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Francis K. Lee, Ph.D. Senior Service Fellow (Research Microbiologist) Newborn Screening Translation Research Initiative, CDC Emeritus Professor of Pediatrics,

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Presentation on theme: "Francis K. Lee, Ph.D. Senior Service Fellow (Research Microbiologist) Newborn Screening Translation Research Initiative, CDC Emeritus Professor of Pediatrics,"— Presentation transcript:

1 Francis K. Lee, Ph.D. Senior Service Fellow (Research Microbiologist) Newborn Screening Translation Research Initiative, CDC Emeritus Professor of Pediatrics, Emory University School of Medicine Newborn Screening Molecular Workshop June 28-30, 2011 Model Proficiency Evaluation Survey for TREC Assay in Newborn Screening for SCID National Center for Environmental Health · Division of Laboratory Sciences Newborn Screening and Molecular Biology Branch

2 DBS Specimen Preparation DNA on DBS (2mm punch) DNA Extract (from 3 mm punch) QUANTITATIVE ASSAY Real time PCREndpoint PCR Calibrator PlasmidsTransfected Cells 96 /386 well format Primers & Probes SingleplexMultiplex Decisions!

3 Classification of TREC Assay Results for NBS Below Cutoff Above Cutoff  TREC Content Control Gene Below Cutoff Above Cutoff Screen Positive Indeterminate / Unsatisfactory Screen Negative F/U Required (Result cannot be reliably interpreted) F/U Required (SCID-like)

4 Newborn Screening for SCID Model Performance Evaluation Surveys CDC Reference Materials

5 DBS Reference Materials for the TREC Assay 1.DBS for Screen Normal Phenotype (3 types) –TREC above lower limit of expected range (H, M, L) –Control Gene above lower limit of expected range 2.DBS for SCID-Like Phenotype –TREC below lower limit of expected range (Very low or undetectable) –Control Gene above lower limit of expected range 3.DBS for Indeterminate/Unsatisfactory Results –TREC below lower limit of expected range –Control Gene below lower limit of expected range

6 DBS Reference Materials for TREC Assay DBS made from Serial Dilutions of Cord Blood –Cord blood at median of expected range for TREC –Dilute into lymphocyte-depleted blood (No detectable TREC) –Equal-volume serial dilutions: 100%, 50%, 25%, 12%, 6%, 3% Assay development; Calibration; Assess Comparability of Decision Ranges

7 No Follow-up Required Follow-up Required

8 Model Performance Evaluation Surveys (“MPES” = Pilot PT) Sendouts at 4-6 week interval Five DBS w/ prior consensus categorization Additional DBS included for RM evaluation 11 Laboratories currently participating >500 results to date

9 Sample ID TREC Final Categorical Result Reference Gene: Beta-actin Comments Cq Copy Number No F/UF/U action required Cq Copy Number Valueper Rxn per µ L Bld TREC NLTREC ↓ Ref gene NL Ref gene ↓ Valueper Rxn per µ L Bld A X B XX C XX D X E X FUndet.00 XX G XX HUndet.00 X X Cutoff60 If TREC↓selected, indicate ref erence gene category Cutoff10000 Report from MPES Lab# 999

10 MPES#22 Sample ID Sample No F/UF/U required CodeTREC NLTREC ↓Ref gene NLRef gene ↓ Higher NormalE 9 Lower NormalA 9 SCID -likeF 99 Leuko-depletedB 9 9 CB-cal 4 (25%)G 811 CB-cal 5 (12.5%)C 4532 CB-cal 6 (6.3%)H 954 CB-cal 7 (3.1%)D 963 CDC Report - Summary of Results

11 CDC NSTRI – SCID Project Team Robert Vogt ( Francis Lee ( Jennifer Taylor ( Golriz Yazdanpanah (


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