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 ( 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)