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Laboratory Testing: Innate and Adaptive Immune Systems

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:

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Laboratory Testing: Innate and Adaptive Immune Systems

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  1. Laboratory Testing: Innate and Adaptive Immune Systems Thomas A. Fleisher, M.D., FAAAAI, FACAAI National Institutes of Health Bethesda, MD, USA

  2. Dr. Fleisher has no conflicts of interest related to this presentation

  3. 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

  4. 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

  5. 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 • Total immunoglobulin level (for each class) is the net of production, consumption, and loss 2.5% controls below 2.5% controls above

  6. 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

  7. 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 post- antibody levels

  8. 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

  9. 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

  10. 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

  11. Flow Cytometric Evaluation of a Child withAgammaglobulinemia B cells =0.2% T cells =95% Low or absent B cells = XLA or AR agammaglobulinemia;

  12. 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+

  13. 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

  14. Evaluating Innate Immunity

  15. 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

  16. 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

  17. Evaluating TLR Response I- Activation in vitro TLR specific ligand 37ºC 5% CO2 MC MC MC PBMC 2.0 x 105 cells/well Supernatant Cells II- Quantify Response MC MC Multiplex assay TNF ELISA CD62L shedding (Flow) Von Bernuth, et al, Pediatrics, 118:2498-2503, 2006; Deering and Orange, Clin Vaccine Immunol, 12:68-76, 2006

  18. 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)

  19. 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

  20. DHR Assay to Diagnose CGD Phox91 deficient CGD (X-linked) Normal Phox47 deficient CGD (AR) X-linked CGD carrier

  21. Flow Cytometric Analysis of CD18 Surface Expression in Unstimulated Cells Normal LAD1 patient Absent/markedly decreased CD18 expression = LAD type I Lymphocytes PMNs

  22. 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

  23. 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

  24. 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 patho-biology of innate immune deficiencies. Curr Allergy Asthma Rep. 2011, 11:369.

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