1. Prevent Disease – Promote Wellness – Improve Quality of Life Michigan update for T Cell Receptor Excision Circle (TREC) analysis for detection of primary immune deficiency syndromes (PID s ). Heather Wood Michigan Department of Community Health Bureau of Laboratories

2. Prevent Disease – Promote Wellness – Improve Quality of Life What are TRECs? T Cell Receptor Excision circle- TRECs are small, episomal pieces of DNA that are formed during the differentiation of T-cells in the thymus as a result of rearrangement of the T cell receptor genes. Screening for the absence of TRECs identifies babies who cannot generate functional T cells TREC assay will differentiate between autologous T cells and maternal T cells.

3. Prevent Disease – Promote Wellness – Improve Quality of Life Overall Analysis Scheme 3 µl DBS is punched into a 96 well plate Automated DNA extraction using an epMotion 5075 Automated set up of real time qPCR using plasmid standard curves in a 384 well format using the epMotion 5075 Duplex PCR amplification and analysis (TREC and beta actin) on a 7900HT.

4. Prevent Disease – Promote Wellness – Improve Quality of Life Standard curve *Now only using TREC for standard curve

5. Prevent Disease – Promote Wellness – Improve Quality of Life Began screening all MI newborns for SCID Oct 1, 2011 As of Sept 2012 we have screened over 100,000 babies. Approximately a 0.3% “abnormal” rate. »This includes strong positive, borderline positive, and inconclusive MI Update

6. Prevent Disease – Promote Wellness – Improve Quality of Life Strong positives reported: a majority resolved by either repeat DBS or flow cytometry. SCID reported October 1, 2011 through July 20, 2012 155 infants with strong positive SCID determination 1 "Severe" Partial DiGeorge; no 22q11 deletion; hypoplastic L heart 1Charge syndrome with DiGeorge (T - B + NK +); no 22q11 deletion 2Partial DiGeorge, with 22q11 deletion 1 22q11 microdeletion confirmed. Congenital absent thymus. Work-up for DiGeorge still ongoing 1 T-Cell related lymphocyte deficiency; hepatoblastoma; bone marrow supressed by malignancy; infant expired 2 Secondary transient t-cell lymphocyte deficiency; secondary to chylothorax 4 Secondary immune deficiency; confirmed + thymectomy; post cardiac surgery 1 Cardiac surgery - thymectomy; no previous newborn screen; follow-up still ongoing 42Resolved with normal flow cytometry results 62Resolved with normal repeat newborn screen 17Expired 21Pending

7. Prevent Disease – Promote Wellness – Improve Quality of Life Reasons to change the TREC method in MI High repeat rate in the lab (extra work and extra cost). High “abnormal” rate reported to follow up (0.3%). Mostly “inconclusives” which requires another DBS. Too many tips used= too much $. Extraction method time consuming. Noticed low beta actin levels early on after going live. CDC SCID grant aims included: »A new extraction method that is faster, less expensive, and more efficient. »Utilizing the DNA extract for other molecular methods in the MI NBS lab (cystic fibrosis).

8. Prevent Disease – Promote Wellness – Improve Quality of Life New Method Validation To compare a new and improved DNA extraction method to the old DNA extraction method used for TREC analysis and for Cystic fibrosis. To compare Gene Expression master mix to Environmental master mix. Part 2 includes validation of the DNA extraction for cystic fibrosis assay. Implementation of “cherry picking” using the epMotion 5075.

9. Prevent Disease – Promote Wellness – Improve Quality of Life Comparison of the two DNA extraction methods 2 washes using Generation DNA purification solution (aka solution 1). 1 wash using Generation DNA elution solution (aka solution 2). –Note: a 10 minute shake at room temperature 500 rpm followed each wash step. 50 µl of elution solution added Incubation at 95 degrees C for 15 minutes Old extraction 1 wash using Generation DNA elution solution (solution 2) followed by a 10 min shake at 60 degrees C at 1000 rpm 50 µl of elution solution added Incubation at 95 degrees C for 30 minutes New extraction

10. Prevent Disease – Promote Wellness – Improve Quality of Life Comparison of the two DNA extraction methods –75 minutes on Epmotion and 15 minutes in thermocycler. –Tips used per 96 well plate = 404 –Average repeat rate in lab for repeating initial samples= 10% –*Average cost per sample= $3.18 *cost per sample includes reagents and consumables for both the extraction and PCR Old extraction –20 minutes on Epmotion and 30 minutes in thermocycler. –Tips used per 96 well plate= 112 –Average repeat rate in lab for repeating initial samples= 3% –*Average cost per sample= $2.50 New extraction

11. Prevent Disease – Promote Wellness – Improve Quality of Life Comparison of master mixes from ABI *Each data point is an average of 6 DBS replicates with various heparin levels with 2 master mixes. Environmental master mix much better! Gene Expression Master Mix Environmental Master Mix Summary of the comparison of two master mixes from ABI. Heparin in Units/mL of blood B actin quantity per µl of bloodBactin_CT 1056552421829.3427.11 2036951687830.0527.88 3038372130729.9827.27 4043832223129.7227.21 5062752271029.1527.17 6058852198229.2727.22 7082252151128.8627.26 8087532066828.6827.34 9079362472228.8327.05 10090252275628.6227.17 20055162216329.4327.26 30050832377129.5127.12 40030242382030.3127.12 50017512562131.4227.00 60010602169031.9327.26 7008291895432.6227.47 8003791974434.2227.40 9005541860333.7327.49 10001901724435.2527.62

12. Prevent Disease – Promote Wellness – Improve Quality of Life Accuracy All samples from CDC, New York, and previously positive MI samples were categorized clinically correctly 100%

13. Prevent Disease – Promote Wellness – Improve Quality of Life New Algorithm Strong positive= refer to clinic for flow analysis Borderline and inconclusive= request another DBS Two B+ for a baby =referral to clinic for flow analysis Three inconclusive samples= referral to clinic for flow analysis

14. Prevent Disease – Promote Wellness – Improve Quality of Life New method with CDC cord blood samples Table 3: New method with new Cord Blood samples 2012 Sample name % cord bloodLog 10 (%) Average TREC Ct Average TREC Quantity per ul of blood Average beta actin Ct Average beta actin quantity per ul of blood CB cal 12002.3031.1248026.2931274 CB cal 21002.0032.3220327.1117632 CB cal 3501.7033.827028.546755 CB cal 4251.4034.434628.736237 CB cal 512.51.1035.082928.588873 CB cal 66.250.8036.231428.915453 CB cal 73.1250.4936.431228.486963

15. Prevent Disease – Promote Wellness – Improve Quality of Life CDC Cord blood samples with old method Old method not as good as the new method for reproducibility of extraction

16. Prevent Disease – Promote Wellness – Improve Quality of Life Cord blood calibrator dilutions and TREC cutoff analysis Table 3: New method with new Cord Blood samples 2012 Sample name % cord bloodLog 10 (%) Average TREC Ct Average TREC Quantity per ul of blood Average beta actin Ct Average beta actin quantity per ul of blood CB cal 12002.3031.1248026.2931274 CB cal 21002.0032.3220327.1117632 CB cal 3501.7033.827028.546755 CB cal 4251.4034.434628.736237 CB cal 512.51.1035.082928.588873 CB cal 66.250.8036.231428.915453 CB cal 73.1250.4936.431228.486963

17. Prevent Disease – Promote Wellness – Improve Quality of Life True positives and Proficiency sample results Table 6: True positive samples from New York Diagnosis TREC Quantity per µl of blood B actin quantity per µl of bloodMI result NY1: X linked SCID010372Positive NY2: JAK3 SCID024525Positive NY3: ADA SCID010576Positive NY4: Unknown SCID015284Positive NY5: T-B+NK+SCID02694Positive Table 7: NSQAP PT samples from CDC Samples from CDC TREC Quantity per µl of blood B actin quantity per µl of bloodMI result PCDC112R1-SCID Like04736Positive PCDC112R2-Normal37422275Negative PCDC112R3-Normal8722960Negative PCDC112R4-Unsat0562Unsat PCDC112R5-Normal22218227Negative PCDC212R1-Normal36828217Negative PCDC212R2-Unsat0441Unsat PCDC212R3-Normal9120883Negative PCDC212R4-Normal23712256Negative PCDC212R5-SCID Like022886Positive 100% clinical correlation Beta actin Ct value =29.79 Beta actin Ct value =29.00

18. Prevent Disease – Promote Wellness – Improve Quality of Life All samples were consented samples Reference Range

19. Prevent Disease – Promote Wellness – Improve Quality of Life Summary table of population data for old vs new method with initial values Table 20: Average and Median TREC and beta actin copies for population study with "old" method Table 21: Average and median TREC and beta actin copies for population study with New method All sample s NBW 2500 grams or greater LBW less than 2500 grams All sample s NBW 2500 grams or greater LBW less than 2500 grams TREC N50544711343 TRE C N50544711343 Mean181184140Mean183186137 Median152154111Median153155111 B actin N50544711343 B acti n N50544711343 Mean134951370310638Mean221902250417859 Median11113113298956Median181171830515334

20. Prevent Disease – Promote Wellness – Improve Quality of Life Algorithm change from old to new method TREC Quantity β-Actin Ct value Result: New Algorithm Normal Birth Weight(≥ 2500 grams) or Missing or (Low Birth Weight (<2500 grams) <20 ≤ 30.00 Strong Positive 20-<30 ≤ 30.00 Borderline Positive <30>30.00Inconclusive

21. Prevent Disease – Promote Wellness – Improve Quality of Life Where do we go from here? Began new TREC method Sept 1. May adjust algorithm after first three months. Complete the cystic fibrosis portion of method validation. Implement “cherry” picking using epMotion 5075 from extracted DNA to use for CF mutation analysis. Receive a VIIA 7 in lab Host other NBS labs for training and assist with SCID startup

22. Prevent Disease – Promote Wellness – Improve Quality of Life Acknowledgements MI NBS laboratory This work was partially funded by a research cooperative agreement from CDC (Grant # 01EH000936) and does not represent the official view of CDC. MAP team members NY & WI laboratories CDC/NCEH Other NBS labs