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Ann Allergy Asthma Immunol 118 (2017) 655e663

Contents lists available at ScienceDirect

CME Review

Advanced clinical testing of the adaptive immune system


Stephanie Erdle, MD *; Anne K. Ellis, MD, FRCP(C) y; Julia E.M. Upton, MD, FRCP(C) z
* Department of Paediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada
y
Division of Allergy and Immunology, Department of Medicine, Queen’s University, Kingston, Ontario, Canada
z
Division of Immunology and Allergy, Department of Pediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada

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

Mitchell H. Grayson, MD (CME Series Editor, Deputy Editor)


Gailen D. Marshall, Jr, MD, PhD (Editor-in-Chief)
Disclosure Policy
As required by the Accreditation Council for Continuing Medical Education (ACCME) and in accordance with the American College of
Allergy, Asthma and Immunology (ACAAI) policy, all educational planners, presenters, instructors, moderators, authors, reviewers, and
other individuals in a position to control or influence the content of an activity must disclose all relevant financial relationships with any
commercial interest that have occurred within the past 12 months. All identified conflicts of interest must be resolved and the
educational content thoroughly vetted for fair balance, scientific objectivity, and appropriateness of patient care recommendations. It is
required that disclosure be provided to the learners prior to the start of the activity. Individuals with no relevant financial relationships
must also inform the learners that no relevant financial relationships exist. Learners must also be informed when off-label, experimental/
investigational uses of drugs or devices are discussed in an educational activity or included in related materials. Disclosure in no way
implies that the information presented is biased or of lesser quality. It is incumbent upon course participants to be aware of these factors
in interpreting the program contents and evaluating recommendations. Moreover, expressed views do not necessarily reflect the
opinions of ACAAI.
All identified conflicts of interest have been resolved.
Disclosure of Relevant Financial Relationships:
A. K. Ellis, S. Erdle, J. E. M. Upton have no relevant financial relationships to disclose. J.A. Bernstein e PI, Consultant, Speaker for
AstraZeneca, CSL Behring, Novartis/Genentech, and Shire, he received research grants and fees; Speaker for Baxalta, he received fees;
Consultant for Imedics, he received fees; PI and Consultant for Boehringer Ingelheim and GlaxoSmithKline, he received research grants
and fees. G. Krishnaswamy e Clinical Research for CSL Behring, he received a research grant.
J. Oppenheimer e Consultant for DBV Technologies, GlaxoSmithKline, and Kaleo, he received other financial gains; Clinical research for
AstraZeneca, Boehringer Ingelheim, and Novartis, he received research grants. M.H. Grayson e Clinical research for Polyphor, Ltd., he
received a research grant. G.D. Marshall e Clinical Research for Stallergens and Sanofi, he received research grants. Any unapproved/
investigative uses of therapeutic agents/devices discussed are appropriately noted.
Recognition of Commercial Support: This activity has not received external commercial support.
Copyright Statement: Copyright Ó 2017 American College of Allergy, Asthma & Immunology. All rights reserved.
CME Inquiries: Contact the American College of Allergy, Asthma & Immunology at [email protected] or 847-427-1200.

Introduction evaluation. We will discuss the application and interpretation of


immunoglobulin levels, vaccine titers, and flow cytometry for B cell
Primary immunodeficiencies (PIDs), although rare, collectively
phenotyping and protein detection and genetic evaluation of hu-
occur in as many as 1 of 2,000 live births and can result in recurrent
moral immunodeficiency.
and/or severe infections, autoimmunity, or autoinflammation.1
Immunodeficiencies often present with overlapping signs and
Immunoglobulin Levels
symptoms, making knowledge and application of available labo-
ratory investigations critical to confirm a diagnosis or for direct Quantitative measurement of immunoglobulin levels is routine
additional testing. This article explores the indications, methodol- in cases of suspected antibody deficiency and is very familiar to the
ogy, and interpretation of clinically available tests used in the allergist and immunologist. Serum immunoglobulin levels are
evaluation of T and B cell number and function and provides an interpreted according to age-matched reference intervals in
approach to using and understanding the tests performed by im- the pediatric population.2,3 During the first months of life, IgGs
munologists once a clinical suspicion of T and B cell immunodefi- are predominantly maternal in origin.3,4 IgG subclasses can be
ciencies exists (Fig 1 and Table 1). measured, although this is controversial and not routinely
recommended.1
Illustrative Case 1 The pattern of immunoglobulins can aid in diagnosis5 but
occasionally can be misleading. For example, most patients with
A 13-year-old boy was referred for further immune evaluation. He
increased IgE do not have a genetic hyper-IgE syndrome and would
has had 3 radiologically proven pneumonias during the past 3 years.
be far more likely to have atopic disease or a parasitic infection. In
He had been diagnosed previously by hematology with idiopathic
addition, it was recently reported that a patient with a
thrombocytopenia since early childhood. He has received many
recombination-activating gene (RAG) 2 mutation had a very high
steroid treatments for treatment of idiopathic thrombocytopenia,
IgM level and was initially misdiagnosed as having hyper-IgM
although none recently. Immunizations were up to date. There was
syndrome.6 It also has been reported that patients can have
no family history of immunodeficiency or autoimmunity. Physical
severe immunodeficiencies with increased, rather than decreased,
examination was significant for splenomegaly. Basic blood work,
IgG levels.7
including complete blood cell count and differential, C-reactive
protein, renal and hepatic function, and total protein and albumin,
Vaccine Titers (Tetanus or Diphtheria and Pneumococcus)
showed normal values. Immunoglobulin (Ig) G, IgA, and IgM were
slightly low and tetanus titer showed a low protective level. Antibody function can be examined by evaluating a patient’s
Lymphocyte immunophenotyping showed low but present CD19þ response to 2 general types of antigens: protein antigens (T-
and CD20þ B cells, with normal CD3þ, CD4þ, and CD8þ levels. dependent) and polysaccharide antigens (T-independent).8
Humoral immunodeficiencies typically present with recurrent Antibodies to tetanus or diphtheria toxoid are commonly used
bacterial infections of the sinopulmonary tract and can have other to measure the antibody response to protein antigens.4 If basal
features such as autoimmunity. Determining a diagnosis of hu- antibody titers are low or non-protective, a boosting immunization
moral immunodeficiency and whether a patient requires immu- can be performed with repeat antibody levels obtained 4 to 6
noglobulin replacement is easy in the extremes but often difficult weeks later.4,8
when results are mixed, such as when a patient has only a slightly The unconjugated pneumococcal polysaccharide vaccine is used
low IgG, has mixed antibody responses, or if treatments hinder the to test the antibody response to polysaccharide antigens in
S. Erdle et al. / Ann Allergy Asthma Immunol 118 (2017) 655e663 657

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

Immunodeficiency type Clinical clues Initial evaluation Advanced testing

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

Absent or extremely decreased B cell numbers are usually sec-


ondary to a block in B cell development.1 Other immunodefi-
ciencies affecting the humoral system can have low or normal
B cells and it can be difficult to distinguish primary from secondary
PID. Immunophenotyping techniques were recently applied to
differentiate secondary hypogammaglobulinemia from CVID. The
investigators reported a maintenance of the memory B cell popu-
lation in patients undergoing high-dose steroid treatment, which
was in contrast to changes seen in a CVID population.13 Results from
the EUROclass trial12 suggested that memory phenotyping, espe-
cially for switched memory subsets (IgD-IgM-CD27þ), could be
helpful to phenotype patients with CVID. Although these results are
encouraging, phenotyping has not allowed for robust treatment
decisions and therefore many physicians still rely on clinical in-
formation and vaccine responses.10
Figure 2. Titers to individual serotypes of 23-valent pneumococcal polysaccharide
After the initial evaluation of humoral immunodeficiency, if a
vaccine. Titers can boost poorly, increase and then decrease quickly, or be sustained. specific condition is suspected, then this can be targeted by disease-
Analysis of multiple serotypes classifies the response (severe, moderate, mild, specific flow cytometric methods. Flow cytometry can diagnose
memory, or normal). X-linked agammaglobulinemia, X-linked hyper-IgM syndrome
(CD40 ligand [CD40L] deficiency), and lipopolysaccharide-
responsive beige-like anchor (LRBA) deficiency, among others
concerning which vaccines should be assessed, protocol vari- (Table 2).14 Furthermore, detection of protein is useful to assess for
ability, protective titers, and the importance of differentiating a genotype-phenotype correlation in cases in which genetic testing
what is a protective vs a normal response to vaccination. In shows a variant of uncertain clinical significance.14
addition, many patients with CVID would have generated
vaccine-specific memory B cells before the development of their
late-onset antibody dysfunction, making protective antibody Genetic Evaluation
levels misleading. A recent discussion of the difficulty in deter-
mining when a patient needs immunoglobulin replacement Until recently, genetic evaluation for humoral PIDs was largely
outlined the use of reimmunization and the importance of used when there was a clear phenotype suspected, such as testing
clinical factors to decide treatment. It highlighted that sometimes for CD40L in patients with hypogammaglobinemia and high or
the “test” used in the humoral evaluations is a trial of antibiotic normal IgM or for identifying a transplantable condition such as
prophylaxis or immunoglobulin replacement because laboratory X-linked lymphoproliferative disease. The heterogeneity in phe-
testing is not always sufficient to differentiate patients.10 notypes made identifying new underlying genetic mutations
Overall, the evaluation of humoral immunodeficiency remains challenging.15 There were few genetic conditions known to account
suboptimal. for CVID. Next-generation sequencing (NGS) approaches allow for
more molecular diagnoses in this population.
The methodology of NGS has recently been reviewed in detail.16
Briefly, NGS involves sequencing hundreds of millions of small DNA
Lymphocyte Immunophenotyping by Flow Cytometry
fragments in parallel. The sequencing data are compared with those
In the evaluation of humoral immunodeficiency, the presence or in a public database to filter out high-frequency polymorphisms
absence of B cells can be established using flow cytometry, usually that are unlikely to cause disease. This can be used to perform
by evaluating the CD19þ subpopulation of lymphocytes. Flow targeted PID gene panels, whole-exome sequencing (WES), or
cytometry uses monoclonal antibodies attached to fluorescent tags whole-genome sequencing.16
to identify cell populations that have cell surface markers called Findings are classified as “disease-causing mutations,” “likely
clusters of differentiation (CDs). Some laboratories can further disease-causing mutations,” “disease-associated functional poly-
identify naïve cells and memory B cells; for example, memory B morphisms,” “disease-associated polymorphisms,” “functional
cells can be identified by CD27þ.11 The B cells also can be pheno- polymorphisms,” or a “frameshift or truncating variant.”17 Therefore,
typed by which antibodies are on their surface and other markers.12 results are not always clearly pathologic or benign.

Table 2
Select Disease-specific Flow Cytometry Testing of Cellular, Humoral, and Combined Immunodeficiencies

Immunodeficiency Clinical presentation Flow cytometry test

X-linked agammaglobulinemia Humoral immunodeficiency Bruton tyrosine kinase protein expression


X-linked lymphoproliferative syndrome Humoral immunodeficiency; exaggerated Epstein-Barr infection Signaling lymphocyte activation moleculeeassociated
protein expression
Wiskott-Aldrich syndrome Thrombocytopenia, eczema, recurrent infections with encapsulated Wiskott-Aldrich syndrome protein expression
organisms
DOCK8 Cellular immunodeficiency; allergy DOCK8 protein expression
ZAP70 Cellular immunodeficiency; severe combined immunodeficiency ZAP70 protein expression
LRBA deficiency Autoimmunity; inflammatory bowel disease; variable humoral and Flow cytometry for LRBA protein
cellular immunodeficiency
CD40 ligand deficiency Combined humoral and cellular immunodeficiency Flow cytometry for expression of CD40 ligand (CD154);
CD40 ligand function

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

Abbreviations: BrdU, 5-bromo-20 -deoxyuridine; CFSE, carboxy fluorescein


diacetate; EDU, 5-ethynyl-20 -deoxyuridine.

and ethnic background were not shown to significantly affect


lymphocyte subpopulations.33 Ideally, immunophenotyping should
be performed when the patient is clinically stable, because in-
fections or immunosuppressive treatment can affect results.
Lymphocyte subpopulations can be normal even with signifi-
cant immune dysfunction. In patients with Omenn syndrome, a
form of leaky SCID in which some T cells develop, expand oligo-
clonally, and are poorly functional, flow cytometry can be
misleading because the proportion and number of CD3þ T cells can
be normal.34 The number of CD4þ and CD8þ cells can vary and also
be normal.34 Oligoclonal expansion of T cells leads to a limited di-
versity and profound cellular immunodeficiency.34 Lymphocyte
immunophenotyping for naïve and memory populations will be
abnormal. A healthy infant should have primarily naïve CD45RAþ
cells,2,4 whereas a patient with Omenn syndrome might have oli-
goclonal T cell expansion with a skewed distribution of mature
memory CD45ROþ cells and limited naïve T cells.35 Figure 3. Progressive evaluation of T cell quantity. From top to bottom, lymphocytes
Other important cell populations that can be identified by flow are enumerated morphologically, by the presence/absence of receptors and then by
cytometry include positive TH17 cells, which are particularly their antigen receptor specificity. CBC, complete blood cell count; Hgb, hemoglobin;
important in candida defense,36 and regulatory T cell populations, Plt, plate count; TCR, T cell receptor; WBC, white blood cell count.
of which the best characterized are those of thymic origin and can
be identified by the cell surface markers CD25, CTLA4, and FOXP3.37
However, for some proteins, expression and function can be eval-
The role of regulatory T cells in immunologic and allergic diseases
uated. For example, flow cytometry can accurately evaluate the
was recently reviewed,37 highlighting their important role in
expression and function of CD40L, providing more rapid results for
autoimmunity and tolerance of foreign proteins.
CD40L deficiency than genetic testing.14
Immunophenotyping using memory or regulatory markers
could have an increasing role in differentiating diseases. For
T Cell Functional Testing: T Cell Proliferation to Mitogens and
example, it would be helpful to identify the immunodeficiencies
Antigens
associated with eczema-like presentations from those of atopic
dermatitis. Recently, it was recognized that flow cytometric bio- T cell functional testing is beneficial in a number of circum-
markers could help differentiate dedicator of cytokinesis 8 (DOCK8) stances, including the evaluation of patients with suspected
deficiency from severe atopic dermatitis, thereby identifying pa- immunodeficiency and to evaluate for reconstitution after
tients who would benefit from further specialized diagnostic hematopoietic stem cell transplantation.39
testing.38 A profile of low CD3þ and CD4þ T cells and decreased T cell proliferation is a surrogate of T cell function because T cells
naïve CD8þ T cells with a normal total B cell percentage and need to be able to replicate to mount an effective immune response.
decreased memory B cells was strongly suggestive of DOCK8 defi- Mitogens use the intrinsic mechanisms of the T cell to cause pro-
ciency rather than atopic dermatitis. liferation independent of T cell specificity. The most common
In addition to enumerating subsets of cells, flow cytometry can mitogen used is plant lectin phytohemagglutinin.40 Concanavalin A
detect cell surface and intracellular proteins.14 Flow cytometric and pokeweed are other mitogens used in some centers.40 Antigen
detection methods, rather than genetic testing in DOCK8 defi- stimulation relies on a memory T cell population and thus the pa-
ciency, can be particularly useful given the large size of the gene.14 tient needs to have previously encountered the antigen. Common
Detection of the protein does not rule out disease because the antigens used to assess T cell function are tetanus toxoids or
protein might be present but poorly functional or nonfunctional. Candida albicans. The variability of antigen responses with age has
S. Erdle et al. / Ann Allergy Asthma Immunol 118 (2017) 655e663 661

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,
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