Tandem mass spectrometry qa qc for newborn screening routine operations l.jpg
This presentation is the property of its rightful owner.
Sponsored Links
1 / 23

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


  • 449 Views
  • Updated On :
  • Presentation posted in: General

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

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

Download Presentation

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

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Tandem mass spectrometry qa qc for newborn screening routine operations l.jpg

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

Mark A. Morrissey, PhD

Wadsworth Center

Department of Health

State of New York


Outline l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

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.


Documentation preparation l.jpg

Documentation -- Preparation


Documentation ms ms analysis l.jpg

Documentation -- MS/MS Analysis


Derivatized method challenges or hints l.jpg

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 l.jpg

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 l.jpg

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.


Evaluation of quality control samples l.jpg

Evaluation of Quality Control Samples.


Evaluation of abnormal results l.jpg

Evaluation of Abnormal Results


Evaluation of results l.jpg

Evaluation of Results


Analysis of trends l.jpg

Analysis of Trends


Summary l.jpg

Summary

  • Start with well defined rules and written Standard Operating Procedures

  • Be consistent

  • Follow long-term trends as well as the daily results.


  • Login