Hemoglobin Variant Analysis via Direct Surface Sampling of Dried Blood Spots Coupled with High-Resolution Mass Spectrometry (original) (raw)
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2021
ABSTRACTHemoglobin (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), hemo...
Journal of The American Society for Mass Spectrometry, 2012
We have previously shown that liquid microjunction surface sampling of dried blood spots coupled with high resolution top-down mass spectrometry may be used for screening of common hemoglobin variants HbS, HbC, and HbD. In order to test the robustness of the approach, we have applied the approach to unknown hemoglobin variants. Six neonatal dried blood spot samples that had been identified as variants, but which could not be diagnosed by current screening methods, were analyzed by direct surface sampling top-down mass spectrometry. Both collision-induced dissociation and electron transfer dissociation mass spectrometry were employed. Four of the samples were identified as β-chain variants: two were heterozygous Hb D-Iran, one was heterozygous Hb Headington, and one was heterozygous Hb J-Baltimore. The fifth sample was identified as the α-chain variant heterozygous Hb Phnom Penh. Analysis of the sixth sample suggested that it did not in fact contain a variant. Adoption of the approach in the clinic would require speed in both data collection and interpretation. To address that issue, we have compared manual data analysis with freely available data analysis software (ProsightPTM). The results demonstrate the power of top-down proteomics for hemoglobin variant analysis in newborn samples.
Automated Analysis of Hemoglobin Variants Using NanoLC–MS and Customized Databases
Journal of Proteome Research, 2013
Unambiguous analysis of hemoglobin variants is critical in the diagnosis of hemoglobinopathies. In diagnostic laboratories, alkaline gel electrophoresis and automated HPLC are used in identifying variants. In specific instances, comigration of hemoglobin variant bands in gel and coelution of different variants or elution of variants with unmatched library information in HPLC can result in ambiguities in interpretation. Hemoglobin variants mostly arise from point mutations leading to very high sequence homology between normal and variant hemoglobin. In addition, unavailability of a variant database compatible with proteomics data analysis software makes mass spectrometry based variant analysis very challenging. In the present study, we standardized a nanoLC−MS based method for variant analysis to achieve substantially high sequence coverage. We developed three hemoglobin variant databases, specific to three different proteolytic enzymes, compatible with proteomics search engine software. The above nanoLC−MS method and the compatibility of the customized databases were validated by analysis of a sickle hemoglobin variant. Six other hemoglobin variants were characterized wherein diagnosis reports based on conventional tools were ambiguous. The novelty of our method lies in its simplicity and accuracy of the analysis with minimal manual intervention. The presently described method may be used in the future for the routine hemoglobin variant diagnosis.
Newborn Screening for Sickle Cell Disease Using Tandem Mass Spectrometry
2008
BACKGROUND: Neonatal screening programs for sickle cell disease are now widespread in North American and European countries. Most programs apply isoelectric focusing or HPLC to detect hemoglobin variants. Because tandem mass spectrometry (MS/MS) is being used for screening of inherited metabolic disorders and allows protein identification, it was worth testing for hemoglobinopathy screening.
Analytical Chemistry, 2003
Mass spectrometry has a basic limitation when human hemoglobin variants are analyzed, because it cannot resolve two globin chains that differ in mass by <6 Da. Several common -chain variants differ by 1 Da from normal and, hence, when present in heterozygotes, are not resolved from the normal -chain. Normal and variant chains appear together in the spectrum as a single entity, whose mass is the abundance weighted mean of the two chains. Here we show that such heterozygotes can be detected in 500-fold diluted blood by accurately measuring the mass of the -chain using an electrospray ionization quadrupole instrument and the r-chain for internal mass calibration. A statistical analysis of the normal -chain mass (n ) 86) showed that the standard deviation (SD) of the mean was <(0.05 Da (<(3.2 ppm). Hence, at the 95% confidence level ((2 SD), an abnormal r-or -chain differing by 1 Da from normal should be detectable in a heterozygote provided its abundance is >10% of total r-or -chains, respectively. Variants whose masses lay between 1 and 4 Da from normal were detected in 19 heterozygotes. Moreover, the proportion of each variant estimated from the mass change correlated with the proportion determined by cation-exchange HPLC. Variants were assigned to the r-or -chain by combining the sign of the mass change with the polarity change inferred from electrophoretic data. This procedure could be used for screening clinically significant hemoglobin variants. Green, B. N.; Cooper, E. K.; Lalloz, M. R. A.; Erten, S.; Stephens, A. D.; Layton, D. M. Blood Cells Mol. Dis. 2001, 27, 691-704. (3) Shackleton, C. H. L.; Falick, A. M.; Green, B. N.; Witkowska, H. E.
Phenotype determination of hemoglobinopathies by mass spectrometry
Clinical Biochemistry, 2009
Nowadays, nearly 1000 hemoglobin (Hb) variants are known. The standard biochemical techniques used in Hb analysis are mainly: isoelectric focusing, cation-exchange liquid chromatography (LC) and reversed-phase LC. In addition to this approach, a protein analysis is achieved by mass spectrometry (MS) and additional DNA studies are performed. The aim of this review is to emphasize the significance of MS methods applied to Hb variants analysis.To perform Hb studies, different MS techniques are currently used such as electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI) and tandem mass spectrometry (MS/MS). As shown here, MS is an efficient tool for identification of all types of variants (substitution of a single amino acid residue, several substitutions in the same globin chain, insertions/deletions, fusion Hbs). The use of MS in neonatal screening of Hb variants is also presented.MS is a powerful technique for Hb analysis. It appears as being an important additional method in the set of biochemical techniques.
Biochemical genetics, 2016
Hemoglobinopathies are caused by point mutation in globin gene that results in structural variant of hemoglobin. While 7 % of world populations are carrier of hemoglobinopathies, the prevalence of the disease varies between 3 to 17 % across different population groups in India. In a diagnostic laboratory, alkaline gel electrophoresis and cation exchange-based HPLC (CE-HPLC) are most widely used techniques for characterization of hemoglobin variants. In the above methods, the differential surface charge of hemoglobin molecule in variants is exploited for their characterization. Sometime, co-migration of variants in gel electrophoresis and co-elution or elution with unknown retention time in automated CE-HPLC might lead to ambiguity in the analysis of hemoglobinopathies. Under such circumstances, it is necessary to use other analytical methods that provide unambiguous results. Mass spectrometry-based proteomics approach and DNA sequence analysis are examples of such alternative method...
Indian Journal of Hematology and Blood Transfusion, 2021
Screening of newborns for the presence of sickle hemoglobin (HbS) is aimed at reducing the morbidity and mortality associated with sickle cell disease in early childhood. The high cost and limited availability of dedicated high performance liquid chromatography (HPLC) systems specially designed for screening of dry blood spots (DBS), however, restrict a wider application of this preventive approach. Therefore, we examined the ability of a commonly used HPLC system for detection of hemoglobinopathies in DBS samples in order to find an alternative for the dedicated newborn screening (NBS) HPLC system. DBS samples from 7522 newborns were first examined by Variant NBS HPLC system (Bio Rad, USA) for the presence of hemoglobinopathies. Positive samples were then analysed by Variant II system (Bio Rad, USA), another platform commonly used for hemoglobinopathy screening of anticoagulated blood samples. Eighty six newborns (1.1%) showed the presence of hemoglobinopathies (HbS 28, HbE 21, HbD 27, HbQ India 9 and Hb Barts 1) by Variant NBS system-all in heterozygous state. There was 100% correlation between the two sets of results obtained by the two HPLC systems. Newborns with HbQ India showed an additional Hb peak in HPLC resulting from combination of the abnormal alpha globin chain of HbQ India with the normal gamma chain of HbF-'HbF Q India'. Variant II HPLC system, used for routine hemoglobinopathy screening in anticoagulated blood, can also be used for screening DBS samples. This obviates the need for a dedicated NBS system for hemoglobinopathy screening in newborns. We also demonstrated that both the systems are equally competent in detecting non-sickle Hb variants in DBS samples.
The Journal of molecular diagnostics : JMD, 2009
A number of common mutations in the hemoglobin beta (HBB) gene cause beta-thalassemia, a monogenic disease with high prevalence in certain ethnic groups. As there are 30 HBB variants that cover more than 99.5% of HBB mutant alleles in the Thai population, an efficient and cost-effective screening method is required. Three panels of multiplex primer extensions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were developed. The first panel simultaneously detected 21 of the most common HBB mutations, while the second panel screened nine additional mutations, plus seven of the first panel for confirmation; the third panel was used to confirm three HBB mutations, yielding a 9-Da mass difference that could not be clearly distinguished by the previous two panels. The protocol was both standardized using 40 samples of known genotypes and subsequently validated in 162 blind samples with 27 different genotypes (including a normal control), comprising ...