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1 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Newborn Screening forSevere Combined Immunodeficiencies
(SCID) -- 2008 Wisconsin Perspective
Mei Baker, C. Brokopp, G. Hoffman, R. Laessig, D. Kurtycz, W. Grossman,
J. Routes, M. Cogley, T. Litsheim, D. Mickle
University of Wisconsin School of Medicine and Public HealthMedical College of Wisconsin – Childrens Hospital of WI
Newborn Screening ProgramWisconsin State Laboratory of Hygiene
6 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Outline ---- A laboratory director’s view of SCID
Overview of Wisconsin SCID project
Background information on SCID and TRECs
Newborn screening for SCID
TREC assay development and validation
Newborn screening for SCID in Wisconsin --Results
7 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Wisconsin NBS -- SCID InitiativeNov. 2006: Routes and Grossman at NBS Umbrella CommitteeJan. 2007: Financial support from the Jeffrey Modell Foundation, Children’s Hospital of Wisconsin (CHW) and WI State Laboratory of Hygiene (WSLH): [$250,000 each]Mar. 2007: Mei Baker rejoins NBS program full time SCID
Goal: to establish a sensitive, cost-effective NBS for SCID.Method: measuring TRECs by Real-time PCR using DNA extracted from newborn screening specimens.
Oct.2007: Workable PCR Based screening method validatedDec.2007: Dept. Health adds Immunodeficiencies to NBS PanelJan. 2008: Wisconsin began routine newborn SCID screeningOct.2008: CDC Grant funding begins
8 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Severe Combined Immunodeficiency (SCID)• SCID is often called "bubble boy disease".
SCID became widely known during the 1970'sand 80's, when the world learned of David Vetter, a boy with X-linked SCID, who lived for 12 years in a plastic, germ-free bubble.
• "What we're saying is that essentially every baby with SCID could be cured if diagnosedearly enough. SCID should be considered apediatric emergency."
Dr. Rebecca Buckley,
chief of Duke's division of pediatric allergy and immunology
9 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Severe Combined Immunodeficiency (SCID)• SCID is often called "bubble boy disease".
SCID became widely known during the 1970'sand 80's, when the world learned of David Vetter, a boy with X-linked SCID, who lived for 12 years in a plastic, germ-free bubble.
• "What we're saying is that essentially every baby with SCID could be cured if diagnosed early enough. SCID should be considered a pediatric emergency."
Dr. Rebecca Buckley,
chief of Duke's division of pediatric allergy and immunology
10 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Severe Combined Immunodeficiency (SCID)
Infections in first year of life recurrent,ANY and ALL types!! (viral, bacterial, fungal)
persistent, despite routine treatmentsevere, including sepsis, meningitis
Severe, sometimes fatal reactions to vaccination
100 % fatal without immune reconstitution
11 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Prevalence of the disease (1:100,000 or greater)SCID: 1:66,000 (conservative estimate)
Does the disease cause serious medical complications?SCID: Universally fatal within the first year of life
Is there potential for successful treatment?SCID: Hematopoietic stem cell transplantation
Does technology exist to screen for the disease?SCID: Yes: Real time PCR measurement of TREC cells
What is the cost of the test?SCID: Estimated at $6.00 per baby screened. Comparable to existing diseases on the current screening panel.
Is there a confirmatory test?SCID: Yes: Lymphocyte subpopulation analysis (flow cytometry)
Does SCID fulfill NBS criteria? ….YES!!
12 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Does SCID fulfill NBS criteria? ….YES!!
Can the disorder be detected by routine physical exam?SCID: No, a SCID baby appears normal at birth.
Does the disorder have a short asymptomatic period after birth?SCID: Yes, SCID baby can be protected by maternal immune materials
Does early intervention leads better outcome?SCID: Yes!
13 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
A
0 2 4 6 8 10 12 14 16 18 20 22 240
10
20
30
40
50
60
70
80
90
100
Y t t l t ti
Per
cent
sur
vivi
ng
46 SCID infants with HSCT at than 3.5 months of age or less, Duke Univ.
96%
SCID - Early Diagnosis/BMT is curative!
113 SCID infants with HSCT at greater than 3.5 months of age, Duke Univ.
66%
Courtesy of R. Buckley(Duke)
14 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Molecular Defects in SCID
IL2Rγ T- B+ NK-JAK3 T- B+ NK-IL7Rα T- B+ NK+CD45 T- B+ NK+RAG1 T- B- NK+RAG2 T- B- NK+ARTEMIS T- B- NK+ADA T- B- NK-Reticular Dysgenesis T- B+ NK+SCID, multiple bowel atresias T-B+/- NK+SCID, congenital abnormalities T-B+/- NK+Severe DiGeorge Syndrome T-B+/- NK+CD3 Deficiency T+/- B+ NK+CD8 Deficiency T+B+ NK+Severe Ataxia Telangiectasia T+/- B+/- NK+Unknown: ~25%
Common Feature: ABSENT/NON-FUNCTIONAL T CELLS
15 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
TRECs are reduced in nearly ALL forms of SCID
16 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
T Cell Receptor Recombination During Development in the Thymus
Generation of T cell receptor excision circles (TRECs) occur in >70% of all new (naïve) T cells
and can be detected by PCR
17 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
TRECs and SCID
Quantification of TREC in peripheral blood by real-time PCR, and using TRECs as markers for recent thymic emigrants
Douek DC. Lancet 2000; 355:1875-81
Measurement of TRECs by real-time PCR can accurately identify infants with SCID
Chan K, Puck JM. J Allergy Clin Immunol. 2005 Feb;115(2):391-8
18 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Overall analysis scheme:
DBS →1/8” Punch →96 well plate →→DNA Extraction →RT PCR →→(TREC Amplification) → Analysis
19 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Fluorescence Emission(report dye) vs cycle number
Calibrators (l-r):1M, 100K, 10K, 1K, 100, 50(19-37 cycles)96 well plate, 82 samples
Threshold (Ct) is horizontal line
20 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Calibration Curve:TRECs/ul vs Cycle Number
1 10 100 1000 10K 100K 1M
CYCLE
NUM BER
21 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Plot of slope and R² for ≈ 75 consecutive analytical runs
AverageSlope 3.520 ± 0.070
AverageR² 0.996 ± 0.002
22 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Distribution of TRECs results, 0-3900 copies/ul
Frequency January-March 2008
0
500
1000
1500
2000
2500
3000
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39
TRECs / uL (0.0 to 3900 copies)
# of
Pat
ient
s
Series1
(Approximately 18,000 Specimens Screened)
23 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Distribution of TREC results, 0-210 Copies/ul
FREQUENCY DISTRIBUTION 0-200 TREC UNITS
0
100
200
300
400
500
600
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
TREC/uL x 0.1
# O
F P
ATIE
NTS
Series1
(Cutoff at 25 TRECs/ul, approximately 18,000 screened)
24 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
0.02%1.40%Screening Positive Rate
<75/DBS¹<15/DBSScreening Positive Cut-
off
708Median TREC/DBS
827510Average TREC/DBS
5,766239Sample Size
WI DataPuck & Chan
TREC Assay Results: Comparison
¹ Equivalent to ≈25 TRECs/ul of whole blood(Higher cut-off includes more newborns with low number of TRECs)
25 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
TREC Assay: SCID identification• Recent identification of a 1 week old SCID patient at CHW (age
range for proposal).
• Patient sample was de-identified, spotted on a NBS card, and sent to WSLH for TREC screening.
• Results are ‘proof in principle’ that the methodology works.
SCID TREC result:
26 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Screening data from 1/1/08 to 8/31/2008:
1. Total screened: 47,250
2. Abnormal ResultsPremature (<37 weeks) 11 (0.023%)Full Term 9 (0.019%)¹
3. Inconclusive ResultsPremature (<37 weeks) 57 (0.121%)Full Term 19 (0.04%)²
¹ Flow Cytometry Whole Blood Specimen² Second Filter Paper Specimen
27 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
+TRECs
Algorithm for SCID Screening
-TRECs/below cut-off
Re-punch card (X2) & repeat TREC w/ βactin
-TRECs/-βactin
poor card;request new card
Normal;Stop
+TRECs/+βactin
-TRECs or below cut-off/+βactin
Notify CHW physician*
Talk to PCP to getblood for flow cytometry
* Bill GrossmanJames VerbskyJack Routes
28 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
NBS-SCID Flow Cytometry Panel
CD45RO
CD56
IgG1/2
FL4
CD45
CD45
CD45
FL3
T, CD4/CD8, Lymphoid,CD4 & CD8 Memory Status
CD4/CD8CD3
T, B, Lymphoid, NKCD19CD3
Isotype AntibodiesIgG1/2IgG1/2/M
FL2FL1
Associated LymphocyteSubsets
Fluorochrome/Channel
29 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
normalcounts
Algorithm for SCID Screening, cont.
‘SCID’ panel(24hrs)
low/absent counts(c/w SCID); contact PCP& family for immediatereferral and evaluation
low counts (non-SCID);evaluate in IDC*within 1 month
Stop;Inform PCP
& WSLH
Continue on withconfirmatory testing
* Immune Deficiency Clinic at CHW
30 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Abnormal Results Reported*
1 DiGeorge’s Syndrome1 Down’s Syndrome with Sepsis at Birth1 Idiopathic T-Cell lymphopenia1 Leukocyte migration defect4 Normal flow cytometry results (F Pos)9 Normal results on repeat filter paper2 Pending cases1 Child expired(*10 months, 57,000 screened)
31 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• Decreased medical costs
- CHW Experience:
1) Five SCID babies diagnosed late: (only 3 survivors)Average hospital bill: ~$2,200,000/child
2) One early diagnosed SCID baby (sibling)Hospital bill: ~$350,000 (successfully transplanted)
3) Est. cost of WI NBS for SCID $450,000/yr(70,000 per year)
- JMF Study: $40 billion cost to Healthcare System for unrecognized PI patients
Cost-benefit – SCID Screening
32 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
NBS-SCID (November 2008)
10 Months routine screening (≈57,000)No SCID, multiple immunodeficienciesMethod is “manual,” two FTE to performAmenable to Automation of stepsRate of False Positives acceptableCost $6.00/newborn → $4.00 w/automationShare technology, experience, QC/QA
33 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
NBS-SCID (November 2008)
10 Months routine screening (≈57,000)No SCID, multiple immunodeficienciesMethod is “manual,” two FTE to performAmenable to Automation of stepsRate of False Positives acceptableCost $6.00/newborn → $4.00 w/automationShare technology, experience, QC/QAInverse of Kung Fu Panda
34 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Inverse Kung Fu Panda
I’m not a slow, fat, pandaI’m THE slow fat panda
35 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Inverse Kung Fu Panda
I’m not a slow, fat, pandaI’m THE slow fat panda
We’re not THE SCID screening methodWe’re a SCID screening method
36 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Inverse Kung Fu Panda
I’m not a slow, fat, pandaI’m THE slow fat panda
We’re not THE SCID screening methodWe’re a SCID screening method(or maybe immunodeficiencies)
37 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Acknowledgement
WSLH-Charles Brokopp, Dr. P.H.Mei Baker, MD Ron Laessig, PhDDan Kurtycz, MDGary Hoffman, BSMichael Cogley, BSDavid Mickle, BS
DHFS-Murray Katcher, MD, PhDAlexandria Meyer, MS
CHW -Jack Routes, MDBill Grossman, MD, PhD
JMF - Vicki Modell
Fred Modell
We Thank Dr. Daniel Douek at NIAID for kindly sharing TREC plasmids with us and Dr. Jennifer Puck for assisting us in all aspects of our start up program.
38 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
QuestionsComments
Ron Laessig
Thank you!