1. Tandem Mass Spectrometry Newborn Screening Quality Assurance and Control Instrument and Method Validation Gary Hoffman Wisconsin Newborn Screening Laboratory State Laboratory of Hygiene Madison WI

2. Training Issues Instrument vendor site training - Instrument operation - Instrument troubleshooting Visit other MS/MS laboratories - 20 + MS/MS testing programs - spit equally between vendors APHL and NNSGRC sponsored courses - Baylor Institute of Metabolic Diseases - Duke Medical Center Write standard operating procedure Develop training and competency logs

3. Instrument Performance Issues Mass Calibration – Establish operating range of instrument Typical mass range 59 mu to 1800 mu – Materials used Polypropylene (PPG) NaI / RbI solution

4. Instrument Performance Issues Unit Resolution – MS/MS unit resolution voltages 0.7 of a mass unit at 50% peak height – Frequency Initial instrument set up Schedules vary

5. Instrument Performance Issues Sample loop size – Provides consistent injection volumes – Best results when loop size equals injection volume. – Typical loop sizes used 10 - 30 µL Probe rinses – Minimize Carryover

6. Instrument Performance Issues Detection Optimization – Front End (ESI, cone, orifice, ring) voltages. Allows analyte ionization Minimize fragmentation – Collision chamber, 2 nd quadrapole, detector voltages Maximize output response detector voltage decrease needs adjustment.

7. Instrument Performance Issues State files – Mass calibration – Voltages for each experiment (precursor ion, neutral loss, MRM) Method files – Scan Parameters – Analytical run time Help minimize carryover Long enough to return to baseline Typical run times: 1.5 to 3.0 minutes/specimen

8. Instrument Performance Issues Data Reduction Software – Calibration Table Analyte & internal standard masses – Designates which masses will be calculated from with internal standard – Example: DC8 for C5DC, C10, C10:1, C10:2, C6DC Internal standard concentration Blood spot volume – small changes are significant – 0.2 µL change – 20% change in control results – standard blood spot volume needed

9. Instrument Performance Issues Calibration Table (Cont) Extraction volume – Accounts for specimen dilution – V olume added before extraction is critical – After extraction, exact volume is less important Analyte cut off levels Analyte ratios – Phe/Tyrosine – C8/C10 Internal standard count thresholds

10. Method Validation Issues Establish linearity – Prepare spiked blood spots Six to eight levels – Lowest level: endogenous – Highest level: expected in affected babies Plot observed vs expected results – linearity is the straight part of the line

11. Method Validation Issues Establish intra and inter run precision – Materials Two analyte(s) spike levels – First level: Medical decision level – Second level: 4 X first level

12. Method Validation Issues Intra run precision – extract and prepare a set of blood spots. Minimum of 20 replicate analysis – Analyze in the same run on the same day. – Calculate Coefficient of Variation (CV) Expected coefficient of variation: < 10%.

13. Method Validation Issues Inter run precision – Multiple day analysis – Prepare 2 extracts for each spiked pool daily – Analyze in runs for a minimum of 10 days. – Calculate Coefficient of Variation (CV) Expected coefficient of variation (CV) : 15 – 20%

14. Method Validation Issues Establish Accuracy – Recovery Calculate recovery from intra run precision data – Observed value/expected value X100 – Acceptable recoveries: > 85% – Known disease cases Specimens on disease cases (metabolic clinics) Contact MS/MS colleagues

15. Method Validation Issues Non-Peer Reviewed methods – Direct comparison with established methods Analyze a minimum of 500 specimens by both methods May have to “spike” blood for some analytes Calculate slope and intercept for each analyte – Slopes greater than 0.900 are acceptable

16. Routine Specimen Analysis Analysis of routine blood spots specimens – Test all specimens received Establish a reporting policy – Test limited number of specimens Obtain blinded specimens from MS/MS colleagues Liability issues eliminated

17. Interferences/Contamination Interferences – TPN Amino Acids Acylcarnitines: C5, and C18:2 – Reporting As potential disorder – potentially confusing Unsatisfactory – Request repeat after TPN discontinued – Closely review results

18. Interferences/Contamination Contaminations – Floor wax Leucine interference – Detergent surfactants Baseline increase – Glassware contamination Hemoglobin testing stain

19. Establishing Analyte Cutoffs Pilot Testing – Do a literature search – Contact existing MS/MS programs – Manufacture of instrument or reagents Routine Testing Cutoffs – Analyze several thousand normal specimens – Calculate mean and standard deviation

20. Establishing Analyte Cutoffs Establish Analyte cutoffs Consult metabolic specialist/follow up staff. Typical cutoffs: 3 and 10 sd from mean Compare cutoffs with other MS/MS programs. A balance between false positives/negatives Consider separate ranges for age > 7 days.

21. Quality Control Plan Documents quality control decisions – Imprecision factors Blood spot absorption methodology drift Inconsistent ion flow – Acceptable plate quality control Control results within ± 3 sd Allow some number outside ± 3 sd

22. Quality Control Plan Quality control review – Daily Checked by analyst and supervisor – Monthly Reviewed by Supervisor Long term documentation

23. Quality Control Plan – Repeat individual specimens No masses detected – No sample injected – Electronic errors Abnormal results profiles – Example: C6, C8, C10:1, C8/C10 – Some secondary markers not reported Poor Sensitivity – d-Phe, d-C8, d-Cit below sensitivity threshold – Latitude in decision making – Document decisions