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Simon G, Boemer F, Luis G, Gothot A, Tassin F, Keutgens A. Diagnostic performance of morphological analysis and red blood cell parameter-based algorithms in the routine laboratory screening of heterozygous haemoglobinopathies. Clin Chem Lab Med 2025:cclm-2025-0210. [PMID: 40418782 DOI: 10.1515/cclm-2025-0210] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2025] [Accepted: 04/30/2025] [Indexed: 05/28/2025]
Abstract
OBJECTIVES The aim of this study was to carry out a cross-analysis of the morphological abnormalities (MA) and the electrophoretic profile (EP) of blood samples suspect for heterozygous haemoglobinopathies (HTZ HGP). Screening for HTZ HGP was based on erythrocyte parameters provided by the Sysmex XN analysers. METHODS A total of 596,000 blood samples was included in the study. According to the results of the mean corpuscular haemoglobin concentration (MCHC), the percentage of microcytes (Micro%) and the standard deviation of the red blood cell distribution width (RDW-SD), 842 different adults were screened as suspect for HTZ HGP and underwent simultaneous morphological analysis of red blood cells (RBCMA) and haemoglobin fraction analysis. RESULTS The majority (72.8 %) of HTZ HGP suspects presented a pathological EP, mostly compatible with a confirmed β-thalassaemia trait (50.1 %) or a heterozygous β-haemoglobin variant (12.2 %). MA were identified in 360 (42.8 %) samples and 70 (8.3 %) of these had 3 or more MA. The most common MA was poikilocytosis (28.1 %). Patients with at least 1 MA detected were more likely to have a pathological EP (p=0.003). However, correlation between the number of MA detected and the type of EP was negligible. CONCLUSIONS Screening for HTZ HGP based on erythrocyte parameters measured on Sysmex XN analysers is a relevant tool with a positive predictive value of 72.8 % and definitely superior to microscopic RBCMA which now appears to be of low added value and obsolete in this indication.
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Affiliation(s)
- Germain Simon
- Department of Laboratory Hematology, University Hospital Center of Liège, Liège, Belgium
| | - François Boemer
- Biochemical Genetics Laboratory, Department of Human Genetics, University Hospital Center of Liège, Liège, Belgium
| | - Géraldine Luis
- Biochemical Genetics Laboratory, Department of Human Genetics, University Hospital Center of Liège, Liège, Belgium
| | - André Gothot
- Department of Laboratory Hematology, University Hospital Center of Liège, Liège, Belgium
| | - Françoise Tassin
- Department of Laboratory Hematology, University Hospital Center of Liège, Liège, Belgium
| | - Aurore Keutgens
- Department of Laboratory Hematology, University Hospital Center of Liège, Liège, Belgium
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Renoux C, Roland E, Ruet S, Zouaghi S, Michel M, Joly P, Feray C, Zhao F, Gavanier D, Gaucherand P, Roumieu F, Cannas G, Merazga S, Connes P, Renom G, Massardier J, Cheillan D. Evaluation of a New Tandem Mass Spectrometry Method for Sickle Cell Disease Newborn Screening. Int J Neonatal Screen 2024; 10:77. [PMID: 39728397 DOI: 10.3390/ijns10040077] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/24/2024] [Revised: 11/12/2024] [Accepted: 11/18/2024] [Indexed: 12/28/2024] Open
Abstract
In France, sickle cell disease newborn screening (SCD NBS) has been targeted to at-risk regions since 1984, but generalization to the whole population will be implemented from November 2024. Although tandem mass spectrometry (MS/MS) is already used for the NBS of several inherited metabolic diseases, its application for SCD NBS has not been widely adopted worldwide. The aim of this study was to evaluate a dedicated MS/MS kit (Targeted MS/MS Hemo, ZenTech, LaCAR Company, Liege, Belgium) for SCD NBS and to compare the results obtained with those from an NBS reference center using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and cation-exchange high-performance liquid chromatography (CE-HPLC, Variant NBS, Biorad Laboratories, Inc., Hercules, CA, USA) as confirmatory method. The MS/MS Hemo kit was used according to the manufacturer's instructions and performed on a Waters Xevo TQ-D (Waters Corporation, USA). The software provided by the manufacturer was used for the calculation and analysis of peptide signal ratios. Among the 1333 samples, the results of 1324 samples were consistent with the HPLC and/or MALDI-TOF results (1263 FA, 50 FAS, 7 FAC, 1 FAO-Arab, and 3 FS). All the discordant results (one FAS on MS/MS vs. FA in CE-HPLC, one FA on MS/MS vs. FAS in CE-HPLC, seven FS on MS/MS vs. FAS in CE-HPLC) were corrected after modifying the peptide signal ratios thresholds, allowing the MS/MS Hemo kit to achieve near-100% sensitivity and specificity for SCD NBS. In conclusion, the MS/MS Hemo kit appears to be an effective method for SCD NBS, particularly for laboratories already equipped with MS/MS technology. However, these results should be confirmed in a larger cohort including a greater number of positive samples for SCD.
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Affiliation(s)
- Céline Renoux
- Laboratoire de Biologie Médicale Multi-Sites (LBMMS), Service de Biochimie et Biologie Moléculaire Grand Est, Hospices Civils de Lyon, 69500 Bron, France
- Centre Régional de Dépistage Néonatal Auvergne Rhône-Alpes (CRDN AuRA), 69003 Lyon, France
- Centre de Référence Constitutif des Maladies Constitutionnelles du Globule Rouge et de l'Erythropoïèse, 69003 Lyon, France
- Inter-University Laboratory of Human Movement Sciences, LIBM, Universite Claude Bernard Lyon 1, UR 7424, 69622 Villeurbanne, France
| | - Estelle Roland
- Laboratoire de Dépistage Périnatal, Pôle de Biologie Pathologie Génétique, Centre Hospitalier Universitaire de Lille, 59000 Lille, France
| | - Séverine Ruet
- Laboratoire de Biologie Médicale Multi-Sites (LBMMS), Service de Biochimie et Biologie Moléculaire Grand Est, Hospices Civils de Lyon, 69500 Bron, France
| | - Sarah Zouaghi
- Laboratoire de Biologie Médicale Multi-Sites (LBMMS), Service de Biochimie et Biologie Moléculaire Grand Est, Hospices Civils de Lyon, 69500 Bron, France
| | - Marie Michel
- Laboratoire de Biologie Médicale Multi-Sites (LBMMS), Service de Biochimie et Biologie Moléculaire Grand Est, Hospices Civils de Lyon, 69500 Bron, France
- Centre Régional de Dépistage Néonatal Auvergne Rhône-Alpes (CRDN AuRA), 69003 Lyon, France
| | - Philippe Joly
- Laboratoire de Biologie Médicale Multi-Sites (LBMMS), Service de Biochimie et Biologie Moléculaire Grand Est, Hospices Civils de Lyon, 69500 Bron, France
- Centre Régional de Dépistage Néonatal Auvergne Rhône-Alpes (CRDN AuRA), 69003 Lyon, France
- Centre de Référence Constitutif des Maladies Constitutionnelles du Globule Rouge et de l'Erythropoïèse, 69003 Lyon, France
- Inter-University Laboratory of Human Movement Sciences, LIBM, Universite Claude Bernard Lyon 1, UR 7424, 69622 Villeurbanne, France
| | - Cécile Feray
- Laboratoire de Biologie Médicale Multi-Sites (LBMMS), Service de Biochimie et Biologie Moléculaire Grand Est, Hospices Civils de Lyon, 69500 Bron, France
- Centre Régional de Dépistage Néonatal Auvergne Rhône-Alpes (CRDN AuRA), 69003 Lyon, France
- Centre de Référence Constitutif des Maladies Constitutionnelles du Globule Rouge et de l'Erythropoïèse, 69003 Lyon, France
| | - Fanny Zhao
- Laboratoire de Biologie Médicale Multi-Sites (LBMMS), Service de Biochimie et Biologie Moléculaire Grand Est, Hospices Civils de Lyon, 69500 Bron, France
- Centre Régional de Dépistage Néonatal Auvergne Rhône-Alpes (CRDN AuRA), 69003 Lyon, France
| | - Déborah Gavanier
- Service de Chirurgie Gynécologique et Cancérologique-Obstétrique, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, 69495 Pierre-Bénite, France
| | - Pascal Gaucherand
- Service d'Obstétrique, Hôpital Femme Mère Enfant, Hospices Civils de Lyon, 69500 Bron, France
| | - Fanny Roumieu
- Service de Gynécologie-Obstétrique, Groupement Hospitalier Nord, Hospices Civils de Lyon, 69004 Lyon, France
| | - Giovanna Cannas
- Centre de Référence Constitutif des Maladies Constitutionnelles du Globule Rouge et de l'Erythropoïèse, 69003 Lyon, France
- Service de Médecine Interne, Hôpital Edouard Herriot, Hospices Civils de Lyon, 69003 Lyon, France
| | - Salima Merazga
- Centre de Référence Constitutif des Maladies Constitutionnelles du Globule Rouge et de l'Erythropoïèse, 69003 Lyon, France
| | - Philippe Connes
- Centre de Référence Constitutif des Maladies Constitutionnelles du Globule Rouge et de l'Erythropoïèse, 69003 Lyon, France
- Inter-University Laboratory of Human Movement Sciences, LIBM, Universite Claude Bernard Lyon 1, UR 7424, 69622 Villeurbanne, France
| | - Gilles Renom
- Laboratoire de Dépistage Périnatal, Pôle de Biologie Pathologie Génétique, Centre Hospitalier Universitaire de Lille, 59000 Lille, France
| | - Jérôme Massardier
- Centre Régional de Dépistage Néonatal Auvergne Rhône-Alpes (CRDN AuRA), 69003 Lyon, France
- Service d'Obstétrique, Hôpital Femme Mère Enfant, Hospices Civils de Lyon, 69500 Bron, France
| | - David Cheillan
- Laboratoire de Biologie Médicale Multi-Sites (LBMMS), Service de Biochimie et Biologie Moléculaire Grand Est, Hospices Civils de Lyon, 69500 Bron, France
- Centre Régional de Dépistage Néonatal Auvergne Rhône-Alpes (CRDN AuRA), 69003 Lyon, France
- Laboratoire Carmen-Inserm U1060, INRAE UMR1397, Université Claude Bernard Lyon 1, 69310 Pierre-Bénite, France
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Sani A, Idrees Khan M, Shah S, Tian Y, Zha G, Fan L, Zhang Q, Cao C. Diagnosis and screening of abnormal hemoglobins. Clin Chim Acta 2024; 552:117685. [PMID: 38030031 DOI: 10.1016/j.cca.2023.117685] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2023] [Revised: 11/21/2023] [Accepted: 11/24/2023] [Indexed: 12/01/2023]
Abstract
Hemoglobin (Hb) abnormalities, such as thalassemia and structural Hb variants, are among the most prevalent inherited diseases and are associated with significant mortality and morbidity worldwide. However, there were not comprehensive reviews focusing on different clinical analytical techniques, research methods and artificial intelligence (AI) used in clinical screening and research on hemoglobinopathies. Hence the review offers a comprehensive summary of recent advancements and breakthroughs in the detection of aberrant Hbs, research methods and AI uses as well as the present restrictions anddifficulties in hemoglobinopathies. Recent advances in cation exchange high performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), isoelectric focusing (IEF), flow cytometry, mass spectrometry (MS) and polymerase chain reaction (PCR) etc have allowed for the definitive detection by using advanced AIand portable point of care tests (POCT) integrating with smartphone microscopic classification, machine learning (ML) model, complete blood counts (CBC), imaging-based method, speedy immunoassay, and electrochemical-, microfluidic- and sensing-related platforms. In addition, to confirm and validate unidentified and novel Hbs, highly specialized genetic based techniques like PCR, reverse transcribed (RT)-PCR, DNA microarray, sequencing of genomic DNA, and sequencing of RT-PCR amplified globin cDNA of the gene of interest have been used. Hence, adequate utilization and improvement of available diagnostic and screening technologies are important for the control and management of hemoglobinopathies.
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Affiliation(s)
- Ali Sani
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Muhammad Idrees Khan
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Saud Shah
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Youli Tian
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China; School of Life Science and Biotechnology, State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Genhan Zha
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Liuyin Fan
- Student Innovation Center, Shanghai Jiao Tong University, Shanghai 200240, China.
| | - Qiang Zhang
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China.
| | - Chengxi Cao
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China; School of Life Science and Biotechnology, State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, 200240, China.
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Molloy MP, Hill C, McKay MJ, Herbert BR. Proteome Analysis of Whole Blood Collected by Volumetric Absorptive Microsampling. Methods Mol Biol 2023; 2628:173-179. [PMID: 36781785 DOI: 10.1007/978-1-0716-2978-9_11] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/25/2023]
Abstract
Proteomic biomarker discovery and analysis from human biofluids using liquid chromatography-mass spectrometry (LC-MS) is an area of intense biomedical research. There is a growing interest to analyze microsampled patient blood specimens as this is potentially more patient-friendly enabling at-home and bedside self-collection of small blood volumes. However, there are limited studies applying LC-MS proteomic analysis of whole blood as it is dominated by red blood cell proteins such as hemoglobin which suppresses the detection of other less abundant proteins. Volumetric absorptive microsampling (VAMS) devices overcome this issue in part by providing a trapping matrix which allows depletion of abundant blood cell proteins through washing, prior to proteolysis and LC-MS. This approach allows the analysis of proteins from erythrocytes, leukocytes, and plasma and leads to deeper proteomic coverage compared to conventional plasma proteomics, increasing the prospects to discover novel biomarker proteins.
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Affiliation(s)
- Mark P Molloy
- Bowel Cancer and Biomarker Laboratory, School of Medical Science, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.
| | - Cameron Hill
- Sangui Bio Pty Ltd, St. Leonards, NSW, Australia
| | - Matthew J McKay
- Bowel Cancer and Biomarker Laboratory, School of Medical Science, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia
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Wooden-Tip Electrospray Mass Spectrometry Characterization of Human Hemoglobin in Whole Blood Sample for Thalassemia Screening: A Pilot Study. MOLECULES (BASEL, SWITZERLAND) 2022; 27:molecules27123952. [PMID: 35745074 PMCID: PMC9228021 DOI: 10.3390/molecules27123952] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/07/2022] [Revised: 06/07/2022] [Accepted: 06/17/2022] [Indexed: 11/17/2022]
Abstract
Traditional analytical methods for thalassemia screening are needed to process complicated and time-consuming sample pretreatment. In recent decades, ambient mass spectrometry (MS) approaches have been proven to be an effective analytical strategy for direct sample analysis. In this work, we applied ambient MS with wooden-tip electrospray ionization (WT-ESI) for the direct analysis of raw human blood samples that were pre-identified by gene detection. A total of 319 whole blood samples were investigated in this work, including 100 α-thalassemia carriers, 67 β-thalassemia carriers, and 152 control healthy samples. Only one microliter of raw blood sample was directly loaded onto the surface of the wooden tip, and then five microliters of organic solvent and a high voltage of +3.0 kV were applied onto the wooden tip to generate spray ionization. Multiply charged ions of human hemoglobin (Hb) were directly observed by WT-ESI-MS from raw blood samples. The signal ratios of Hb chains were used to characterize two main types of thalassemia (α and β types) and healthy control blood samples. Our results suggested that the ratios of charged ions to Hb chains being at +13 would be an indicator for β-thalassemia screening.
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Dua M, Bello-Manga H, Carroll YM, Galadanci AA, Ibrahim UA, King AA, Olanrewaju A, Estepp JH. Strategies to increase access to basic sickle cell disease care in low- and middle-income countries. Expert Rev Hematol 2022; 15:333-344. [PMID: 35400264 PMCID: PMC9442799 DOI: 10.1080/17474086.2022.2063116] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Accepted: 04/04/2022] [Indexed: 12/18/2022]
Abstract
INTRODUCTION Sickle cell disease (SCD) is the most common hemoglobinopathy in the world. Over 90% of those born with SCD live in low- and middle-income countries (LMICs), yet individuals in these settings have much poorer outcomes compared to those in high-income countries. AREAS COVERED This manuscript provides an in-depth review of the cornerstones of basic SCD care, the barriers to implementing these in LMICs, and strategies to increase access in these regions. Publications in English language, peer-reviewed, and edited from 2000 to 2021 were identified on PubMed. Google search was used for gray literature. EXPERT OPINION Outcomes for patients with SCD in high-income countries have improved over the last few decades due to the implementation of universal newborn screening programs and use of routine antimicrobial prophylaxis, increase in therapeutic and curative options, and the adoption of specific measures to decrease risk of stroke. This success has not translated to LMICs due to several reasons including resource constraints. A combination of several strategies is needed to increase access to basic SCD care for patients in these settings.
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Affiliation(s)
- Meghna Dua
- Department of Global Medicine, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - Halima Bello-Manga
- Department of Hematology and Blood Transfusion, Barau Dikko Teaching Hospital/Kaduna State University, Nigeria
| | - Yvonne M. Carroll
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | | | | | - Allison A. King
- in Occupational Therapy, Departments of Pediatrics, Medicine and Surgery, Washington University School of MedicineProgram , St. Louis, USA
| | - Ayobami Olanrewaju
- Department of Global Medicine, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - Jeremie H. Estepp
- Department of Global Medicine, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
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Molloy MP, Hill C, O'Rourke MB, Chandra J, Steffen P, McKay MJ, Pascovici D, Herbert BR. Proteomic Analysis of Whole Blood Using Volumetric Absorptive Microsampling for Precision Medicine Biomarker Studies. J Proteome Res 2022; 21:1196-1203. [PMID: 35166117 DOI: 10.1021/acs.jproteome.1c00971] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Microsampling of patient blood promises several benefits over conventional phlebotomy practices to facilitate precision medicine studies. These include at-home patient blood collection, supporting telehealth monitoring, minimal postcollection processing, and compatibility with nonrefrigerated transport and storage. However, for proteomic biomarker studies, mass spectrometry of whole blood has generally been avoided in favor of using plasma or serum obtained from venepuncture. We evaluated the use of a volumetric absorptive microsampling (VAMS) device as a sample preparation matrix to enable LC-MS proteomic analyses of dried whole blood. We demonstrated the detection and robust quantitation of up to 1600 proteins from single-shot shotgun-LC-MS analysis of dried whole blood, greatly enhancing proteome depth compared with conventional single-shot LC-MS analyses of undepleted plasma. Some proteins not previously reported in blood were detected using this approach. Various washing reagents were used to demonstrate that proteins can be preferentially removed from VAMS devices prior to downstream analyses. We provide a demonstration that archival frozen blood cell pellets housed under long-term storage (exceeding 5 years) are compatible with VAMS to enable quantitation of potential biomarker proteins from biobank repositories. These demonstrations are important steps in establishing viable analysis workflows to underpin large-scale precision medicine studies. Data are available via ProteomeXchange with the identifier PXD028605.
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Affiliation(s)
- Mark P Molloy
- Bowel Cancer and Biomarker Research Laboratory, School of Medical Sciences, The University of Sydney, Sydney 2065, Australia
| | | | - Matthew B O'Rourke
- Bowel Cancer and Biomarker Research Laboratory, School of Medical Sciences, The University of Sydney, Sydney 2065, Australia
| | - Jason Chandra
- Bowel Cancer and Biomarker Research Laboratory, School of Medical Sciences, The University of Sydney, Sydney 2065, Australia
| | - Pascal Steffen
- Bowel Cancer and Biomarker Research Laboratory, School of Medical Sciences, The University of Sydney, Sydney 2065, Australia
| | - Matthew J McKay
- Bowel Cancer and Biomarker Research Laboratory, School of Medical Sciences, The University of Sydney, Sydney 2065, Australia
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Forgrave LM, Wang M, Yang D, DeMarco ML. Proteoforms and their expanding role in laboratory medicine. Pract Lab Med 2022; 28:e00260. [PMID: 34950758 PMCID: PMC8672040 DOI: 10.1016/j.plabm.2021.e00260] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2021] [Revised: 10/31/2021] [Accepted: 11/22/2021] [Indexed: 12/17/2022] Open
Abstract
The term “proteoforms” describes the range of different structures of a protein product of a single gene, including variations in amino acid sequence and post-translational modifications. This diversity in protein structure contributes to the biological complexity observed in living organisms. As the concentration of a particular proteoform may increase or decrease in abnormal physiological states, proteoforms have long been used in medicine as biomarkers of health and disease. Notably, the analytical approaches used to analyze proteoforms have evolved considerably over the years. While ligand binding methods continue to play a large role in proteoform measurement in the clinical laboratory, unanticipated or unknown post-translational modifications and sequence variants can upend even extensively tested and vetted assays that have successfully made it through the medical regulatory process. As an alternate approach, mass spectrometry—with its high molecular selectivity—has become an essential tool in detection, characterization, and quantification of proteoforms in biological fluids and tissues. This review explores the analytical techniques used for proteoform detection and quantification, with an emphasis on mass spectrometry and its various applications in clinical research and patient care including, revealing new biomarker targets, helping improve the design of contemporary ligand binding in vitro diagnostics, and as mass spectrometric laboratory developed tests used in routine patient care.
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Affiliation(s)
- Lauren M. Forgrave
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada
| | - Meng Wang
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada
| | - David Yang
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada
| | - Mari L. DeMarco
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada
- Department of Pathology and Laboratory Medicine, St. Paul's Hospital, Providence Health Care, 1081 Burrard St, Vancouver, V6Z 1Y6, Canada
- Corresponding author. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada.
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Nakajima D, Ohara O, Kawashima Y. Toward proteome-wide exploration of proteins in dried blood spots using liquid chromatography-coupled mass spectrometry. Proteomics 2021; 21:e2100019. [PMID: 34379369 DOI: 10.1002/pmic.202100019] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2021] [Revised: 08/07/2021] [Accepted: 08/09/2021] [Indexed: 11/12/2022]
Abstract
Dried blood spot (DBS) sampling is a method with advantages over conventional blood sampling in relation to collection, cost, storage, and transportation. Such advantages have led to its wide use in newborn screening (NBS). Although target analysis of various biomolecules is conducted in NBS, protein quantification-based NBS is still in its infancy. Thus, it is important to clarify how many proteins could be quantitatively detected in DBS samples using advanced liquid chromatography-mass spectrometry (LC-MS/MS) technologies; a catalog of proteins detectable in DBSs by LC-MS/MS will enable us to judge which causative proteins in genetic diseases can be monitored at the protein level in NBS. In this review, we outline conventional proteome analyses of DBSs with a distinction between target and nontarget approaches. Additionally, we discuss the future perspectives for proteome analysis of DBSs in NBS of genetic diseases. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Daisuke Nakajima
- Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan
| | - Osamu Ohara
- Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan
| | - Yusuke Kawashima
- Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan
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Kambale-Kombi P, Marini Djang'eing'a R, Alworong'a Opara JP, Minon JM, Boemer F, Bours V, Tonen-Wolyec S, Kayembe Tshilumba C, Batina-Agasa S. Management of sickle cell disease: current practices and challenges in a northeastern region of the Democratic Republic of the Congo. ACTA ACUST UNITED AC 2021; 26:199-205. [PMID: 33594960 DOI: 10.1080/16078454.2021.1880752] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
Abstract
BACKGROUND The Democratic Republic of the Congo (DRC) is the third most affected country worldwide by sickle cell disease (SCD). However, this disease is still orphaned in the country; large-scale control actions are rare, and little is known about its management. OBJECTIVE To assess current practices in the management of SCD in Kisangani, DRC. METHODS This cross-sectional study was conducted in six health facilities in Kisangani. It involved 198 presumed sickle cell patients attending the above health facilities. The study focused on the sociodemographic and clinical data of the participants, obtained through a clinical examination and their medical records. Diagnostic confirmation of SCD was made by high-performance liquid chromatography coupled to mass spectrometry. Data were analyzed using SPSS 20.0. RESULTS The diagnosis of SCD was confirmed in 194 (98.0%; 95% CI: 94.9-99.2) participants, while it was not confirmed in 4 (2.0%; 95% CI: 0.8-5.1) participants. The diagnosis was mainly made by the Emmel test (42.9%). 45.8% of participants had previously been transfused with the blood of their parents. Folic acid was taken by 48.5% of participants and the previous intake of hydroxyurea was reported in 5.1% of participants. The participants vaccinated against Pneumococcus were 13.6% and against Haemophilus influenzae type b 28.3%. Penicillin prophylaxis was received by only 1.5% and malaria prophylaxis by 11.6% of participants. CONCLUSION Standard-care practices for SCD patients in Kisangani are insufficient. The Congolese government should regard this disease as a health priority and consider actions to improve its management.
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Affiliation(s)
- Paul Kambale-Kombi
- Faculty of Medicine and Pharmacy, University of Kisangani, Kisangani, Democratic Republic of the Congo
| | - Roland Marini Djang'eing'a
- Faculty of Medicine and Pharmacy, University of Kisangani, Kisangani, Democratic Republic of the Congo.,Faculty of Medicine, University of Liège, Liège, Belgium
| | | | | | | | - Vincent Bours
- Faculty of Medicine, University of Liège, Liège, Belgium
| | - Serge Tonen-Wolyec
- Faculty of Medicine and Pharmacy, University of Kisangani, Kisangani, Democratic Republic of the Congo
| | - Charles Kayembe Tshilumba
- Faculty of Medicine and Pharmacy, University of Kisangani, Kisangani, Democratic Republic of the Congo
| | - Salomon Batina-Agasa
- Faculty of Medicine and Pharmacy, University of Kisangani, Kisangani, Democratic Republic of the Congo
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Technological advancement in dry blood matrix microsampling and its clinical relevance in quantitative drug analysis. Bioanalysis 2020; 12:1483-1501. [DOI: 10.4155/bio-2020-0211] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
In the past few decades, dried blood matrix biosampling has witnessed a marvelous interest among the researcher due to its user-friendly operation during blood sampling in preclinical and clinical applications. It also complies with the basic 3Rs (reduce, reuse and recycle) philosophy. Because of comparative simplicity, a huge number of researchers are paying attention to its technological advancements for widespread application in the bioanalysis and diagnosis arena. In this review, we have explained different approaches to be considered during dried blood matrix based microsampling including their clinical relevance in therapeutic drug monitoring. We have also discussed various strategies for avoiding and minimizing major unwanted analytical interferences associated with this technique during drug quantification. Further, various recent technological advancement in microsampling devices has been discussed correlating their clinical applications.
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12
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Cerqueira LB, Fachi MM, Kawagushi WH, Pontes FLD, de Campos ML, Pontarolo R. New Validated Method for Quantification of Glycated Hemoglobin by LC-QToF-MS: Is HRMS Able to Quantify Clinical Samples? JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2020; 31:1172-1179. [PMID: 32298572 DOI: 10.1021/jasms.9b00049] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
High-resolution mass spectrometry is a powerful tool in clinical analysis but remains less explored due to its lower dynamic range and sensitivity compared to triple quadrupoles. Glycated hemoglobin (HbA1c) is the current gold standard biomarker to monitor the control of diabetes, representing long-term plasma glycemic levels. Due to its clinical importance, several methods have been developed for HbA1c quantification, using different principles; however, the results obtained with these techniques may differ according to the method adopted. Hence, there is a great need to standardize the current methods to quantify glycated hemoglobin. A new UPLC-QToF-MS method was fully validated and tested to quantify HbA1c in human samples. The peptides VHLTPE m/z 695.373 and gly-VHLTPE m/z 857.426, obtained via Glu-C digestion, were the selected peptides for quantification of HbA1c (mmol/mol). Chromatographic separation was obtained in a C18 column, maintained at 40 °C. The mobile phase was composed of water and acetonitrile, both containing 0.02% TFA and 0.1% acetic acid, and eluted in gradient mode. The method was fully validated, being considered linear in the range of 25-107 mmol/mol of HbA1c, and was sensitive, selective, precise, accurate, and free of matrix and carryover effects. The method was successfully applied to real samples, reaching about 90% agreement with reference method results, providing accurate and precise information on peptide mass, without laborious sample preparation. These results support the use of HRMS to improve the quality of quantitative results of HbA1c in health services and demonstrate a possible application of peptide investigation for clinical analysis in the near future.
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Affiliation(s)
- Letícia Bonancio Cerqueira
- Universidade Federal do Paraná, Department of Pharmacy, 632 Lothário Meissner Avenue, 80210-170 Curitiba, PR, Brazil
| | - Mariana Millan Fachi
- Universidade Federal do Paraná, Department of Pharmacy, 632 Lothário Meissner Avenue, 80210-170 Curitiba, PR, Brazil
| | - Wilton Hideki Kawagushi
- Universidade Federal do Paraná, Department of Pharmacy, 632 Lothário Meissner Avenue, 80210-170 Curitiba, PR, Brazil
| | - Flavia Lada Degaut Pontes
- Universidade Federal do Paraná, Department of Pharmacy, 632 Lothário Meissner Avenue, 80210-170 Curitiba, PR, Brazil
| | - Michel Leandro de Campos
- Universidade Federal de Mato Grosso, Health Sciences Institute,1200 Alexandre Ferronato Avenue,78550-728Sinop, MT Brazil
| | - Roberto Pontarolo
- Universidade Federal do Paraná, Department of Pharmacy, 632 Lothário Meissner Avenue, 80210-170 Curitiba, PR, Brazil
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13
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Kasai ET, Boemer F, Djang’eing’a RM, Ntokumunda JK, Agasa SB, Dauly NN, Opara JPA. Systematic Screening of Neonatal Sickle Cell Disease with HemoTypeSC<sup>TM</sup> Kit-Test: Case Study and Literature Review. ACTA ACUST UNITED AC 2020. [DOI: 10.4236/ojbd.2020.101002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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14
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Lassout O, Hartmer R, Jabs W, Clerici L, Tsybin YO, Samii K, Vuilleumier N, Hochstrasser D, Scherl A, Lescuyer P, Coelho Graça D. Clinical method evaluation of hemoglobin S and C identification by top-down selected reaction monitoring and electron transfer dissociation. Clin Proteomics 2019; 16:41. [PMID: 31889938 PMCID: PMC6915975 DOI: 10.1186/s12014-019-9261-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2019] [Accepted: 11/26/2019] [Indexed: 01/21/2023] Open
Abstract
Background Biological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample. Currently, hematology laboratories usually use high-performance liquid chromatography (HPLC), capillary electrophoresis and gel-based methods to characterize Hb variants. Co-elution and co-migration may represent major issues for precise identification of Hb variants, even for the most common ones such as Hb S and C. Methods We adapted a top-down selected reaction monitoring (SRM) electron transfer dissociation (ETD) mass spectrometry (MS) method to fit with a clinical laboratory environment. An automated analytical process with semi-automated data analysis compatible with a clinical practice was developed. A comparative study between a reference HPLC method and the MS assay was performed on 152 patient samples. Results The developed workflow allowed to identify with high specificity and selectivity the most common Hb variants (Hb S and Hb C). Concordance of the MS-based approach with HPLC was 71/71 (100%) for Hb S and 11/11 (100%) for Hb C. Conclusions This top-down SRM ETD method can be used in a clinical environment to detect Hb S and Hb C.
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Affiliation(s)
- Olivier Lassout
- 1Department of Medical Specialties, Faculty of Medicine, University of Geneva, Geneva, Switzerland.,2Division of Laboratory Medicine, Department of Laboratory Medicine, Genetics and Pathology, Geneva University Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland
| | | | | | - Lorella Clerici
- 2Division of Laboratory Medicine, Department of Laboratory Medicine, Genetics and Pathology, Geneva University Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland
| | - Yury O Tsybin
- 4Spectroswiss, EPFL Innovation Park, Lausanne, Switzerland
| | - Kaveh Samii
- 2Division of Laboratory Medicine, Department of Laboratory Medicine, Genetics and Pathology, Geneva University Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland.,5Division of Hematology, Geneva University Hospitals, Geneva, Switzerland
| | - Nicolas Vuilleumier
- 1Department of Medical Specialties, Faculty of Medicine, University of Geneva, Geneva, Switzerland.,2Division of Laboratory Medicine, Department of Laboratory Medicine, Genetics and Pathology, Geneva University Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland
| | - Denis Hochstrasser
- 1Department of Medical Specialties, Faculty of Medicine, University of Geneva, Geneva, Switzerland.,2Division of Laboratory Medicine, Department of Laboratory Medicine, Genetics and Pathology, Geneva University Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland
| | - Alexander Scherl
- 1Department of Medical Specialties, Faculty of Medicine, University of Geneva, Geneva, Switzerland.,2Division of Laboratory Medicine, Department of Laboratory Medicine, Genetics and Pathology, Geneva University Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland
| | - Pierre Lescuyer
- 1Department of Medical Specialties, Faculty of Medicine, University of Geneva, Geneva, Switzerland.,2Division of Laboratory Medicine, Department of Laboratory Medicine, Genetics and Pathology, Geneva University Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland
| | - Didia Coelho Graça
- 1Department of Medical Specialties, Faculty of Medicine, University of Geneva, Geneva, Switzerland.,2Division of Laboratory Medicine, Department of Laboratory Medicine, Genetics and Pathology, Geneva University Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland
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15
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Kumar P, Agrawal P, Chatterjee K. Challenges and opportunities in blood flow through porous substrate: A design and interface perspective of dried blood spot. J Pharm Biomed Anal 2019; 175:112772. [DOI: 10.1016/j.jpba.2019.07.020] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2019] [Revised: 07/09/2019] [Accepted: 07/10/2019] [Indexed: 12/13/2022]
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16
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He L, Rockwood AL, Agarwal AM, Anderson LC, Weisbrod CR, Hendrickson CL, Marshall AG. Diagnosis of Hemoglobinopathy and β-Thalassemia by 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry of Hemoglobin from Blood. Clin Chem 2019; 65:986-994. [PMID: 31040099 DOI: 10.1373/clinchem.2018.295766] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2018] [Accepted: 03/28/2019] [Indexed: 12/15/2022]
Abstract
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).
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Affiliation(s)
- Lidong He
- Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL
| | - Alan L Rockwood
- Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL.,Rockwood Scientific Consulting, Salt Lake City, UT.,University of Utah Health, Salt Lake City, UT
| | - Archana M Agarwal
- University of Utah Health, Salt Lake City, UT.,ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT
| | - Lissa C Anderson
- National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL
| | - Chad R Weisbrod
- National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL
| | - Christopher L Hendrickson
- Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL.,National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL
| | - Alan G Marshall
- Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL; .,National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL
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17
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Naubourg P, El Osta M, Rageot D, Grunewald O, Renom G, Ducoroy P, Périni JM. A Multicentre Pilot Study of a Two-Tier Newborn Sickle Cell Disease Screening Procedure with a First Tier Based on a Fully Automated MALDI-TOF MS Platform. Int J Neonatal Screen 2019; 5:10. [PMID: 33072970 PMCID: PMC7510195 DOI: 10.3390/ijns5010010] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/04/2018] [Accepted: 01/21/2019] [Indexed: 11/17/2022] Open
Abstract
The reference methods used for sickle cell disease (SCD) screening usually include two analytical steps: a first tier for differentiating haemoglobin S (HbS) heterozygotes, HbS homozygotes and β-thalassemia from other samples, and a confirmatory second tier. Here, we evaluated a first-tier approach based on a fully automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform with automated sample processing, a laboratory information management system and NeoSickle® software for automatic data interpretation. A total of 6701 samples (with high proportions of phenotypes homozygous (FS) or heterozygous (FAS) for the inherited genes for sickle haemoglobin and samples from premature newborns) were screened. The NeoSickle® software correctly classified 98.8% of the samples. This specific blood sample collection was enriched in qualified difficult samples (premature newborns, FAS samples, late and very late samples, etc.). In this study, the sensitivity of FS sample detection was found to be 100% on the Lille MS facility and 99% on the Dijon MS facility, and the specificity of FS sample detection was found to be 100% on both MS facilities. The MALDI-MS platform appears to be a robust solution for first-tier use to detect the HbS variant: it is reproducible and sensitive, it has the power to analyze 600-1000 samples per day and it can reduce the unit cost of testing thanks to maximal automation, minimal intervention by the medical team and good overall practicability. The MALDI-MS approach meets today's criteria for the large-scale, cost-effective screening of newborns, children and adults.
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Affiliation(s)
- Pierre Naubourg
- Biomaneo, 22B boulevard Winston Churchill, F-21000 Dijon, France
| | - Marven El Osta
- Biomaneo, 22B boulevard Winston Churchill, F-21000 Dijon, France
| | - David Rageot
- CLIPP, Clinical Innovation Proteomic Platform, Université de Bourgogne Franche Comté, F-21000 Dijon, France
| | - Olivier Grunewald
- Newborn Screening Laboratory, Biology and Pathology Center, Lille University Medical Centre, F-59000 Lille, France
| | - Gilles Renom
- Newborn Screening Laboratory, Biology and Pathology Center, Lille University Medical Centre, F-59000 Lille, France
| | - Patrick Ducoroy
- Biomaneo, 22B boulevard Winston Churchill, F-21000 Dijon, France
| | - Jean-Marc Périni
- Newborn Screening Laboratory, Biology and Pathology Center, Lille University Medical Centre, F-59000 Lille, France
- Correspondence:
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18
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Daniel Y, Elion J, Allaf B, Badens C, Bouva MJ, Brincat I, Cela E, Coppinger C, de Montalembert M, Gulbis B, Henthorn J, Ketelslegers O, McMahon C, Streetly A, Colombatti R, Lobitz S. Newborn Screening for Sickle Cell Disease in Europe. Int J Neonatal Screen 2019; 5:15. [PMID: 33072975 PMCID: PMC7510219 DOI: 10.3390/ijns5010015] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/30/2018] [Accepted: 02/06/2019] [Indexed: 11/22/2022] Open
Abstract
The history of newborn screening (NBS) for sickle cell disease (SCD) in Europe goes back almost 40 years. However, most European countries have not established it to date. The European screening map is surprisingly heterogenous. The first countries to introduce sickle cell screening on a national scale were France and England. The French West Indies started to screen their newborns for SCD as early as 1983/84. To this day, all countries of the United Kingdom of Great Britain and Northern Ireland have added SCD as a target disease to their NBS programs. The Netherlands, Spain and Malta also have national programs. Belgium screens regionally in the Brussels and Liège regions, Ireland has been running a pilot for many years that has become quasi-official. However, the Belgian and Irish programs are not publicly funded. Italy and Germany have completed several pilot studies but are still in the preparatory phase of national NBS programs for SCD, although both countries have well-established concepts for metabolic and endocrine disorders. This article will give a brief overview of the situation in Europe and put a focus on the programs of the two pioneers of the continent, England and France.
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Affiliation(s)
- Yvonne Daniel
- Public Health England, NHS Sickle Cell and Thalassemia Screening Programme, London SE16LH, UK
- Correspondence:
| | - Jacques Elion
- Laboratoire d’Excellence GR-Ex, UMR_S1134, Inserm, Université Paris Diderot, Sorbonne Paris Cité, Institut National de la Transfusion Sanguine, 75015 Paris, France
| | - Bichr Allaf
- NBS Laboratory for Haemoglobinopathies, Hôpital Universitaire Robert-Debré, 75019 Paris, France
| | - Catherine Badens
- Département de génétique médicale, Aix-Marseille Université, Hôpital de la Timone, 13385 Marseille, France
| | - Marelle J. Bouva
- National Institute for Public Health and the Environment, Centre for Health Protection, 3720 Bilthoven, The Netherlands
| | - Ian Brincat
- Pediatric Medicine Laboratory, Department of Pathology, Mater Dei Hospital, Triq Tal-Qroqq, MSD2090 Msida, Malta
| | - Elena Cela
- Department of Pediatric Oncology/Hematology, Hospital Universitario General Gregorio Marañón, Facultad de Medicina, Universidad Complutense Madrid, 28007 Madrid, Spain
| | - Cathy Coppinger
- Public Health England, NHS Sickle Cell and Thalassemia Screening Programme, London SE16LH, UK
| | - Mariane de Montalembert
- Department of Pediatrics, Reference Center for Sickle Cell Disease, AP-HP Hôpital Universitaire Necker-Enfants Malades, 75743 Paris, France
| | - Béatrice Gulbis
- Department of Clinical Chemistry, Cliniques Universitaires de Bruxelles, Hôpital Erasme—ULB, 1070 Bruxelles, Belgium
| | - Joan Henthorn
- Public Health England, NHS Sickle Cell and Thalassemia Screening Programme, London SE16LH, UK
| | - Olivier Ketelslegers
- Laboratoire—Biologie Clinique, Centre Hospitalier Régional de la Citadelle, 4000 Liège, Belgium
| | - Corrina McMahon
- Our Lady’s Children’s Hospital, Crumlin, D12V004 Dublin, Ireland
| | - Allison Streetly
- School of Population Health and Environmental Sciences, Faculty of Life Sciences & Medicine, King’s College London, London WC2R2LS, UK
- Division of Healthcare Public Health, Health Protection and Medical Directorate, Public Health England, London SE18UG, UK
| | - Raffaella Colombatti
- Department of Child and Maternal Health, Clinic of Pediatric Hematology/Oncology, Azienda Ospedaliera-Università di Padova, 35129 Padova, Italy
| | - Stephan Lobitz
- Department of Pediatric Oncology/Hematology, Kinderkrankenhaus Amsterdamer Straße, 50735 Cologne, Germany
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19
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El-Haj N, Hoppe CC. Newborn Screening for SCD in the USA and Canada. Int J Neonatal Screen 2018; 4:36. [PMID: 33072956 PMCID: PMC7548901 DOI: 10.3390/ijns4040036] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/03/2018] [Accepted: 11/20/2018] [Indexed: 01/09/2023] Open
Abstract
Sickle cell disease (SCD) encompasses a group of inherited red cell disorders characterized by an abnormal hemoglobin, Hb S. The most common forms of SCD in the United States and Canada are identified through universal newborn screening (NBS) programs. Now carried out in all fifty U.S. states and 8 Canadian provinces, NBS for SCD represents one of the major public health advances in North America. The current status of NBS programs for hemoglobinopathies and the screening techniques employed in many regions worldwide reflect in large part the U.S. and Canadian experiences. Although the structure, screening algorithms and laboratory procedures, as well as reporting and follow up, vary between NBS programs, the overall workflow is similar. The current review summarized the historical background, current approaches, and methods used to screen newborns for SCD in the United States and Canada.
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Affiliation(s)
| | - Carolyn C. Hoppe
- Department of Hematology-Oncology, UCSF Benioff Children’s Hospital Oakland, Oakland, CA 94609, USA
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20
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Newborn Sickle Cell Disease Screening Using Electrospray Tandem Mass Spectrometry. Int J Neonatal Screen 2018; 4:35. [PMID: 33072955 PMCID: PMC7510227 DOI: 10.3390/ijns4040035] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Accepted: 11/22/2018] [Indexed: 11/17/2022] Open
Abstract
There is a growing demand for newborn sickle cell disease screening globally. Historically techniques have relied on the separation of intact haemoglobin tetramers using electrophoretic or liquid chromatography techniques. These techniques also identify haemoglobin variants of no clinical significance. Specific electrospray ionization-mass spectrometry-mass spectrometry techniques to analyse targeted peptides formed after digestion of the haemoglobin with trypsin were reported in 2005. Since this time the method has been further developed and adopted in several European countries. It is estimated that more than one million babies have been screened with no false-negative cases reported. This review reports on the current use of the technique and reviews the related publications.
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21
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Newborn screening by tandem mass spectrometry confirms the high prevalence of sickle cell disease among German newborns. Ann Hematol 2018; 98:47-53. [DOI: 10.1007/s00277-018-3477-4] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2018] [Accepted: 08/13/2018] [Indexed: 10/28/2022]
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22
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Nys G, Kok MG, Servais AC, Fillet M. Beyond dried blood spot: Current microsampling techniques in the context of biomedical applications. Trends Analyt Chem 2017. [DOI: 10.1016/j.trac.2017.10.002] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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23
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Detemmerman L, Olivier S, Bours V, Boemer F. Innovative PCR without DNA extraction for African sickle cell disease diagnosis. Hematology 2017; 23:181-186. [DOI: 10.1080/10245332.2017.1371470] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
Affiliation(s)
| | | | - V. Bours
- Biochemical Genetics Lab, Human Genetics, CHU of Liege, University of Liège, Liège, Belgium
| | - F. Boemer
- Biochemical Genetics Lab, Human Genetics, CHU of Liege, University of Liège, Liège, Belgium
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24
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Moat SJ, Rees D, George RS, King L, Dodd A, Ifederu A, Ramgoolam T, Hillier S. Newborn screening for sickle cell disorders using tandem mass spectrometry: three years’ experience of using a protocol to detect only the disease states. Ann Clin Biochem 2017; 54:601-611. [DOI: 10.1177/0004563217713788] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Background Tandem mass spectrometry (MS/MS) has recently become an alternative method for the newborn screening of sickle cell disorders (SCD), as it is able to detect haemoglobin (Hb) peptides following digestion of bloodspots with trypsin. Using the SpOtOn Diagnostics Reagent Kit, we previously developed a screening protocol to detect only the disease states of SCD, using action values based on the ratio between the variant Hb peptide to wild-type peptide abundances for the HbS, C, DPunjab, OArab, E and Lepore peptides. Methods Action values using the ratios between the wild type HbA (ßT1-3) peptides and the foetal Hb (γT2) peptide were developed to identify bloodspot samples from premature and transfused infants. An evaluation was undertaken to assess the transferability of the action values onto an additional MS/MS instrument. We report here our experience using this MS/MS protocol. Results During a three-year period, we screened 100,456 babies and identified 10 SCD cases (1 HbS/HPFH, 5 HbS/S and 4 HbS/C) and a case of HbE/ß-thalassaemia that was identified as a by-product. The Hb variant to wild-type peptide ratio action values were transferable to a second MS/MS instrument. Our protocol prevented the identification of an estimated 810 carrier infants. Gestational age-related action values for HbA to HbF peptide ratios were required to minimize the number of samples referred for second-line testing to exclude ß-thalassaemia. Conclusion MS/MS is a robust alternative screening technology for SCD; in addition, it also optimizes the use of equipment and expertise that currently exist in newborn screening laboratories.
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Affiliation(s)
- Stuart J Moat
- Wales Newborn Screening Laboratory, Department of Medical Biochemistry, Immunology & Toxicology, Cardiff University, Cardiff, UK
- School of Medicine, Cardiff University, Cardiff, UK
| | - Derek Rees
- Wales Newborn Screening Laboratory, Department of Medical Biochemistry, Immunology & Toxicology, Cardiff University, Cardiff, UK
| | - Roanna S George
- Wales Newborn Screening Laboratory, Department of Medical Biochemistry, Immunology & Toxicology, Cardiff University, Cardiff, UK
| | - Lawrence King
- Department of Haematology, University Hospital Wales, Cardiff, UK
| | - Alan Dodd
- Wales Newborn Screening Laboratory, Department of Medical Biochemistry, Immunology & Toxicology, Cardiff University, Cardiff, UK
| | - Adeboye Ifederu
- Newborn Screening Laboratory, Department of Chemical Pathology, Great Ormond Street Hospital, London, UK
| | - Tejswurree Ramgoolam
- Newborn Screening Laboratory, Department of Chemical Pathology, Great Ormond Street Hospital, London, UK
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25
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Lehmann S, Picas A, Tiers L, Vialaret J, Hirtz C. Clinical perspectives of dried blood spot protein quantification using mass spectrometry methods. Crit Rev Clin Lab Sci 2017; 54:173-184. [DOI: 10.1080/10408363.2017.1297358] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- Sylvain Lehmann
- Laboratory of Biochemistry and Clinical Proteomics, CHU Montpellier, Institute of Regenerative Medicine & Biotherapy, Montpellier, France
| | - Alexia Picas
- Laboratory of Biochemistry and Clinical Proteomics, CHU Montpellier, Institute of Regenerative Medicine & Biotherapy, Montpellier, France
| | - Laurent Tiers
- Laboratory of Biochemistry and Clinical Proteomics, CHU Montpellier, Institute of Regenerative Medicine & Biotherapy, Montpellier, France
| | - Jerome Vialaret
- Laboratory of Biochemistry and Clinical Proteomics, CHU Montpellier, Institute of Regenerative Medicine & Biotherapy, Montpellier, France
| | - Christophe Hirtz
- Laboratory of Biochemistry and Clinical Proteomics, CHU Montpellier, Institute of Regenerative Medicine & Biotherapy, Montpellier, France
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26
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van den Broek I, Fu Q, Kushon S, Kowalski MP, Millis K, Percy A, Holewinski RJ, Venkatraman V, Van Eyk JE. Application of volumetric absorptive microsampling for robust, high-throughput mass spectrometric quantification of circulating protein biomarkers. CLINICAL MASS SPECTROMETRY (DEL MAR, CALIF.) 2017; 4-5:25-33. [PMID: 39193127 PMCID: PMC11322776 DOI: 10.1016/j.clinms.2017.08.004] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/03/2017] [Revised: 08/23/2017] [Accepted: 08/23/2017] [Indexed: 01/22/2023]
Abstract
Volumetric absorptive micro sampling (VAMS™) allows accurate sampling of 10 µL of blood from a minimally invasive finger prick and could enable remote personalized health monitoring. Moreover, VAMS overcomes effects from hematocrit and sample heterogeneity associated with dried blood spots (DBS). We describe the first application of VAMS with the Mitra® microsampling device for the quantification of protein biomarkers using an automated, high-throughput sample preparation method coupled with mass spectrometric (MS) detection. The analytical performance of the developed workflow was evaluated for 10 peptides from six clinically relevant proteins: apolipoproteins A-I, B, C-I, C-III, E, and human serum albumin (HSA). Extraction recovery from blood with three different levels of hematocrit varied between 100% and 111% for all proteins. Within-day and total assay reproducibility (i.e., 5 replicates on 5 days) ranged between 3.2-10.4% and 3.4-12.6%, respectively. In addition, after 22 weeks of storage of the Mitra microsampling devices at -80 °C, all peptide responses were within ±15% deviation from the initial response. Application to data-independent acquisition (DIA) MS further demonstrated the potential for broad applicability and the general robustness of the automated workflow by reproducible detection of 1661 peptides from 423 proteins (average 15.7%CV (n = 3) in peptide abundance), correlating to peptide abundances in corresponding plasma (R = 0.8383). In conclusion, we have developed an automated workflow for efficient extraction, digestion, and MS analysis of a variety of proteins in a fixed small volume of dried blood (i.e., 10 µL). This robust and high-throughput workflow will create manifold opportunities for the application of remote, personalized disease biomarker monitoring.
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Affiliation(s)
- Irene van den Broek
- Advanced Clinical Biosystems Research Institute, The Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA
| | - Qin Fu
- Advanced Clinical Biosystems Research Institute, The Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA
| | | | | | - Kevin Millis
- Cambridge Isotope Laboratories, Tewksbury, MA, USA
| | - Andrew Percy
- Cambridge Isotope Laboratories, Tewksbury, MA, USA
| | - Ronald J. Holewinski
- Advanced Clinical Biosystems Research Institute, The Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA
| | - Vidya Venkatraman
- Advanced Clinical Biosystems Research Institute, The Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA
| | - Jennifer E. Van Eyk
- Advanced Clinical Biosystems Research Institute, The Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA
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Henderson CM, Bollinger JG, Becker JO, Wallace JM, Laha TJ, MacCoss MJ, Hoofnagle AN. Quantification by nano liquid chromatography parallel reaction monitoring mass spectrometry of human apolipoprotein A-I, apolipoprotein B, and hemoglobin A1c in dried blood spots. Proteomics Clin Appl 2017; 11. [PMID: 28112871 DOI: 10.1002/prca.201600103] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2016] [Revised: 12/20/2016] [Accepted: 01/19/2017] [Indexed: 12/21/2022]
Abstract
PURPOSE Proteomic analysis of blood proteins in dried blood spots (DBS) is gaining attention as a possible replacement for measurements in plasma/serum collected by venipuncture. We aimed to develop and provisionally validate a nanoflow LC-PRM-MS method for clinical use. EXPERIMENTAL DESIGN We used Skyline to develop a nanoflow LC-PRM-MS method to quantify glycated hemoglobin-β, apolipoprotein A-I, and apolipoprotein B in DBS. Precision, linearity, interferences, and stability were determined and the method was used to analyze samples from 36 human volunteers. The method was compared with clinically validated measurements in paired blood collected via venipuncture. RESULTS The method was relatively precise for these proteins (10-11% CV) and linear across the normal concentration ranges of these proteins. Interference from high total serum protein concentration (>8 g/dL) was noted for apolipoprotein A-I and apolipoprotein B. Proteins in DBS were stable for 14 days at temperatures below 25°C and trypsinized samples were stable for 48 h at 7°C. There was moderate correlation with clinical methods (r = 0.783-0.858) and significant bias in individual samples. CONCLUSIONS AND CLINICAL RELEVANCE Although the method had adequate precision and linearity for a biomarker, the accuracy compared with clinically validated assays raises concerns regarding the use of DBS compared with venipuncture for clinical use.
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Affiliation(s)
- Clark M Henderson
- Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
| | - James G Bollinger
- Departments of Genome Sciences, University of Washington, Seattle, WA, USA
| | - Jessica O Becker
- Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
| | - Jennifer M Wallace
- Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
| | - Thomas J Laha
- Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
| | - Michael J MacCoss
- Departments of Genome Sciences, University of Washington, Seattle, WA, USA
| | - Andrew N Hoofnagle
- Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.,Department of Medicine, University of Washington, Seattle, WA, USA
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Traeger-Synodinos J, Harteveld CL. Preconception carrier screening and prenatal diagnosis in thalassemia and hemoglobinopathies: challenges and future perspectives. Expert Rev Mol Diagn 2017; 17:281-291. [DOI: 10.1080/14737159.2017.1285701] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Affiliation(s)
- Joanne Traeger-Synodinos
- Department of Medical Genetics, National and Kapodistrian University of Athens, St. Sophia’s Children’s Hospital, Athens, Greece
| | - Cornelis L. Harteveld
- Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis (LDGA), Leiden, The Netherlands
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Yu C, Huang S, Wang M, Zhang J, Liu H, Yuan Z, Wang X, He X, Wang J, Zou L. A novel tandem mass spectrometry method for first-line screening of mainly beta-thalassemia from dried blood spots. J Proteomics 2017; 154:78-84. [DOI: 10.1016/j.jprot.2016.12.008] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2016] [Revised: 12/08/2016] [Accepted: 12/16/2016] [Indexed: 02/03/2023]
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30
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Opening the toolbox of alternative sampling strategies in clinical routine: A key-role for (LC-)MS/MS. Trends Analyt Chem 2016. [DOI: 10.1016/j.trac.2016.01.030] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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31
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Helmich F, van Dongen JL, Kuijper PH, Scharnhorst V, Brunsveld L, Broeren MA. Rapid phenotype hemoglobin screening by high-resolution mass spectrometry on intact proteins. Clin Chim Acta 2016; 460:220-6. [DOI: 10.1016/j.cca.2016.07.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2016] [Revised: 07/07/2016] [Accepted: 07/08/2016] [Indexed: 01/14/2023]
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Daniel YA, Henthorn J. Newborn screening for sickling and other haemoglobin disorders using tandem mass spectrometry: A pilot study of methodology in laboratories in England. J Med Screen 2016; 23:175-178. [DOI: 10.1177/0969141316631008] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2015] [Accepted: 01/06/2016] [Indexed: 11/16/2022]
Abstract
Objective To determine (i) if electrospray mass spectrometry–mass spectrometry with the SpOtOn Diagnostics Ltd reagent kit for sickle cell screening could be integrated into the English newborn screening programme, under routine screening conditions, and provide mass spectrometry–mass spectrometry results which match existing methods, and (ii) if common action values could be set for all manufacturers in the study, for all assessed haemoglobins, to indicate which samples require further investigation. Methods Anonymised residual blood spots were analysed using the SpOtOn reagent kit as per manufacturer’s instructions, in parallel with existing techniques at four laboratories. Mass spectrometry–mass spectrometry instrumentation at Laboratories A and B was AB Sciex (Warrington, UK) AP4000, and at Laboratories C and D, Waters Micromass (Manchester, UK), Xevo TQMS and Premier, respectively. Results There were 23,898 results accepted from the four laboratories. Excellent specificity at 100% sensitivity was observed for haemoglobin S, haemoglobin C, haemoglobin E and haemoglobin OArab. A common action value was not possible for Hb C, but action values were set by manufacturer. The two haemoglobin DPunjab cases at Laboratory D were not detected using the common action value. Conversely, false-positive results with haemoglobin DPunjab were a problem at the remaining three laboratories. Conclusions This multicentre study demonstrates that it is possible to implement mass spectrometry–mass spectrometry into an established screening programme while maintaining consistency with existing methods for haemoglobinopathy screening. However, one of the instruments investigated cannot be recommended for use with this application.
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Affiliation(s)
- Yvonne A Daniel
- Public Health England, PHE Sickle Cell and Thalassaemia Screening Programme, London, UK
| | - Joan Henthorn
- Public Health England, PHE Sickle Cell and Thalassaemia Screening Programme, London, UK
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Wagner M, Tonoli D, Varesio E, Hopfgartner G. The use of mass spectrometry to analyze dried blood spots. MASS SPECTROMETRY REVIEWS 2016; 35:361-438. [PMID: 25252132 DOI: 10.1002/mas.21441] [Citation(s) in RCA: 162] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/16/2023]
Abstract
Dried blood spots (DBS) typically consist in the deposition of small volumes of capillary blood onto dedicated paper cards. Comparatively to whole blood or plasma samples, their benefits rely in the fact that sample collection is easier and that logistic aspects related to sample storage and shipment can be relatively limited, respectively, without the need of a refrigerator or dry ice. Originally, this approach has been developed in the sixties to support the analysis of phenylalanine for the detection of phenylketonuria in newborns using bacterial inhibition test. In the nineties tandem mass spectrometry was established as the detection technique for phenylalanine and tyrosine. DBS became rapidly recognized for their clinical value: they were widely implemented in pediatric settings with mass spectrometric detection, and were closely associated to the debut of newborn screening (NBS) programs, as a part of public health policies. Since then, sample collection on paper cards has been explored with various analytical techniques in other areas more or less successfully regarding large-scale applications. Moreover, in the last 5 years a regain of interest for DBS was observed and originated from the bioanalytical community to support drug development (e.g., PK studies) or therapeutic drug monitoring mainly. Those recent applications were essentially driven by improved sensitivity of triple quadrupole mass spectrometers. This review presents an overall view of all instrumental and methodological developments for DBS analysis with mass spectrometric detection, with and without separation techniques. A general introduction to DBS will describe their advantages and historical aspects of their emergence. A second section will focus on blood collection, with a strong emphasis on specific parameters that can impact quantitative analysis, including chromatographic effects, hematocrit effects, blood effects, and analyte stability. A third part of the review is dedicated to sample preparation and will consider off-line and on-line extractions; in particular, instrumental designs that have been developed so far for DBS extraction will be detailed. Flow injection analysis and applications will be discussed in section IV. The application of surface analysis mass spectrometry (DESI, paper spray, DART, APTDCI, MALDI, LDTD-APCI, and ICP) to DBS is described in section V, while applications based on separation techniques (e.g., liquid or gas chromatography) are presented in section VI. To conclude this review, the current status of DBS analysis is summarized, and future perspectives are provided.
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Affiliation(s)
- Michel Wagner
- School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Life Sciences Mass Spectrometry, Quai Ernest-Ansermet 30, 1211, Geneva, Switzerland
| | - David Tonoli
- School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Life Sciences Mass Spectrometry, Quai Ernest-Ansermet 30, 1211, Geneva, Switzerland
| | - Emmanuel Varesio
- School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Life Sciences Mass Spectrometry, Quai Ernest-Ansermet 30, 1211, Geneva, Switzerland
| | - Gérard Hopfgartner
- School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Life Sciences Mass Spectrometry, Quai Ernest-Ansermet 30, 1211, Geneva, Switzerland
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Ombrone D, Giocaliere E, Forni G, Malvagia S, la Marca G. Expanded newborn screening by mass spectrometry: New tests, future perspectives. MASS SPECTROMETRY REVIEWS 2016; 35:71-84. [PMID: 25952022 DOI: 10.1002/mas.21463] [Citation(s) in RCA: 95] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/23/2014] [Accepted: 01/09/2015] [Indexed: 05/02/2023]
Abstract
Tandem mass spectrometry (MS/MS) has become a leading technology used in clinical chemistry and has shown to be particularly sensitive and specific when used in newborn screening (NBS) tests. The success of tandem mass spectrometry is due to important advances in hardware, software and clinical applications during the last 25 years. MS/MS permits a very rapid measurement of many metabolites in different biological specimens by using filter paper spots or directly on biological fluids. Its use in NBS give us the chance to identify possible treatable metabolic disorders even when asymptomatic and the benefits gained by this type of screening is now recognized worldwide. Today the use of MS/MS for second-tier tests and confirmatory testing is promising especially in the early detection of new disorders such as some lysosomal storage disorders, ADA and PNP SCIDs, X-adrenoleucodistrophy (X-ALD), Wilson disease, guanidinoacetate methyltransferase deficiency (GAMT), and Duchenne muscular dystrophy. The new challenge for the future will be reducing the false positive rate by using second-tier tests, avoiding false negative results by using new specific biomarkers and introducing new treatable disorders in NBS programs.
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Affiliation(s)
- Daniela Ombrone
- Newborn screening, Clinical Chemistry and Pharmacology Lab, Meyer Children's University Hospital, Viale Pieraccini 24, Florence, 50139, Italy
- Department of Neurosciences, Psychology, Drug Research and Child Health, University of Florence, Viale Pieraccini 6, Florence, 50139, Italy
| | - Elisa Giocaliere
- Newborn screening, Clinical Chemistry and Pharmacology Lab, Meyer Children's University Hospital, Viale Pieraccini 24, Florence, 50139, Italy
| | - Giulia Forni
- Newborn screening, Clinical Chemistry and Pharmacology Lab, Meyer Children's University Hospital, Viale Pieraccini 24, Florence, 50139, Italy
| | - Sabrina Malvagia
- Newborn screening, Clinical Chemistry and Pharmacology Lab, Meyer Children's University Hospital, Viale Pieraccini 24, Florence, 50139, Italy
| | - Giancarlo la Marca
- Newborn screening, Clinical Chemistry and Pharmacology Lab, Meyer Children's University Hospital, Viale Pieraccini 24, Florence, 50139, Italy
- Department of Neurosciences, Psychology, Drug Research and Child Health, University of Florence, Viale Pieraccini 6, Florence, 50139, Italy
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35
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A novel method for quantification of human hemoglobin from dried blood spots by use of tandem mass spectrometry. Anal Bioanal Chem 2015; 407:8121-7. [DOI: 10.1007/s00216-015-8988-4] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Revised: 08/07/2015] [Accepted: 08/17/2015] [Indexed: 10/23/2022]
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Chambers AG, Percy AJ, Yang J, Borchers CH. Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots. Mol Cell Proteomics 2015; 14:3094-104. [PMID: 26342038 DOI: 10.1074/mcp.o115.049957] [Citation(s) in RCA: 73] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2015] [Indexed: 01/19/2023] Open
Abstract
The dried blood spot (DBS) methodology provides a minimally invasive approach to sample collection and enables room-temperature storage for most analytes. DBS samples have successfully been analyzed by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS) to quantify a large range of small molecule biomarkers and drugs; however, this strategy has only recently been explored for MS-based proteomics applications. Here we report the development of a highly multiplexed MRM assay to quantify endogenous proteins in human DBS samples. This assay uses matching stable isotope-labeled standard peptides for precise, relative quantification, and standard curves to characterize the analytical performance. A total of 169 peptides, corresponding to 97 proteins, were quantified in the final assay with an average linear dynamic range of 207-fold and an average R(2) value of 0.987. The total range of this assay spanned almost 5 orders of magnitude from serum albumin (P02768) at 18.0 mg/ml down to cholinesterase (P06276) at 190 ng/ml. The average intra-assay and inter-assay precision for 6 biological samples ranged from 6.1-7.5% CV and 9.5-11.0% CV, respectively. The majority of peptide targets were stable after 154 days at storage temperatures from -20 °C to 37 °C. Furthermore, protein concentration ratios between matching DBS and whole blood samples were largely constant (<20% CV) across six biological samples. This assay represents the highest multiplexing yet achieved for targeted protein quantification in DBS samples and is suitable for biomedical research applications.
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Affiliation(s)
- Andrew G Chambers
- From the ‡University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada
| | - Andrew J Percy
- From the ‡University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada
| | - Juncong Yang
- From the ‡University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada
| | - Christoph H Borchers
- From the ‡University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada; §Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada
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Greene DN, Vaughn CP, Crews BO, Agarwal AM. Advances in detection of hemoglobinopathies. Clin Chim Acta 2014; 439:50-7. [PMID: 25314938 DOI: 10.1016/j.cca.2014.10.006] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2014] [Revised: 10/04/2014] [Accepted: 10/04/2014] [Indexed: 01/19/2023]
Abstract
Hemoglobin disorders are recognized as one of the most common inherited diseases worldwide. Detecting and characterizing variant hemoglobins and thalassemias depends primarily on clinical laboratory methods. Multiple biophysical, biochemical, and genetic assays are available to provide phenotypic or genotypic evidence of pathology. For many years conventional slab-gel electrophoresis and HPLC were the most commonly utilized laboratory methods. However, the field has rapidly expanded to regularly include capillary zone electrophoresis, molecular assays, and, more recently, mass spectrometric assays. Interpretation of these techniques is, in general, complicated because of the involvement of multiple polymorphic genes. Proper characterization of hemoglobin variants is necessary for diagnosis, primary prevention and genetic counseling for underlying disorders. This review provides an overview of the current hemoglobin analysis techniques, and also discusses technologies that have potential to translate into widespread clinical settings.
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Affiliation(s)
- Dina N Greene
- TPMG, Northern California Kaiser Permanente Regional Laboratories, Berkeley, CA, United States.
| | - Cecily P Vaughn
- ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States
| | - Bridgit O Crews
- TPMG, Northern California Kaiser Permanente Regional Laboratories, Berkeley, CA, United States
| | - Archana M Agarwal
- ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States; Department of Pathology, University of Utah Health Sciences, Salt Lake City, UT, United States
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38
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Martin NJ, Cooper HJ. Challenges and opportunities in mass spectrometric analysis of proteins from dried blood spots. Expert Rev Proteomics 2014; 11:685-95. [DOI: 10.1586/14789450.2014.965158] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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Traeger-Synodinos J, Harteveld CL. Advances in technologies for screening and diagnosis of hemoglobinopathies. Biomark Med 2014; 8:119-31. [PMID: 24325233 DOI: 10.2217/bmm.13.103] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Hemoglobinopathies constitute the most common monogenic disorders worldwide, caused by mutations in the globin genes that synthesize the globin chains of hemoglobin. Synthesis may be reduced (thalassemia) or underlie abnormal hemoglobins. Mutation interactions produce a wide range of disorders. For neonatal and antenatal screening, identification of affected newborns or carriers is achieved by hematological tests. DNA analysis supports definitive diagnosis, and additionally facilitates prenatal diagnosis procedures. Most methods used today have been developed over several decades, with few recent advances in hematology methods. However, DNA methods evolve continuously. With global migration and multiethnic societies the trend is from targeted, population-specific methods towards generic methods, such as Sanger sequencing (point mutations) and multiplex ligation probe amplification (deletions). DNA microarrays constitute an advanced DNA method for some mutation categories. The newest DNA technology is next-generation sequencing. Although not completely ready for routine use currently, next-generation sequencing may soon become a reality for some hemoglobin diagnostic laboratories.
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Affiliation(s)
- Jan Traeger-Synodinos
- Medical Genetics, University of Athens, St Sophia's Children's Hospital, Athens 11527, Greece.
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Abstract
Hemoglobin E (Hb E) disorder is an important kind of hemoglobinopathy. It can be seen around the world with the highest prevalence in Southeast Asia. The screening for this disorder becomes the public health policies in many countries. The screening can be performed in several population groups. The newborn screening program for Hb E disorder is an important issue in pediatric genetics. In this brief review, the author discusses on important laboratory tests for screening for Hb E disorder in newborn.
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Moat SJ, Rees D, King L, Ifederu A, Harvey K, Hall K, Lloyd G, Morrell C, Hillier S. Newborn Blood Spot Screening for Sickle Cell Disease by Using Tandem Mass Spectrometry: Implementation of a Protocol to Identify Only the Disease States of Sickle Cell Disease. Clin Chem 2014; 60:373-80. [DOI: 10.1373/clinchem.2013.210948] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Abstract
BACKGROUND
The currently recommended technologies of HPLC and isoelectric focusing for newborn blood spot screening for sickle cell disease (SCD) identify both the disease and carrier states, resulting in large numbers of infants being followed up unnecessarily. Analysis of blood spot tryptic peptides performed by using tandem mass spectrometry (MS/MS) is an alternative technology to detect hemoglobin (Hb) variant disorders.
METHODS
We analyzed 2154 residual newborn blood spots and 675 newborn blood spots from infants with Hb variants by using MS/MS after trypsin digestion. Screening cutoffs were developed by using the ratio between the variant peptide–to–wild-type peptide abundance for HbS, C, DPunjab, OArab, Lepore, and E peptides. A postanalytical data analysis protocol was developed using these cutoffs to detect only the disease states of SCD and not to identify carrier states. A parallel study of 13 249 newborn blood spots from a high-prevalence SCD area were analyzed by both MS/MS and HPLC.
RESULTS
Screening cutoffs developed distinguished the infants with the disease states of SCD, infants who were carriers of SCD, and infants with normal Hb. In the parallel study no false-negative results were identified, and all clinically relevant cases were correctly identified using the MS/MS protocol. Unblinding the data revealed a total of 328 carrier infants that were successfully excluded by the protocol.
CONCLUSIONS
The screening protocol developed correctly identified infants with the disease states of SCD. Furthermore, large numbers of sickle cell carrier infants were successfully not identified, thereby avoiding unnecessary follow-up testing and referral for genetic counseling.
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Affiliation(s)
- Stuart J Moat
- Wales Newborn Screening Laboratory, Department of Medical Biochemistry, Immunology & Toxicology, and
| | - Derek Rees
- Wales Newborn Screening Laboratory, Department of Medical Biochemistry, Immunology & Toxicology, and
| | - Lawrence King
- Department of Haematology, University Hospital Wales, Cardiff, UK
| | - Adeboye Ifederu
- Newborn Screening Laboratory, Department of Chemical Pathology, Great Ormond Street Hospital, London, UK
| | - Katie Harvey
- Newborn Screening Laboratory, Department of Chemical Pathology, Great Ormond Street Hospital, London, UK
| | - Kate Hall
- Newborn Screening & Biochemical Genetics, Birmingham Children's Hospital, Birmingham, UK
| | - Geoff Lloyd
- Wales Newborn Screening Laboratory, Department of Medical Biochemistry, Immunology & Toxicology, and
| | - Christine Morrell
- Directorate for Public Health and Health Professions, Welsh Government, Cardiff, UK
| | - Sharon Hillier
- Screening Division, Public Health Wales, Cathedral Road, Cardiff, UK
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Mavanga NM, Boemer F, Seidel L, Malafu AN, Gothot A, Gerard C. Blood groups, hemoglobin phenotypes and clinical disorders of consanguineous Yansi population. World J Hematol 2013; 2:109-114. [DOI: 10.5315/wjh.v2.i4.109] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/06/2013] [Revised: 10/08/2013] [Accepted: 10/18/2013] [Indexed: 02/05/2023] Open
Abstract
AIM: To study frequency of blood groups, prevalence of sickle-cell anemia trait and glucose-6-phosphate dehydrogenase deficiency (G6PD), among consanguineous Yansi tribe.
METHODS: A total of 525 blood samples were collected, of which 256 among the Yansi population, and 269 for the unrelated control group in the Bandundu province of Democratic Republic of Congo. Blood group antigens were determined in the following systems: ABO, Rh, Kell, Duffy, Kidd and MNS. Blood grouping and extended phenotype tests were performed according to standard immunohematological procedures. Spot tests and tandem mass spectrometry were used respectively for the assessment of G6PD and sickle-cell anemia trait.
RESULTS: The frequency of ABO phenotypes conformed to the following order O>A>B>AB with notably 62.5%, 23.8%, 12.1% and 1.6% for the Yansi, and 54.6%, 27.5%, 14.1% and 3.7% for the unrelated control group, respectively (P = 0.19). As for the Rh phenotypes, the most frequent were ccD.ee, ccD.Ee, CcD.ee, corresponding to 71.5%, 12.1% and 12.1% for the Yansi, and 70.6%, 15.6% and 8.2%, for the unrelated control group (P = 0.27). The frequency of MN and Ss phenotypes were statistically different between groups (P = 0.0021 and P = 0.0006). G6PD was observed in 11.3% of subjects in the Yansi group, and in 12.4% of controls (P = 0.74). The sickle-cell anemia trait was present in 22.4% of Yansi subjects and 17.8% in the control group (P = 0.24). Miscarriages and deaths in young age were more common among Yansi people.
CONCLUSION: This study shows a significant difference in MNS blood group distribution between the Yansi tribe and a control population. The distribution of other blood groups and the prevalence of hemoglobinopathies did not differ in the Yansi tribe.
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Chambers AG, Percy AJ, Hardie DB, Borchers CH. Comparison of proteins in whole blood and dried blood spot samples by LC/MS/MS. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2013; 24:1338-1345. [PMID: 23821375 DOI: 10.1007/s13361-013-0678-x] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/13/2013] [Revised: 05/08/2013] [Accepted: 05/09/2013] [Indexed: 06/02/2023]
Abstract
Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77%-92% within replicates and the majority of these repeated proteins (70%) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.
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Affiliation(s)
- Andrew G Chambers
- Genome British Columbia Proteomics Centre, University of Victoria, Vancouver Island Technology Park, Victoria, BC, Canada
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Martin NJ, Bunch J, Cooper HJ. Dried blood spot proteomics: surface extraction of endogenous proteins coupled with automated sample preparation and mass spectrometry analysis. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2013; 24:1242-9. [PMID: 23728546 PMCID: PMC3713260 DOI: 10.1007/s13361-013-0658-1] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/01/2013] [Revised: 04/18/2013] [Accepted: 04/24/2013] [Indexed: 05/09/2023]
Abstract
Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.
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Affiliation(s)
- Nicholas J. Martin
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT UK
| | - Josephine Bunch
- School of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT UK
| | - Helen J. Cooper
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT UK
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Abstract
MS allows for the unequivocal diagnosis of hemoglobin variants, or hemoglobinopathies. Hemoglobinopathies are the most common inherited disorder and there is a need for rapid detection of clinically significant variants, such as sickle hemoglobin, which is responsible for sickle cell disease. In this review, we describe the development of MS approaches for the determination of hemoglobin variants from both whole blood samples and dried blood spots. MS approaches that are suitable for population screening are discussed, as are recent advances in direct surface analysis of dried blood spots.
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Chambers AG, Percy AJ, Yang J, Camenzind AG, Borchers CH. Multiplexed quantitation of endogenous proteins in dried blood spots by multiple reaction monitoring-mass spectrometry. Mol Cell Proteomics 2013; 12:781-91. [PMID: 23221968 PMCID: PMC3591668 DOI: 10.1074/mcp.m112.022442] [Citation(s) in RCA: 78] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2012] [Revised: 11/26/2012] [Indexed: 11/06/2022] Open
Abstract
Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R(2) value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at -20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins.
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Affiliation(s)
- Andrew G. Chambers
- From the ‡University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101, 4464 Markham St., Victoria, BC V8Z 7X8, Canada
| | - Andrew J. Percy
- From the ‡University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101, 4464 Markham St., Victoria, BC V8Z 7X8, Canada
| | - Juncong Yang
- From the ‡University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101, 4464 Markham St., Victoria, BC V8Z 7X8, Canada
| | - Alexander G. Camenzind
- From the ‡University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101, 4464 Markham St., Victoria, BC V8Z 7X8, Canada
| | - Christoph H. Borchers
- From the ‡University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101, 4464 Markham St., Victoria, BC V8Z 7X8, Canada
- §Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada
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Affiliation(s)
- Plamen A. Demirev
- Johns Hopkins University Applied Physics Laboratory, Laurel,
Maryland 20723, United States
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Wolff F, Cotton F, Gulbis B. Screening for haemoglobinopathies on cord blood: laboratory and clinical experience. J Med Screen 2012; 19:116-22. [DOI: 10.1258/jms.2012.011107] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Objectives Blood from the umbilical cord (cord blood) is screened for haemoglobinopathies in several neonatal screening programmes, as well as before banking as a source of stem cells. We investigated the pre-analytical and analytical aspects of neonatal screening for haemoglobinopathies on liquid cord blood using the Sebia Haemoglobin kit. We give an interpretation of the results as well as a proposed scheme for reporting of the results. Methods A neonatal screening programme on liquid cord blood has been performed in all labour wards in Brussels since 1994. Using that material, the screening methods of isoelectric focusing and capillary zone electrophoresis were compared using 962 cord blood samples. From December 2008 to December 2010, 47,388 neonatal samples were analysed by capillary electrophoresis as the first-line method for neonatal screening. High-performance liquid chromatography was used as the second-line method. Results Capillary zone electrophoresis on liquid cord blood enabled the detection of all clinically significant haemoglobin variants, significant levels of Hb Bart's, and β-thalassaemia major. Among the 47,388 neonatal samples tested, 362 (0.7%) were suspected to be contaminated with maternal blood, but no diagnostic error was reported retrospectively for a major haemoglobinopathy. Recommendations for the interpretation and reporting of results of neonatal screening for haemoglobinopathies using the Sebia Haemoglobin kit are proposed. Conclusions A routine capillary electrophoresis kit adapted to neonatal screening and liquid cord blood is reliable for screening for haemoglobinopathies. It enables early detection and reporting of all major haemoglobinopathies and most minor ones. It also enables use of a simple scheme to report the results.
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Affiliation(s)
| | | | - Béatrice Gulbis
- Laboratory of Clinical Chemistry, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium
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Abstract
Hemoglobin disorders consist of two different groups, the structural hemoglobin variants and the thalassemias. The structural hemoglobin variants typically are based on the point mutations in the alpha- or beta-globin chain that results in a single-amino acid substitution in the corresponding globin chain, whereas thalassemias are caused by quantitative reduction in globin chain synthesis. Various techniques are applied for the laboratory investigation of these diseases, among them mass spectrometry (MS) for the detection and identification of structural hemoglobin variants and array techniques for the thalassemias. In this review, we present in the first part the most important mass spectrometric techniques applied in hemoglobin variant detection and identification and discuss several important aspects of this analysis technique in hematology. In the second part, the DNA analysis techniques used in hemoglobin analysis, such as reverse hybridization or microarray-based comparative genomic hybridization (CGH) techniques, are briefly discussed.
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Affiliation(s)
- Heinz Troxler
- Division of Clinical Chemistry and Biochemistry, University Children's Hospital Zurich, Zurich, Switzerland
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