model proficiency evaluation survey for trec assay in newborn screening for scid
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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 . - PowerPoint PPT PresentationTRANSCRIPT
Francis K. Lee, Ph.D.Senior Service Fellow (Research Microbiologist)
Newborn Screening Translation Research Initiative, CDCEmeritus Professor of Pediatrics, Emory University School of Medicine
Newborn Screening Molecular WorkshopJune 28-30, 2011
Model Proficiency Evaluation Survey for
TREC Assay in Newborn Screening for SCID
National Center for Environmental Health · Division of Laboratory SciencesNewborn Screening and Molecular Biology Branch
DBS Specimen Preparation
DNA on DBS (2mm punch)DNA Extract (from 3 mm punch)
QUANTITATIVE ASSAY
Real time PCR Endpoint PCR
Calibrator
Plasmids Transfected Cells
96 /386 well format
Primers & Probes
Singleplex Multiplex
Decisions! Decisions!
Classification of TREC Assay Results for NBS
Below Cutoff Above Cutoff -----------------------
TREC Content
Con
trol
Gen
e
B
elow
Cut
off
A
bove
Cut
off
ScreenPositive
Indeterminate/
Unsatisfactory
Screen Negative
F/U Required(Result cannot be
reliably interpreted)
F/URequired(SCID-like)
Newborn Screening for SCID
Model Performance Evaluation Surveys
CDC Reference Materials
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
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 RequiredFollow-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
Sample ID
TREC------- Final Categorical Result --------
Reference
Gene: Beta-actin
CommentsCq Copy Number No F/U F/U action required Cq Copy Number
Value per Rxnper µL
Bld TREC NL TREC ↓Ref gene
NLRef gene
↓ Value per Rxn per µL Bld
A 35.3 121 121 X
B 37.3 23 23 X X 28.1 16300 16300
C 36.6 40 40 X X 27.8 18800 18800
D 34.7 196 196 X
E 35.6 95 95 X
F Undet. 0 0 X X 27.7 20600 20600
G 36.3 48 48 X X 28.2 15200 15200
H Undet. 0 0 X X 33.4 557 557
Cutoff 60 If TREC↓selected, indicate ref erence gene category Cutoff 10000
Report from MPES Lab# 999
MPES#22
Sample IDSample No F/U F/U required
Code TREC NL TREC ↓ Ref gene NL Ref gene ↓
Higher Normal E 9
Lower Normal A 9
SCID -like F 9 9
Leuko-depleted B 9 9
CB-cal 4 (25%) G 8 1 1
CB-cal 5 (12.5%) C 4 5 3 2
CB-cal 6 (6.3%) H 9 5 4
CB-cal 7 (3.1%) D 9 6 3
CDC Report - Summary of Results
CDC NSTRI – SCID Project Team
Robert Vogt ( [email protected] 770-488-7895)Francis Lee ( [email protected] 770-488-7946)Jennifer Taylor ( [email protected] 770-488-7893)Golriz Yazdanpanah ( [email protected] 770-488-7893)