Laboratory diagnosis of primary immunodeficiencies

Laboratory Diagnosis of

Primary Immunodeficiencies

Suda Sibunruang, M.D.

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

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

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 immunologic assays, thus providing improvements in the diagnosis and treatment of PID


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


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


Scope • Systematic approach of the evaluation

of a suspected PID

• Comprehensive list of testing options and their results


Outline • Disorders of Humoral Immunity







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

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

Definition Hypogammaglobulinemia

• IgG value is less than 2 SD below

age-adjusted normal

Agammaglobulinemia

• IgG of less than 100 mg/dL


If either of these findings is found, then further immunologic workup should be pursued

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 of titers 4–6 weeks later is warranted



There is controversy regarding “normal” response to vaccination, particularly to polysaccharide vaccine

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

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


Howabout individuals receiving immunoglobulin therapy ? Since immunoglobulin contain detectable titers to common vaccines

(Keyhole limpet hemocyanin)

Natural IgM antibodies; Blood group antibodies

Titer > 1:8 in age above 1 year

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 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


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

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

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


(Inducible T-cell co-stimulator)

Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI)

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


TACI signals intracellularly through TNF receptor-associated factors (TRAF) to induce nuclear factor-κ-B activation

Calcium modulator and cyclophilin ligand (CAML)


(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

Laboratory investigations for CVID in the patients who are HIV-negative in whom other causes of recurrent

infections have been ruled out (1)


Laboratory investigations for CVID in the patients who are HIV-negative in whom other causes of recurrent

infections have been ruled out (2)


Cunningham-Rundles C. et al. Nature Reviews Immunology 2005;5:880-92

X-Linked Agammaglobulinemia

Genetic defect in Bruton’s Tyrosine Kinase (BTK) gene

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 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

de Vries E. et al. Eur J Pediatr 2001;160:583–91 Absent B cell, T cell & NK cell - present

de Vries E. et al. Eur J Pediatr 2001;160:583–91 Few B cell but mostly immature B cell (CD10+, CD19+,CD20-)


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

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


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 recombination excision circles

Circular DNA segments generated in T and B cells during their maturation in the thymus and bone marrow

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

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)

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 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

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.

Stracker TH et al. Front Genet 2013;25:1-54

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


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, lymphoblastoid

cell lines, and PBMC

- Increased γδ T-cell

- Low number of CD4+/CD45RA+

• KREC analysis


Slatter MA. Expert Rev Mol Med 2010 Mar 18;12:e9

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


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



Measures a cellular mediated memory response to a previously seen antigen

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


Ahmed AR and Blose DA. Arch Dermatol 1983;119:934-45

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


induce response in T cells

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

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

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

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)


Flow cytometry aids in diagnosis of SCID

Confirmation of SCID requires sequence analysis of suspected genes

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

Gennery AR, Cant AJ. J Clin Pathol 2001;54:191–5

Clinical characteristics • Erythroderma • Eosinophilia • Elevated IgE levels • Lymphadenopathy • Hepatosplenomegaly


Normal CD4+ CD45RA/RO+ profile Skewed CD4+ CD45RA/RO+ profile

T-cell repertoire analysis will show a restricted pattern of T-cell receptors

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


Immunophenotype of MHC Class II Deficiency


• 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)

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

http://www.ufrgs.br/

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

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

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 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


Picture from www.diseasesforum.com Access 12 February 2015

DiGeorge Syndrome

22q11 deletion syndrome - Thymic aplasia/hypoplasia - Decreased T cells

http://www.diseasesforum.com/

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 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 %

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

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 diminished levels of IgA and IgG

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)

Etzioni A, Ochs HD. Pediatr Res 2004;56:519-25

Qamar N, Fuleihan RL. Clinic Rev Allerg Immunol 2014;46:120–30


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

Qamar N, Fuleihan RL. Clinic Rev Allerg Immunol 2014;46:120–30

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

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


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


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

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

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

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


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

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

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

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

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

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

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

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

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

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 cytometry

• Genetic mutational analysis is necessary for

a definitive diagnosis


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

Picture from www.immunochemistry.com

Test effector function

Culture target cells with PBMCs Measuring markers of cell death i.e., annexin V, 7-ADD

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)


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

X-Linked Lymphoproliferative Syndrome (XLP)

• Fatal hemophagocytosis

• Hypogammaglobulinemia

• Lymphoma

• Severe infectious mononucleosis occurs

in 2/3 of all XLP patients


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

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

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

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


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

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.

Wen L. et al. J Allergy Clin Immunol 2004;113:585-93

Access from www.worldallergy.org , February 19, 2015

Immune Dysregulation, Polyendocrinopathy,

Enteropathy, X-Linked Syndrome (IPEX)


• 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

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

Autoimmune lymphoproliferative syndrome (ALPS)


• 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


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

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


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


Conclusion (3) • As the knowledge of the immune system

expands, new testing will become available to aid in the diagnosis of primary immunodeficiencies


Thank you for your attention

Laboratory diagnosis of primary immunodeficiencies

Health & Medicine

Transcript of Laboratory diagnosis of primary immunodeficiencies