This document summarizes the diagnostic approach to primary immunodeficiency disorders. It describes the major components of the immune system that can be deficient and classifies primary immunodeficiencies according to the affected component. Evaluation involves assessing the patient history, physical exam findings, and laboratory tests including blood counts, immunoglobulin levels, vaccine response, and flow cytometry of lymphocyte subsets. Specific immunodeficiencies are characterized by their clinical features, age of onset, infectious pathogens involved, affected organs, and genetic or molecular abnormalities identified.

1. DIAGNOSTIC APPROACH TO PRIMARY
IMMUNODEFIDIENCY DISORDER
By- DR.PRERNA CHOUDHARY

2. INTRODUCTION
• Immunodeficiency can be due to any of the four major components
of immune system
• The deficiencies can be either Congenital or Acquired.
B Cells T Cells Complement system Phagocytes

3. Primary immunodeficiency disorders
• Genetic diseases with primary Mendelian inheritance
• >300 conditions
• ˜ 150 genes
• Overall prevalence 5 / 1,00,000
• Classified according to the component of immune system primarily
involve

4. types disease
1. Combined T & B cell ID
heterogenous disorders characterized by lack of T cell-mediated immunity
associated with impaired B cell function.
SCID
Omenn syndrome
2. Syndrome associated combined ID Ataxia telangiectasia
Di George syndrome
Wiskott Aldrich syndrome
3. Predominantly antibody deficiencies
The spectrum of PADs is broad, ranging from patients with a severe reduction
of all serum immunoglobulin classes (Ig) and totally absent B cells to patients
who have a selective antibody deficiency with normal serum
immunoglobulin.
Agammaglobulinemia (XL & AR)
CVID
Selective IgA deficiency
Hyper IgM syndrome
Transient hypergammaglobulinemia of
infancy
4. Immunodeficiencies associated with Lymphoproliferative disorders Chediak Higashi syndrome
Familial Hemophagocytic
Lymphoproliferative syndrome

5. types diseases
5. Phagocyte defect
Defects of neutrophil function and/or differentiation, defects
of motility, and defects of respiratory burst are the main sub-
classification of phagocytes defects.
Chronic granulomatous disease
Leukocyte adhesion defect
Severe congenital neutropenia
Cyclic neutropenia
6. Defects of innate immunity Defect in TLR(tolllike receptors) signalling
Mendelian susceptibility to Mycobacterial disease
7.Complement deficiencies Deficiencies of complements of classical / alternate pathway
C1 inhibitor deficiency
8. Other ID Hyper IgE syndrome
IPEX syndrome (Immunodysregulation polyendocrinopathy
enteropathy X-linked)
Chronic mucocutaneous candidiasis
Ectodermal dysplasia
Heriditary/ Cong Asplenia / Hyposplenia

7. When to suspect ?

8. CHARACTERIS
TIC
PREDOMINANT T-CELL
DEFECT
PREDOMINANT B-CELL
DEFECT
GRANULOCYTE DEFECT COMPLEMENT DEFECT
Age at the
onset of
infection
Early onset, usually 2-6
months of age
Onset after maternal
antibodies diminish, usually
after 5-7 mo of age,
laterchildhood to adulthood
Early onset Onset at any age
Specific
pathogens
involved
Bacteria: common Gram
positive and Gram
negative bacteria and
Mycobacteria
Viruses: CMV, EBV,
Adenovirus,
parainfluenza3, varicella,
enterovirus
Fungi: Candida and
Pneumocystis jiroveci
Protozoa:
cryptosporidium species
Bacteria: pneumococci,
streptococci, staphylococci,
Haemophilus,
Campylobacter, Mycoplasma
Fungi and parasites: giardia,
cryptosporidia
Enterovirus: ECHO, polio
Bacteria: staphylococci,
Pseudomonas, Serratia, Klebsiella,
Salmonella Viruses: enterovirus
Fungi and parasites: Candida,
Nocardia, Aspergillus
Early complement
defect: (C1, C2, C3, C4)-
pyogenic infection
late complement
system (C5-9) result in
susceptibility to
neisserial infections
Affected
organs
Extensive
mucocutaneous
candidiasis, lungs, failure
to thrive, sepsis,
protracted diarrhea
Recurrent sinopulmonary
infections, chronic
gastrointestinal symptoms,
malabsorption, arthritis,
enteroviral
meningoencephalitis (CNS)
Skin: abscesses, impetigo, cellulitis
Lymph nodes: suppurative adenitis
Oral cavity: gingivitis, mouth ulcers
Internal organs: abscesses,
Osteomyelitis (bone)
Infections: meningitis,
arthritis, septicemia,
recurrent
sinopulmonary
infections

9. FEATURES DIAGNOSIS
Hypocalcemia, unusual facies & ears, heart disease, characterized by thymic
aplasia/hypoplasia , ch.22q11.2 deletion
Di George anamoly (t cell defect)
Defect in cellular adhesion molecules, Delayed umbilical cord seperation,
leukocytosis, reccurent infections with no pus formations
Leukocyte adhesion defect (phagocyte
defect)
AR inheritance, caused by Adenosine deaminase deficiency necessary for T-cell
survival. Persistent thrush, FTT, pneumonia, diarrhoea
SCID (combined B&T)
Triad of bleeding, eczema and recurrent infections , thrombocytopenia, bloody
stools, draining ears.
Wiscott Aldrich Syndrome (combined
B&T)
Pneumocystis jeroveci pneumonia, neutropenia, reccurrent infectons
Lymphoid hyperplasia
Defective CD40 signalling via B cells affecting class switch recombination and
somatic hypermutation
Hyper IgM syndrome (B cell defect)
eczematous dermatitis, enteropathy, and endocrinopathies, is the prototypical
presentation. Enteropathy typically manifests as profuse watery diarrhea within the
first few months of life. Intestinal biopsies have noted villous atrophy
X-Linked Syndrome (IPEX)
(T-cell defect)
In newborns and young Infants (o-6months)
Characteristic patterns in some PIDs

10. FEATURES DIAGNOSIS
Severe progressive infectious mononucleosis
fatal hemophagocytosis, hypogammaglobulinemia, or
lymphoma. Defective NK cells and cytotoxic T cell
function
Lymphoproliferative syndrome (phagocyte defect)
Reccurrent staph abscess, staph pneumonia with
pneumatoceles, coarse features, pruritic dermatitis
Mutation in STAT3 (AD), DOCK8(AR)
Hyper IgE syndrome
Oculocutaneous albinism, reccurrent infection Chediak Higashi syndrome
Abscess, suppurative LNpathy, antral outlet
obstruction, pneumonia, osteomyelitis
Defect in NADPH oxidase complex of Phagocytes
Chronic Granulomatous Disease
IN INFANTS AND YOUNG CHILDREN (6MON- 5 YR)
FEATURES

11. FEATURES DIAGNOSIS
Progressive dermatomyositis with chr entero virus
encephalitis. Btk mutation present
Agammaglobulinemia (B cell defect)
Sinopulmonary infection, neurologic deterioration,
telangiectasias
Ataxia telangiectasia
Recurrent Neisserial meningitis C6,C7 & C8 deficiency (complement defect)
sinopulmonary infection, splenomegaly,
autoimmunity, malabsorption
CVID (B cell defect)
IN OLDER CHILDREN(>5YRS) AND ADULTS FEATURES

12. Evaluation of suspected ID
• History
• Diagnosis of PID must be entertained when an individual has a family
h/o PID or H/O severe unusual , frequent or difficult to treat
infections.
• Detailed H/O infections including site, severity, frequency & organism
involved.

13. Laboratory workup
Age specific and age adjusted norms to be used.
1. CBC & Differential
Lymphocyte count – normal
Lymphopenia
absolute lymphocyte count (ALC) (As a
general guideline, an infant with an ALC
of less than 3,000/mm3 should prompt
evaluation for a possible immune
defect)
rules out T cell defect
SCID
Leukocytosis –LAD
Neutropenia primary neutropenia
XL Hyper IgM syndrome
25 % of XLA patients
Neutrophils with Giant granules Chediak Higashi syndrome
Platelet count- normal rules out WAS
Thrombocytopenia WAS, IgA Deficiency, CVID

14. Eosinophilia, ANEMIA, THROMBOCYTOPENIA,
Neutopenia
IPEX
Eosinophilia Hyper IgE
Anemia, thrombocytopenia HLH

15. 2. Nitroblue tetrazolium (NBT) test - chronic granulomatous disease-
drop of whole blood is placed on a microscope slide coated with an
activating agent, such as lipopolysaccharide or phorbol ester.
Phagocytes adhering to the slide are activated and develop blue inclusions
on incubation with NBT.
The number of NBT-positive cells is scored under a microscope.
This test can be useful in identifying X-linked carrier female individuals
when peripheral phagocytes consist of 2 cell populations: one that reduces
NBT to formazan and one that does not.

16. 3. Biochemical parameters
Digeorge anamoly- hypocalcemia, reduced parathyroid hormones
IPEX- type1 DM (high glucose level in early months),
hyper/hypothyroidism
HLH- increased liver function test results; hypofibrinogenemia; and
hypertriglyceridemia. High ferritin

17. 4. Quantitative serum immunoglobulin assay –
• for antibody deficiency & functional adequacy of humoral immunity.
• Vaccines like tetanus, diphtheria, H influenza B & Pneumococcus to
evaluate antibody response – it determines serum antibody

18. • CVID - low IgG with a low IgA and/or IgM, as well as defective antibody
response to vaccination
• SCID- Low levels of IgG, IgA, IgM, IgE but IgG normal in initial life (from mother)
• X-Linked Agammaglobulinemia- low levels of IgG, IgA, and IgM; and absent
immunization titers
• ATAXIA TELANGECTACIA- decreased serum immunoglobulins and poor cell
mediated responses
• Hyper IgM syndrome (HIGM) is associated with defects in B cell class switching
and somatic hypermutation, which leads to normal/high IgM levels, but low
levels of IgG, IgA, IgE
• IPEX- Quantitative immunoglobulins may be normal, elevated, or low due to
severe protein-losing enteropathy. IgE levels are typically elevated, which may
relate to severe food allergies noted in some of these patients
• Hyper IgE Serum IgG, IgA, IgM are typically normal, IgE is increased
• Major Histocompatibility Complex Class II Deficiency B cells are present and
they express high levels of IgM and IgD

19. 5. FLOW CYTOMETRY
• SCID
Absent T cell, and variable number of B or NK cell

20. Nk cell
T and b cell
CD132 (common g chain) expression by
CD19þ B cells. Patient cells lack CD132
expression.
Gray shaded area, isotype control; black
line, anti-CD132 staining

21. Flow cytometric evaluation of
STAT5 phosphorylation
following IL-2 stimulation.
Patient cells fail to respond
, Flow cytometric evaluation of STAT3
phosphorylation following IL-21
stimulation. Patient cells fail to
respond. Gray shaded area, no
stimulant; black line, 20 min post-IL-
21 stimulation

22. X-Linked Agammaglobulinemia-
• Lymphocyte subset analysis will demonstrate a low/absent circulating B cell
population with normal T-cell counts
• Bruton’s Tyrosine Kinase (BTK) gene DEFECT

23. Di George Anomaly, Omenn syndrome-
• There is defects in T-cell development, this may be masked due to
transplacental transfer of maternal T lymphocytes.
• Therefore, analysis of markers of cell activation should be analyzed
since the transferred T cells will activate and expand in the infant.
• B cell is absent
• Typically, maternal T cells will display a memory (CD45RO+) phenotype
is expressed whereas a healthy infant should have predominantly naïve
CD45RA+ T cells

25. • Ataxia-Telangiectasia- large portion of these patients have a relative
increase in the γδ T-cell population and an inappropriate expansion of T-
cell clones leading to a low number of CD4+ /CD45RA+ T lymphocytes in
these patients
• Wiskott-Aldrich Syndrome- Flow cytometric detection of WASp

26. • HYPER Ig M- The most prevalent
cause of hyper IGM syndrome the X-
linked form due to a deficiency in
CD40 ligand (CD40L, CD154).
Flow cytometry can be used as a
screening test for the AR form of
HIGM by evaluating for the absence of
CD40, which is constitutively
expressed on B cells, monocytes, and
dendritic cells

27. Major Histocompatibility Complex Class II
Deficiency - .
• Immunophenotyping of these patients
demonstrates normal numbers of both
CD8+ T cells and B cells, with reduced or
absent CD4+ numbers.
• no detectable MHC class II proteins (HLA-
DR, HLA-DP, HLA-DQ, or HLA-DM)

28. • Leukocyte adhesion defect
LAD type 1 is the result of a mutation in the β2-integrin
protein CD18, which is shared by a number of integrin
molecules, including leukocyte function-associated
protein (LFA-1 or CD11a/CD18), Mac-1 (CD11b/CD18),
and p150/95 (CD11c/CD18). The diminished levels of the
CD11abc or CD18 expression by flow cytometry is a
diagnostic screen for LAD type 1
LAD type 2 is due to a defect in fucose metabolism,
which results in the absence of sialyl lewis X (CD15s)
which is a carbohydrate ligand on the cell surface of
neutrophils that binds to the E- and P-selectins on
activated endothelial cells. Abnormal CD15s expression
on neutrophils by flow cytometry is indicative of LAD type
2.
LAD type 3 is characterized by increased bleeding risk,
recurrent infections, and leukocytosis. Detection is from
mutation analysis

29. X-Linked Lymphoproliferative Syndrome-
• XLP-1 decreased/absent numbers of
invariant natural killer T cells .
decrease intracellular SAP in NK
and CD8 + T cells
• 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 or XIAP expression

30. • Familial HLH, Chediak-Higashi, and Griscelli
syndrome
• Lysosome-associated membrane protein-1
(LAMP1 or CD107a) is normally expressed
on the internal membrane of cytotoxic cell
granules, which contain perforin and
granzyme.
• These granules are transported to the cell
surface and fuse with the target cell in
order to release their contents.
• Perforin leads to pore formation and
osmotic lysis, whereas granzymes induce
apoptosis.
• Defective CD107a expression has been
used as a biomarker for disorders of
degranulation

31. • IPEX- This disease is caused by defects which affect the forkhead box P3
(Foxp3) protein, Flow cytometry can be used to identify Foxp3-expressing CD4+
T cells
Chronic granulomatous diseases.
• Fluorescent detection using Dihydro rhodamine 123 dye known as DHR assay
• Analysis of neutrophil oxidative burst
• Mutations in five components (gp91phox, p22phox, p47phox, p67phox, and
p40phox) of the NADPH complex

33. Autoimmune Lymphoproliferative Syndrome-
• This syndrome is caused by mutations in genes which induce lymphocyte
apoptosis.
• These mutations occur in the genes encoding for FAS (CD95), FAS ligand,
and caspase 10.
• One diagnostic criteria for this disease includes an increased percentage
of double-negative T cells (DNT, CD3+ CD4- CD8- TCRαβ+ )
• These cells also express B220 and CD27
• Other significant findings include decreased CD4+ CD25+ T cells, expanded
CD3+ HLADR+ T-cell population, decreased levels of CD27+ B cells,
increased CD5+ B-cell count, increased CD8+ CD57+ T-cell numbers

38. 6. Evaluation of complement system components
Screening for disorders of the complement system
CH50(total hemolytic complement) Classical pathway
AH50 Alternate pathway
C1, C2, or C4 deficiency - low CH50, but normal AH50
factor B, factor D, or properdin deficiency - low AH50, but normal CH50
shared complement component deficiency C3 or C5-C9 - decrease in both CH50
and AH50

39. SCREENING TESTS ADVANCED TESTS
B-CELL DEFICIENCY
IgG, IgM, IgA, and IgE levels Isohemagglutinin
titers
Ab response to vaccine antigens (e.g., tetanus,
diphtheria, pneumococci, Haemophilus
influenzae)
B-cell enumeration (CD19 or CD20)
Ab responses to boosters or to new vaccines
T-CELL DEFICIENCY
Lymphocyte count
Chest x-ray examination for thymic size
Delayed skin tests (e.g., Candida, tetanus
toxoid)
T-cell subset enumeration (CD3, CD4, CD8)
Proliferative responses to mitogens, antigens,
allogeneic cells
HLA typing
Chromosome analysis
PHAGOCYTIC DEFICIENCY
WBC count, morphology Respiratory burst
assay
Adhesion molecule assays (e.g., CD11b/CD18,
selectin ligand) Mutation analysis
COMPLEMENT DEFICIENCY
CH50 activity C3 level,C4 level
AH50,activity Component assays Activation
assays (e.g., C3a, C4a, C4d, C5a)

40. GENETIC TESTS – ADVANCED TESTS
• FISH using commercial probes: The
most common technique used to
detect 22q11.2 deletion (DiGeorge
Syndrome) utilizing a HIRA
(TUPLE1) probe
• OMENN- Omenn is classically
caused by hypomorphic mutations
in the RAG1 or RAG2 genes
• Western blotting – Ataxia
telengectasia - Immunoblotting is
preffered technique for detection
of ATM DNA damage

41. PCR- T cell defect- TREC detection- newborn
screening test.
It is performed on DNA isolated from the Guthrie
card blood spots. Decreased TRECs as a measure of
decreased thymopoiesis are seen in infants with
congenital T-cell defects - such as SCID, idiopathic
T lymphopenia, ataxia telangiectasia, di goerge
syndrome.
B cell defect – KREC detection- KRECs are
episomal DNA fragments generated during the
rearrangement process of the kappa light-chain
genes during B-cell development - Decreased
KREC is seen in X-linked agammaglobulinemia

42. • Targeted gene Sequencing-

49. CONCLUSION
• Diagnosis of specific PID from a large spectrum of disorders requires
expertise in clinical and laboratory evaluation.
• .Wide array of assays are available for evaluation of immune system which
help immensely in the diagnosis of PIDs.
• Knowledge of clinical presentation of these disorders, correct
interpretation of initial results of immunophenotyping of lymphocytes The
use of the laboratory in
• Evaluating the immune system should not follow a shotgun approach but
rather should be a focused evaluation using specific testing in an orderly
process based on the clinical history is essential for choosing the
appropriate test for specific diagnosis.

50. REFERECE
• Madkaikar M, Mishra A, Ghosh K. Diagnostic approach to primary immunodeficiency
disorders. Indian pediatrics. 2013 Jun 1;50(6):579-86.
• Locke BA, Dasu T, Verbsky JW. Laboratory diagnosis of primary immunodeficiencies.
Clinical reviews in allergy & immunology. 2014 Apr 1;46(2):154-68.
• Oliveira JB, Fleisher TA. Laboratory evaluation of primary immunodeficiencies. Journal of
Allergy and Clinical Immunology. 2010 Feb 1;125(2):S297-305.
• Kanegane H, Hoshino A, Okano T, Yasumi T, Wada T, Takada H, Okada S, Yamashita M, Yeh
TW, Nishikomori R, Takagi M. Flow cytometry-based diagnosis of primary
immunodeficiency diseases. Allergology International. 2018;67(1):43-54.
• Ladomenou, F., & Gaspar, B. (2016). How to use immunoglobulin levels in investigating
immune deficiencies. Archives of Disease in Childhood - Education & Practice Edition,
101(3), 129–135. doi:10.1136/archdischild-2015-309060
• Chinen J, Lawrence M, Dorsey M, Kobrynski LJ. Practical approach to genetic testing for
primary immunodeficiencies. Annals of Allergy, Asthma & Immunology. 2019 Nov
1;123(5):433-9.

51. THANK YOU