Erdle 2017
Erdle 2017
Erdle 2017
CME Review
A R T I C L E I N F O
Article history:
Received for publication October 24, 2016.
Received in revised form April 5, 2017.
Accepted for publication April 6, 2017.
INSTRUCTIONS
Credit can now be obtained, free for a limited time, by reading the review article and completing all activity components. Please note the
instructions listed below:
Review the target audience, learning objectives and all disclosures.
Complete the pre-test.
Read the article and reflect on all content as to how it may be applicable to your practice.
Complete the post-test/evaluation and claim credit earned. At this time, physicians will have earned up to 1.0 AMA PRA Category 1
CreditTM. Minimum passing score on the post-test is 70%.
Overall Purpose
Participants will be able to demonstrate increased knowledge of the clinical treatment of allergy/asthma/immunology and how new
information can be applied to their own practices.
Learning Objectives
At the conclusion of this activity, participants should be able to:
Interpret immunological testing of T and B-cells
Discuss the limitations of immunological testing techniques
Release Date: June 1, 2017
Expiration Date: May 31, 2019
Target Audience
Physicians involved in providing patient care in the field of allergy/asthma/immunology
Accreditation
The American College of Allergy, Asthma & Immunology (ACAAI) is accredited by the Accreditation Council for Continuing Medical
Education (ACCME) to provide continuing medical education for physicians.
Designation
The American College of Allergy, Asthma & Immunology (ACAAI) designates this journal-based CME activity for a maximum of 1.0 AMA
PRA Category 1 CreditTM. Physicians should claim only the credit commensurate with the extent of their participation in the activity.
Planning Committee Members
Anne K. Ellis, MD, FRCP(C) (Author)
Stephanie Erdle, MD (Author)
Julia E. M. Upton, MD, FRCP(C) (Author)
Jonathan A. Bernstein, MD (CME Subcommittee)
Guha Krishnaswamy, MD (CME Subcommittee)
John J. Oppenheimer, MD (CME Subcommittee, Associate Editor)
Reprints: Julia E.M. Upton, MD, FRCP(C), Division of Immunology and Allergy,
Department of Pediatrics, The Hospital for Sick Children, 555 University Avenue,
Toronto, ON, M5G 1X8, Canada; E-mail: [email protected].
Disclosures: Dr Ellis declares a grant to her institution from Green Cross. Dr Upton
is a member of the medical advisory board of Immunodeficiency Canada.
http://dx.doi.org/10.1016/j.anai.2017.04.007
1081-1206/Ó 2017 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
656 S. Erdle et al. / Ann Allergy Asthma Immunol 118 (2017) 655e663
Figure 1. A simplified general approach to the clinical laboratory evaluation of suspected humoral and cellular PIDs. See text for further details. Ab, antibiotic; CBC, complete
blood cell count; CVID, common variable immune deficiency; FTT, failure to thrive; HIV, human immunodeficiency virus; IgG, immunoglobulin G; IVIG, intravenous immu-
noglobulin; IgM, immunoglobulin M; NK, natural killer; PID, primary immunodeficiency; SCID, severe combined immunodeficiency; TCR, T cell receptor.
individuals older than 2 years.8 Serum isohemagglutinins, pre- Including the normal response, there are 5 pneumococcal vaccine
dominantly IgM antibodies against ABO blood group antigens that response patterns (Fig 2). The response can be (1) no more than 2
occur naturally in response to gut flora polysaccharide antigens, protective serotypes (severe phenotype), (2) no more than 70% of
also have been used. protective serotypes (moderate phenotype), (3) mildly decreased
The interpretation of vaccine responses can be quite difficult. number of expected protective responses (mild phenotype), (4)
A working group report has provided some guidance and lists the initially appropriate responses but not sustained for 6 months
protective levels of multiple vaccines.8 For example, the protective (memory phenotype), or (5) normal.
level of tetanus is reported as 0.15 IU/mL. The protective titer for The interpretation of vaccine booster responses remains a
each pneumococcal serotype also is controversial and has been challenge and was recently reviewed as part of a larger consid-
reviewed in detail.8 Most use a protective titer of 1.3 mg/mL for at eration of common variable immunodeficiency (CVID) diagnostic
least 70% of serotypes in adults and 50% of serotypes in children.8 criteria.9 Several concerns were detailed, including controversies
Table 1
Clinical Clues, Initial Evaluation, and Advanced Testing for Suspected Cellular and Humoral Immunodeficiencies
þ
Humoral Sinopulmonary infections with 1. Immunophenotyping (CD19 ) 1. Flow cytometry: disease specific
encapsulated organisms
Frequent need for IV antibiotics 2. Immunoglobulin levels (IgG, IgM, IgA, IgE) 2. Advanced immunophenotyping
(eg, memory and naïve B cells)
Enterovirus infections 3. Vaccine titers 3. Genetic testing
4. Response to booster vaccines
Cellular Opportunistic infections 1. CBC count with differential 1. T cell proliferation in vitro assays
FTT 2. Review NBS 2. TCR Vb repertoire
Chronic diarrhea 3. Immunophenotyping (CD4þ and CD8þ) 3. Disease-specific flow cytometry
Fungal infections 4. HIV test 4. Genetic testing
Consanguinity
Family history of immunodeficiency
Abbreviations: CBC, complete blood cell; FTT, failure to thrive; HIV, human immunodeficiency virus; IV, intravenous; NBS, newborn screen; TCR, T cell receptor.
658 S. Erdle et al. / Ann Allergy Asthma Immunol 118 (2017) 655e663
Table 2
Select Disease-specific Flow Cytometry Testing of Cellular, Humoral, and Combined Immunodeficiencies
Abbreviations: DOCK8, dedicator of cytokinesis 8; LRBA, lipopolysaccharide-responsive beige-like anchor; ZAP70, z-chaineassociated protein kinase 70.
S. Erdle et al. / Ann Allergy Asthma Immunol 118 (2017) 655e663 659
It was recently described that an NGS targeted search of 269 development, induces T cell apoptosis, or blocks T cell maturation in
genes in 50 patients with severe clinical manifestations of CVID the thymus can result in a low TREC.
established a likely disease-causing mutation in 30%18 in genes Because patients with SCID typically have no T cells, few T cells,
such as NFKB1, STAT3, CTLA4, and LRBA. The identification of some or oligoclonal expansion of T cells, they will typically have low or
genetic conditions has prognostic and treatment implications. absent TREC levels.20 TREC assay cutoff values differ among cen-
Various CVID molecular panels are available commercially and the ters.25 During the first year of screening, Wisconsin set screening to
probability of finding a molecular diagnosis in patients with a se- detect no more than 25 TRECs/mL of blood, which represented the
vere phenotype warrants consideration for this testing by lowest 1.1% of TREC values from the blood spots during the devel-
immunologists. opment of the assay.26 This value was increased to 40 TRECs/mL
because of the small number of full-term infants with abnormal
Case 1 Revisited TRECs.26
In this case example of a child with multiple infections, auto- The primary targets of TREC NBS include typical SCID, leaky
immunity, and splenomegaly, the IgG, IgA, and IgM levels were SCID, and Omenn syndrome.25 TREC levels are low in neonates with
slightly low, baseline vaccine responses were low but protective, most forms of SCID, including those caused by mutations in RAG1/2,
and a tetanus toxoid boost showed only a small increase. CD19þ ADA, CD3D, JAK3, and IL2RA.19,27,28 A positive TREC result also can
cells were slightly low. Switched memory B cells were low. CD4þ identify non-SCID syndromes associated with T cell lymphopenia,
and CD8þ cells were normal. The patient was diagnosed with CVID such as DiGeorge syndrome, ataxia telangiectasia, trisomy 21,
and started on intravenous immunoglobulin replacement. This is a CHARGE (coloboma, heart defects, atresia choanae, growth retar-
case that could be considered for NGS panels to guide monitoring, dation, genital abnormalities, ear abnormalities) syndrome, and
genetic counseling, and the consideration of directed treatments. CD3 lymphopenia of unknown significance.20,25,29 A positive TREC
also can be seen in patients with T cell lymphopenia from other
Illustrative Case 2 medical conditions, including congenital heart disease, lymphatic
malformations, gastrointestinal malformations, neonatal leukemia,
A 9-month-old Mennonite girl was referred to immunology for prenatal administration of glucocorticoids, and neonatal sepsis.25
assessment of recurrent oral thrush, chronic diarrhea, and failure to Premature infants can have T cell lymphopenia with low TRECs at
thrive. She was born at term at the 50th percentile for weight. She birth, which usually improves as they approach term age.25
had a negative newborn screen (NBS) result for severe combined However, a negative TREC screen result does not rule out the
immunodeficiency (SCID). She was growing and developing well possibility of PID.19,20,25 Because TRECs only examine T cells, PIDs
until 5 months of age and then developed recurrent oral thrush, limited to humoral immunity, natural killer (NK) cells, neutrophils,
chronic nonbloody diarrhea, growth deceleration, and a skin rash. or complement will not be detected by TREC. There are additional
There was no family history of immunodeficiency. She had negative circumstances in which TREC cannot identify disorders of T cell
human immunodeficiency virus test results. Complete blood cell function. FOXP3 deficiency, interleukin-10 receptor defects, partial
count and differential showed a normal lymphocyte count and adenosine deaminase deficiency, and some forms of interleukin-2
eosinophilia. receptor g-chain might not be detected by TREC.30,31 Similarly,
A history of thrush, chronic diarrhea, and failure to thrive raises genetic defects that affect T cell development after VDJ recombi-
a high index of suspicion for SCID and yet the NBS result was nation in the thymus can lead to normal TREC values but func-
normal. A number of tests will be considered, including reviewing tionally impaired T cells. These include MHC Class I deficiency and
the NBS results, lymphocyte immunophenotyping, T cell Vb CD40L deficiency.27 A child with z-chaineassociated protein ki-
repertoire analysis, functional testing of T cells, and genetic nase 70 (ZAP70) deficiency was recently reported to have normal
evaluation. TREC results as a newborn, which then decreased to undetectable
at 9 months of age.19 Children with ZAP70 deficiency have sig-
NBS Analysis of T Cell Receptor Excision Circles
nificant numbers of functionally abnormal CD4 T cells and mark-
T cell receptor excision circles (TRECs) are measured as part of edly decreased CD8þ T cells. As NBS continues, it is expected that
NBS programs for SCID.19,20 Introduced initially as a pilot program the range of conditions that are detected and are not detected by
in Wisconsin, NBS for SCID has been recommended by the USA TREC will increase.
Advisory Committee on Heritable Disorders.19 The number of
centers practicing NBS continues to increase, and a current list of
Lymphocyte Immunophenotyping and Protein Detection by Flow
states offering NBS can be found through the website of the
Cytometry
Immune Deficiency Foundation.21 In Canada, Ontario was the first
province to screen for SCID and more have followed.22 NBS aims to The assessment of lymphocyte subpopulations by flow cytom-
identify children before they develop symptoms of SCID to decrease etry can provide clues to the underlying diagnosis. It also can be
mortality through expedited diagnosis and treatment. used to enumerate regulatory cell populations when autoimmunity
T cell differentiation in the thymus is characterized by organized or immuno-dysregulation is suspected and memory and naïve
rearrangement steps in the T cell receptor (TCR) genes, resulting in phenotypes when evaluating suspected T cell disorders. Further-
the joining of the variable (V), diversity (D), and joining (J) gene more, flow cytometry can be used in a very directed way to ascer-
segments (VDJ recombination) to create a diverse repertoire of tain specific diagnoses by identifying missing proteins.14
unique TCRs with different antigen specificities.23 During each Patterns of low, normal, or high subsets of CD4, CD8, and NK
rearrangement step, DNA fragments are deleted as circular excision cells are well known to immunologists.11 For instance, low T cells
products, known as TRECs. TRECs persist within the cell as extra with normal B and NK cells are seen in disorders of TCR recombi-
genomic pieces of DNA and cannot replicate, therefore becoming nation, low T and NK cells with normal B cells are seen in classic
diluted as T cells proliferate.23 Analysis of TRECs provides an X-linked SCID because of mutations in interleukin-2 receptor
assessment of thymic function by indicating T cells that have g-chain, and low CD8þ cells are seen in ZAP70 and MHC Class II
recently emerged from the thymus.24 TRECs can be measured by deficiency. An extensive table of the patterns commonly seen in
polymerase chain reaction using DNA isolated from infant dried particular PIDs has been recently published.32
blood spots collected at NBS,25 although analytical methods vary The relative and absolute size of lymphocyte subpopulations
across different states. Any genetic defect that disrupts T cell varies with age.33 In a limited North American sample, sex, race,
660 S. Erdle et al. / Ann Allergy Asthma Immunol 118 (2017) 655e663
Table 3
Clinical Methods of Measuring Lymphocyte Proliferation
Method Details
Scintillation counter
(radioactive)
Label newly Incubate cells with a stimulant of cellular division and a
synthesized DNA radioactive nucleotide (tritiated thymidine) that
incorporates into the DNA of dividing cells
Flow cytometry
Label newly Incubate cells with a stimulant of cellular division and a
synthesized DNA label, such as BrdU or EdU, which incorporates into the
DNA of dividing cells, and then quantify proliferation by
labeling with a fluorescent tagged antibody (BrdU) or by
directly fluorescing the label (EdU)
Serial dilution of Label cells with the cytoplasmic fluorescent dye CFSE;
intracellular label stimulate cellular division and with each cell division
the label is diluted 50%; can determine the number of
generations of cells up to 8e10 divisions
Label cell markers Incubate cells with a stimulant of cellular division and
of division then label 1 cell surface marker, such as Ki-67,
indicative of cellular division
Figure 4. (A) Normal infant: multiple TRECS per total T cells, TCR diversity, naïve cells > memory cells, good proliferation. (B) Omenn syndrome: few TRECs per total T cells,
restricted repertoire, memory > naïve cells, poor proliferation. SCID, severe combined immunodeficiency; TCR, T cell receptor; TREC, T cell receptor excision circle.
not been as well studied as mitogen response,40 and the greater proliferation test results. In addition, patients could have a history
variability of control responses compared with mitogen responses of severe infections and yet have normal mitogen stimulation, as
makes this test more difficult to interpret.39 documented in some patients with CVID.39
Newborns generally have higher mitogen responses than Some clinically available laboratory techniques of measuring
adults.41 Values decrease to adult levels during childhood and then lymphocyte proliferation are presented in Table 3. Cellular prolif-
wane further with advancing age.40 Proliferation below the fifth eration can be evaluated by a radioactive method39,40 or by flow
percentile of healthy controls is generally used as a cutoff for sig- cytometric methods.44e47 A major benefit to flow cytometry-based
nificant functional impairment in the interpretation of mitogen and methods is that other cellular markers can be evaluated simulta-
antigen stimulation testing. Steroid use and concurrent infection neously. This allows for phenotyping of the proliferating cells.
also can lead to decreased responses.
Most non-SCID T cell defects have moderately depressed pro-
T Cell Receptor Vb Repertoire
liferation.39,40 Although most cases of SCID have markedly impaired
mitogen proliferation responses, there are some exceptions. Origi- T cell receptor Vb repertoire testing has been used to study T cell
nally described in 199742 and recently reviewed with 10 additional selective responses in conditions such as autoimmunity and pri-
cases,43 the R222C mutation of interleukin-2 receptor g-chain can mary immunodeficiency and after hematopoietic stem cell
lead to clinical SCID with normal or only mildly decreased mitogen transplantation.48
662 S. Erdle et al. / Ann Allergy Asthma Immunol 118 (2017) 655e663
The TCR repertoire characterizes collections of T cells into circulating CD3þ, CD4þ, CD19þ and CD56þ but a markedly
“families” based on their specific receptor (Fig 3). T cell differenti- decreased CD8þ count. T cell proliferation was low. Given this
ation in the thymus is characterized by organized rearrangement in presentation, in conjunction with the normal NBS result and the
the TCR genes, generating a diverse repertoire of unique TCRs with Mennonite heritage, ZAP70 could be evaluated by flow cytometry
different antigen specificities.24,49 A good TCR Vb repertoire is or genetic methods to confirm the diagnosis of ZAP70 deficiency.
necessary for an effective adaptive immune response against new
antigens.50 The TCR Vb repertoire can be measured by flow
cytometry or by polymerase chain reaction, which is referred to as Conclusion
spectratyping.51 The laboratory evaluation in the workup of T and B cell immu-
The Vb repertoire should be polyclonal, with each Vb family nodeficiencies has progressed significantly in recent years. NBS
demonstrating a Gaussian distribution.2 Restricted TCR Vb reper- identifies infants with SCID and we continue to learn about
toires can be seen in cases of poorly functional VDJ recombination. important limitations of this test. Flow cytometry allows for
For instance, in Omenn syndrome, the TCR Vb repertoire would detailed immunophenotyping and functional assays but cannot
show oligo-clonality and therefore a very distorted distribution.4,34 always discriminate disease states. Using multiple approaches to
The TREC, flow cytometry for memory and naïve cells, TCR Vb perform T cell proliferation allows this important test of function to
repertoire, and T cell proliferation are different ways of evaluating be more widely available than radioactive methods. Humoral im-
the T cell. High TRECs, high naïve cells, and a diverse repertoire munity evaluation has been recognized to be suboptimal in some
suggest good T cell generation. Figure 4 shows a synthesis of these cases and literature exists to support decision making. Genetic
tests and displays their expected results for Omenn syndrome. testing by NGS finds answers to previously unsolved cases of sus-
pected PID yet generates questions about new variants that in turn
Next-Generation Sequencing require functional testing to understand their clinical significance.
Traditionally, based on a patient’s phenotype, at least 1 candi- Many of these advanced tests are readily available to the practicing
date gene would be evaluated through Sanger sequencing, which is immunologist and their appropriate use and interpretation will
time consuming and often cannot identify a genetic diagnosis.52,53 continue to advance the diagnosis and care of the patient with PID.
In cases in which a phenotypic or genetic diagnosis is not apparent,
NGS is becoming increasingly popular.14e16,52,54 NGS is a rapid,
accurate, and relatively low-cost high-throughput sequencing References
technology used to identify novel disease-causing mutations [1] Bonilla FA, Khan DA, Ballas ZK, et al. Practice parameter for the diagnosis and
through targeted PID gene panels, WES, and whole-genome management of primary immunodeficiency. J Allergy Clin Immunol. 2015;136:
1186e1205.
sequencing.54,55
[2] Oliveira JB, Fleisher TA. Laboratory evaluation of primary immunodeficiencies.
Targeted PID gene panels evaluate specific genes or regions of J Allergy Clin Immunol. 2010;125(suppl 2):S297eS305.
interest. Gene panels have shown that known PIDs have more [3] Fried AJ, Bonilla FA. Pathogenesis, diagnosis, and management of primary
phenotypic variation than previously appreciated; a targeted gene antibody deficiencies and infections. Clin Microbiol Rev. 2009;22:396e414.
[4] Notarangelo LD. Primary immunodeficiencies. J Allergy Clin Immunol. 2010;
panel resulted in a genetic diagnosis for 35 of 139 patients consid- 125(suppl 2):S182eS194.
ered “unsolved cases,” most of which were phenotypic variants of [5] Bousfiha AA, Jeddane L, Ailal F, et al. A phenotypic approach for IUIS PID
known PIDs.56 However, in large cohort studies, PID panels could be classification and diagnosis: guidelines for clinicians at the bedside. J Clin
Immunol. 2013;33:1078e1087.
used to reach a genetic diagnosis in only approximately 15% of [6] Chou J, Hanna-Wakim R, Tirosh I. A novel homozygous mutation in recom-
patients.52,55 In addition, this technique cannot identify novel bination activating gene 2 in 2 relatives with different clinical phenotypes:
disease-causing genes.16 Therefore, an unbiased approach to eval- Omenn syndrome and hyper-IgM syndrome. J Allergy Clin Immunol. 2012;130:
1414e1416.
uating PID with WES is becoming increasingly popular.16 Of the [7] Upton J. Immunodeficiencies with hypergammaglobulinemia: a review.
entire genome, although only 1% (20,000e25,000 genes) represents Lymphosign J. 2015;2:57e73.
the exome, the exome contains 85% of the known disease-causing [8] Orange JS, Ballow M, Stiehm ER, et al. Use and interpretation of diagnostic
vaccination in primary immunodeficiency: a working group report of the
mutations, making WES relatively efficient and cost-effective.53
Basic and Clinical Immunology Interest Section of the American Academy of
There are some concerns with WES. Exome capture is designed Allergy, Asthma & Immunology. J Allergy Clin Immunol. 2012;130(suppl):
from known sequences and therefore has the potential to miss S1eS24.
[9] Ameratunga R, Brewerton M, Slade C, et al. Comparison of diagnostic criteria
poorly mapped areas.57 Therefore, WES is gradually being replaced
for common variable immunodeficiency disorder. Front Immunol. 2014;5:415.
with whole-genome sequencing, which provides better coverage of [10] Jolles S, Chapel H, Litzman J. When to initiate immunoglobulin replacement
exons, in addition to intronic sequences and regulatory regions. therapy (IGRT) in antibody deficiencyda practical approach [published online
Interpretation of these tests remains a constant challenge ahead of print December 2016]. Clin Exp Immunol. http://dx.doi.org/10.1111/
cei.12915.
because of the volume of information produced and individual [11] Fleisher TA, Madkaikar M, Rosenzweig SD. Application of flow cytometry in
variation.58 In addition, sequencing techniques can show medically the evaluation of primary immunodeficiencies. Indian J Pediatr. 2016;83:
important information about other genes and thus genetic testing 444e449.
[12] Wehr C, Kivioja T, Schmitt C, et al. The EUROclass trial: defining subgroups in
requires careful counseling and consent. common variable immunodeficiency. Blood. 2008;111:77e85.
The application of NGS to PID has resulted in a cost-effective [13] Wirsum C, Glaser C, Gutenberger S, Keller B, Unger S. Secondary antibody
diagnostic tool and a powerful research tool to look for multiple deficiency in glucocorticoid therapy clearly differs from primary antibody
deficiency. J Clin Immunol. 2016;36:406e412.
suspected genetic conditions at once and to discover new genetic [14] Abraham RS, Aubert G. Flow cytometry, a versatile tool for diagnosis and
causes of PID. In recent years, most new PID genes discovered have monitoring of primary immunodeficiencies. Clin Vaccine Immunol. 2016;23:
been using NGS, and each year at least 10 to 15 new genetic defects 254e271.
[15] van Schouwenburg PA, Davenport EE, Kienzler AK, et al. Application of whole
associated with PIDs are identified.16,55
genome and RNA sequencing to investigate the genomic landscape of com-
mon variable immunodeficiency disorders. Clin Immunol. 2015;160:301e314.
Case 2 Revisited [16] Meyts I, Bosch B, Bolze A, Boisson B, Itan Y. Exome and genome sequencing
for inborn errors of immunity. J Allergy Clin Immunol. 2016;138:957e969.
The clinical suspicion of SCID in this case was high given the [17] Dewey FE, Grove ME, Pan C, et al. Clinical interpretation and implications of
physical examination results described, even with the normal NBS whole-genome sequencing. JAMA. 2014;311:1035e1044.
[18] Maffucci P, Filion CA, Boisson B, et al. Genetic diagnosis using whole
result and no lymphopenia with complete blood cell counts. exome sequencing in common variable immunodeficiency. Front Immu-
Lymphocyte immunophenotyping showed normal numbers of nol. 2016;7:220.
S. Erdle et al. / Ann Allergy Asthma Immunol 118 (2017) 655e663 663
[19] Grazioli S, Bennett M, Hildebrand KJ, Vallance H, Turvey SE, Junker AK. Lim- [38] Janssen E, Tsitsikov E, Al-Herz W, et al. Flow cytometry biomarkers distin-
itation of TREC-based newborn screening for ZAP70 severe combined im- guish DOCK8 deficiency from severe atopic dermatitis. Clin Immunol. 2014;
munodeficiency. Clin Immunol. 2014;153:209e210. 150:220e224.
[20] Buckley RH. The long quest for neonatal screening for severe combined im- [39] Stone KD, Feldman HA, Huisman C, Howlett C, Jabara HH, Bonilla FA. Analysis
munodeficiency. J Allergy Clin Immunol. 2012;129:597e604. of in vitro lymphocyte proliferation as a screening tool for cellular immu-
[21] Immune Deficiency Foundation. SCID newborn screening: current status of nodeficiency. Clin Immunol. 2009;131:41e49.
implementation map. http://primaryimmune.org/wp-content/uploads/2016/ [40] Bonilla FA. Interpretation of lymphocyte proliferation tests. Ann Allergy
10/IDF-SCID-Map-10-01-2016-Large.png. Accessed January 22, 2017. Asthma Immunol. 2008;101:101e104.
[22] Newborn Screening Ontario. A first in Canada, Ontario newborns to be [41] Hicks MF, Jones JF, Thies AC, Weigle KA, Minnich LL. Age-related changes in
screened for fatal but curable genetic disorder. Screening for severe combined mitogen-induced lymphocyte function from birth to old age. Am J Clin Pathol.
immune deficiency (SCID) expected to save lives. http://immunodeficiency.ca/ 1983;80:159e163.
wp-content/uploads/2013/08/SCID-Press-Release-FINAL.pdf. Accessed January [42] Sharfe N, Shahar M, Roifman CM. An interleukin-2 receptor gamma chain
22, 2017. mutation with normal thymus morphology. J Clin Invest. 1997;100:
[23] Kwan A, Puck JM. History and current status of newborn screening for severe 3036e3043.
combined immunodeficiency. Semin Perinatol. 2015;39:194e205. [43] Fuchs S, Rensing-Ehl A, Erlacher M, et al. IL-2 receptor g chain deficiency have
[24] Six A, Mariotti-Ferrandiz ME, Chaara W, et al. The past, present, and future of differentially-impaired cytokine signaling resulting in severe combined im-
immune repertoire biologydthe rise of next-generation repertoire analysis. munodeficiency. Eur J Immunol. 2014;44:3129e3140.
Front Immunol. 2013;4:413. [44] Lyons AB. Analysing cell division in vivo and in vitro using flow cytometric
[25] Kwan A, Abraham RS, Currier R, et al. Newborn screening for severe combined measurement of CFSE dye dilution. J Immunol Methods. 2000;243:147e154.
immunodeficiency in 11 screening programs in the United States. JAMA. [45] Rosenzweig SD, Fleisher TA. Laboratory evaluation for T-cell dysfunction.
2014;312:729e738. J Allergy Clin Immunol. 2013;131:622e623.e4.
[26] Verbsky JW, Baker MW, Grossman WJ, et al. Newborn screening for severe [46] Yu Y, Arora A, Min W, Roifman CM, Grunebaum E. EdU incorporation is an
combined immunodeficiency; the Wisconsin experience (2008e2011). J Clin alternative non-radioactive assay to [3H]thymidine uptake for in vitro mea-
Immunol. 2012;32:82e88. surement of mice T-cell proliferations. J Immunol Methods. 2009;350:29e35.
[27] Roifman CM, Somech R, Kavadas F, et al. Defining combined immunodefi- [47] Lastovicka J, Rataj M, Bartunkova J. Assessment of lymphocyte proliferation
ciency. J Allergy Clin Immunol. 2012;130:177e183. for diagnostic purpose: comparison of CFSE staining, Ki-67 expression and
[28] Schroeder ML, Zelinski T, Al-Mousa H, et al. An infant with ZAP-70 deficiency 3H-thymidine incorporation. Hum Immunol. 2016;77:1215e1222.
with disseminated mycobacterial disease. J Allergy Clin Immunol. 2016;137: [48] McLean-Tooke A, Barge D, Spickett GP, Gennery AR. T cell receptor Vb
103e106. repertoire of T lymphocytes and T regulatory cells by flow cytometric analysis
[29] Mallott J, Kwan A, Church J, et al. Newborn screening for SCID identifies pa- in healthy children. Clin Exp Immunol. 2007;151:190e198.
tients with ataxia telangiectasia. J Clin Immunol. 2013;33:540e549. [49] Plotkin SA. Correlates of protection induced by vaccination. Clin Vaccine
[30] Walkovich K, Connelly JA. Seminars in fetal & neonatal medicine primary Immunol. 2010;17:1055e1065.
immunodeficiency in the neonate: early diagnosis and management. Semin [50] Britanova OV, Putintseva EV, Shugay M, et al. Age-related decrease in TCR
Fetal Neonatal Med. 2016;21:35e43. repertoire diversity measured with deep and normalized sequence profiling.
[31] la Marca G, Canessa C, Giocaliere E, Romano F, Duse M. Tandem mass spec- J Immunol. 2014;192:2689e2698.
trometry, but not T-cell receptor excision circle analysis, identifies newborns [51] Rosenzweig S, Fleisher T. Laboratory evaluation for T-cell dysfunction.
with late-onset adenosine deaminase deficiency. J Allergy Clin Immunol. 2013; J Allergy Clin Immunol. 2014;131:622e623.
131:1604e1610. [52] Stoddard JL, Niemela JE, Fleisher TA, Rosenzweig SD. Targeted NGS: a
[32] Picard C, Al-Herz W, Bousfiha A, Casanova J, Gaspar HB. Primary immuno- cost-effective approach to molecular diagnosis of PIDs. Front Immunol. 2014;
deficiency diseases: an update on the classification from the International 5:531.
Union of Immunological Societies Expert Committee for Primary Immuno- [53] Gallo V, Dotta L, Giardino G, et al. Diagnostics of primary immunodeficiencies
deficiency 2015. J Clin Immunol. 2015;35:696e726. through next-generation sequencing. Front Immunol. 2016;7:466.
[33] Shearer WT, Rosenblatt HM, Gelman RS, et al. Lymphocyte subsets in healthy [54] Mousallem T, Urban TJ, McSweeney KM, et al. Clinical application of whole-
children from birth through 18 years of age: The pediatric AIDS clinical trials genome sequencing in patients with primary immunodeficiency. J Allergy
group P1009 study. J Allergy Clin Immunol. 2003;112:973e980. Clin Immunol. 2015;136:476e479.
[34] Villa A, Notarangelo LD, Roifman CM. Omenn syndrome: Inflammation in [55] Nijman IJ, Van Montfrans JM, Hoogstraat M, et al. Targeted next-generation
leaky severe combined immunodeficiency. J Allergy Clin Immunol. 2008;122: sequencing: a novel diagnostic tool for primary immunodeficiencies.
1082e1086. J Allergy Clin Immunol. 2014;133:529e534.e1.
[35] Matthews AG, Briggs CE, Yamanaka K, et al. Compound heterozygous muta- [56] Al-Mousa H, Abouelhoda M, Monies D. Unbiased targeted next-generation
tion of Rag1 leading to Omenn syndrome. PLoS One. 2015;10:e0121489. sequencing molecular approach for primary immunodeficiency diseases.
[36] Saijo S, Ikeda S, Yamabe K, Kakuta S, Ishigame H. Dectin-2 recognition of J Allergy Clin Immunol. 2016;137:1780e1787.
a-mannans and induction of Th17 cell differentiation is essential for host [57] Chou J, Ohsumi TK, Geha RS. Use of whole exome and genome sequencing in
defense against Candida albicans. Immunity. 2010;32:681e691. the identification of genetic causes of primary immunodeficiencies. Curr Opin
[37] Pellerin L, Jenks JA, Bégin P, Bacchetta R, Nadeau KC. Regulatory T cells and Allergy Clin Immunol. 2012;12:623e628.
their roles in immune dysregulation and allergy. Immunol Res. 2014;58: [58] Conley ME, Casanova JL. Discovery of single-gene inborn errors of immunity
358e368. by next generation sequencing. Curr Opin Immunol. 2014;30:17e23.