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Tandem Mass Spectrometry QA/QC for Newborn Screening: Routine Operations. Mark A. Morrissey, PhD Wadsworth Center Department of Health State of New York. Outline. Routine, but not daily considerations Daily analysis of samples i.    Sample Preparation 1.      Derivatized

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tandem mass spectrometry qa qc for newborn screening routine operations

Tandem Mass Spectrometry QA/QC for Newborn Screening: Routine Operations.

Mark A. Morrissey, PhD

Wadsworth Center

Department of Health

State of New York

outline
Outline
  • Routine, but not daily considerations
  • Daily analysis of samples

i.    Sample Preparation

1.      Derivatized

2.      Un-derivatized

  ii.    Documentation

  • Daily Data Evaluation

   i.     Data Reduction

ii.     Evaluation of QC results

iii. Evaluation of abnormal samples

d. Summary

routine but not daily considerations
Routine, but not daily considerations

Instrument Maintenance

  • Follow the manufacturers recommendations.
  • Most labs use a check-off sheet with signature and date
  • Activities may include: clean cone, ballast the rough pump and check oil level, check chiller temp, etc…
  • Maintained in the instrument logbook
  • Ultimately up to the area supervisor and laboratory director
routine but not daily considerations4
Routine, but not daily considerations

Non-routine maintenance

  • Documented with nature of the problem, who found the problem, resolution and the person fixing the problem.
  • We include preventative maintenance from the manufacturer. They have their own specification for function checks and mass calibration.

Tuning

  • Concentrated Standard Solution. May not include all the applicable analytes (C14:1, C5OH, etc). .
  • Collected material from a used plate (not documented or traceable). We do this as needed if the QC results are out of range.
reagent preparation
Reagent Preparation
  • HPLC grade, or Reagent grade, or better
  • To filter, or not to filter?
  • Hazards
    • Acetonitrile -- toxic, flammable
    • Methanol -- toxic, flammable
    • Butanol -- flammable, irritant
    • Acetyl Chloride -- corrosive, water reactive. Use a chilled bath and mix with butanol under argon.
internal standard preparation
Internal Standard Preparation
  • MMWR recommends a deuterated standard to correspond to each analyte.
  • Pre-prepared Internal Standards
    • Available from Perkin-Elmer or Cambridge Isotope Laboratories.
    • Concentration and Expiration are provided by the supplier
internal standard preparation7
Internal Standard Preparation
  • In-house Prepared Internal Standards
    • Available from Dr. H.J. ten Brink (acylcarnitines) and Cambridge Isotope Labs (amino acids and acylcarnitines).
    • Cheaper, but requires more labor
    • I estimate that for 250,000 samples it requires:
      • Approximately 80 hours to prepare the standard
      • 40 hours to go through a qualification
internal standard preparation procedure
Internal Standard Preparation Procedure
  • Weigh individual deuterated stds into a suitable container.
  • Dissolve in 0.01N HCl (d-C16 in MeOH) for an exact concentration of 1.0 mg/mL or 0.1 mg/mL.
  • Add the appropriate amount of each to individual 15-mL polypropylene centrifuge tubes. (Adds up to about 7.3 mL). We make about 40 tubes.
  • Seal with Teflon tape and store at –70 C. Stable for at least 1-year
  • Should check concentrations and adjust, if necessary
internal standard preparation procedure9
Internal Standard Preparation Procedure
  • To prepare working internal standard dissolve the entire tube in 2-L of methanol. Store at –20C. Lasts about 10-days, (10,000 samples).
  • 2-years worth of standards costs us approximately $10,000 (500,000 samples)
preparation of quality control samples
Preparation of Quality Control Samples
  • Standards available from Life Science Resources, H.J. ten Brink (carnitines) and Sigma (amino acids).
  • Blood obtained from the Red Cross.
  • Individual stock standards of amino acids and acylcarnitines are prepared in saline.
  • Added individually to 50 mL aliquots of whole blood. Stored frozen along with a desiccant. Lasts approximately six months. (24/day at each level)
preparation of quality control samples11
Preparation of Quality Control Samples
  • One level is approximately equal to the request for a repeat specimen level. The other level is approximately 2 to 3 times the concentration of the first.
  • Determination of control limits.
  • QC Samples available from CDC (bi-annually) for periodic use.
sample preparation and analysis
Sample Preparation and Analysis
  • Underivatized - fewer steps, faster
    • Add Internal Standard (critical step)
    • Cover plates and extract for 20 –30 minutes
    • Transfer
    • Analyze by MS/MS
  • Derivatized - published, more sensitive
  • Both Methods require care and consistency in sample preparation
derivatized method
Derivatized Method
  • Add Internal Standard (critical step) we use 12-channel pipetter.
  • Extract 20 minutes on a shaker (room temperature).
  • Transfer extract to a fresh plate. (We keep original plate).
  • Evaporate Solvent using warm air and gentle warming.
  • Add Butanolic HCl, cover (we use Teflon plates) and heat to approximately 60 C for 20 minutes (ovens corrode over time).
  • Evaporate Butanolic HCl using warm air.
  • Add reconstitution solvent.
  • Cover with Aluminum foil and analyze by MS/MS.
derivatized method challenges or hints
Derivatized Method, Challenges or Hints
  • We have seen high C4 when samples were extracted in polystyrene plates.
  • Overheating can cause high leucine results (QC results may appear normal)
  • We have observed low response for Methionine internal standard. This is under investigation.
data reduction and evaluation
Data Reduction and Evaluation
  • Objectives
    • Determine Concentration of Analytes
    • Incorporate Results into a Database of Samples
    • Compare Values to a set of Rules
    • Produce Reports of Normal and Abnormal Samples
data reduction and evaluation18
Data Reduction and Evaluation
  • First Step –review all the “chromatograms.”
    • Missed injections
    • Failure of the pump program
  • Data Conversion
    • Neolynx generates a tab-delimited file that can be read by Excel or a database program.
    • Ratios can be calculated by the MS software, Excel, or the database.
summary
Summary
  • Start with well defined rules and written Standard Operating Procedures
  • Be consistent
  • Follow long-term trends as well as the daily results.
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