1. 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

2. 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

3. 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

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

5. 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

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. 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

10. 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

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