factors to consider in the newborn screening … · factors to consider in the newborn screening...

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 SymposiumMarch 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

Presenter

Presentation Notes

Following analysis of initial screen specimens, all specimens will have one of two initial results according to our initial screening algorithm. Specimens with a TREC quantity greater than 200 TRECs per uL will be considered Normal and will not be screened any further. Specimens with TREC quantities less than or equal to 200 TRECs per uL are flagged for retest and will be retested in duplicate. All three replicates (initial and two retest replicates) will be considered when making the final result determination.

RETEST SCREEN ALGORITHM

Presenter

Presentation Notes

For those specimens meeting retest criteria, the algorithm for final result determination is described in this slide. Any baby—regardless of birthweight—with a TREC quantity greater than 150 TRECs per uL will be resulted as Normal. On the other end, if the TREC quantity is less than or equal to 150 TRECs per uL and RNaseP results are above our cutoff, suggesting either a poor extraction or low white blood cell count, the specimen will receive an unsat code—uFC. For normal birthweight babies—babies weighing more than 2000g—if the TREC quantity is less than or equal to 150 TRECs per uL with an acceptable RNaseP value, the specimen is coded as abnormal. low birthweight babies—those babies weighing less than 2000g—can be coded as either borderline or abnormal. If the TREC quantity is between 110 to 150 TRECs per uL, the specimen receives a borderline result code. Low birthweight babies with a TREC quantity less than or equal to 110 TRECs per uL are considered abnormal. We are hoping to get your feedback as to the benefit of continuing to use the Borderline designation for low birthweight babies, but we plan to discuss this further in a later slide.

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).

Presenter

Presentation Notes

After all Newborn Screening teams have released results for a specimen, mailers are then sent to the providers with the screen results as well as any additional instructions for the provider to follow if the result is anything but normal. If the SCID result was normal, no additional notes for our screening assay are included. Specimens with an abnormal result will have an additional note indicating that the specimen had very low numbers of TRECS and asks the provider to follow any recommendations received from Clinical care coordination. For borderline results, an additional note indicating that the specimen had Borderline low numbers of TRECs and requesting that another specimen to be collected within 7 days. uFC result codes have an additional note indicating that the specimen was inadequate for accurate detection of TRECs and requesting for another specimen to be resubmitted within 7 days.

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 4,954 (0.25%)

# of Newborns Referred 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

Positive Predictive Value = 46.4%

Screen Positive

SCID and Secondary Conditions 316

Cleared 365

Total 681

ALL BIRTH WEIGHT

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TREC copies/µl whole blood

Presenter

Presentation Notes

7000 specimens, TREC ranges from 28 to 5,399 with a medium of 1,159

LOW BIRTH WEIGHT ONLY

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TREC copies/ µl whole blood

Presenter

Presentation Notes

Of the 212 specimens from low birth weight newborns, TREC ranges from 38 to 2,753 with a medium of 864

0%10%20%30%40%50%60%70%80%90%

100%

All Newborns SCID PresumptivePositives

Normal Birth WtLow Birth Wt (<2,000g)

BIRTH WEIGHT

Presumptive Positive Rate: Normal birth weight 0.14%Low birth weight 4%

Presenter

Presentation Notes

Of all the newborns screened, about 3.3% were low birth weight. However, almost half of the presumptive positive results we reported out were from low birth weight babies. As I mentioned in the previous slide, our presumptive positive rate is 0.25%. For normal birth weight babies, the presumptive positive rate was much lower, 0.14%. For low birth weight babies, the presumptive positive rate was 30 times higher, 4.0%.

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 with Chromosomal Defects• 4 T-cell Syndrome• 4 Congenital Heart Defects• 1 Gastrointestinal disorder• 1 Lymphatic imbalance• 4 Multiple congenital anomalies with T-cell defect• 3 Pulmonary disorders• 2 Preterm and no other recognizable disorder• 3 Preterm with complications

• No SCID cases with a Borderline result

AGE AT SPECIMEN COLLECTION

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Age at Collection (Days)

Presenter

Presentation Notes

no significant differences were found regarding TREC quantities and age

0%10%20%30%40%50%60%70%80%90%

100%

All Newborns SCID PresumptivePositives

Sick w/TransfusionSick w/o TransfusionNormal (Healthy)

CLINICAL STATUS

Presumptive Positive Rate: Normal status 0.09%Sick w/o transfusion 1.3%Sick w/ transfusion 13.3%

Presenter

Presentation Notes

Of all the newborns screened, around 90% of newborns were health babies. Sick newborns account for less than 10% of all birth but 70% of SCID presumptive positive. For babies with normal status, the presumptive positive rate was 0.09%. For babies sick w/o transfusion, the presumptive positive rate jump close to 15 times to 1.3%. For babies sick w/transfusion, the presumptive positive rate increased another 10 folds, 13.3%.

AGE OF SPECIMENS

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Days after Collection

Average TREC Quantity

Average TREC Quantity

TREC cutoff

Newborn TREC Quantity Mean During theStudy Period

Presenter

Presentation Notes

Eighty-eight newborn DBS specimens, received in NBS check-in within 2 days after Date of Collection (DOC), were selected and amplification of TREC and RNaseP was tracked over the course of 365 days. Specimens were stored at room temperature throughout the entire study period. For each day tested, specimens were extracted on the same Biomek FXp (Batman) and run on the same QuantStudio™ 12K Flex (Jack) the following day. Average TREC quantities and RNaseP Ct’s for all 88 specimens were then calculated and plotted against time to assess TREC and RNaseP stability throughout the course of the year. The variations in average TREC quantity and RNaseP Ct observed during the study period, especially on days 8 and 92, are probably caused by spot to spot variability, standard curve shifts, and reagent lot to lot differences.

CALIBRATOR LOT

Average Median TREC Quantity: 1073 ± 173

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Median TREC Quantity

Presenter

Presentation Notes

The arrows indicate changes in Standard Curve lots, and with many of these changes, you can see that there are shifts in median TREC quantities ranging from 800 TRECs per uL to 1400 TRECs/ul.

REAGENT LOT

Average Median TREC (copies/µL): 33.859 ± 0.273

32.500

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Ct

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Median TREC Ct

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 1st screens is ~15% lower than 2nd screensDuring 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 1st Normal and 2nd Non-normal

1ST SCREEN VS. 2ND SCREEN

Screen Age of Collection

SCID Screening Results

First 1 day Normal

Second 7 days Unsatisfactory

Third 15 days Abnormal

Fourth 36 days Abnormal

CASE #1 – SCID WITH UNKNOWN MUTATION

Screen Age of Collection

SCID Screening Results

First* ~3 days Normal

Second 159 days Abnormal

Third 180 days Abnormal

CASE #2 – ZAP70

* Collected by birthing hospital and tested in newborn screening laboratory out of Texas

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

Alternative source of TREC calibrator Reagent lot variability Evaluate an alternate approach to determining

our cutoff (e.g. MoM, floating) SCID cases can have 1st screen normal results. 2nd screens can aid in identifying certain types

of SCID and secondary T-cell lymphopenia

LESSON LEARNED (CONT.)

THANK YOU!

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