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