newborn screening for severe combined immunodeficiencies

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


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


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


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


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


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


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!!


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!


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)


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


TRECs are reduced in nearly ALL forms of SCID


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


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


Overall analysis scheme:

DBS →1/8” Punch →96 well plate →→DNA Extraction →RT PCR →→(TREC Amplification) → Analysis


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


Calibration Curve:TRECs/ul vs Cycle Number

1 10 100 1000 10K 100K 1M

CYCLE

NUM BER


Plot of slope and R² for ≈ 75 consecutive analytical runs

AverageSlope 3.520 ± 0.070

AverageR² 0.996 ± 0.002


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)


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)


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)


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:


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


+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


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


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


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)


• 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


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


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


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




We’re not THE SCID screening methodWe’re a SCID screening method(or maybe immunodeficiencies)


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.


QuestionsComments

Ron Laessig

Thank you!

newborn screening for severe combined immunodeficiencies

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