# Presentation: 564038

##  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

##  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