Tandem Mass Spectrometry Newborn Screening Quality Assurance and Control Instrument and Method Validation Gary Hoffman Wisconsin Newborn Screening Laboratory.
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Tandem Mass SpectrometryNewborn Screening
Quality Assurance and Control
Instrument and Method Validation
Gary Hoffman
Wisconsin Newborn Screening Laboratory
State Laboratory of Hygiene
Madison WI
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
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
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
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
Instrument Performance Issues
• Detection Optimization– Front End (ESI, cone, orifice, ring) voltages.
• Allows analyte ionization
• Minimize fragmentation
– Collision chamber, 2nd quadrapole, detector voltages
• Maximize output response• detector voltage decrease needs adjustment.
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
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
Instrument Performance Issues
• Calibration Table (Cont)
• Extraction volume– Accounts for specimen dilution– Volume 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
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
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
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%.
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%
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
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
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
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
Interferences/Contamination • Contaminations
– Floor wax • Leucine interference
– Detergent surfactants• Baseline increase
– Glassware contamination• Hemoglobin testing stain
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
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.
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
Quality Control Plan
• Quality control review
– Daily
• Checked by analyst and supervisor
– Monthly
• Reviewed by Supervisor• Long term documentation
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
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