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Laboratory Tests in PID

Learn about specific laboratory tests used in immune system evaluation including antibody deficiency disorders, T-cell immunodeficiency, phagocyte dysfunction, and more, emphasizing the importance of focused testing. Discover the role of flow cytometry and genetic testing in diagnosing primary immunodeficiency disorders.

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Laboratory Tests in PID

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  1. Laboratory Tests in PID What is available? Daniel maina

  2. Introduction • The use of the laboratory in evaluating the immune system should not follow a shotgun approach but rather should be a focused evaluation using specific testing in an orderly process based on the clinical history

  3. Layout • Evaluating Suspected Antibody Deficiency Disorders • Evaluating Suspected T-Cell or Combined T- and B-Cell Immunodeficiency • Evaluating Suspected Phagocyte Dysfunction Syndromes • Evaluating Suspected Natural Killer and Cytotoxic T-Cell Defects • Evaluating Suspected Defects Involving the Adaptive-Innate Immunity Interface • Evaluating Suspected Complement Disorders • Evaluating Suspected Immune Dysregulation Disorders • Flow Cytometry, a Versatile tool for diagnosis and monitoring of PIDs

  4. Evaluating Suspected Antibody Deficiency Disorders • Measuring the levels of the major immunoglobulin classes IgG, IgA, IgM, and IgE • An additional and readily available test is quantitation of IgG subclass* levels. • Measurement of specific antibody responses~ is useful in confirming defective antibody production • Normal responses typically consist of finding at least a 4-fold increase in antibody levels and/or protective antibody levels after immunization

  5. Immunoanalyser

  6. Evaluating Suspected T-Cell or Combined T- and B-Cell Immunodeficiency • Careful analysis of the white blood cell count and differential is of utmost importance • The absolute lymphocyte count must be compared with age-matched control ranges for proper interpretation* • Assessment of cellular immunity - immunophenotyping of T cells by means of flow cytometry together with - in vitro functional testing (e.g., proliferation and cytokine production assays) • Fluorescence in situ hybridization for the 22q11 microdeletion found in the majority of patients with DiGeorge syndrome

  7. Evaluating Suspected Phagocyte Dysfunction Syndromes • Screening studies directed at the evaluation of neutrophil function should start with a leukocyte count, differential, and morphologic review • The diagnosis of cyclic neutropenia* requires multiple absolute neutrophil counts 2 to 3 times a week for at least 4 to 6 weeks • Bone marrow analysis is useful to exclude insufficient production because of neoplasia or other causes and to document other abnormalities, such as the maturation arrest typical of Kostmann syndrome^ • If neutropenia and morphologic abnormalities are ruled out- assess assays that provide functional information

  8. Cont’d • LAD - flow cytometric assessment of the neutrophil adhesion molecules CD11 and CD18, - expression of which is absent or decreased on neutrophils from patients with LAD1 • Neutrophil oxidative burst pathway - screened with either the nitrobluetetrazolium* tests or a flow cytometric assay (dihydrorhodamine 123 [DHR]), - results of both of which are abnormal in patients with chronic granulomatous disease

  9. Evaluating Suspected Natural Killer and Cytotoxic T-Cell Defects • NK cell function - immunophenotyping NK cells by means of flow cytometry • Assaying cytotoxicity with standard in vitro assays - demonstrate absent invariant-chain NK T cells in peripheral blood of XLP1 patients, as measured by CD3+Vα24+Vβ11+ staining. • Intracellular flow cytometry can be used to evaluate for expression of SAP (SLAM-associated protein) and XIAP (X-linked inhibitor of apoptosis), the proteins defective in XLP1 and XLP2 respectively

  10. Evaluating Suspected Complement Disorders • Classical complement component deficiency will result in virtual absence of hemolysis on CH50* testing • A decreased AH50^ test result suggests a deficiency in factor B, factor D, or properdin. • A decrease in both CH50 and AH50 test results suggests deficiency in a shared complement component (from C3 to C9)

  11. FLOW CYTOMETRY • Flow cytometric assays -qualitative & quantitative (relative and absolute) and phenotyping -functional -assessing specific protein expression, cell viability, apoptosis and death, cellular interactions and cell enrichment

  12. X-linked hyper-IgM syndrome

  13. Chronic granulomatous disease (CGD)

  14. Genetic testing • ~150 PIDs, >200 genes (both growing) - increasing role for mutation testing (PIDs defined by genes) - novel technologies (whole genome/exome) • One disease multiple genes (overlapping phenotypes) - CMC, MSMD, HSE • One gene, multiple diseases - STAT1, NEMO • Inheritance: AR, AD, XL - AR/AD IFNGR1

  15. Gene Panels ACP5, ACTB, ADA, AGA, AICDA, AIRE, AK2, ALG13, AP3B1, AP4E1, APOL1, ATM, B2M, BLM, BLNK, BLOC1S3, BLOC1S6, BTK, C1QA, C1QB, C1QC, C1R, C1S, C2, C3, C4A, C4B, C5, C6, C7, C8A, C8B, C9, CARD11, CARD9, CASP10, CASP8, CD19, CD247, CD27, CD3D, CD3E, CD3G, CD40, CD40LG, CD46, CD55, CD59, CD79A, CD79B, CD81, CD8A, CEBPE, CFB, CFD, CFH, CFHR1, CFHR3, CFHR5, CFI, CFP, CHD7, CIITA, CLEC7A, COLEC11, CORO1A, CR2, CREBBP, CSF2RA, CSF3R, CTSC, CXCR4, CYBA, CYBB, DCLRE1C, DHFR, DKC1, DNMT3B, DOCK8, DTNBP1, ELANE, EPG5, ERCC2, ERCC3, F12, FADD, FAS, FASLG, FCGR1A, FCGR3A, FCN3, FERMT3, FOXN1, FOXP3, G6PC, G6PC3, G6PD, GATA2, GFI1, GJC2, GTF2H5, HAX1, HPS1, HPS3, HPS4, HPS5, HPS6, ICOS, IFNGR1, IFNGR2, IGHG2, IGHM, IGKC, IGLL1, IKZF1, IL10, IL10RA, IL10RB, IL12B, IL12RB1, IL17F, IL17RA, IL1RN, IL2, IL21, IL21R, IL2RA, IL2RG, IL36RN, IL7R, INSR, IRAK4, IRF8, ITCH, ITGB2, ITK, JAK2, JAK3, KMT2D, KRAS, LAMTOR2, LCK, LIG1, LIG4, LPIN2, LRBA, LRRC8A, LYST, MAGT1, MALT1, MAN2B1, MANBA, MASP1, MASP2, MBL2, MC2R, MCM4, MEFV, MLPH, MPO, MRE11A, MS4A1, MSH6, MTHFD1, MVK, MYD88, MYO5A, NBN, NCF1, NCF2, NCF4, NCSTN, NFKB1, NFKB2, NFKBIA, NHEJ1, NHP2, NKX2-5, NLRP12, NLRP3, NOD2, NOP10, NRAS, ORAI1, PCCA, PCCB, PEPD, PGM3, PIGA, PIK3CD, PIK3R1, PLCG2, PLG, PMM2, PMS2, PNP, PRF1, PRKCD, PRKDC, PRPS1, PSENEN, PSMB8, PSTPIP1, PTPN11, PTPRC, PTRF, RAB27A, RAC2, RAG1, RAG2, RASGRP2, RBCK1, RECQL4, RFX5, RFXANK, RFXAP, RNASEH2A, RNASEH2B, RNASEH2C, RNF168, RORC, RPSA, RTEL1, SAMHD1, SBDS, SERAC1, SERPING1, SH2D1A, SKIV2L, SLC35A1, SLC35C1, SLC37A4, SLC39A4, SLC46A1, SMARCAL1, SP110, SPINK5, STAT1, STAT2, STAT3, STAT4, STAT5B, STIM1, STK4, STX11, STXBP2, TAP1, TAP2, TAPBP, TAZ, TBX1, TCIRG1, TCN2, TERT, TFRC, THBD, TICAM1, TINF2, TLR3, TMC6, TMC8, TNFRSF11A, TNFRSF13B, TNFRSF13C, TNFRSF1A, TNFRSF4, TRAC, TRAF3, TREX1, TTC37, TYK2, UNC119, UNC13D, UNC93B1, UNG, USB1, VPS13B, VPS45, WAS, WIPF1, WRAP53, XIAP, ZAP70, ZBTB24

  16. Advantages of Molecular analysis for PID diagnosis. • Distinguishing genetic from acquired disorders • Confirming the clinical diagnosis • Identifying novel presentations of PIDs • Identifying atypical presentations of PIDs

  17. Conclusion • Laboratory testing serves as the critical approach necessary for evaluating immune function in the setting of a patient with a history of recurrent infections, unusual infections, or both. • The appropriate and directed use of immune function testing provides not only critical diagnostic information but also directs decisions regarding the most appropriate therapy.

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