2. Define immunodeficiency
Classification
B cell/Antibody deficiency
T Cell/ cellular deficiency
Phagocyte deficiency
Complement deficiency
Application of flow
cytometry
Diagnostic evaluation of PID
3.
4. DEFINITIO
N
It is the absence or failure of normal
function of one or more elements of the
immune system
Results in immunodeficiency disease
5. 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
7. 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
8. The immune system functional
compartments
The B-lymphocyte/ Antibody system
The T-lymphocyte/ cellular system
The Phagocytic system
The Complement system
9. Clinical Manifestations of the Primary
Immunodeficiency Diseases
INFECTIOUS DISEASES
AUTOIMMUNE DISEASES
GASTROINTESTINAL DISEASE
HEMATOLYMPHOID DISEASES
10. 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
22. 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
23. 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
25. 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
26. 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
29. 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
32. 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
33. 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
34. 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
37. • 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
38. 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
39. Scoring system is esp for the diagnosis
of Type 1 HIES
Definitive diagnosis : genetic analysis of
STAT3 and DOCK8 genes
41. 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
42. 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
43. 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
46. 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
47. 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
48. So, for exact diagnosis :
demonstration of CD40L gene
mutation
49. ATAXIA
TELANGIECTASIA
Mutation in ATM gene (11q)
This gene is required for cell repair after
DNA damage
2 important presenting features:
50. • 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
51.
52. 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
53. 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
54. 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
55. • Thymic hypoplasia
• T-cell number and
maturation defect
• So, increased
susceptibility to
infections
THYMUS
GLAND
• Underdeveloped and
hypoparathyroidism
occurs
• Hypocacemia occurs
PARATHYR
OID GLAND
59. X-linked immune dysregulation and
polyendocrinopathy syndrome (IPEX)
Hoyeraal-Hereidarson syndrome
(dyskeratosis congenita)
Immunodeficiency with centromeric instability
anomalies (ICF)
Comel-netherton syndrome(C-NS)
60. 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
67. 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
68.
69. COMMON VARIABLE IMMUNE
DEFICIENCY
• Since it is relatively
frequent
COMMON
• Since degree and
type of Ig deficiency
varies from patient
to patient
VARIABLE
73. 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
74.
75. 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
76. 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)
78. 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
79. 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
80.
81. 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
82. MC is IgG2 or IgG3 subclass deficiency
May be associated with :
1. IgA deficiency
2. WAS
3. A-T
83. 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
84. 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
85. 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
86. 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
87. Warts, hypogammaglobulinemia,
infection and myelokathexis syndrome
(WHIM)
Kappa chain deficiency
Heavy chain deficiency
Post meiotic segregation
disorder(PMS2)
Unspecified hypogammaglobulinemia
90. 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
91. 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
93. 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
94. 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
102. 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
104. 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)
107. 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
108. 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
109. 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
111. 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
112. 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)
114. 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
115. How to diagnose
PIDs?
First Step:
Think of PID
Second Step:
Think of PID
Third Step:
Think of PID
Careful look at the CBC
116. 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).
117. 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)
118. 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)
119. 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)
120. 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
121. 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
122. Treatment options incude:
Prophylactic antibiotics
IV immunoglobulin
Stem cell or bone marrow transplantation
New biologicals
Gene therapy
123. Outcomes for children with
immunodeficiency dependent depends on
the following:
Timely recognition
Adequate therapy and surveillance
The nature of the underlying disease