Immunology, Asthma and Allergy Research Institute (IAARI)
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1 Immunology, Asthma and Allergy Research Institute (IAARI)
2 Prenatal and postnatal genetic diagnosis of PIDs: From routine methods to new molecular assessments for newborn screening Maryam Nourizadeh (PhD in Immunology) Assistant Prof. Immunology, Asthma and Allergy Research Institute (IAARI) Tehran University of Medical Sciences (TUMS) 21 April 2016 (02/02/95) Immunology, Asthma and Allergy Research Institute (IAARI)
3 Patients with recurrent infections Flowchart of managing PID patients in IAARI Patients history and visiting by pediatric sub specialist in immunology 10 warning signs of PIDs for children Screening of PIDs (CBC, Igs, PPD, CH50, C3, C4,NBT/DHR, ) Diagnosis and confirmation by Subspecialist Advanced immunological evaluations (LTT, immunophenotyping, ) Genetic analysis and sequencing the suspected genes for final confirmation Immunology, Asthma and Allergy Research Institute (IAARI)
4 Immunology, Asthma and Allergy Research Institute (IAARI)
5 PIDs single-gene defects (genes associated with >60 PIDs have been identified). Diagnosing different forms of PID which have similar symptoms Early diagnosis and treatment confirming diseases, identification of carrier cases and also prenatal diagnosis in family with positive history Immunology, Asthma and Allergy Research Institute (IAARI)
6 Immunology, Asthma and Allergy Research Institute (IAARI)
7 Immunology, Asthma and Allergy Research Institute (IAARI)
8 Immunology, Asthma and Allergy Research Institute (IAARI)
9 Immunology, Asthma and Allergy Research Institute (IAARI)
10 2. Why do we need to screen the newborns for PIDs especially T/B deficiencies (e.g SCID)? Higher incidence than that estimated before (app. 1:1200 in USA, Buckley, 2007, J Clin Immunol, 27: ) Often appear normal at birth with no family history of immunodeficiency Not identified until life-threatening and serious infections occur (Ca. albicans, P. carinii, adenovirus, parainfluenza, CMV, EBV, BCG, ) Improve the treatment and survival by early diagnosis (Successful HSCT up to 95% if performed before months) Important: if SCID suspected, child should not receive any live vaccines. Immunology, Asthma and Allergy Research Institute (IAARI)
11 cellular assessments: CBC percentage of lymphocyte subsets T cell proliferation (LTT ) Genetic et analysis a s to find the genetic et defects Early screening, 3-5 days after birth using dot blood spots of Guthrie cards, TREC/KREC assessment Immunology, Asthma and Allergy Research Institute (IAARI)
12 Multiplex quantitative Real Time PCR Immunology, Asthma and Allergy Research Institute (IAARI)
13 T cell receptor excision circles (TRECs): stable episomal, non replicative e DNA circles cesgenerated e during T cell receptor (TCR) α chain rearrangement in thymus Immunology, Asthma and Allergy Research Institute (IAARI)
14 κ deleting recombination excision circles (KRECs) during B cell development as a result of light chain rearrangements Immunology, Asthma and Allergy Research Institute (IAARI)
15 Immunology, Asthma and Allergy Research Institute (IAARI)
16 TREC/KREC assay on dried blood spots Immunology, Asthma and Allergy Research Institute (IAARI)
17 Simple diagram of TREC/KREC assay Immunology, Asthma and Allergy Research Institute (IAARI)
18 Immunology, Asthma and Allergy Research Institute (IAARI)
19 An experience in Sweden to determine the cutoffs Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex real-time PCR by Stephan Borte, Ulrika von Döbeln, Anders Fasth, Ning Wang, Magdalena Janzi, Jacek Winiarski, Ulrich Sack, Qiang Pan-Hammarström, Michael Borte, and Lennart Hammarström Blood Volume 119(11): March 15, by American Society of Hematology
20 Flow chart of the triplex RT-qPCR assay, including results of 2560 freshly collected, anonymized regular Guthrie cards from Swedish newborns. Stephan Borte et al. Blood 2012;119: by American Society of Hematology
21 TREC and KREC copy numbers in dried blood spot samples (DBSS) from anonymized Guthrie cards and samples of SCID, XLA, AT, NBS, X-HIGM, CVID, or IgAD patients. Stephan Borte et al. Blood 2012;119: by American Society of Hematology
22 Determining the TREC/KREC cutoffs in IRAN An international project between IAARI, Stockholm University and Leipzig University, Germany ( ) Immunology, Asthma and Allergy Research Institute (IAARI)
23 Method: A multiplex quantitative Real time PCR 3 primer probes probes used for ACTB, TREC, KREC (Commercial kits) Immunology, Asthma and Allergy Research Institute (IAARI)
24 We could find the cut offs of TREC and KREC by evaluating more than 2000 newborn Guthrie card samples and positive PID patients as controls (preparing p rin for publication). Immunology, Asthma and Allergy Research Institute (IAARI)
25 Immunology, Asthma and Allergy Research Institute (IAARI) Real time PCR results
26 This project was designed to be done in separate 3 phases: 1. Determining the cutoffs (has been done) 2. Field study to show the feasibility of the test t and finding the prevalence in some provinces 3. Convincing i the government to establish tblihthe test in the national screening program Immunology, Asthma and Allergy Research Institute (IAARI)
27 Newborn screening for SCID (TREC only) in 37 states and planning to begin screening in 2016 in 5 states Established in USA and Taiwan. Pilot screening programs have recently been initiated in Sweden, G Germany, UK UK, FFrance, Norway N and d Spain. S i Borte et al, Int. J. Neonatal Screen. 2015, 1, Immunology, Asthma and Allergy Research Institute (IAARI)
28 "Berlin Declaration" Mandates Newborn Screening Globally 212world renowned experts in Primary Immunodeficiency from 78 countries signed: a "Berlin Declaration" calling for: global implementation of newborn screening for Severe Combined Immunodeficiency (SCID). Immunology, Asthma and Allergy Research Institute (IAARI)
29 Immunology, Asthma and Allergy Research Institute (IAARI)
30 Immunology, Asthma and Allergy Research Institute (IAARI)
31 Immunology, Asthma and Allergy Research Institute (IAARI)
32 Complements IMIGIA IgM, IgG, IgA Iso Hemagglutinin LTT(PHA_Candida_BCG_Loop) DTH(PPD_Candida_DT) DT) CD markers (Flow cytometry) Humoral immunity Cellular immunity NBT/DHR Opsonization Chemotaxis Phagocytic assay Immunology, Asthma and Allergy Research Institute (IAARI) Others Blood Smear for Giant Granulecyte Hair Study Protein Study(Western)GP91,P47,P67,P22, WAS,Btk Galactomannan (ELISA)
33 Immunology, Asthma and Allergy Research Institute (IAARI)
34 CH 50 C1q C 3 Humoral Immunity&Complement RIDA Allergy Diagnostic tests (Specific IgE) Type 2(IAARI) Penicilloyl G HSA Type 4 Penicilloyl V HAS C 4 C1 INH Q C1 INH F Weeds Asprin HAS Amoxicilloyl Ibuprofen Penicilloyl G Ciprofloxacin IgA(Saliva) IgA Insulin human Amoxicillin Protamine Cephalosp. IgM Latex Diclofenac IgG Rice IgG 1 Soy bean IgG 2 DP IgG 3 DF IgG 4 Honney Bee IgD WASP venum Total IgE Trees Grass Iso Hemagglutinin Milk Protein Electrophoresis Wheat Phagocytosis assay Egg White DHR NBT Egg Yolk Opsonization Chemotaxis Peper Cell function assays Nuts LTT DTH Aspergillous IgE (PHA_Candida_BCG_Loop) (PPD_Candida_DT) Flowcytometery CD16+56,CD11a _ CD11b _ CD11c _ CD18 fic IgE ImmunoCap ImmunoCap Specif I Aspergillous IgG Others CD 3 CD4 CD 8 CD 19 CD 15 Atomic Absorption Cu _ Se _ Zn Blood Smear for Giant Granulecyte Hair Study Protein Study(Western)GP91 P47 P67 P22 Immunology, Galactomannan Asthma and (ELISA) Allergy Research BTK(Broton)(Western) Institute (IAARI) DNA Extraction
35 Immunology, Asthma and Allergy Research Institute (IAARI)
Short Communication. Int Arch Allergy Immunol 2016;171: DOI: /
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