Laboratory Diagnosis of

Primary Immunodeficiencies

Suda Sibunruang, M.D.

 Outline
• Introduction
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer and Cytotoxic T-Cell Defects
• Adaptive-Innate Immunity Defects
• Disorders of Complement System
• Immune Dysregulation Disorders
Primary Immunodeficiencies (PID)
• Heterogeneous group of disorders
• Careful history to delineate the pattern
of infectious organisms and other
complications is important to guide
the workup
• Then, focused laboratory evaluation is
essential to the diagnosis

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Ochs HD et al. Ann Allergy Asthma Immunol 2014;112: 489-95
Oliveira JB, Fleisher TA. J Allergy Clin Immunol 2010;125:S297-305
 Translating basic science findings
to patients
• Recent advancements in immunology have
led to the development of novel
immunologic assays, thus providing
improvements in the diagnosis and
treatment of PID

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Immunologic assays
• Flow cytometric-based assays
- Immune cell function
(e.g., neutrophil oxidative burst, NK cytotoxicity)
- Intracellular cytokine production (e.g., Th17)
- Cellular signaling pathways
(e.g., phosphorylated-STAT analysis)
- Protein expression (e.g., BTK, Foxp3)
• Genetic testing
• Mass sequencing technologies

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 In order to utilize these
complex assays…
One must have…
• Firm understanding of the immunologic
assay
• How the results are interpreted
• Pitfalls in the assays
• How the test affects treatment decisions

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Scope
• Systematic approach of the evaluation
of a suspected PID
• Comprehensive list of testing options
and their results

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer and Cytotoxic T-Cell Defects
• Adaptive-Innate Immunity Defects
• Disorders of Complement System
• Immune Dysregulation Disorders
Disorders of Humoral Immunity

50–60 % of all identified primary immunodeficiencies

Ballow M. Global Atlas of Allergy 2014; 248-50

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
It is important that the results of IgG levels be
compared to age-adjusted normal values

Agarwal S. et al. Ann Allergy Asthma Immunol 2007;99:281-3

 Definition
Hypogammaglobulinemia
• IgG value is less than 2 SD below
age-adjusted normal
Agammaglobulinemia
• IgG of less than 100 mg/dL
If either of these findings is found, then further
immunologic workup should be pursued
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Orange JS. J Allergy Clin Immunol 2012;130:S1-24
Specific antibody response to vaccination

• If any serum vaccination titers are below

normal, revaccination and assessment of
titers 4–6 weeks later is warranted

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 There is controversy regarding “normal” response
to vaccination, particularly to polysaccharide vaccine

Orange JS. J Allergy Clin Immunol 2012;130:S1-24

Criteria for adequate vaccination response

• A measured protective titer per lab normals

• A 4-fold increase in post-vaccination
compared to pre-vaccination titer
• A measured response to >50 % of
polysaccharide serotypes tested from
ages 2–5 years old or a response of >70 %
in patients > 5 years of age

Paris K and Sorensen RU. Ann Allergy Asthma Immunol 2007;99:462–4

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Validation of current joint American Academy of Allergy, Asthma & Immunology and American
College of Allergy, Asthma and Immunology guidelines for antibody response to the 23-valent
pneumococcal vaccine using a population of HIV-infected children

• Using individual post-PPV

titer of ≥4-fold increase over
pre-vaccination as a positive
response, yielded the highest
accuracy as measured by AUC
• Numbers of serotypes with
positive responses of ≤5 of 12
serotypes measured yielded
72.7% sensitivity and
56.8% specificity in detecting
antibody-deficient subjects
• The minimal positive responses
should be at least 50% of serotypes
tested

Kamchaisatian W. et al. J Allergy Clin Immunol 2006;118:1336-41

 Howabout individuals receiving immunoglobulin therapy ?
Since immunoglobulin contain detectable titers to common vaccines

(Keyhole limpet hemocyanin)

Orange JS. J Allergy Clin Immunol 2012;130:S1-24

Natural IgM antibodies; Blood group antibodies

Titer > 1:8 in age above 1 year

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Common Variable Immunodeficiency (CVID)

Park MA et al. Lancet 2008;372: 489–502

 Diagnosis of CVID
ESID/Pan-American Group for Immunodeficiency (PAGID) criteria
• male or female patient with a marked decrease in IgG
levels (>2 SDs less than the mean for age)
and a marked decrease in levels of at least 1 of
the isotypes IgM or IgA
and fulfilling all of the following criteria:
• 1. onset of immunodeficiency at greater than 4 years
of age;
• 2. absent isohemagglutinins, poor response to
vaccines, or both
• 3. exclusion of defined causes of
hypogammaglobulinemia.
Conley M. et al. Clin Immunol 1999;93:190-7
www.esid.org/clinicalsummarymeeting-
on-how-to-update-diagnostic-criteria-in-pid-368-0
Park MA et al. Lancet 2008;372: 489–502
Park MA et al. Lancet 2008;372: 489–502
Park MA et al. Lancet 2008;372: 489–502
 Unswitched memory
Switch memory B
cells (IgD- CD27+) B cells (IgD+ CD27+)

Naïve B cells Low numbers of

(IgD+ CD27-) switch memory B cells

Flow cytometry can be used to evaluate

maturational state of B lymphocytes
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Memory T-cell compartment can also demonstrate abnormalities,
with a reduced CD4+/CD8+ ratio and diminished percentage
of CD4+ Tcells expressing CD45RA

Baumert E et al. Clin Exp Immunol 1992;90:25–30

Wehr C. et al. Blood 2008;111:77–85
Classification of CVID by B-cell phenotyping (EUROclass)

Wehr C. et al. Blood 2008;111:77–85

CD21- B cells is seen in patients who are
more likely to develop autoimmunity
Isnardi I. et al. Blood 2010;115:5026-36
Rosel AL et al. J Allergy Clin Immunol 2015;135:198-208
 Transmembrane activator and calcium-modulating
cyclophilin ligand interactor (TACI)

(Inducible T-cell co-stimulator)

Park MA et al. Lancet 2008;372: 489–502

Receptors for BAFF and APRIL control
B cell development and function

TNFR family members

- BR3 or BAFFR
- TACI
- BCMA

TNF-like ligands
- BAFF or BLys
- APRIL

Martin F and Dixit VM. Nat Genet 2005;37:829–34

Calcium modulator and cyclophilin ligand (CAML)

TACI signals intracellularly through

TNF receptor-associated factors (TRAF)
to induce nuclear factor-κ-B activation

Park MA et al. Lancet 2008;372: 489–502

Park MA et al. Lancet 2008;372: 489–502
 Transmembrane activator and calcium-modulating
cyclophilin ligand interactor (TACI)

(Inducible T-cell co-stimulator)

Flow cytometry can be used to demonstrate reduced

expression of protein markers TACI, CD19, and BAFFR,
but this should be paired with genetic evaluation

Park MA et al. Lancet 2008;372: 489–502

 Laboratory investigations for CVID in the patients
who are HIV-negative in whom other causes of recurrent
infections have been ruled out (1)

Park MA et al. Lancet 2008;372: 489–502

 Laboratory investigations for CVID in the patients
who are HIV-negative in whom other causes of recurrent
infections have been ruled out (2)

Park MA et al. Lancet 2008;372: 489–502

X-Linked Agammaglobulinemia

Genetic defect in Bruton’s Tyrosine Kinase (BTK) gene

Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92
Ponader S. and Burger JA. J Clin Oncol 2014;32:1830-9
X-Linked Agammaglobulinemia

• Low levels of IgG, IgA, and IgM

• Absent immunization titers
• Low/absent circulating B cell population with normal T-cell counts

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92
Absent B cell, T cell & NK cell - present de Vries E. et al. Eur J Pediatr 2001;160:583–91
Few B cell but mostly immature B cell (CD10+, CD19+,CD20-) de Vries E. et al. Eur J Pediatr 2001;160:583–91
Intracellular expression of BTK in monocytes

This modality does not pick up

all mutations, only those that
affect protein stability.
For this reason, it is recommended
Detection of monocytes and B cells to perform BTK sequence analysis

BTK expression in monocytes

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Baker MW et. al. Public Health Rep 2010;125:S88–95
T-cell receptor excision circles Kappa-deleting recombination excision circles

Circular DNA segments generated in T and B cells

during their maturation in the thymus and bone marrow

Serana F. et al. Journal of Translational Medicine 2013;11:119

PCR of newborn blood spots to quantitate
these non-replicative DNA elements

Nijmegen-breakage
-syndrome

TREC and KREC copy numbers in dried blood spot

samples (DBSS) from anonymized Guthrie cards
Borte S et. al. Blood 2012;119:2552–5
Ataxia telangiectasia (AT)

Multisystem syndrome
- Results from mutation of ATM
(ataxia telangiectasia,mutated)
- Hallmark: debilitating progressive
neurodegeneration
Other characteristics
- Ocular and facial telangiectasia
- Immunodeficiency
- Extreme radiosensitivity
- Sterility
- Predisposition to cancer
(haematopoietic malignancy)

McKinnon PJ. EMBO reports 2004;5:772–6

 ATM
• A protein kinase that responds to
DNA damage
• Controls cell-cycle checkpoints

McKinnon PJ. EMBO reports 2004;5:772–6

Stracker TH et al. Front Genet 2013;25:1-54
• ATM in inactive multimeric complex
undergoes autophosphorylation to
active monomer
• Histone H2AX, present within chromatin,
becomes phosphorylated and serves as
tethering platform for repair factors
• MRE11–RAD50–NBS1 complex
and BRCA1 locates to the DNA lesion
• Assembly of this complex facilitates
coordinated co-localization of
active ATM together with other factors

McKinnon PJ. EMBO reports 2004;5:772–6

Slatter MA. Expert Rev Mol Med 2010 Mar 18;12:e9
In humans, approximately 10–15 %
of histone H2A is made up of H2AX.
After exposure to ionizing radiation,
DNA repair mechanisms induce
phosphorylation of H2AX to γ-H2AX.
Due to ATM gene defects in patients
with AT, they do not recognize DNA defects
and thus do not phosphorylate H2AX.

McKinnon PJ. EMBO reports 2004;5:772–6

Slatter MA. Expert Rev Mol Med 2010 Mar 18;12:e9
Stracker TH et al. Front Genet 2013;25:1-54
 Ataxia-Telangiectasia
• Markedly decreased serum
immunoglobulins and poor cell mediated
responses
• Sensitivity to γ-radiation which normally
disrupts cell cycle checkpoints and induces
DNA repair mechanisms
• Radiation sensitivity testing has a long
turnaround time, is not widely available,
and abnormal results are not specific for AT
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Serum alpha-fetoprotein

Waldmann TA McIntire KR. Lancet 1972;2:1112-5

 Laboratory
• Flow cytometry
- Alterations of ATM protein or
phosphorylated histone H2AX
- γ-H2AX in T-cell lines, lymphoblastoid
cell lines, and PBMC
- Increased γδ T-cell
- Low number of CD4+/CD45RA+
• KREC analysis

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Slatter MA. Expert Rev Mol Med 2010 Mar 18;12:e9
 Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer and Cytotoxic T-Cell Defects
• Adaptive-Innate Immunity Defects
• Disorders of Complement System
• Immune Dysregulation Disorders
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Absolute lymphocyte count (ALC)
• Compared to age-adjusted normal since
infants have much higher lymphocyte
count than adults
• If presented with decreasing
lymphocyte count, possible HIV
infection must be evaluated

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Transplacentally acquired
maternal T lymphocytes
• Low T cells are typically observed in majority
of defects in T-cell development
• However, this may be masked due to
transplacental transfer of maternal T
lymphocytes
• Typically, maternal T cells will display a
memory (CD45RO+) phenotype, whereas a
healthy infect should have predominantly
naïve CD45RA+ T cells

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Measures a cellular mediated memory
response to a previously seen antigen

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Some caveats for DTH testing
• Requires that there has been previous exposure
to antigen
• Not recommended to perform on children
< 1 year of age as they are frequently
unresponsive due to immunologic maturity
• Various infections and medications can result
in falsely negative
• Positive test to some antigens does not ensure
normal cellular immunity to all antigens

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Ahmed AR and Blose DA. Arch Dermatol 1983;119:934-45
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
 Common mitogens used
• Phytohemagglutinin (PHA) induce response
• Concanavalin (ConA) in T cells
• Anti-CD3 antibodies
• Pokeweed (PWM) - stimulate both T & B cells
• Escherichia coli lipopolysaccharide (LPS) –
stimulates only B cells

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
TREC are surrogate marker of naïve T cells
Although this is typically used for newborn screening,
patients with T-cell defects can also have low TRECs at any age

Serana F. et al. Journal of Translational Medicine 2013;11:119

T-cell repertoire analysis
• In normal individuals, TCR repertoire is stable
and polyclonal
• Clonal populations are the hallmark of
malignancy
• Clonal or oligoclonal populations of T and B
cells may occur in non-malignant conditions
including: HIV and EBV infections with
specificity for virus, elderly, autoimmunity,
CVID, SCID, Omenn’s SCID variant
Hodges E et al. J Clin Pathol 2003;56:1–11
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Flow cytometric T cell receptor  variable gene (TCRVB) profiles

% V positive T cells
Healthy

% V positive T cells
T cell
lymphoma

Hodges E et al. J Clin Pathol 2003;56:1–11

Pilch H et al. Clin Diagn Lab Immunol 2002;9:257–66
Severe Combined Immunodeficiency (SCID)

Durandy A, Kracker S, Fischer A. Nat Rev Immunol 2013;13:519-33

Flow cytometry aids in diagnosis of SCID

Confirmation of SCID
requires sequence
analysis of
suspected genes

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Predominant CD45RO+
Hypomorphic mutations in the RAG1 or RAG2 Increased % of γδ T cells
genes, but can be observed with other gene defects

Kato M. et al. Allergology International 2006;55:115-9

Clinical characteristics
• Erythroderma
• Eosinophilia
• Elevated IgE levels
• Lymphadenopathy
• Hepatosplenomegaly

Gennery AR, Cant AJ. J Clin Pathol 2001;54:191–5

 Normal CD4+ CD45RA/RO+ profile Skewed CD4+ CD45RA/RO+ profile

T-cell repertoire analysis will show a restricted pattern of T-cell receptors

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Major Histocompatibility Complex
Class II Deficiency
• Rare autosomal recessive disease
• Loss of expression of MHC class II proteins
• These proteins are normally found on APC
and thymic epithelium and are required for
development of CD4+ T cells
• MHC class I protein expression and TCR
expression is typically preserved

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Immunophenotype of
MHC Class II Deficiency

• Normal numbers of both CD8+ T cells and

B cells, with reduced or absent CD4+ numbers
• B cells express high levels of IgM and IgD,
with no detectable MHC class II proteins
(HLA-DR, HLA-DP, HLA-DQ, or HLA-DM)

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Wiskott-Aldrich Syndrome (WAS)

Rare X-linked recessive disease caused by mutation in WAS gene

Variable clinical phenotypes that correlate with type of mutations
in WAS protein (WASP) gene Picture from www.ufrgs.br, access 12 Feb 2015
Nonsense mutations, insertions, deletions, and complex
mutations are distributed throughout the WAS gene
and result in WAS

Massaad MJ et. al. Ann N Y Acad Sci 2013 May;1285:26-43

 WASP
• Key regulator of actin polymerization in
hematopoietic cells
• Involve in signaling, cell locomotion, and
immune synapse formation
• Facilitate nuclear translocation of nuclear
factor kB
• Play an important role in lymphoid
development and in the maturation and
function of myeloid monocytic cells

Ochs HD, Thrasher AJ. J Allergy Clin Immunol 2006;117:725-38

Ochs HD, Thrasher AJ. J Allergy Clin Immunol 2006;117:725-38
Flow cytometric analysis of WASP expression
Yamada M. et al. Blood 1999;93:756-9
 Wiskott-Aldrich Syndrome
• Presence of WASp, however, does not
exclude the diagnosis, and sequencing
analysis should be sent if WAS is suspected
despite normal protein expression
• There has been reported WASp mutation
reversions in 11 % of WAS patients whose
cells were previously WASp-negative
• Possibility of gene therapy as a potential
treatment option

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

DiGeorge Syndrome

22q11 deletion syndrome

- Thymic aplasia/hypoplasia
- Decreased T cells

Picture from www.diseasesforum.com

Access 12 February 2015
Maggadottir SM, Sullivan KE. J Allergy Clin Immunol Pract 2013;1:589-94
 Flow cytometry
Decrease
• CD3+, CD4+, andCD8+ T cells
• Αβ T cells
Normal numbers
• γδ T cells

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Fluorescent in situ hybridization (FISH)
utilizing a HIRA (TUPLE1) probe

False-negative rate of 5 % Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Picture from www.mcqs.leedsmedics.org.uk
Multiplexed quantitative real-time PCR to detect 22q11.2
deletion in patients with congenital heart disease

• Mutation detect utilizes

quantitative real-time PCR
• Sensitivity & specificity of 100 %
• Decreased turnaround time

Tomita-Mitchell A. et al. Physiol Genomics 2010;42:52–60

Maggadottir SM, Sullivan KE. J Allergy Clin Immunol Pract 2013;1:589-94
 Hyper IgM Syndrome

Normal/high IgM levels, but diminished levels of IgA and IgG

Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92

Defects in T Cell–Dependent B Cell Activation

X-linked hyper-IgM syndrome is caused by mutations in the gene

encoding the T cell effector molecule CD40 ligand (CD154)

Abbas AK et al. Cellular and Molecular immunology. Eighth Edition

Etzioni A, Ochs HD. Pediatr Res 2004;56:519-25
Qamar N, Fuleihan RL. Clinic Rev Allerg Immunol 2014;46:120–30
 CD40 ligand (CD154) expression

Sensitivity can be enhanced to 90 % Lack of CD40L expression

by using a biotinylated CD40-Ig fusion protein after activation
which binds to a functional CD40L receptor complex

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Qamar N, Fuleihan RL. Clinic Rev Allerg Immunol 2014;46:120–30
 Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer and Cytotoxic T-Cell Defects
• Adaptive-Innate Immunity Defects
• Disorders of Complement System
• Immune Dysregulation Disorders
 Disorders of Neutrophils
• Neutropenia
• Defect in adhesion: LAD
• Defect in killing: CGD
• Defect in signaling: IL-12, IL-12R, IFNGR
• Defect in specific granule: neutrophil specific
granule deficiency
 Disorders of Neutrophils
Typically present as
• Recurrent skin and respiratory tract
infections due to either bacteria or fungi
(especially Candida and Aspergillus)
• Delayed umbilical cord separation, omphalitis
• Deepseeded abscess formation
• Poor wound healing
• Recurrent oral stomatitis
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
 Initial screening
• CBC, absolute neutrophil count (ANC) and
morphology of neutrophils
• High ANC can be seen in response to
infections, as well as in leukocyte adhesion
• Low or absent ANC is seen in defects
involving neutrophil development or
maturation

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Leukocyte Adhesion Deficiency

- Typically presents with

recurrent bacterial infections
CD18,11
with no pus formation
- Delayed umbilical cord
separation or omphalitis

LAD3 diagnosis requires specialized testing

absence of sialyl lewis of integrin function in platelets or leukocytes
X (CD15s) or by molecular methods

Zimmerman GA. Blood 2009;113:4485-6

Chronic Granulomatous Disease

Granulomatous inflammation occurs due to failure to clear

the infections, and also due to an inherent propensity for
increased inflammation in these patients

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Picture from www.immunopaedia.org.za
Lack of NADPH oxidase
which is made up of
• One X-linked gene
- Cytochrome b-245, beta polypeptide (CYBB)

• Three autosomal genes

- Cytochrome c-245, alpha polypeptide (CYBA)
- Neutrophil cytosolic factor 1 (NCF1)
- Neutrophil cytosolic factor 2 (NCF2)

Decreased/absent oxidative
burst and production of reactive
oxygen intermediates

Hampton MB. et al. Blood 1998;92:3007-17

Nitroblue tetrazolium test

Measure ability of phagocytic cells to ingest and reduce

a soluble yellow dye to an intracellular blue crystal
Lekstrom-Himes JA, Gallin JI. N Engl J Med 2000;343:1703-14
 Neutrophil oxidative burst in CGD

Normal activated neutrophils produce

superoxides that oxidize DHR resulting in
increased fluorescence as depicted by
shift of histogram peak to the right

CGD patients cannot generate

oxidative burst and, therefore,
do not oxidize DHR

CGD carriers (usually mothers of affected

male patients) demonstrate bimodal
induction of neutrophil oxidative burst due to
random X inactivation

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Dihydrorhodamine 123 (DHR) assay
 Myeloperoxidase Deficiency

• MPO plays a role in formation

of reactive oxidative intermediates
• More than 95 % of MPO-deficient
patients are asymptomatic
• In vitro testing has shown that
MPO-deficient neutrophils
retain killing potential,
but at a slower rate

Hampton MB. et al. Blood 1998;92:3007-17

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Definitive diagnosis is by histochemical staining for MPO

DHR assay is also a reasonable testing option; however,

it can be falsely negative in cases of complete MPO deficiency
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Picture from www.pathologyoutlines.com
Glucose-6-Phosphate Dehydrogenase Deficiency

• G6P reduces NADP to NADPH

• NADPH is needed for
production of gluthatione
• Gluthatione is needed in
maintain of RBC membrane
from oxidant stress

Ho HY et. al. Free Radical Research 2014; 48: 1028–48

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
G6PD deficiency is rarely caused an impaired neutrophilic respiratory burst.
This defect can occur when there is < 5 % enzymatic activity in neutrophils
and is overcome when there is > 20 % activity

Ardati KO et al. Acta Haematol 1997;97:211–5

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
 Detection of G6PD Deficiency
• DNA tests
• Measurement of NADPH production capacity of
G6PD
- Fluorescent spot test
- Spectrophotometric assay
- Cytochemical Assay
• Brilliant Cresyl Blue (BCB) dye test
• Metosulfate-3-(4,5-dimethylthiazal-2-yl)-2,5-
diphenyltetrazolim bromide test (PMS/MTT test)

Peters AL, Van Noorden C. J Histochem Cytochem 2009;57:1003–11

 Hyper IgE syndrome (HIES)

- Recurrent S. aureus
infections of the skin and
pulmonary tract
- High IgE
- Eosinophilia
- Eczema
- Mucocutaneous
candidiasis

Buckley RH. and Orange JS. Middleton’s Allergy 8th edition, 2014, 1144-74
 Wart

predisposition to malignancies
at a young age

STAT3 deficiency DOCK8 deficiency

Majority of HIES have a heterozygous,

dominant-negative mutation in STAT3
which is critical for inducing RORγt

Buckley RH. and Orange JS. Middleton’s Allergy 8th edition, 2014, 1144-74
Mechanism of DOCK8 mutations is not entirely understood
Engelhardt KR et al. J Allergy Clin Immunol 2012;129:294–305
 Laboratory
• Decrease in IL-17-producing T cells (TH17)
• Screen percentage of Th17 cells in the
peripheral blood by flow cytometry
• Genetic mutational analysis is necessary for
a definitive diagnosis

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer and Cytotoxic T-Cell Defects
• Adaptive-Innate Immunity Defects
• Disorders of Complement System
• Immune Dysregulation Disorders
 Natural Killer (NK) cells
NK cell • Play a key role in defending
against viral infections
• If defects, typically present
Perforin with recurrent, severe herpetic
infections
• Defects in NK and cytotoxic T
lymphocyte (CTL) function
result in hemophagocytic
lymphohistiocytosis (HLH)
Screening should begin with immunophenotyping to
Erythroleukemia cell confirm the presence or absence of NK cells

Orange JS, Ballas ZK. Clin Immunol 2006;118:1–10

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Test effector function

Culture target cells with PBMCs Measuring markers of cell death i.e., annexin V, 7-ADD

Picture from www.immunochemistry.com

Chromium Release Assays

Access from www.cai.md.chula.ac.th , February 19, 2015

Intracellular cytotoxic proteins (i.e., perforin/granzyme)
Orange JS, Ballas ZK. Clin Immunol 2006;118:1–10
NK-cell lysosomal-associated membrane protein-1 (LAMP-1)/
CD107a expression (marker of degranulation)
Co-culture with K562
Erythroleukemic cell line

Percent of NK cells
Expressing CD107a

No upregulation of CD107a on
NK cells of a patient with a
MUNC13 mutation (FHLH3)

Defective CD107a expression

- Chediak-Higashi
- Griscelli syndrome

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 X-Linked Lymphoproliferative Syndrome (XLP)

• Fatal hemophagocytosis
• Hypogammaglobulinemia
• Lymphoma
• Severe infectious mononucleosis occurs
in 2/3 of all XLP patients

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 There are two different forms of XLP which are caused
by two distinct genetic mutations

• XLP-1 accounts for 60 % of XLP cases due to mutation in SH2 domain containing 1A
(SH2D1A), a signaling lymphocyte activation molecule (SLAM)-associated protein (SAP).
Immunophenotyping is able to demonstrate decreased/absent numbers of invariant
natural killer T cells in XLP-1
• XLP-2, is due to a mutation in the X-linked inhibitor of apoptosis gene
(XIAP, also known as BIRC4)
• Flow cytometry can be used to detect intracellular SAP
Marsh RA et al. J Immunol Methods 2010;362:1-9
or XIAP expression Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
 Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer and Cytotoxic T-Cell Defects
• Adaptive-Innate Immunity Defects
• Disorders of Complement System
• Immune Dysregulation Disorders
 Inherited Susceptibility to
Mycobacterial Disease

Main regulatory pathway

of cell-mediated immunity

• Production of cytokines and chemokines

• Enhancement TNF- production
• Upregulation of MHC class II expression
• Enhancement antigen processing
• Production of reactive oxygen and
nitrogen intermediates (in mice)

Dorman SE and Holland SM Cytokine & Growth Factor Reviews 2000;11;321-33

 Six genes have been found to be mutated

IL12 p40 IL12RB1

NEMO

STAT 1

IFNGR1 & IFNGR2

Flow cytometry for IL12RB1 and IFNGR1

Casanova JL. Swiss Med Wkly 2001;131:445-54
Muhsen SA and Casanova JL. J Allergy Clin Immunol 2008;122:1043-51
Measure phosphorylated STAT1
after IFN-γ stimulation and
Phosphorylated STAT4
by IL-12 stimulation

Decrease STAT1 phosphorylation

suggests defect in either STAT1,
IFNGR1, or IFNGR2

Decrease STAT4 phosphorylation

suggests defect in IL12R

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Abbas AK et al. Cellular and Molecular immunology. Eighth Edition
Evaluation of STAT1 and STAT4

Normal phosphorylation
of STAT1 in response to IFN-γ
stimulation in monocytes

Normal phosphorylation
of STAT4 in response to IL-12 in
PHA-blasted lymphocytes

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer and Cytotoxic T-Cell Defects
• Adaptive-Innate Immunity Defects
• Disorders of Complement System
• Immune Dysregulation Disorders
Disorders of Complement System

Screening for disorders of the complement system

Total hemolytic complement (CH50) test
measures function of classic complement cascade
Alternative pathway (AH50) test
measures the function of the alternative complement pathway.

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Wen L. et al. J Allergy Clin Immunol 2004;113:585-93
Wen L. et al. J Allergy Clin Immunol 2004;113:585-93
Access from www.worldallergy.org , February 19, 2015
 Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer and Cytotoxic T-Cell Defects
• Adaptive-Innate Immunity Defects
• Disorders of Complement System
• Immune Dysregulation Disorders
Immune Dysregulation, Polyendocrinopathy,
Enteropathy, X-Linked Syndrome (IPEX)

• Caused by defects which affect

forkhead box P3 (Foxp3) protein
• Only 50 % of patients have
FOXP3 gene mutations
• Foxp3 is involved in function of Treg
which help control autoreactive T cells

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92
Flow cytometry can be used to identify Foxp3-expressing CD4+ T cells.
However, expression of Foxp3 is not sufficient to rule out IPEX,
and sequence analysis of the FOXP3 gene should be evaluated
if the clinical picture is consistent. Other proteins which affect Treg development
and function, such as CD25 or STAT5 deficiency, can also result in
IPEX-like phenotype

D’Hennezel E et. Al. N Engl J Med 2009;361:1710–3

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Autoimmune lymphoproliferative
syndrome (ALPS)
• Caused by mutations in genes which
induce lymphocyte apoptosis e.g., FAS (CD95),
FAS ligand, and caspase 10
• One diagnostic criteria includes
increased % of double-negative T cells
(DNT, CD3+CD4-CD8- TCRαβ+)
• These cells also express B220 and CD27
• Other significant findings:
- decreased CD4+CD25+ Tcells
- expandedCD3+HLADR+T-cell population
- decreased levels of CD27+ B cells
- increased CD5+ B-cell count
- increased CD8+CD57+ T-cell
- hypergammaglobulinemia

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92
 Double-negative T cells (DNTs)

Normally, less than 2 % of TCRαβ+ T cells do not Increased DNT cells in a patient with ALPS
express either the CD4 or CD8 co-receptors

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Conclusion (1)
• Stepwise, systematic Laboratory evaluation
of PID is important to reach a correct
diagnosis without unnecessary testing
• Moreover, interpretation of these results
should always be placed within the context
of patient and their clinical scenario

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Conclusion (2)
• There have been many new innovations in
immunodiagnostic studies over the last
several years with new protocols on the
horizon
• Current diagnostic laboratories are no longer
limited to evaluating cellular markers, but are
now able to perform diverse functional
assays

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

 Conclusion (3)
• As the knowledge of the immune system
expands, new testing will become available
to aid in the diagnosis of primary
immunodeficiencies

Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68

Thank you for your attention