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Laboratory Diagnosis of
Primary Immunodeficiencies
Suda Sibunruang, M.D.
Outline
• Introduction
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
•...
Primary Immunodeficiencies (PID)
• Heterogeneous group of disorders
• Careful history to delineate the pattern
of infectio...
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
im...
Immunologic assays
• Flow cytometric-based assays
- Immune cell function
(e.g., neutrophil oxidative burst, NK cytotoxicit...
In order to utilize these
complex assays…
One must have…
• Firm understanding of the immunologic
assay
• How the results a...
Scope
• Systematic approach of the evaluation
of a suspected PID
• Comprehensive list of testing options
and their results...
Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer...
Disorders of Humoral Immunity
Ballow M. Global Atlas of Allergy 2014; 248-50
Locke BA et al. Clinic Rev Allerg Immunol 201...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Agarwal S. et al. Ann Allergy Asthma Immunol 2007;99:281-3
It is important that the results of IgG levels be
compared to a...
Definition
Hypogammaglobulinemia
• IgG value is less than 2 SD below
age-adjusted normal
Agammaglobulinemia
• IgG of less ...
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...
Orange JS. J Allergy Clin Immunol 2012;130:S1-24
There is controversy regarding “normal” response
to vaccination, particul...
Criteria for adequate vaccination response
• A measured protective titer per lab normals
• A 4-fold increase in post-vacci...
Kamchaisatian W. et al. J Allergy Clin Immunol 2006;118:1336-41
Validation of current joint American Academy of Allergy, A...
Orange JS. J Allergy Clin Immunol 2012;130:S1-24
Howabout individuals receiving immunoglobulin therapy ?
Since immunoglobu...
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
Park MA et al. Lancet 2008;372: 489–502
Common Variable Immunodeficiency (CVID)
Diagnosis of CVID
ESID/Pan-American Group for Immunodeficiency (PAGID) criteria
• male or female patient with a marked dec...
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
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Naïve B cells
(IgD+ CD27-)
Unswitched memory
B cells (IgD+ CD27+)...
Baumert E et al. Clin Exp Immunol 1992;90:25–30
Memory T-cell compartment can also demonstrate abnormalities,
with a reduc...
Wehr C. et al. Blood 2008;111:77–85
Wehr C. et al. Blood 2008;111:77–85
Classification of CVID by B-cell phenotyping (EUROclass)
Isnardi I. et al. Blood 2010;115:5026-36
CD21- B cells is seen in patients who are
more likely to develop autoimmunity
Rosel AL et al. J Allergy Clin Immunol 2015;135:198-208
Park MA et al. Lancet 2008;372: 489–502
(Inducible T-cell co-stimulator)
Transmembrane activator and calcium-modulating
cy...
Martin F and Dixit VM. Nat Genet 2005;37:829–34
Receptors for BAFF and APRIL control
B cell development and function
TNFR ...
Park MA et al. Lancet 2008;372: 489–502
TACI signals intracellularly through
TNF receptor-associated factors (TRAF)
to ind...
Park MA et al. Lancet 2008;372: 489–502
Park MA et al. Lancet 2008;372: 489–502
(Inducible T-cell co-stimulator)
Transmembrane activator and calcium-modulating
cy...
Laboratory investigations for CVID in the patients
who are HIV-negative in whom other causes of recurrent
infections have ...
Laboratory investigations for CVID in the patients
who are HIV-negative in whom other causes of recurrent
infections have ...
Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92
X-Linked Agammaglobulinemia
Genetic defect in Bruton’...
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...
de Vries E. et al. Eur J Pediatr 2001;160:583–91Absent B cell, T cell & NK cell - present
de Vries E. et al. Eur J Pediatr 2001;160:583–91Few B cell but mostly immature B cell (CD10+, CD19+,CD20-)
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Intracellular expression of BTK in monocytes
Detection of monocyt...
Misdiagnosed as CVID
• Some patients with BTK mutations have been
misdiagnosed as CVID patients who may
present later with...
Baker MW et. al. Public Health Rep 2010;125:S88–95
Serana F. et al. Journal of Translational Medicine 2013;11:119
T-cell receptor excision circles Kappa-deleting recombinati...
Borte S et. al. Blood 2012;119:2552–5
TREC and KREC copy numbers in dried blood spot
samples (DBSS) from anonymized Guthri...
McKinnon PJ. EMBO reports 2004;5:772–6
Multisystem syndrome
- Results from mutation of ATM
(ataxia telangiectasia,mutated)...
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
McKinnon PJ. EMBO reports 2004;5:772–6
Slatter MA. Expert Rev Mol Med 2010 Mar 18;12:e9
• ATM in inactive multimeric compl...
McKinnon PJ. EMBO reports 2004;5:772–6
Slatter MA. Expert Rev Mol Med 2010 Mar 18;12:e9
In humans, approximately 10–15 %
o...
Stracker TH et al. Front Genet 2013;25:1-54
Ataxia-Telangiectasia
• Markedly decreased serum
immunoglobulins and poor cell mediated
responses
• Sensitivity to γ-radia...
Waldmann TA McIntire KR. Lancet 1972;2:1112-5
Serum alpha-fetoprotein
Laboratory
• Flow cytometry
- Alterations of ATM protein or
phosphorylated histone H2AX
- γ-H2AX in T-cell lines, lymphobl...
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...
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 adu...
Transplacentally acquired
maternal T lymphocytes
• Low T cells are typically observed in majority
of defects in T-cell dev...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Measures a cellular mediated memory
response to a previously seen...
Some caveats for DTH testing
• Requires that there has been previous exposure
to antigen
• Not recommended to perform on c...
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)
• Concanavalin (ConA)
• Anti-CD3 antibodies
• Pokeweed (PWM) - stimulate b...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Serana F. et al. Journal of Translational Medicine 2013;11:119
TREC are surrogate marker of naïve T cells
Although this is...
T-cell repertoire analysis
• In normal individuals, TCR repertoire is stable
and polyclonal
• Clonal populations are the h...
Hodges E et al. J Clin Pathol 2003;56:1–11
Healthy
T cell
lymphoma
Flow cytometric T cell receptor  variable gene (TCRVB)...
Pilch H et al. Clin Diagn Lab Immunol 2002;9:257–66
Durandy A, Kracker S, Fischer A. Nat Rev Immunol 2013;13:519-33
Severe Combined Immunodeficiency (SCID)
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Flow cytometry aids in diagnosis of SCID
Confirmation of SCID
req...
Kato M. et al. Allergology International 2006;55:115-9
Hypomorphic mutations in the RAG1 or RAG2
genes, but can be observe...
Gennery AR, Cant AJ. J Clin Pathol 2001;54:191–5
Clinical characteristics
• Erythroderma
• Eosinophilia
• Elevated IgE lev...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Normal CD4+ CD45RA/RO+ profile Skewed CD4+ CD45RA/RO+ profile
T-c...
Major Histocompatibility Complex
Class II Deficiency
• Rare autosomal recessive disease
• Loss of expression of MHC class ...
Immunophenotype of
MHC Class II Deficiency
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
• Normal numbers of bo...
Picture from www.ufrgs.br, access 12 Feb 2015
Wiskott-Aldrich Syndrome (WAS)
Rare X-linked recessive disease caused by mut...
Massaad MJ et. al. Ann N Y Acad Sci 2013 May;1285:26-43
Nonsense mutations, insertions, deletions, and complex
mutations a...
WASP
• Key regulator of actin polymerization in
hematopoietic cells
• Involve in signaling, cell locomotion, and
immune sy...
Ochs HD, Thrasher AJ. J Allergy Clin Immunol 2006;117:725-38
Yamada M. et al. Blood 1999;93:756-9
Flow cytometric analysis of WASP expression
Wiskott-Aldrich Syndrome
• Presence of WASp, however, does not
exclude the diagnosis, and sequencing
analysis should be se...
Picture from www.diseasesforum.com
Access 12 February 2015
DiGeorge Syndrome
22q11 deletion syndrome
- Thymic aplasia/hypo...
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 ...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Picture from www.mcqs.leedsmedics.org.uk
Fluorescent in situ hybr...
Tomita-Mitchell A. et al. Physiol Genomics 2010;42:52–60
Multiplexed quantitative real-time PCR to detect 22q11.2
deletion...
Maggadottir SM, Sullivan KE. J Allergy Clin Immunol Pract 2013;1:589-94
Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92
Hyper IgM Syndrome
Normal/high IgM levels, but dimini...
Abbas AK et al. Cellular and Molecular immunology. Eighth Edition
Defects in T Cell–Dependent B Cell Activation
X-linked h...
Etzioni A, Ochs HD. Pediatr Res 2004;56:519-25
Qamar N, Fuleihan RL. Clinic Rev Allerg Immunol 2014;46:120–30
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
CD40 ligand (CD154) expression
Lack of CD40L expression
after act...
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...
Disorders of Neutrophils
• Neutropenia
• Defect in adhesion: LAD
• Defect in killing: CGD
• Defect in signaling: IL-12, IL...
Disorders of Neutrophils
Typically present as
• Recurrent skin and respiratory tract
infections due to either bacteria or ...
Initial screening
• CBC, absolute neutrophil count (ANC) and
morphology of neutrophils
• High ANC can be seen in response ...
Zimmerman GA. Blood 2009;113:4485-6
Leukocyte Adhesion Deficiency
- Typically presents with
recurrent bacterial infections...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Picture from www.immunopaedia.org.za
Chronic Granulomatous Diseas...
Hampton MB. et al. Blood 1998;92:3007-17
Lack of NADPH oxidase
which is made up of
• One X-linked gene
- Cytochrome b-245,...
Nitroblue tetrazolium test
Lekstrom-Himes JA, Gallin JI. N Engl J Med 2000;343:1703-14
Measure ability of phagocytic cells...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Neutrophil oxidative burst in CGD
Normal activated neutrophils pr...
Hampton MB. et al. Blood 1998;92:3007-17
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Myeloperoxidase Deficien...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Picture from www.pathologyoutlines.com
Definitive diagnosis is by...
Ho HY et. al. Free Radical Research 2014; 48: 1028–48
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
• G6P reduc...
Ardati KO et al. Acta Haematol 1997;97:211–5
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
G6PD deficiency is r...
Detection of G6PD Deficiency
• DNA tests
• Measurement of NADPH production capacity of
G6PD
- Fluorescent spot test
- Spec...
Peters AL, Van Noorden C. J Histochem Cytochem 2009;57:1003–11
Cells lack G6PD activity, do not contain formazan crystals,...
Buckley RH. and Orange JS. Middleton’s Allergy 8th edition, 2014, 1144-74
Hyper IgE syndrome (HIES)
- Recurrent S. aureus
...
Buckley RH. and Orange JS. Middleton’s Allergy 8th edition, 2014, 1144-74
STAT3 deficiency DOCK8 deficiency
Majority of HI...
Engelhardt KR et al. J Allergy Clin Immunol 2012;129:294–305
Mechanism of DOCK8 mutations is not entirely understood
Laboratory
• Decrease in IL-17-producing T cells (TH17)
• Screen percentage of Th17 cells in the
peripheral blood by flow ...
Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer...
Orange JS, Ballas ZK. Clin Immunol 2006;118:1–10
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
NK cell
Erythrol...
Picture from www.immunochemistry.com
Test effector function
Culture target cells with PBMCs Measuring markers of cell deat...
Chromium Release Assays
Access from www.cai.md.chula.ac.th , February 19, 2015
Orange JS, Ballas ZK. Clin Immunol 2006;118:1–10
Intracellular cytotoxic proteins (i.e., perforin/granzyme)
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
NK-cell lysosomal-associated membrane protein-1 (LAMP-1)/
CD107a ...
X-Linked Lymphoproliferative Syndrome (XLP)
• Fatal hemophagocytosis
• Hypogammaglobulinemia
• Lymphoma
• Severe infectiou...
Marsh RA et al. J Immunol Methods 2010;362:1-9
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
There are two diff...
Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer...
Dorman SE and Holland SM Cytokine & Growth Factor Reviews 2000;11;321-33
• Production of cytokines and chemokines
• Enhanc...
Casanova JL. Swiss Med Wkly 2001;131:445-54
Muhsen SA and Casanova JL. J Allergy Clin Immunol 2008;122:1043-51
Six genes h...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Abbas AK et al. Cellular and Molecular immunology. Eighth Edition...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Evaluation of STAT1 and STAT4
Normal phosphorylation
of STAT1 in ...
Outline
• Disorders of Humoral Immunity
• Cellular and Combined Immune Defects
• Disorders of Neutrophils
• Natural Killer...
Disorders of Complement System
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Wen L. et al. J Allergy Clin Immun...
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...
Immune Dysregulation, Polyendocrinopathy,
Enteropathy, X-Linked Syndrome (IPEX)
Locke BA et al. Clinic Rev Allerg Immunol ...
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
Flow cytometry ...
Autoimmune lymphoproliferative
syndrome (ALPS)
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Cunningham-Rundles...
Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
Double-negative T cells (DNTs)
Normally, less than 2 % of TCRαβ+ ...
Conclusion (1)
• Stepwise, systematic Laboratory evaluation
of PID is important to reach a correct
diagnosis without unnec...
Conclusion (2)
• There have been many new innovations in
immunodiagnostic studies over the last
several years with new pro...
Conclusion (3)
• As the knowledge of the immune system
expands, new testing will become available
to aid in the diagnosis ...
Thank you for your attention
Laboratory diagnosis of primary immunodeficiencies
Laboratory diagnosis of primary immunodeficiencies
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Laboratory diagnosis of primary immunodeficiencies

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Laboratory diagnosis of primary immunodeficiencies

Presented by Suda Sibunruang, MD.

February 20, 2015

Published in: Health & Medicine

Laboratory diagnosis of primary immunodeficiencies

  1. 1. Laboratory Diagnosis of Primary Immunodeficiencies Suda Sibunruang, M.D.
  2. 2. 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
  3. 3. 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
  4. 4. Ochs HD et al. Ann Allergy Asthma Immunol 2014;112: 489-95
  5. 5. Oliveira JB, Fleisher TA. J Allergy Clin Immunol 2010;125:S297-305
  6. 6. 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
  7. 7. 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
  8. 8. 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
  9. 9. 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
  10. 10. 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
  11. 11. Disorders of Humoral Immunity Ballow M. Global Atlas of Allergy 2014; 248-50 Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 50–60 % of all identified primary immunodeficiencies
  12. 12. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
  13. 13. Agarwal S. et al. Ann Allergy Asthma Immunol 2007;99:281-3 It is important that the results of IgG levels be compared to age-adjusted normal values
  14. 14. Definition Hypogammaglobulinemia • IgG value is less than 2 SD below age-adjusted normal Agammaglobulinemia • IgG of less than 100 mg/dL Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 If either of these findings is found, then further immunologic workup should be pursued
  15. 15. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
  16. 16. Orange JS. J Allergy Clin Immunol 2012;130:S1-24
  17. 17. 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
  18. 18. Orange JS. J Allergy Clin Immunol 2012;130:S1-24 There is controversy regarding “normal” response to vaccination, particularly to polysaccharide vaccine
  19. 19. 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
  20. 20. Kamchaisatian W. et al. J Allergy Clin Immunol 2006;118:1336-41 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
  21. 21. Orange JS. J Allergy Clin Immunol 2012;130:S1-24 Howabout individuals receiving immunoglobulin therapy ? Since immunoglobulin contain detectable titers to common vaccines (Keyhole limpet hemocyanin)
  22. 22. Natural IgM antibodies; Blood group antibodies Titer > 1:8 in age above 1 year
  23. 23. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
  24. 24. Park MA et al. Lancet 2008;372: 489–502 Common Variable Immunodeficiency (CVID)
  25. 25. 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
  26. 26. Park MA et al. Lancet 2008;372: 489–502
  27. 27. Park MA et al. Lancet 2008;372: 489–502
  28. 28. Park MA et al. Lancet 2008;372: 489–502
  29. 29. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Naïve B cells (IgD+ CD27-) Unswitched memory B cells (IgD+ CD27+) Switch memory B cells (IgD- CD27+) Low numbers of switch memory B cells Flow cytometry can be used to evaluate maturational state of B lymphocytes
  30. 30. Baumert E et al. Clin Exp Immunol 1992;90:25–30 Memory T-cell compartment can also demonstrate abnormalities, with a reduced CD4+/CD8+ ratio and diminished percentage of CD4+ Tcells expressing CD45RA
  31. 31. Wehr C. et al. Blood 2008;111:77–85
  32. 32. Wehr C. et al. Blood 2008;111:77–85 Classification of CVID by B-cell phenotyping (EUROclass)
  33. 33. Isnardi I. et al. Blood 2010;115:5026-36 CD21- B cells is seen in patients who are more likely to develop autoimmunity
  34. 34. Rosel AL et al. J Allergy Clin Immunol 2015;135:198-208
  35. 35. Park MA et al. Lancet 2008;372: 489–502 (Inducible T-cell co-stimulator) Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI)
  36. 36. Martin F and Dixit VM. Nat Genet 2005;37:829–34 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
  37. 37. Park MA et al. Lancet 2008;372: 489–502 TACI signals intracellularly through TNF receptor-associated factors (TRAF) to induce nuclear factor-κ-B activation Calcium modulator and cyclophilin ligand (CAML)
  38. 38. Park MA et al. Lancet 2008;372: 489–502
  39. 39. Park MA et al. Lancet 2008;372: 489–502 (Inducible T-cell co-stimulator) Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) Flow cytometry can be used to demonstrate reduced expression of protein markers TACI, CD19, and BAFFR, but this should be paired with genetic evaluation
  40. 40. 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
  41. 41. 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
  42. 42. Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92 X-Linked Agammaglobulinemia Genetic defect in Bruton’s Tyrosine Kinase (BTK) gene
  43. 43. Ponader S. and Burger JA. J Clin Oncol 2014;32:1830-9
  44. 44. 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
  45. 45. de Vries E. et al. Eur J Pediatr 2001;160:583–91Absent B cell, T cell & NK cell - present
  46. 46. de Vries E. et al. Eur J Pediatr 2001;160:583–91Few B cell but mostly immature B cell (CD10+, CD19+,CD20-)
  47. 47. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Intracellular expression of BTK in monocytes Detection of monocytes and B cells BTK expression in monocytes This modality does not pick up all mutations, only those that affect protein stability. For this reason, it is recommended to perform BTK sequence analysis
  48. 48. Misdiagnosed as CVID • Some patients with BTK mutations have been misdiagnosed as CVID patients who may present later with low B-cell counts and low-level antibody production • Recognize and evaluate for BTK mutation in cases which may clinically correlate with CVID Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
  49. 49. Baker MW et. al. Public Health Rep 2010;125:S88–95
  50. 50. Serana F. et al. Journal of Translational Medicine 2013;11:119 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
  51. 51. Borte S et. al. Blood 2012;119:2552–5 TREC and KREC copy numbers in dried blood spot samples (DBSS) from anonymized Guthrie cards PCR of newborn blood spots to quantitate these non-replicative DNA elements Nijmegen-breakage -syndrome
  52. 52. McKinnon PJ. EMBO reports 2004;5:772–6 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) Ataxia telangiectasia (AT)
  53. 53. ATM • A protein kinase that responds to DNA damage • Controls cell-cycle checkpoints McKinnon PJ. EMBO reports 2004;5:772–6
  54. 54. Stracker TH et al. Front Genet 2013;25:1-54
  55. 55. McKinnon PJ. EMBO reports 2004;5:772–6 Slatter MA. Expert Rev Mol Med 2010 Mar 18;12:e9 • 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
  56. 56. 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.
  57. 57. Stracker TH et al. Front Genet 2013;25:1-54
  58. 58. 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
  59. 59. Waldmann TA McIntire KR. Lancet 1972;2:1112-5 Serum alpha-fetoprotein
  60. 60. 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
  61. 61. Slatter MA. Expert Rev Mol Med 2010 Mar 18;12:e9
  62. 62. 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
  63. 63. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
  64. 64. 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
  65. 65. 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 infant should have predominantly naïve CD45RA+ T cells Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
  66. 66. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Measures a cellular mediated memory response to a previously seen antigen
  67. 67. 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
  68. 68. Ahmed AR and Blose DA. Arch Dermatol 1983;119:934-45
  69. 69. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
  70. 70. Common mitogens used • Phytohemagglutinin (PHA) • Concanavalin (ConA) • 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 induce response in T cells
  71. 71. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68
  72. 72. Serana F. et al. Journal of Translational Medicine 2013;11:119 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
  73. 73. 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
  74. 74. Hodges E et al. J Clin Pathol 2003;56:1–11 Healthy T cell lymphoma Flow cytometric T cell receptor  variable gene (TCRVB) profiles % V positive T cells % V positive T cells
  75. 75. Pilch H et al. Clin Diagn Lab Immunol 2002;9:257–66
  76. 76. Durandy A, Kracker S, Fischer A. Nat Rev Immunol 2013;13:519-33 Severe Combined Immunodeficiency (SCID)
  77. 77. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Flow cytometry aids in diagnosis of SCID Confirmation of SCID requires sequence analysis of suspected genes
  78. 78. Kato M. et al. Allergology International 2006;55:115-9 Hypomorphic mutations in the RAG1 or RAG2 genes, but can be observed with other gene defects Predominant CD45RO+ Increased % of γδ T cells
  79. 79. Gennery AR, Cant AJ. J Clin Pathol 2001;54:191–5 Clinical characteristics • Erythroderma • Eosinophilia • Elevated IgE levels • Lymphadenopathy • Hepatosplenomegaly
  80. 80. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Normal CD4+ CD45RA/RO+ profile Skewed CD4+ CD45RA/RO+ profile T-cell repertoire analysis will show a restricted pattern of T-cell receptors
  81. 81. 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
  82. 82. Immunophenotype of MHC Class II Deficiency Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 • 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)
  83. 83. Picture from www.ufrgs.br, access 12 Feb 2015 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
  84. 84. Massaad MJ et. al. Ann N Y Acad Sci 2013 May;1285:26-43 Nonsense mutations, insertions, deletions, and complex mutations are distributed throughout the WAS gene and result in WAS
  85. 85. 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
  86. 86. Ochs HD, Thrasher AJ. J Allergy Clin Immunol 2006;117:725-38
  87. 87. Yamada M. et al. Blood 1999;93:756-9 Flow cytometric analysis of WASP expression
  88. 88. 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
  89. 89. Picture from www.diseasesforum.com Access 12 February 2015 DiGeorge Syndrome 22q11 deletion syndrome - Thymic aplasia/hypoplasia - Decreased T cells
  90. 90. Maggadottir SM, Sullivan KE. J Allergy Clin Immunol Pract 2013;1:589-94
  91. 91. 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
  92. 92. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Picture from www.mcqs.leedsmedics.org.uk Fluorescent in situ hybridization (FISH) utilizing a HIRA (TUPLE1) probe False-negative rate of 5 %
  93. 93. Tomita-Mitchell A. et al. Physiol Genomics 2010;42:52–60 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
  94. 94. Maggadottir SM, Sullivan KE. J Allergy Clin Immunol Pract 2013;1:589-94
  95. 95. Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92 Hyper IgM Syndrome Normal/high IgM levels, but diminished levels of IgA and IgG
  96. 96. Abbas AK et al. Cellular and Molecular immunology. Eighth Edition 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)
  97. 97. Etzioni A, Ochs HD. Pediatr Res 2004;56:519-25
  98. 98. Qamar N, Fuleihan RL. Clinic Rev Allerg Immunol 2014;46:120–30
  99. 99. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 CD40 ligand (CD154) expression Lack of CD40L expression after activation Sensitivity can be enhanced to 90 % by using a biotinylated CD40-Ig fusion protein which binds to a functional CD40L receptor complex
  100. 100. Qamar N, Fuleihan RL. Clinic Rev Allerg Immunol 2014;46:120–30
  101. 101. 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
  102. 102. 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
  103. 103. 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
  104. 104. 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
  105. 105. Zimmerman GA. Blood 2009;113:4485-6 Leukocyte Adhesion Deficiency - Typically presents with recurrent bacterial infections with no pus formation - Delayed umbilical cord separation or omphalitis CD18,11 absence of sialyl lewis X (CD15s) LAD3 diagnosis requires specialized testing of integrin function in platelets or leukocytes or by molecular methods
  106. 106. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Picture from www.immunopaedia.org.za 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
  107. 107. Hampton MB. et al. Blood 1998;92:3007-17 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
  108. 108. Nitroblue tetrazolium test Lekstrom-Himes JA, Gallin JI. N Engl J Med 2000;343:1703-14 Measure ability of phagocytic cells to ingest and reduce a soluble yellow dye to an intracellular blue crystal
  109. 109. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 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 Dihydrorhodamine 123 (DHR) assay
  110. 110. Hampton MB. et al. Blood 1998;92:3007-17 Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 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
  111. 111. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Picture from www.pathologyoutlines.com 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
  112. 112. Ho HY et. al. Free Radical Research 2014; 48: 1028–48 Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 • G6P reduces NADP to NADPH • NADPH is needed for production of gluthatione • Gluthatione is needed in maintain of RBC membrane from oxidant stress Glucose-6-Phosphate Dehydrogenase Deficiency
  113. 113. Ardati KO et al. Acta Haematol 1997;97:211–5 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
  114. 114. 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
  115. 115. Peters AL, Van Noorden C. J Histochem Cytochem 2009;57:1003–11 Cells lack G6PD activity, do not contain formazan crystals, show strong autofluorescence The more formazan crystals are present in cells, the less fluorescence can be observed
  116. 116. Buckley RH. and Orange JS. Middleton’s Allergy 8th edition, 2014, 1144-74 Hyper IgE syndrome (HIES) - Recurrent S. aureus infections of the skin and pulmonary tract - High IgE - Eosinophilia - Eczema - Mucocutaneous candidiasis
  117. 117. Buckley RH. and Orange JS. Middleton’s Allergy 8th edition, 2014, 1144-74 STAT3 deficiency DOCK8 deficiency Majority of HIES have a heterozygous, dominant-negative mutation in STAT3 which is critical for inducing RORγt Wart predisposition to malignancies at a young age
  118. 118. Engelhardt KR et al. J Allergy Clin Immunol 2012;129:294–305 Mechanism of DOCK8 mutations is not entirely understood
  119. 119. 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
  120. 120. 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
  121. 121. Orange JS, Ballas ZK. Clin Immunol 2006;118:1–10 Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 NK cell Erythroleukemia cell Perforin Natural Killer (NK) cells • Play a key role in defending against viral infections • If defects, typically present 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 confirm the presence or absence of NK cells
  122. 122. Picture from www.immunochemistry.com Test effector function Culture target cells with PBMCs Measuring markers of cell death i.e., annexin V, 7-ADD
  123. 123. Chromium Release Assays Access from www.cai.md.chula.ac.th , February 19, 2015
  124. 124. Orange JS, Ballas ZK. Clin Immunol 2006;118:1–10 Intracellular cytotoxic proteins (i.e., perforin/granzyme)
  125. 125. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 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
  126. 126. 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
  127. 127. Marsh RA et al. J Immunol Methods 2010;362:1-9 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 or XIAP expression
  128. 128. 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
  129. 129. Dorman SE and Holland SM Cytokine & Growth Factor Reviews 2000;11;321-33 • 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) Main regulatory pathway of cell-mediated immunity Inherited Susceptibility to Mycobacterial Disease
  130. 130. Casanova JL. Swiss Med Wkly 2001;131:445-54 Muhsen SA and Casanova JL. J Allergy Clin Immunol 2008;122:1043-51 Six genes have been found to be mutated IFNGR1 & IFNGR2 STAT 1 IL12 p40 IL12RB1 NEMO Flow cytometry for IL12RB1 and IFNGR1
  131. 131. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Abbas AK et al. Cellular and Molecular immunology. Eighth Edition 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
  132. 132. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 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
  133. 133. 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
  134. 134. Disorders of Complement System Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Wen L. et al. J Allergy Clin Immunol 2004;113:585-93 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.
  135. 135. Wen L. et al. J Allergy Clin Immunol 2004;113:585-93
  136. 136. Access from www.worldallergy.org , February 19, 2015
  137. 137. 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
  138. 138. Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked Syndrome (IPEX) Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92 • 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
  139. 139. 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 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
  140. 140. Autoimmune lymphoproliferative syndrome (ALPS) Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92 • 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
  141. 141. Locke BA et al. Clinic Rev Allerg Immunol 2014;46:154–68 Double-negative T cells (DNTs) Normally, less than 2 % of TCRαβ+ T cells do not express either the CD4 or CD8 co-receptors Increased DNT cells in a patient with ALPS
  142. 142. 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
  143. 143. 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
  144. 144. 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
  145. 145. Thank you for your attention

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