FACTORS TO CONSIDER IN THE
NEWBORN SCREENING
ALGORITHM FOR SEVERE
COMBINED IMMUNODEFICIENCY
RACHEL LEE
M. Nolen, C. Corrigan, D. Luna, D. Freedenberg, MD, PhD,
S.M. Tanksley, PhD
Texas Department of State Health Services, Austin, TX
2016 APHL Newborn Screening and Genetic Testing
Symposium
March 1, 2016
SCREENING FOR SCID IN TEXAS
 Screen for absence of T-cell Receptor
Excision Circles (TREC)
 Multiplex Real-Time PCR based assay
 Automated DNA extraction followed by Real-Time PCR
 Each SCID reaction amplifies two targets of interest:
 T-cell receptor excision circle (TREC)
 RNaseP reference gene extraction control
 TX is a 2-screen state. Testing specimens:
All newborns, follow-ups, requested
repeats
INITIAL SCREENING ALGORITHM
RETEST SCREEN ALGORITHM
SCID RESULT NOTES
 Abnormal Result Code:
 Very low number of T-cell receptor excision circles (TREC).
Please follow recommendations received from the DSHS
newborn screening Clinical Care Coordination team .
 Borderline Result Code:
 Borderline low number of T-cell receptor excision circles
(TREC). Please repeat the newborn screen within 7 days .
 uFC (SCID unsat) Result Code:
 Unsatisfactory - Please resubmit within 7 days: Specimen
inadequate for accurate detection of TREC (T-cell receptor
excision circles).
SCID SCREENING DATA
December 2012 – June 2015
# of Specimens Screened
1,952,400
# of 1st Screen Specimens (~ # of Newborns)
1,010,063
# of Abnormal/Borderline Specimens
# of Newborns Referred
4,954 (0.25%)
681 (0.07%)
# of Diagnosed SCID Cases
25 (1:40,403)
# of Secondary Diagnosed Cases **
322 (1:3,137)
**Does not include Preterm alone
SENSITIVIT Y, SPECIFICIT Y, FALSE POSITIVE RATE,
FALSE NEGATIVE RATE, AND POSITIVE PREDICTIVE
VALUE—SCID DIAGNOSED
Screen Positive
Screen Negative
Total
SCID Diagnosed
25
0
25
Cleared
656
1,009,382
1,010,038
Total
681
1,009,382
1,010,063
Sensitivity = 100%
Specificity = 99.9%
False Positive Rate = 0.065%
False Negative Rate = 0%
Positive Predictive Value = 3.7%
POSITIVE PREDICTIVE VALUE—SCID &
SECONDARY CONDITIONS, REFERRED
Screen Positive
SCID and Secondary Conditions
316
Cleared
365
Total
681
Positive Predictive Value = 46.4%
TREC copies/µl whole blood
4900
4775
4650
4525
4400
4275
4150
4025
3900
3775
3650
3525
3400
3275
3150
3025
2900
2775
2650
2525
2400
2275
2150
2025
1900
1775
1650
1525
1400
1275
1150
1025
900
775
650
525
400
275
150
25
Frequency
ALL BIRTH WEIGHT
160
140
120
100
80
60
40
20
0
0
25
125
225
325
425
525
625
725
825
925
1025
1125
1225
1325
1425
1525
1625
1725
1825
1925
2025
2125
2225
2325
2425
2525
2625
2725
2825
2925
3025
3125
3225
3325
3425
3525
3625
3725
3825
3925
4025
4125
4225
4325
4425
4525
4625
4725
4825
4925
(blank)
Frequency
LOW BIRTH WEIGHT ONLY
10
9
8
7
6
5
4
3
2
1
TREC copies/ µl whole blood
BIRTH WEIGHT
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Normal Birth Wt
Low Birth Wt (<2,000g)
All Newborns
SCID Presumptive
Positives
Presumptive Positive Rate: Normal birth weight 0.14%
Low birth weight 4%
BORDERLINE
• Low birthweight babies (<2000g) with TREC
quantities between 110 TREC copies/µL and 150
TREC copies/µL
• 23 Secondary diagnosed cases with one or more
Borderline result
•
•
•
•
•
•
•
•
•
1
4
4
1
1
4
3
2
3
with Chromosomal Defects
T-cell Syndrome
Congenital Heart Defects
Gastrointestinal disorder
Lymphatic imbalance
Multiple congenital anomalies with T-cell defect
Pulmonary disorders
Preterm and no other recognizable disorder
Preterm with complications
• No SCID cases with a Borderline result
AGE AT SPECIMEN COLLECTION
TREC Copy #
4000
3000
2000
1000
0
0
500
1000
Age at Collection (Days)
1500
CLINICAL STATUS
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Sick w/Transfusion
Sick w/o Transfusion
Normal (Healthy)
All Newborns
SCID Presumptive
Positives
Presumptive Positive Rate: Normal status 0.09%
Sick w/o transfusion 1.3%
Sick w/ transfusion 13.3%
AGE OF SPECIMENS
Mean TREC Quantity (TRECs/uL Whole Blood)
Average TREC Quantity
2,000
Average TREC Quantity
1,800
TREC cutoff
1,600
Newborn TREC Quantity Mean During the
Study Period
1,400
1,200
1,000
800
600
400
200
0
0
25
50
75
100
125
150 175 200 225
Days after Collection
250
275
300
325
350
375
CALIBRATOR LOT
Median TREC Quantity
1800
TREC/uL Whole Blood
1600
1400
1200
1000
800
600
400
200
Month
Average Median TREC Quantity: 1073 ± 173
REAGENT LOT
Median TREC Ct
35.500
35.000
Ct
34.500
34.000
33.500
33.000
32.500
Month
Average Median TREC (copies/µL): 33.859 ± 0.273
1 ST SCREEN VS. 2 ND SCREEN
 Texas Newborn Screening Laboratory tests all 2nd
screens even if the 1st screen was normal.
 Median RNaseP Ct for 1st screens is 0.75-1 lower
than 2nd screens
 Median TREC quantity for 1 st screens is ~15%
lower than 2 nd screens
During 12/1/2012 – 4/30/2014, 737
newborns had 1st Normal/2nd Non-normal.
 Majority (89.8%) were cleared by additional screens.
 10.2% had secondary diagnoses.
 Since then, 2 SCID cases have been identified
with 1 st Normal and 2 nd Non-normal
CASE #1 – SCID WITH UNKNOWN
MUTATION
Screen
First
Age of
Collection
1 day
SCID Screening
Results
Normal
Second
7 days
Unsatisfactory
Third
15 days
Abnormal
Fourth
36 days
Abnormal
CASE #2 – ZAP70
Screen
Age of
Collection
SCID Screening
Results
First*
~3 days
Normal
Second
159 days
Abnormal
Third
180 days
Abnormal
* Collected by birthing hospital and tested in
newborn screening laboratory out of Texas
LESSON LEARNED
 low birth weight and transfusion status have an
effect on SCID screening results.
 The median TREC value seems to decrease with
age; however, statistical analysis performed
indicated there was no significant difference.
 DBS specimens are stable for SCID screening at
least 1 year after collection.
LESSON LEARNED (CONT.)
 Alternative source of TREC calibrator
 Reagent lot variability
 Evaluate an alternate approach to determining
our cutoff (e.g. MoM, floating)
 SCID cases can have 1 st screen normal results.
 2 nd screens can aid in identifying certain types
of SCID and secondary T-cell lymphopenia
THANK YOU!