Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.

Biomedical Research Networking Centre Consortium of Oncology CB 16 12 00233 Instituto Carlos 3 Madrid Spain

Department of Immunohematology and Blood Transfusion Leiden University Medical Center Leiden Netherlands

Department of Immunology and Pathology Central Clinical School Alfred Hospital Monash University Melbourne VIC Australia

Department of Immunology Erasmus MC Rotterdam Netherlands

Department of Immunology Hospital Universitario La Paz Madrid Spain

Department of Laboratory Medicine University Hospital Ghent Ghent Belgium

Department of Medicine Cancer Research Centre Salamanca Spain

Department of Pediatric Hematology and Oncology University Hospital Motol Charles University Prague Czechia

Department of Pediatrics Leiden University Medical Center Leiden Netherlands

Experimental Medicine Division Nuffield Department of Medicine University of Oxford Oxford United Kingdom

Faculdade de Medicina Instituto de Medicina Molecular Universidade de Lisboa Lisbon Portugal

Institute of Clinical Immunology and Allergology St Anne's University Hospital Brno Masaryk University Brno Czechia

Zobrazit více v PubMed

Picard C, Bobby Gaspar H, Al-Herz W, Bousfiha A, Casanova JL, Chatila T, et al. . International Union of Immunological Societies: 2017 Primary Immunodeficiency Diseases Committee Report on Inborn Errors of Immunity. J Clin Immunol. (2018) 38:96–128. 10.1007/s10875-017-0464-9 PubMed DOI PMC

Bousfiha A, Jeddane L, Picard C, Ailal F, Bobby Gaspar H, Al-Herz W, et al. . The 2017 IUIS phenotypic classification for primary immunodeficiencies. J Clin Immunol. (2018) 38:129–43. 10.1007/s10875-017-0465-8 PubMed DOI PMC

ESID European Society for Immunodeficiencies: ESID Database Statistics 2004-2014. Available online at: https://esid.org/Working-Parties/Registry/ESID-Database-Statistics (accessed May 25, 2019)

Kindle G, Gathmann B, Grimbacher B. The use of databases in primary immunodeficiencies. Curr Opin Aller Clin Immunol. (2014) 14:501–8. 10.1097/ACI.0000000000000113 PubMed DOI

ESID European Society for Immunodeficiencies: ESID Registry - Clinical diagnosis criteria of PID. Available online at: https://esid.org/Working-Parties/Registry/Diagnosis-criteria (accessed May 25, 2019)

de Vries E. Patient-centred screening for primary immunodeficiency, a multi-stage diagnostic protocol designed for non-immunologists: 2011 update. Clin Exp Immunol. (2012) 167:108–19. 10.1111/j.1365-2249.2011.04461.x PubMed DOI PMC

Bonilla FA, Khan DA, Ballas ZK, Chinen J, Frank MM, Hsu JT, et al. . Practice parameter for the diagnosis and management of primary immunodeficiency. J Allergy Clin Immunol. (2015) 136:1186–205.e1-78. 10.1016/j.jaci.2015.04.049 PubMed DOI

Dorsey MJ, Dvorak CC, Cowan MJ, Puck JM. Treatment of infants identified as having severe combined immunodeficiency by means of newborn screening. J Allergy Clin Immunol. (2017) 139:733-42. 10.1016/j.jaci.2017.01.005 PubMed DOI PMC

Stray-Pedersen A, Sorte HS, Samarakoon P, Gambin T, Chinn IK, Coban Akdemir ZH, et al. . Primary immunodeficiency diseases: Genomic approaches delineate heterogeneous Mendelian disorders. J Allergy and clinical immunology. (2017) 139:232-45. 10.1016/j.jaci.2016.05.042 PubMed DOI PMC

Abolhassani H, Chou J, Bainter W, Platt CD, Tavassoli M, Momen T, et al. . Clinical, immunologic, and genetic spectrum of 696 patients with combined immunodeficiency. J Allergy Clin Immunol. (2018) 141:1450-8. 10.1016/j.jaci.2017.06.049 PubMed DOI

Schuetz C, Pannicke U, Jacobsen EM, Burggraf S, Albert MH, Honig M, et al. . Lesson from hypomorphic recombination-activating gene (RAG) mutations: Why asymptomatic siblings should also be tested. J Allergy Clin Immunol. (2014) 133:1211-5. 10.1016/j.jaci.2013.10.021 PubMed DOI

Volk T, Pannicke U, Reisli I, Bulashevska A, Ritter J, Bjorkman A, et al. . DCLRE1C (ARTEMIS) mutations causing phenotypes ranging from atypical severe combined immunodeficiency to mere antibody deficiency. Hum Mol Genet. (2015) 24:7361–72. 10.1093/hmg/ddv437 PubMed DOI PMC

IJspeert H, Driessen GJ, Moorhouse MJ, Hartwig NG, Wolska-Kusnierz B, Kalwak K, et al. . Similar recombination-activating gene (RAG) mutations result in similar immunobiological effects but in different clinical phenotypes. J Allergy Clin Immunol. (2014) 133:1124–33. 10.1016/j.jaci.2013.11.028 PubMed DOI PMC

Kwan A, Abraham RS, Currier R, Brower A, Andruszewski K, Abbott JK, et al. . Newborn screening for severe combined immunodeficiency in 11 screening programs in the United States. JAMA. (2014) 312:729–38. 10.1001/jama.2014.9132 PubMed DOI PMC

Ogonek J, Kralj Juric M, Ghimire S, Varanasi PR, Holler E, Greinix H, et al. . Immune Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation. Front Immunol. (2016) 7:507. 10.3389/fimmu.2016.00507 PubMed DOI PMC

Heimall J, Logan BR, Cowan MJ, Notarangelo LD, Griffith LM, Puck JM, et al. . Immune reconstitution and survival of 100 SCID patients post-hematopoietic cell transplant: a PIDTC natural history study. Blood. (2017) 130:2718–27. 10.1182/blood-2017-05-781849 PubMed DOI PMC

Ameratunga R, Woon ST, Gillis D, Koopmans W, Steele R. New diagnostic criteria for common variable immune deficiency (CVID), which may assist with decisions to treat with intravenous or subcutaneous immunoglobulin. Clin Exp Immunol. (2013) 174:203–11. 10.1111/cei.12178 PubMed DOI PMC

Barmettler S, Price C. Continuing IgG replacement therapy for hypogammaglobulinemia after rituximab–for how long? J Allergy Clin Immunol. (2015) 136:1407–9. 10.1016/j.jaci.2015.06.035 PubMed DOI

Rabbani B, Mahdieh N, Hosomichi K, Nakaoka H, Inoue I. Next-generation sequencing: impact of exome sequencing in characterizing Mendelian disorders. J Hum Genet. (2012) 57:621–32. 10.1038/jhg.2012.91 PubMed DOI

Stessman HA, Bernier R, Eichler EE. A genotype-first approach to defining the subtypes of a complex disease. Cell. (2014) 156:872–7. 10.1016/j.cell.2014.02.002 PubMed DOI PMC

Jansen S, Hoischen A, Coe BP, Carvill GL, Van Esch H, Bosch DGM, et al. . A genotype-first approach identifies an intellectual disability-overweight syndrome caused by PHIP haploinsufficiency. Eur J Hum Genet. (2018) 26:54-63. 10.1038/s41431-017-0039-5 PubMed DOI PMC

D'Amore A, Tessa A, Casali C, Dotti MT, Filla A, Silvestri G, et al. . Next generation molecular diagnosis of hereditary spastic paraplegias: an Italian cross-sectional study. Front Neurol. (2018) 9:981. 10.3389/fneur.2018.00981 PubMed DOI PMC

Chae JH, Vasta V, Cho A, Lim BC, Zhang Q, Eun SH, et al. . Utility of next generation sequencing in genetic diagnosis of early onset neuromuscular disorders. J Med Genet. (2015) 52:208–16. 10.1136/jmedgenet-2014-102819 PubMed DOI

Waldrop MA, Pastore M, Schrader R, Sites E, Bartholomew D, Tsao CY, et al. . Diagnostic utility of whole exome sequencing in the neuromuscular clinic. Neuropediatrics. (2019) 50):96–102. 10.1055/s-0039-1677734 PubMed DOI

Al-Mousa H, Abouelhoda M, Monies DM, Al-Tassan N, Al-Ghonaium A, Al-Saud B, et al. . Unbiased targeted next-generation sequencing molecular approach for primary immunodeficiency diseases. J Allergy Clin Immunol. (2016) 137:1780–7. 10.1016/j.jaci.2015.12.1310 PubMed DOI

Gallo V, Dotta L, Giardino G, Cirillo E, Lougaris V, D'Assante R, et al. . Diagnostics of primary immunodeficiencies through next-generation sequencing. Front Immunol. (2016) 7:466. 10.3389/fimmu.2016.00466 PubMed DOI PMC

Stoddard JL, Niemela JE, Fleisher TA, Rosenzweig SD. Targeted NGS: A cost-effective approach to molecular diagnosis of PIDs. Front Immunol. (2014) 5:531. 10.3389/fimmu.2014.00531 PubMed DOI PMC

Chi ZH, Wei W, Bu DF, Li HH, Ding F, Zhu P. Targeted high-throughput sequencing technique for the molecular diagnosis of primary immunodeficiency disorders. Medicine. (2018) 97:e12695. 10.1097/MD.0000000000012695 PubMed DOI PMC

Cifaldi C, Brigida I, Barzaghi F, Zoccolillo M, Ferradini V, Petricone D, et al. . Targeted NGS platforms for genetic screening and gene discovery in primary immunodeficiencies. Front Immunol. (2019) 10:36. 10.3389/fimmu.2019.00316 PubMed DOI PMC

Rae W, Ward D, Mattocks C, Pengelly RJ, Eren E, Patel SV, et al. . Clinical efficacy of a next-generation sequencing gene panel for primary immunodeficiency diagnostics. Clin Genet. (2018) 93:647–55. 10.1111/cge.13163 PubMed DOI

Abolhassani H, Kiaee F, Tavakol M, Chavoshzadeh Z, Mahdaviani SA, Momen T, et al. Fourth update on the iranian national registry of primary immunodeficiencies: integration of molecular diagnosis. J Clin Immunol. (2018) 38:816–32. 10.1007/s10875-018-0556-1 PubMed DOI

Al-Herz W, Chou J, Delmonte OM, Massaad MJ, Bainter W, Castagnoli R, et al. . Comprehensive genetic results for primary immunodeficiency disorders in a highly consanguineous population. Front Immunol. (2018) 9:3146. 10.3389/fimmu.2018.03146 PubMed DOI PMC

Xia Y, He T, Luo Y, Li C, Lim CK, Abolhassani H, et al. . Targeted next-generation sequencing for genetic diagnosis of 160 patients with primary immunodeficiency in south China. Pediatr Allergy Immunol. (2018) 29:863–72. 10.1111/pai.12976 PubMed DOI

Al-Mousa H, Al-Saud B. Primary immunodeficiency diseases in highly consanguineous populations from middle east and north Africa: epidemiology, diagnosis, and care. Front Immunol. (2017) 8:678. 10.3389/fimmu.2017.00678 PubMed DOI PMC

Bogaert DJ, Dullaers M, Lambrecht BN, Vermaelen KY, De Baere E, Haerynck F. Genes associated with common variable immunodeficiency: one diagnosis to rule them all? J Med Genet. (2016) 53:575–90. 10.1136/jmedgenet-2015-103690 PubMed DOI

Mahlaoui N, Picard C, Bach P, Costes L, Courteille V, Ranohavimparany A, et al. . Genetic diagnosis of primary immunodeficiencies: a survey of the French national registry. J Allergy Clin Immunol. (2019) 143:1646–9. 10.1016/j.jaci.2018.12.994 PubMed DOI

van der Burg M, van Zelm MC, Driessen GJ, van Dongen JJ. New frontiers of primary antibody deficiencies. Cell Mol Life Sci. (2012) 69:59–73. 10.1007/s00018-011-0836-x PubMed DOI PMC

van der Burg M, van Zelm MC, Driessen GJ, van Dongen JJ. Dissection of B-cell development to unravel defects in patients with a primary antibody deficiency. Adv Exp Med Biol. (2011) 697:183–96. 10.1007/978-1-4419-7185-2_13 PubMed DOI

Heimall JR, Hagin D, Hajjar J, Henrickson SE, Hernandez-Trujillo HS, Tan Y, et al. Use of Genetic testing for primary immunodeficiency patients. J Clin Immunol. (2018) 38:320–9. 10.1007/s10875-018-0489-8 PubMed DOI

Abolhassani H, Wang N, Aghamohammadi A, Rezaei N, Lee YN, Frugoni F, et al. . A hypomorphic recombination-activating gene 1 (RAG1) mutation resulting in a phenotype resembling common variable immunodeficiency. J Allergy Clin Immunol. (2014) 134:1375–80. 10.1016/j.jaci.2014.04.042 PubMed DOI PMC

Notarangelo LD, Kim MS, Walter JE, Lee YN. Human RAG mutations: biochemistry and clinical implications. Nat Rev Immunol. (2016) 16:234–46. 10.1038/nri.2016.28 PubMed DOI PMC

Rao VK, Webster S, Dalm V, Sediva A, van Hagen PM, Holland S, et al. . Effective “activated PI3Kdelta syndrome”-targeted therapy with the PI3Kdelta inhibitor leniolisib. Blood. (2017) 130:2307–16. 10.1182/blood-2017-08-801191 PubMed DOI PMC

O'Gorman MR, Zollett J, Bensen N. Flow cytometry assays in primary immunodeficiency diseases. Methods Mol Biol. (2011) 699:317–35. 10.1007/978-1-61737-950-5_15 PubMed DOI

Boldt A, Borte S, Fricke S, Kentouche K, Emmrich F, Borte M, et al. . Eight-color immunophenotyping of T-, B-, and NK-cell subpopulations for characterization of chronic immunodeficiencies. Cytom B Clincytom. (2014) 86:191–206. 10.1002/cytob.21162 PubMed DOI

Takashima T, Okamura M, Yeh TW, Okano T, Yamashita M, Tanaka K, et al. . Multicolor flow cytometry for the diagnosis of primary immunodeficiency diseases. J Clin Immunol. (2017) 37:486–95. 10.1007/s10875-017-0405-7 PubMed DOI

Abraham RS, Aubert G. Flow cytometry, a versatile tool for diagnosis and monitoring of primary immunodeficiencies. Clinical Vacc Immunol. (2016) 23:254–71. 10.1128/CVI.00001-16 PubMed DOI PMC

Oliveira JB, Notarangelo LD, Fleisher TA. Applications of flow cytometry for the study of primary immune deficiencies. Curr Opinion Allergy Clin Immunol. (2008) 8:499–509. 10.1097/ACI.0b013e328312c790 PubMed DOI

Kanegane H, Hoshino A, Okano T, Yasumi T, Wada T, Takada H, et al. . Flow cytometry-based diagnosis of primary immunodeficiency diseases. Allergol Int. (2018) 67:43–54. 10.1016/j.alit.2017.06.003 PubMed DOI

Warnatz K, Wehr C, Drager R, Schmidt S, Eibel H, Schlesier M, et al. . Expansion of CD19(hi)CD21(lo/neg) B cells in common variable immunodeficiency (CVID) patients with autoimmune cytopenia. Immunobiology. (2002) 206:502–13. 10.1078/0171-2985-00198 PubMed DOI

Wehr C, Kivioja T, Schmitt C, Ferry B, Witte T, Eren E, et al. . The EUROclass trial: defining subgroups in common variable immunodeficiency. Blood. (2008) 111:77–85. 10.1182/blood-2007-06-091744 PubMed DOI

Biancotto A, Fuchs JC, Williams A, Dagur PK, McCoy JP, Jr. High dimensional flow cytometry for comprehensive leukocyte immunophenotyping (CLIP) in translational research. J Immunol Method. (2011) 363:245–61. 10.1016/j.jim.2010.06.010 PubMed DOI PMC

Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the human immunology project. Nat Rev Immunol. (2012) 12:191–200. 10.1038/nri3158 PubMed DOI PMC

Streitz M, Miloud T, Kapinsky M, Reed MR, Magari R, Geissler EK, et al. . Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study. Transplant Res. (2013) 2:17. 10.1186/2047-1440-2-17 PubMed DOI PMC

Duffy D, Rouilly V, Libri V, Hasan M, Beitz B, David M, et al. . Functional analysis via standardized whole-blood stimulation systems defines the boundaries of a healthy immune response to complex stimuli. Immunity. (2014) 40:436–50. 10.1016/j.immuni.2014.03.002 PubMed DOI

Veluchamy JP, Delso-Vallejo M, Kok N, Bohme F, Seggewiss-Bernhardt R, van der Vliet HJ, et al. . Standardized and flexible eight colour flow cytometry panels harmonized between different laboratories to study human NK cell phenotype and function. Sci Rep. (2017) 7:43873. 10.1038/srep43873 PubMed DOI PMC

Blanco E, Perez-Andres M, Arriba-Mendez S, Contreras-Sanfeliciano T, Criado I, Pelak O, et al. . Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood. J Allergy Clin Immunol. (2018) 141:2208–19.e16. 10.1016/j.jaci.2018.02.017 PubMed DOI

Blanco E, Perez-Andres M, Arriba-Mendez S, Serrano C, Criado I, Pino-Molina LD, et al. Defects in memory B-cell and plasma cell subsets expressing different immunoglobulin-subclasses in CVID and Ig-subclass deficiencies. J Allergy Clin Immunol. (2019). [Epub ahead of print]. 10.1016/j.jaci.2019.02.017 PubMed DOI

Blanco E, Perez-Andres M, Sanoja-Flores L, Wentink M, Pelak O, Martin-Ayuso M, et al. . Selection and validation of antibody clones against IgG and IgA subclasses in switched memory B-cells and plasma cells. J. Immunol Method. (2017). [Epub ahead of print]. 10.1016/j.jim.2017.09.008 PubMed DOI

van der Burg M, Kalina T, Perez-Andres M, Vlkova M, Lopez-Granados E, Blanco E, et al. . The EuroFlow PID orientation tube for flow cytometric diagnostic screening of primary immunodeficiencies of the lymphoid system. Front Immunol. (2019) 10:246. 10.3389/fimmu.2019.00246 PubMed DOI PMC

van der Velden VH, Flores-Montero J, Perez-Andres M, Martin-Ayuso M, Crespo O, Blanco E, et al. . Optimization and testing of dried antibody tube: The EuroFlow LST and PIDOT tubes as examples. J Immunol Method. (2017). [Epub ahead of print]. 10.1016/j.jim.2017.03.011 PubMed DOI

van Dongen JJ, Lhermitte L, Bottcher S, Almeida J, van der Velden VH, Flores-Montero J, et al. . EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia. (2012) 26:1908–75. 10.1038/leu.2012.120 PubMed DOI PMC

Kalina T, Flores-Montero J, van der Velden VH, Martin-Ayuso M, Bottcher S, Ritgen M, et al. . EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols. Leukemia. (2012) 26:1986–2010. 10.1038/leu.2012.122 PubMed DOI PMC

Theunissen P, Mejstrikova E, Sedek L, van der Sluijs-Gelling AJ, Gaipa G, Bartels M, et al. . Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia. Blood. (2017) 129:347–57. 10.1182/blood-2016-07-726307 PubMed DOI PMC

Flores-Montero J, Sanoja-Flores L, Paiva B, Puig N, Garcia-Sanchez O, Bottcher S, et al. . Next generation flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma. Leukemia. (2017) 31:2094–103. 10.1038/leu.2017.29 PubMed DOI PMC

Lhermitte L, Mejstrikova E, van der Sluijs-Gelling AJ, Grigore GE, Sedek L, Bras AE, et al. . Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia. Leukemia. (2018) 32:874-81. 10.1038/leu.2017.313 PubMed DOI PMC

Pedreira CE, Costa ES, Lecrevisse Q, van Dongen JJ, Orfao A. Overview of clinical flow cytometry data analysis: recent advances and future challenges. Trends Biotechnol. (2013) 31:415–25. 10.1016/j.tibtech.2013.04.008 PubMed DOI

van Dongen JJ, van der Velden VH, Bruggemann M, Orfao A. Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies. Blood. (2015) 125:3996–4009. 10.1182/blood-2015-03-580027 PubMed DOI PMC

Novakova M, Glier H, Brdickova N, Vlkova M, Santos AH, Lima M, et al. . How to make usage of the standardized EuroFlow 8-color protocols possible for instruments of different manufacturers. J Immunol Method. (2017). [Epub ahead of print]. 10.1016/j.jim.2017.11.007 PubMed DOI

Diks A. M., Bonroy C., Teodosio C., Groenland R. J., De Mooij B., De Maertelaere E., et al. (in press). Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research. J. Immunol. Method. 10.1016/j.jim.2019.06.007 PubMed DOI

Bottcher S, van der Velden VHJ, Villamor N, Ritgen M, Flores-Montero J, Murua Escobar H, et al. . Lot-to-lot stability of antibody reagents for flow cytometry. J Immunol Method. (2017). [Epub ahead of print]. 10.1016/j.jim.2017.03.018 PubMed DOI

Kalina T, Flores-Montero J, Lecrevisse Q, Pedreira CE, van der Velden VH, Novakova M, et al. . Quality assessment program for EuroFlow protocols: summary results of four-year (2010–2013) quality assurance rounds. Cytom A. (2015) 87:145–56. 10.1002/cyto.a.22581 PubMed DOI

Kalina T, Brdickova N, Glier H, Fernandez P, Bitter M, Flores-Montero J, et al. Frequent issues and lessons learned from EuroFlow QA. Journal of immunological methods. (2018). [Epub ahead of print]. 10.1016/j.jim.2018.09.008 PubMed DOI

Frenkel J, Neijens HJ, den Hollander JC, Wolvers-Tettero IL, van Dongen JJ. Oligoclonal T cell proliferative disorder in combined immunodeficiency. Pediatric Res. (1988) 24:622–7. 10.1203/00006450-198811000-00017 PubMed DOI

Harville TO, Adams DM, Howard TA, Ware RE. Oligoclonal expansion of CD45RO+ T lymphocytes in Omenn syndrome. J Clin Immunol. (1997) 17:322–32. 10.1023/A:1027330800085 PubMed DOI

Chan K, Puck JM. Development of population-based newborn screening for severe combined immunodeficiency. J Allergy Clin Immunol. (2005) 115:391–8. 10.1016/j.jaci.2004.10.012 PubMed DOI

Brown L, Xu-Bayford J, Allwood Z, Slatter M, Cant A, Davies EG, et al. . Neonatal diagnosis of severe combined immunodeficiency leads to significantly improved survival outcome: the case for newborn screening. Blood. (2011) 117:3243–6. 10.1182/blood-2010-08-300384 PubMed DOI

Thakar MS, Hintermeyer MK, Gries MG, Routes JM, Verbsky JW. A practical approach to newborn screening for severe combined immunodeficiency using the t cell receptor excision circle assay. Front Immunol. (2017) 8:1470. 10.3389/fimmu.2017.01470 PubMed DOI PMC

Berkowska MA, Heeringa JJ, Hajdarbegovic E, van der Burg M, Thio HB, van Hagen PM, et al. . Human IgE(+) B cells are derived from T cell-dependent and T cell-independent pathways. J Allergy Clin Immunol. (2014) 134:688–97.e6. 10.1016/j.jaci.2014.03.036 PubMed DOI

JMF Jeffrey Modell Foundation: 4 Stages of Testing for Primary Immunodeficiency. Available online at: http://downloads.info4pi.org/pdfs/Physician-Algorithm-2-.pdf (accessed May 25, 2019)

ESID European Society for Immunodeficiencies: The 6 ESID Warning Signs for ADULT Primary Immunodeficiency Diseases. Available online at: https://esid.org/Education/6-Warning-Signs-for-PID-in-Adults (accessed May 25, 2019)

Criado I, Blanco E, Rodriguez-Caballero A, Alcoceba M, Contreras T, Gutierrez ML, et al. . Residual normal B-cell profiles in monoclonal B-cell lymphocytosis versus chronic lymphocytic leukemia. Leukemia. (2018) 32:2701–5. 10.1038/s41375-018-0164-3 PubMed DOI PMC

Tviblindi algorithm identifies branching developmental trajectories of human B-cell development and describes abnormalities in RAG-1 and WAS patients

Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative

Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children

Dissection of the Pre-Germinal Center B-Cell Maturation Pathway in Common Variable Immunodeficiency Based on Standardized Flow Cytometric EuroFlow Tools