royalties: SCD Biochip (patent, no royalties); Hemex Health, Inc.: Patents & Royalties: Microfluidic electropheresis (patent, no royalties); NHLBI: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees. Gurkan:Hemex Health, Inc.: Consultancy, Current Employment, Patents & Royalties, Research Funding; BioChip Labs: Patents & Royalties; Xatek Inc.: Patents & Royalties; Dx Now Inc.: Patents & Royalties.

Are hemoglobin A1c point-of-care analyzers fit for purpose? The story continues

ObjectivesPoint-of-care (POC) analyzers are playing an increasingly important role in diabetes management but it is essential that we know the performance of these analyzers in order to make appropriate clinical decisions. Whilst there is a growing body of evidence around the more well-known analyzers, there are many ‘new kids on the block’ with new features, such as displaying the presence of potential Hb-variants, which do not yet have a proven track record.MethodsThe study is a comprehensive analytical and usability study of six POC analyzers for HbA1c using Clinical and Laboratory Standards Institute (CLSI) protocols, international quality targets and certified International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and National Glycohemoglobin Standardization Program (NGSP) Secondary Reference Measurement Procedures (SRMP). The study includes precision (EP-5 and EP-15), trueness (EP-9), linearity (EP-6), sample commutability (fresh, frozen and lyophilized), interference of Hb-variants (fresh and frozen samples).ResultsOnly two of the six analyzers performed to acceptable levels over the range of performance criteria. Hb-variant interference, imprecision or variability between lot numbers are still poor in four of the analyzers.ConclusionsThis unique and comprehensive study shows that out of six POC analyzers studied only two (The Lab 001 and Cobas B101) met international quality criteria (IFCC and NGSP), two (A1Care and Innovastar) were borderline and two (QuikReadgo and Allegro) were unacceptable. It is essential that the scientific and clinical community are equipped with this knowledge in order to make sound decisions on the use of these analyzers.

Compound heterozygosity for hemoglobin variant Hb-Broomhill and the Southeast Asian α-thalassemia deletion does not worsen outcome: a case report of two unrelated patients

We report two unrelated cases of compound heterozygosity for hemoglobin (Hb) variant Broomhill and the Southeast Asian (- - SEA/) α-thalassemia deletion, whose clinical features and laboratory findings have never been reported. Hematological analyses revealed abnormal values for both cases as α-thalassemia traits, and capillary electrophoresis suggested an abnormal peak that was incompletely separated from the Hb A peak. A suspension array system and Sanger sequencing were used to characterize the genotypes. Sanger sequencing confirmed the presence of Hb Broomhill [α114(GH2)Pro→Ala; HBA1: c.343C>G]. Eventually, both cases were accurately diagnosed as compound heterozygotes for Hb Broomhill and the (- - SEA/) α-thalassemia deletion, which is the first known report of these variants. This information will be useful when providing appropriate genetic counselling and prenatal diagnosis.

Why is the novel Hb Miguel Servet visualised by CE-HPLC newborn and not by the CE-HPLC β-thalassaemia programme?

Screening of haemoglobinopathies is indicated for the detection of sickle cell anaemia; thus, neonates can benefit from early and adequate treatment that prevents neurological damage, reduces morbidity and mortality associated with the disease. These types of programmes sometimes lead to unexpected findings. We present a new haemoglobin (Hb) variant (Hb Miguel Servet) detected by newborn screening. During neonatal screening of haemoglobinopathies by cation-exchange high-performance liquid chromatography (CE-HPLC) newborn, an Hb variant was detected. An analysis at 8 months of age using capillary zone electrophoresis (CZE) confirmed the presence of this new Hb. The molecular characterisation was performed by automatic sequencing of the α and β globin genes in an ABI PRISM 3100 Genetic Analyzer. Hb analysis by CE-HPLC β-thalassaemia short programmedid not indicate the presence of abnormal Hbs. By CZE showed a peak in the zone 12 zone comprising 3.3% of the total Hb. A new analysis by CE-HPLC on a Tosoh G8-2 (Horiba) shown a peak, in the region of HbA1b, did not interfere with the quantification of HbA1c. Sequencing of the β gene revealed the substitution of a guanine for a thymine (GGT >TGT) in codon 69 of the second exon, resulting in substitution of cysteine for the amino acid glutamine (HBB:c.208G>T). Hb Miguel Servet is a β-chain globin variant detected by CE-HPLC newborn (BioRad), by CZE and by CE-HPLC-CE Tosoh G8-2 (Horiba), but no by CE-HPLC-CE β-thalassaemia short programme (BioRad). In fact, for all the techniques that are visualised, what would be detected would be the glutathione variant of Hb (Miguel Servet).

Diagnosis of Hemoglobinopathy and β-Thalassemia by 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry of Hemoglobin from Blood

BACKGROUND Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current screening methods include cation-exchange HPLC and electrophoresis, the results of which can be ambiguous because of limited resolving power. Subsequently, laborious genetic testing is required for confirmation. METHODS We performed a top-down tandem mass spectrometry (MS/MS) approach with a fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavage by use of positive electrospray ionization 21 Tesla Fourier transform ion cyclotron resonance–tandem mass spectrometry (21 T FT-ICR MS/MS) for hemoglobin (Hb) variant de novo sequencing and β-thalassemia diagnosis. RESULTS We correctly identified all Hb variants in blind analysis of 18 samples, including the first characterization of homozygous Hb Himeji variant. In addition, an Hb heterozygous variant with isotopologue mass spacing as small as 0.0194 Da (Hb AD) was resolved in both precursor ion mass spectrum (MS1) and product ion mass spectrum (MS2). In blind analysis, we also observed that the abundance ratio between intact δ and β subunits (δ/β) or the abundance ratio between intact δ and α subunits (δ/α) could serve to diagnose β-thalassemia trait caused by a mutation in 1 HBB gene. CONCLUSIONS We found that 21 T FT-ICR MS/MS provides a benchmark for top-down MS/MS analysis of blood Hb. The present method has the potential to be translated to lower resolving power mass spectrometers (lower field FT-ICR mass spectrometry and Orbitrap) for Hb variant analysis (by MS1 and MS2) and β-thalassemia diagnosis (MS1).