Seminar primary immunodeficiency syndrome

 Define immunodeficiency
 Classification
 B cell/Antibody deficiency
 T Cell/ cellular deficiency
 Phagocyte deficiency
 Complement deficiency
 Application of flow
cytometry
 Diagnostic evaluation of PID

DEFINITIO
N
 It is the absence or failure of normal
function of one or more elements of the
immune system
 Results in immunodeficiency disease

Immunodeficiency
Diseases
 Primary: Usually
congenital, resulting from
genetic defects in some
components of the immune
system.
Secondary (Acquired):
as a result of other
diseases or conditions
such as:
»HIV infection
»malnutrition
»immunosuppression

PRIMARY
IMMUNODEFICIENCIES
 Primary immunodeficiencies are inherited
defects of the immune system
 They are classified according to IUIS , 2013

INTERNATIONAL UNION OF
IMMUNOLOGICAL SOCIETIES
(IUIS:2013)
Combined immunodeficiencies
Combined immunodeficiencies with associated or syndromic
features
Predominantly Ab deficiency
Disease of immune dysregulation
Defects in phagocytosis
Defects in innate immunity
Autoinflammatory disorders
Complement deficiencies

The immune system functional
compartments
 The B-lymphocyte/ Antibody system
 The T-lymphocyte/ cellular system
 The Phagocytic system
 The Complement system

Clinical Manifestations of the Primary
Immunodeficiency Diseases
 INFECTIOUS DISEASES
 AUTOIMMUNE DISEASES
 GASTROINTESTINAL DISEASE
 HEMATOLYMPHOID DISEASES

INFECTIOUS
DISEASES
 An increased susceptibility to infection is the
hallmark of the primary immunodeficiency
diseases (PID)
 In most patients, this is manifested by
recurrent infections

HEMATOLYMPHOID
DISEASES
 Anemia, thrombocytopenia, or leukopenia are
seen frequently in patients with PID
 Patients have increased chances of
malignancy especially of lymphoid organs

CELLULAR
DEFICIENCIES
SCID and Combined immune deficiency
Wiskott-Aldrich syndrome
Hyper-IgM syndrome
Ataxia-telangiectasia
Di-George syndrome
Other primary cellular
immunodeficiencies

SEVERE COMBINED
IMMUNODEFICIENCY (SCID)
 Fatal PID
 Combined absence
of T and B
lymphocytes
 13 different genetic
defects that can
cause SCID

MOST COMMON
TYPES
SCID
XSCID (X
linked )
Adenosine
deaminase
(ADA)

T-B+NK- phenotype
SCID
Deficiency of common gamma
chain of TCR (X-SCID)
Deficiency of Janus kinase 3

Deficiency of common gamma
chain of TCR (X-SCID)
 MC form
 Common gamma chain (γc ) – a component shared
by TCR and other growth factor receptors
 Mutation in gene encoding γc
 Result in T-B+NK- phenotype
 XR – so only males are affected

Deficiency of Janus kinase
3
 Mutation in gene encoding Jak3
 Required for function of γc

 So phenotype is T-B+NK- ( same as X-SCID)
 But this is AR – can affect both boys and girls

T-B+NK+ phenotype
SCID
Deficiency of α chain of IL-7 receptor
Deficiency of CD3 chains
Deficiency of CD45

Deficiency of α chain of IL-7
receptor
 3rd MC cause
 Mutation in gene encoding IL-7Rα – a component
of growth factor receptor
 T-B+NK+ phenotype
 However, B cells do not function due to lack of T
cells
 AR

Deficiency of CD3
chains
 CD3 is a receptor complex on T cells and c/o :
CD3δ , CD3ε and ζ-chain
 3 forms of SCID are due to mutations in genes
encoding these 3 chains of CD3 complex

Deficiency of
CD45
 T-B+NK+ phenotype
 AR

T-B-NK- phenotype
SCID
Adenosine deaminase
deficiency
Reticular dysgenesis

Adenosine Deaminase
deficiency
 2nd MC SCID
 Mutation in gene encoding ADA enzyme
 ADA is essential for T-cell function : Its absence
cause accumulation of toxic metabolites within
lymphocytes that cause cells to die
 T-B-NK- phenotype

T-B-NK+ phenotype SCID
RAG1 and RAG2 gene mutation
Artemis deficiency
Cernunnos deficiency
Ligase 4 deficiency

 Life threatening infections : most dangerous
organisms are-
1. Pneumocystis jiroveci
2. Chicken pox
3. CMV
4. Herpes simplex
 Live vaccines should not be given to SCID
patients : they may contract infection from
vaccine viruses
 So if family history of SCID is +ve : avoid live
vaccines

Diagnosi
s
 Easiest way to diagnose: Absolute lymphocyte
count(ALC)
 Normally ALC >4000/cu mm; 70% of which are
T cells
 SCID have ALC < 1500/cu mm
 If ALC found low Repeat test again
If low again specific tests to be done to
count T cells and measure T cell function

COMBINED
IMMUNODEFICIENCIES
 Group of rare genetic disorders that result in
combined immunodeficiency but do not reach a
clinical severity level to qualify as SCID
 7 types

Bare lymphocyte syndrome
Purine nucleosidase phosphorylase deficiency
ZAP70 deficiency
CD25 deficiency
Cartilage hair hypoplasia
Coronin 1A deficiency
MHC classI deficiency

HYPER IgE
SYNDROME
 Aka Job syndrome/ Buckley syndrome
 Characterised by :
1. Recurrent eczema
2. Skin abscesses: particularly by S.aureus
3. Lung infection
4. Eosinophilia
5. Increased IgE levels

• AD
• STAT3 mutation
• Connective tissue and
skeletal abnormality
• Typical facial appearance,
hyper extensibility of joints,
bone fractures after minor
trauma
TYPE1
• AR
• DOCK8 mutation
• Recurrent and severe viral
infection esp by herpes and
molluscum
• Do not have connective
tissue or skeletal abnormality
TYPE2

Diagnosi
s
 Increased IgE levels
 Normal IgG,A,M
 Increased peripheral blood eosinophils
 HIES scoring system by National Institute of
Health(NIH) :
Score : 0-15= unaffected
16-39= possibly affected
40-59= probably affected
>60 = definitely affected

 Scoring system is esp for the diagnosis
of Type 1 HIES
 Definitive diagnosis : genetic analysis of
STAT3 and DOCK8 genes

WISKOTT ALDRICH
SYNDROME
TRIAD :
1.Increased tendency to bleed: due to
small, dysfunctional and decreased
number of platelets
2.Recurrent infection
3.Eczema

 Associated with WAS gene mutation
 Was gene produces WAS protein (WASp)
 If mutation is severe: complete absence of
WAS protein : known as classic WAS
 If mutation is mild : some mutated WAS protein
present : known as milder form of WAS

Diagnosis
1. Platelet abnormality : decreased number and
small size : characteristic
2. Increased IgE
3. Sequencing of WAS gene to identify mutation :
definitive diagnosis
4. Determine WAS protein expression in blood
cells

HYPER IgM
SYNDROME
 Inability to switch from production of Ab of IgM
type to Abs of IgG, A or E types
 Normal B cells can produce IgM on their own
but require Help from T cells to switch from IgM
to IgG,A,E
 HIGM results from defect in interaction between
T and B cells

Genetic
defects
CD40L
def.
CD40
def.
AID
def.
UNG
def
NEMO
defect

 CD40L (CD154) : deficiency of this ligand is the
most common form of HIGM syndrome
 XR
 So only boys are affected
 Defect in NEMO gene : Known as ectodermal
dysplasia
 Associated with sparse hair and conical teeth

DIAGNOSI
S
 Characteristic : failure to express CD40L on
activated T cells – can be assessed by flow
cytometry
 CD40 L deficiency is due to mutation in CD40L
gene
 If gene is normal and CD40L is deficient : not
HIGM syndrome

So, for exact diagnosis :
demonstration of CD40L gene
mutation

ATAXIA
TELANGIECTASIA
 Mutation in ATM gene (11q)
 This gene is required for cell repair after
DNA damage
 2 important presenting features:

• Abnormality in cerebellum
• Can be confused with
cerebral palsy(CP)
• In AT neurologic
deterioration occurs with
age (but not in CP)
• Needs wheelchair by 10-12
yrs age
ATAXIA
• Dilated and corkscrew shaped
vessels esp in white of eyes
TELANGI
ECTASIA

DIAGNOSI
S
 Clinical feature is very important but difficult to
diagnose at early age as telangiectasia occurs
only by 5 yrs of age
Most imp test : AFP levels in
blood – 95% have increased
levels

Other tests:
Absence of ATM protein on western
blot
Abnormal DNA sequence (mutation)
of ATM gene
Increased chromosomal breakage
after exposure of blood cells to X rays
Increased CA 125

DI GEORGE
SYNDROME
 Defect : microdeletion in 22q11.2
 So aka : 22q11.2 syndrome
 Aka : velocardiofacial syndrome, conotruncal
anomaly face syndrome
 MC microdeletion syndrome
 2 imp gland abnormality

• Thymic hypoplasia
• T-cell number and
maturation defect
• So, increased
susceptibility to
infections
THYMUS
GLAND
• Underdeveloped and
hypoparathyroidism
occurs
• Hypocacemia occurs
PARATHYR
OID GLAND

DIAGNOSIS
 FISH analysis to identify
22q11.2 deletion

OTHER PRIMARY CELLULAR
IMMUNODEFICIENCIES
Chronic mucocutaneous candidiasis
Cartilage hair hypoplasia
X-linked lymphoproliferative syndrome 1 & 2
Veno occlusive disease
Schimke syndrome

X-linked immune dysregulation and
polyendocrinopathy syndrome (IPEX)
Hoyeraal-Hereidarson syndrome
(dyskeratosis congenita)
Immunodeficiency with centromeric instability
anomalies (ICF)
Comel-netherton syndrome(C-NS)

Chronic mucocutaneous
candidiasis
 Severe and persistent candida infection of
mucous membrane, scalp, skin and nails
 MC abnormality : negative delayed
hypersensitivity skin test to candida Ag despite
widespread candidial infection

2 important gene defects
STAT1 IL-17

Hereditary form of
CMC
 APECED syndrome : autoimmune
polyendocrinopathy candidiasis ectodermal
dysplasia syndrome
 Associated with AIRE gene defect

• X-linked lymphoproliferative
syndrome 1 & 2
XLP1: SH2DIA
XLP2: XIAP
• X-linked immune dysregulation
and polyendocrinopathy
syndrome (IPEX)
FOX P3
• Immunodeficiency with
centromeric instability
anomalies (ICF)
DNMT3B
• Schimke syndromeSMARCL1

ANTIBODY
DEFICIENCIES
Agammaglobulinemia
Common variable immune deficiencies
Selective IgA deficiency
IgG subclass deficiency
Specific antibody deficiency
Transient hypogammaglobulinemia of infancy
Other Ab deficiency disorders

AGAMMAGLOBULINE
MIA
• Aka bruton’s/congenital
agammaglobulinemia
• BTK gene defect (on X chr)
• Pre B cells cannot develop into immature
B cells
• So no Ab formation
XLA
• Both boys and girls are affected
• Gene defect : μ heavy chain, λ5, Igα, Igβ,
BLNK
• These encode proteins that work with
BTK to convert pre B to mature B cells
ARA

COMMON VARIABLE IMMUNE
DEFICIENCY
• Since it is relatively
frequent
COMMON
• Since degree and
type of Ig deficiency
varies from patient
to patient
VARIABLE

• Granulomatous,
lymphoproliferative,
interstitial lung
disease
GLILD

DIAGNOS
IS
 Low levels of serum Igs : IgG, IgA, IgM
CHARACTERISTIC
 Normal number of B-cells, but these cells fail to
mature into plasma cells – can be assessed by
flow cytometry based immunophenotyping of
B-cells

Diagnostic criteria by European
Society of immunodeficiency (ESID)
Plasma IgG levels<2 SD below mean for
age + marked decrease in eithe IgA or IgM
Age of onset of immunodeficiency >2yrs of
age
Absent isohemeagglutinins or poor
response to vaccines
Defined causes of
hypoagammaglobulinemia have been
excluded

SELECTIVE IgA
DEFICIENCY
 Undetectable level of IgA in blood and
secretions, but no other Ig deficiency
IgA
Present in
serum
Present in secretions
(known as secretory
IgA)

SECRETORY
IgA
Composed of:
•IgA dimer
•J-chain
•Secretory piece

DIAGNOSI
S
 Undetectable levels of IgA (<5-7mg/dl)
 Normal levels of other Igs
 Normal B and T lymphocytes
•Nomal IgA levels in adults: 50-200mg/dl
•In 20% cases associated with low levels of IgG2 or IgG4
subclass

 Subjects >4 years of age with serum IgA
consistently <7mg/dl but normal IgG and IgM
levels are considered Selective IgA deficient
 Defect – impaired differentiation from naïve B-cells
to IgA producing plasma cells
 Patients have anaphylactic reactions during
infusion of blood products due to sensitisation to
IgA which behaves as foreign antigen

IgG SUBCLASS
DEFICIENCY
 IgG is 2nd most common circulating protein
 4 IgG subclasses : IgG1,2,3,4
 When 1 or more of these subclasses are
persistently low and total IgG is normal, a subclass
defiency is present
 IgG(60-70%) > IgM> IgA> IgD > IgE
 IgG1(20-30%) > IgG2 (5-8%) > IgG3 (1-3%) > IgG4

 MC is IgG2 or IgG3 subclass deficiency
 May be associated with :
1. IgA deficiency
2. WAS
3. A-T

TRANSIENT
HYPOGAMMAGLOBULINEMIA OF
INFANCY
 Unborn baby makes no IgG
 At 6 months of pregnancy, maternal IgG via
placenta goes to fetus
This increases during the last trimester of pregnancy
At term, baby has IgG level equal to that of mother
This IgG dissapears completely by 6 months of age

 Fetus doesn’t get any maternal IgM, A or E as
they do not cross placenta
So, if IgM is found: s/o in utero infection
 Between 3-6 months of age :
• Maternal IgG starts falling
• Infant’s IgG starts forming
• As a result IgG levels are low: k/a physiologic
hypogammaglobulinemia of infancy

 In some infants, period of
hypogammaglobulinemia is more severe and
prolonged beyond 6 months of age : k/a Transient
hypogammaglobulinemia of infancy
 Definition :
1. Infants >6 months
2. IgG< 2 SD below the mean for age (typically
<400mg/dl)
 Mostly IgG correction occurs by 2 yrs of age

OTHER AB DEFICIENCY
DISORDERS
Antibody deficiency with normal or
elevated Igs
Selective IgM deficiency
Immunodeficiency with thymoma
(Good’s syndrome)
Transcobalamin II deficiency
Drug induced Ab deficiency

Warts, hypogammaglobulinemia,
infection and myelokathexis syndrome
(WHIM)
Kappa chain deficiency
Heavy chain deficiency
Post meiotic segregation
disorder(PMS2)
Unspecified hypogammaglobulinemia

CHRONIC GRANULOMATOUS
DISEASE
 Phagocytes (neutrophils and monocytes) form
phagosome within cell
 Formation of NADPH oxidase complex
generate burst of reactive oxygen species
activates proteases which destroy ingested
bacteria
 Here, mutations occur which affect formation of
NADPH oxidase

2 different types of CGD:
1.X-linked : MC form
only boys affected
mutation in CYBB gene
2. AR forms : Mutations in CYBA, NCF1, NCF2 or
NCF4 genes

INFECTIONS
Staphyloco
ccus
aureus
Burkholderia
cepacia
complex
Serratia
marcescensNocardia
Aspergillus

DIAGNOSIS
 Any patient of any age with a CGD type infection
should be tested for CGD unless there is a good
reason not to
 Previously used test – Nitroblue Tetrazolium slide
Test (NBT)
 Most accurate test – Dihydrorhodamine reduction
test (DHR) : Measures amount of H2O2 produced
in phagocytes

NITROBLUE TETRAZOLIUM
SLIDE TEST
 Neutrophils make reactive
oxygen species which reduces
colorless NBT dye into a blue
colored formazan salt
 In CGD these reactive oxygen
species are not formed,
so dye is not reduced to blue
color

OTHER PHAGOCYTIC CELL
DISORDERS
Neutropenias
Phagocyte killing defects
Leukocyte adhesion
deficiencies
Specific granule deficiency

Glycogen storage disease
typeIb
Beta-actin deficiency
Chediak higashi
syndrome
Griscelli syndrome

Neutropenia
 <500cells/microl (normally >2000cells)

DIFFERENT FORMS OF
NEUTROPENIA
• Severe congenital neutropenia
(Kostmann syndrome)
HAX1
gene
• Cyclic neutropenia
ELA2
gene
• Benign chronic neutropenia
• Immune neutropenia------

Leukocyte adhesion
deficiencies
LAD1
• CD18
mutation
LAD2
• Mutation in
enzymes
that attach
fucose to
proteins
LAD3
• FERMT3
mutation

CHEDIAK HIGASHI SYNDROME
(CHS)
 Mutation in LYST gene
 Characteristics:
1. Giant granules within
neutrophils
2. Partial albinism
3. Frequent infections

 Most patients reach a stage known as :
accelerated phase/lymphoma like syndrome –
triggered by EBV
 Here, the defective WBCs divide
uncontrollably and invade many organs

GRISCELLI
SYNDROME
• MYO5A gene defectGS TYPE1
• RAB27A gene defect
• a PID disorder
• Partial albinism, recurrent
infection, neutropenia and
thrombocytopenia
GS TYPE2
• Melanophilin (MLPH) gene
defectGS TYPE3

Difference from CHS :
1. Morphology of neutrophils : Normal in
GS (giant granules in CHS)
2. Leukocyte specific protease activity :
Normal in GS (low in CHS)

 Classical pathway : Activated by Abs that are bound
to Ags
 Lectin pathway : initiated by Mannose binding lectin
(MBL), Ficolins and collectin; associated with
enzyme- MASPs (MBL- associated serine
proteases)
 Alternative pathway : initiated by fragments of C3,
Factor B, Factor D, and properdin

 Terminal pathway : forms MAC – membrane
attack complex
 Components are - C3, C5,6,7,8,9
 Terminal complement complex (TCC) : fluid
phase of MAC
 Found in circulation when complement is
activated
 Useful lab marker for complement activation

Deficiency of classical
pathway
 Def. in C1q, r, s, C2, C4 : ass with SLE and RA
 Deficiency in C1 esterase inhibitor: it
inactivates classical pathway component to
prevent from uncontrolled activation
 Its def. causes hereditary angioedema

Deficiency in lectin
pathway
 Def. in MBL, M-ficolin, L-ficolin, H- ficolin,
MASPs :
Increased bacterial infection

Deficiency of alternative
pathway
 Factor B and Factor D def : very rare
 Properdin : only X-linked complement protein
 Synthesised by monoctes, granulocytes and T
cells
 Def. causes increased susceptibility to
Neisseria, esp; N. meningitidis

 Factor H : it is alternative pathway control
protein
 If def. – uncontrolled activation of alternative
pathway and so depletion of C3
 Associated with atypical HUS(aHUS) and age
related macular edema (AMD)

This test is based on the principle that
nonfluorescent DHR
(dihydrorhodamine) 123 when
phagocytosed by normal activated
neutrophils (after stimulation with PMA
– phorbol myristate acetate) can be
oxidized by hydrogen peroxide,
produced during the activated
neutrophil respiratory oxidative burst, to
rhodamine 123, a green fluorescent
compound, which can be detected by
flow cytometry.
Rho 123 is excitable at 488nm and
emits at 515 nm

How to diagnose
PIDs?
 First Step:
 Think of PID
 Second Step:
 Think of PID
 Third Step:
 Think of PID
 Careful look at the CBC

Initial laboratory screening of PID
 CBC including TLC, DLC, Platelets (size)
 Quantitative serum Immunoglobulins (nephelometry)-
IgG, IgA, IgM +/- IgE (ELISA)
 Lymphocyte subset analysis by flowcytometry for
 Quantitation of total T cells (CD3+)
 T cell subsets (CD4+, CD8+)
 B cells (CD19+, CD20+)
 NK cells (CD16+, CD56+)
 HLA-DR to rule out MHC II deficiency
 Dihydrorhodamine to rule out CGD
 Isohemagglutinin titres
 IgG antibodies to
 Known exposure (VZ)
 Known Immunizations (tetanus, diphtheria, Hib, meningococcus, polio,
rubella).

Comprehensive Laboratory Evaluation B cell
deficiency
 Quantitative IgG, IgM, IgA, IgE
 IgG subclass
 B cell numbers in peripheral blood (CD19, CD20)
 Isohemagglutinin titres
 Antibody response to test immunizations, e.g. D/T,
Pneumovax/Typhum Vi.
 Antibody response to neoantigens e.g., KLH, bacteriophage
ØX174
 In vitro IgG synthesis by mitogen-stimulated PBL or purified B
cells cultured in the presence of anti-CD-40 and lymphokines
 In vitro proliferation of B cells in response to anti-CD40 and IL-4
Biopsies: rectal mucosa, lympho nodes (if appropriate)
 Molecular and mutation analysis (e.g. Btk, µ heavy chain)

Comprehensive Laboratory Evaluation T cell
deficiency
 Absolute lymphocyte count
 T cell, T subset, and NK cell enumeration (CD3, CD4, CD8; also CD16,
56)
 Delayed-type hypersensitivity skin tests (only in older children and
adults) (Candida, tetanus toxoid, mumps).
 In vitro proliferation of lymphocytes to mitogens (PHA, ConA), allogenic
cells, and specific antigens (Candida, tetanus toxoid).
 Production of cytokines by activated lymphocytes (ELISA or flow)
 Expression of activation markers (e.g. CD40L, CD69) and lymphokine
receptors (e.g. IL-2Rγc, IFN- γR) after mitogenic stimulation
 Enumeration of MHC I and MHC II expressing lymphocytes
 Chromosome analysis (probe for 22q11)
 Enzyme assays (ADA, PNP). Caution: ? Recent transfusion
 Biopsies: Skin, lymph node, thymus (if appropriate)
 Molecular and mutation analysis (e.g. CD40L, γc chain, Jak 3, ZAP-70)

Comprehensive Laboratory Evaluation
Deficiency of Phagocyte system
 Absolute neutrophil count (serially to rule out cyclic neutropenia)
 WBC turnover
 Anti-neutrophil antibody
 Bone marrow biopsy
 Chemotaxis, assessment of adhesion in vivo and in vitro
 CD11/CD18 assessment for LAD1 (flowcytometry)
 Bombay phenotype/Sialyl Lewis-X/ CD15 (LAD2)
 Phagocytosis (Baker’s yeast)
 NBT Slide test; metabolic bursts (DHR flowcytometry);
chemiluminescence; bacterial assays
 Enzyme assays: MPO, G6PD, Glutathione peroxidase, NADPH
Oxidase
 Mutation analysis (e.g. gp91phox; p22phox; p47phox; p67phox; β
integrin)

Complement deficiency
 CH50 : The CH50 is a screening assay for the activation of
the classical complement pathway
 Sheep RBCs are coated with rabbit anti sheep RBC Ab. To it
test serum is added to see if complement can lyse these
RBCs
 If complement is absent : CH50 will be 0
 If complement is decreased : CH50 will decrease
 AH50
 Analysis of quantity and function of C components
 Chemotactic activity of complement split products, e.g.,
C3a, C5a

CONCLUSIO
N
 Immunodeficiency disorders are fairly infrequent
 Some are transient with improvement over time
 More severe forms of immunodeficiency are
associated with shortened life span without bone
marrow transplantation
 A genetic cause has been identified for a
substantial portion of these disorders

 Treatment options incude:
 Prophylactic antibiotics
 IV immunoglobulin
 Stem cell or bone marrow transplantation
 New biologicals
 Gene therapy

 Outcomes for children with
immunodeficiency dependent depends on
the following:
 Timely recognition
 Adequate therapy and surveillance
 The nature of the underlying disease

Which type of PID is associated
with this cartoon

Seminar primary immunodeficiency syndrome

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