Download Fleisher WAC immune lab testing

Laboratory Testing: Innate and
Adaptive Immune Systems
Thomas A. Fleisher, M.D., FAAAAI, FACAAI
National Institutes of Health
Bethesda, MD, USA
Dr. Fleisher has no conflicts of
interest related to this presentation
Principles of Immune Testing
STARTING POINT INVOLVES A PATIENT:
• Medical history of recurrent infections
including microorganism(s), frequency,
site(s), therapy required
• Family history with attention to early deaths
and recurrent infections
• Physical examination
Evaluation of Adaptive Immunity:
B Cell Function Screening Tests
• History of recurrent sinopulmonary
infections with encapsulated bacteria
• Screening Tests
• Immunoglobulin (IgG, IgA, IgM, IgE) levels
• Specific antibody response
• Protein antigens
• CHO antigens
• HIV testing
Immunoglobulin Levels
• Age related differences are significant and must
be considered for each evaluation (i.e. use age
specific reference intervals to evaluate values)
• Normal range = 95% confidence interval
2.5% controls below
2.5% controls above
• Total immunoglobulin level (for each class) is the
net of production, consumption, and loss
IgG Subclass Levels
• Test is relatively expensive
• Frequency of isolated IgG subclass deficiency
remains controversial
• May be useful in evaluating IgA deficient
patients with recurrent infections
• Generally still need to establish a failure to
produce specific antibody before initiating
IgG replacement therapy
Specific Antibody Response
• Represents the “ standard” regarding the
capacity to mount an antibody response
• “Natural antibody” screen: testing for
isohemagglutinins (<1-2 yrs unreliable),
antibody level to prior immunization
• Provocative testing: immunize with protein
and CHO vaccines, obtain pre- and postantibody levels
Evaluation of Adaptive Immunity:
Screening of T Cell Function
• History recurrent opportunistic infections
often with failure to thrive
• Screening Tests
– HIV test
– Lymphocyte count (T cells = ~75% of lymphs)
– DTH testing (used less frequently in USA)
• Specific response to recall antigens in vivo: antigen
specific T cell activation, cytokine production,
inflammatory cell migration
• Lack of response: anergy versus no prior exposure
Secondary Adaptive Immune
Testing: Primary Focus on T cells
In vitro assays that help identify the cellular
level or degree of T cell dysfunction
• Immune cell and specific protein identification:
flow cytometry
• Immune cell function (ordered based on strong
history of a cellular immune defect)
• T cell proliferation/cytokine assay (mitogens,
recall antigens)
• T cell cytotoxicity assays: used infrequently
in clinical evaluation
Flow Cytometry in the Evaluation of
Adaptive Immunity:
Cell Directed Evaluation
• Evaluation for absence of a lymphocyte population/
subpopulation (e.g. B cells - XLA, B & NK cells - XSCID)
• Evaluation for a specific cell surface protein (e.g. CD40
ligand/CD154 [activated CD4+], IFNgR [monocytes])
• Test for an intracellular protein: specific disease screen
(e.g. BTK – XLA [monocytes], FOXP3 – IPEX [Tregs])
Assessment for Biologic Effect
• Memory/naive T cells (CD45RO/RA, CD62L)
• Memory B cells (CD27)
• B cell isotype switch
Flow Cytometric Evaluation of a Child
with Agammaglobulinemia
B cells =0.2%
T cells =95%
Low or absent B cells = XLA or AR agammaglobulinemia;
Flow Cytometric Evaluation of an
Infant with Opportunistic Infections
No NK cells
Normal B cells
Very low T cells
T-/B+/NK- XSCID; flow allows four cell based phenotypes
of SCID: T-/B-/NK-; T-/B-/NK+; T-/B+/NK-; T-/B+/NK+
Lymphocyte Function Testing
• Response to mitogens, recall antigens, allogeneic
cells
– Proliferation assessed by 3 H-thymidine incorporation
– Cytokine release into culture supernatant
– Activation antigen upregulation (e.g. CD69 by flow)
– Cell division(e.g. CFSE) or cell cycle (e.g. BrDU)
• Cytotoxicity:
– Antigen specific: requires presensitization, initiated thru
TcR recognition of viral (or other) peptide on MHC
• 51Cr release from target cells
• Flow cytometry test for CD107a expression by cytotoxic T cell
Evaluating Innate Immunity
Evaluate NK cells
• Very few patients identified with isolated NK
cell absence
• HLH and XLP are associated with functional
NK cell deficiency
• Enumerate NK cells using flow cytometry
• Evaluate NK cell functional activity using in
vitro cytotoxicity assay with K562 targets
– 51Cr release from target cells
– Flow cytometry test for CD107a expression by NK cells
Evaluating TLR Function
• Clinical phenotype of TLR defects
– Recurrent pyogenic infections with limited
systemic symptoms (minimal fever, normal or
low CRP, etc)
– Herpes simplex encephalitis
• Screening tests of TLRs utilize stimulation by
ligands for specific TLRs
• Currently this testing has relatively limited
availability in clinical laboratories
Evaluating TLR Response
I- Activation in vitro
TLR specific ligand
MC
PBMC
37ºC
5% CO2
MC
MC
2.0 x 105
cells/well
Cells
Supernatant
II- Quantify Response
MC
MC
CD62L shedding (Flow)
Multiplex
assay
TNF ELISA
Von Bernuth, et al, Pediatrics, 118:2498-2503, 2006; Deering and Orange, Clin Vaccine Immunol, 12:68-76, 2006
Neutrophil Immunodeficiency
• History of recurrent/chronic infections with
bacteria and fungi involving the skin, lungs,
bone, liver and oral cavity
• Most often is 2o to neutropenia
• Defined genetic defects primarily impact
microbial killing (CGD) or neutrophil
migration (LAD)
Screening of Neutrophil Function
• Absolute neutrophil count (ANC)
• Evaluation of oxidative burst
– CGD results from defective oxidative burst
– DHR test, NBT test, chemiluminescence
• Evaluation of adhesion molecules
– Leukocyte adhesion deficiency (LAD) I results
from defective CD18 (b2 integrin) expression
– Test for CD18 (+ CD11a,b,c) expression
DHR Assay to Diagnose CGD
Normal
Phox47
deficient
CGD (AR)
Phox91
deficient
CGD (Xlinked)
X-linked
CGD
carrier
Flow Cytometric Analysis of CD18
Surface Expression in Unstimulated Cells
Lymphocytes
PMNs
Normal
LAD1 patient
Absent/markedly
decreased CD18
expression =
LAD type I
Complement Deficiency
• Clinical presentation
• Recurrent encapsulated bacterial infections:
early component deficiency (C1q, C2, C4, C3)
• Recurrent meningococcal infections: deficiency
of terminal complement components (C5-C9)
• Screening test: CH50 (functional assay of
total hemolytic complement activity)
• Additional testing: AP50 and complement
component assays
Summary
• Laboratory testing is crucial in evaluating
patients for possible immune defects
• The choice of tests should be determined
based on the specific clinical history of
recurrent and/or chronic infections
• There remain patients with a clinical history
strongly suggesting an immune defect that
using current testing remain undiagnosed
• Genetic testing is emerging as an important
diagnostic test in resolving possible PID
References
• Notarangelo LD, et al. Primary immunodeficiencies: 2009
update. J Allergy Clin Immunol. 2009, 124:1161.
• Oliveira JB, Fleisher TA. Laboratory evaluation of primary
immunodeficiencies. J Allergy Clin Immunol. 2010, 125:S297.
• Oliveira JB, Fleisher TA. Molecular and flow cytometry-based
diagnosis of primary immunodeficiency disorders. Curr Allergy
Asthma Rep. 2010, 10:460.
• Rosenzweig SD, Holland SM. Recent insights into the pathobiology of innate immune deficiencies. Curr Allergy Asthma
Rep. 2011, 11:369.