Tandem Mass Spectrometry Newborn Screening Quality Assurance and Control Instrument and Method...

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