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PRIMARY IMMUNODEFICIENCY DISEASES
EKTA JAJODIA
Define immunodeficiency Classification B cell/Antibody deficiency T Cell/ cellular deficiency Phagocyte deficiency Complement deficiency Application of flow
cytometry Diagnostic evaluation of PID
DEFINITION
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
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 TYPESSCID
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 SCIDRAG1 and RAG2 gene mutation
Artemis deficiency
Cernunnos deficiency
Ligase 4 deficiency
Life threatening infections : most dangerous organisms are-
1. Pneumocystis jiroveci2. Chicken pox3. CMV4. 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
Diagnosis
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 eczema2. Skin abscesses: particularly by S.aureus3. Lung infection4. Eosinophilia5. 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
Diagnosis
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
Diagnosis1. 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
DIAGNOSIS 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
TELANGIECTASIA
DIAGNOSIS
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
THYMU
S GLAND
• Underdeveloped and hypoparathyroidism occurs
• Hypocacemia occurs
PARATHYROID 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
ANTIBODY DEFICIENCIES
Agammaglobulinemia
Common variable immune deficiencies
Selective IgA deficiency
IgG subclass deficiency
Specific antibody deficiency
Transient hypogammaglobulinemia of infancy
Other Ab deficiency disorders
AGAMMAGLOBULINEMIA
• 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 cellsARA
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
DIAGNOSIS
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
DIAGNOSIS
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 deficiency2. WAS3. 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 months2. 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
PHAGOCYTE DEFICIENCY
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
Staphylococcus
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 neutropeniaELA2 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 neutrophils2. Partial albinism3. 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)
COMPLEMENT DEFICIENCIES
Complement deficiencies
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)
APPLICATION OF FLOW CYTOMETRY
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 EvaluationDeficiency 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
CONCLUSION 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 antibioticsIV immunoglobulinStem cell or bone marrow transplantationNew biologicalsGene therapy
Outcomes for children with immunodeficiency dependent depends on the following:Timely recognitionAdequate therapy and surveillanceThe nature of the underlying disease
Which type of PID is associated with this cartoon
THANK YOU