Model Proficiency Evaluation Survey for TREC Assay in Newborn Screening for SCID 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 National Center for Environmental Health · Division of Laboratory Sciences Newborn Screening and Molecular Biology Branch

DBS Specimen Preparation DNA Extract (from 3 mm punch) Decisions! Decisions! DNA Extract (from 3 mm punch) DNA on DBS (2mm punch) Quantitative ASSAY Primers & Probes Singleplex Multiplex Real time PCR Endpoint PCR 96 /386 well format Calibrator Plasmids Transfected Cells

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

Newborn Screening for SCID CDC Reference Materials Model Performance Evaluation Surveys

DBS Reference Materials for the TREC Assay 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 DBS for SCID-Like Phenotype TREC below lower limit of expected range (Very low or undetectable) DBS for Indeterminate/Unsatisfactory Results Control Gene below lower limit of expected range

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

No Follow-up Required Follow-up Required

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

Report from MPES Lab# 999 No F/U F/U action required TREC Sample ID TREC ------- Final Categorical Result -------- Reference Gene: Beta-actin Comments Cq Copy Number No F/U F/U action required Value per Rxn per µL Bld TREC NL TREC ↓ Ref gene NL Ref gene ↓ A 35.3 121 X   B 37.3 23 28.1 16300 C 36.6 40 27.8 18800 D 34.7 196 E 35.6 95 F Undet. 27.7 20600 G 36.3 48 28.2 15200 H 33.4 557 Cutoff 60 If TREC↓selected, indicate ref erence gene category 10000

CDC Report - Summary of Results MPES#22 Sample ID Sample No F/U F/U required Code TREC NL TREC ↓ Ref gene NL Ref gene ↓ Higher Normal E 9   Lower Normal A SCID -like F Leuko-depleted B CB-cal 4 (25%) G 8 1 CB-cal 5 (12.5%) C 4 5 3 2 CB-cal 6 (6.3%) H CB-cal 7 (3.1%) D 6

CDC NSTRI – SCID Project Team Robert Vogt ( rfv1@cdc.gov 770-488-7895) Francis Lee ( flee1@cdc.gov 770-488-7946) Jennifer Taylor ( vjf6@cdc.gov 770-488-7893) Golriz Yazdanpanah ( czu1@cdc.gov 770-488-7893)