implementation of tandem mass spectrometry: quality

Implementation of Tandem Mass Spectrometry: Quality Control/Quality

Assurance of the New York State Newborn Screening Program.

Mark A. Morrissey, PhDWadsworth CenterDepartment of Health

State of New York

Brand Names

Important:

The use of brand names in this presentations is for purposes of description only.

No endorsement is implied or intended.

Definitions

Quality Assurance is a comprehensive set of policies, procedures, and practices necessary to assure that the laboratory’s results are reliable.

Quality Control is a set of laboratory procedures designed to ensure that the test method is working properly.

From NYS DOH, Clinical Laboratory Evaluation Program (CLEP)

Outline

a. Administrative Preparation

b. Instrument Set-up/Method Validation

c. Routine/Daily Operations

a. Administrative Preparation

• Appropriate space

• Power and air conditioners

• Delineation of test panel

• Qualified staff

• Assay validation

• Lab certification

• Proficiency testing arrangements

More…

• Follow-up considerations

• Available medical specialists and diagnostic lab services

• Adequate notification of physician community

• Educational materials: lay and professional

• Advisory committee

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

• Develop training and competency logs

b. Instrument Set-up/Method Validation

Instrument Performance

Method Validation

Establishment of Cut-off values

Instrument Performance Issues

• Mass Calibration and Resolution– Establish operating range of instrument

• Probe rinses– Minimize Carryover

• Detection Optimization– Front End (Probe, cone, extractor, etc…) voltages.– Collision chamber, 2nd quadrapole, detector voltages

Instrument Performance Issues

• State files– Mass calibration

• Method files– Scan Parameters– Analytical run time (HPLC method)

• Data Reduction Software– Calibration Table

• Analyte & internal standard masses• Internal standard concentration• Blood spot volume

Instrument Performance Issues

• Mass Calibration

– Establish operating range of instrument

• Typical mass range 59amu to 1800 amu• We use 50 to 1100 amu, quarterly

– Materials used

• Polypropylene (PPG)• NaI / RbI solution

Method Validation Protocol

Method Validation

Requirements from CLIA or CLEP– Accuracy– Precision– Reportable Range– Specificity and Sensitivity– Reference Intervals– “Any other performance characteristics required for

test performance”

Method Validation

Establish Accuracy• Calculate recovery from analysis of fortified

samples (6 to 8 levels recommended)– [(S – C)/F] X 100

» Where: S is the observed concentrationC is the background concentrationF is the fortification concentration

• Acceptable recoveries: > 80%• Range is the demonstrated range of acceptable

recoveries (for example: C8 0.06 to 100 µmole/L)

Recovery (4 instruments, 4 Levels, 4 days)

Method Validation

• Intra run precision

– extract and prepare a set of blood spots.• Minimum of 10 replicate analyses

– Analyze in the same run on the same day.

– Calculate Coefficient of Variation (CV)

• Expected coefficient of variation: < 10%.

Method Validation

• Inter run precision

– Multiple day analysis (4 days)

– Prepare 4 extracts for each spiked pool daily

– Calculate Coefficient of Variation (CV)

• Expected coefficient of variation (CV): 5 – 15%

Method Validation(intra/inter run precision)

Establishment of Cut-off Values

• 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– Recalculate after several thousand samples

Establishment of Cut-off Values

• 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.

Establishment of Cut-off Values

c. Routine Operations

a. Routine, but not daily considerationsb. Daily analysis of samples

i. Sample Preparation1. Derivatized2. Un-derivatized

ii. Documentationc. Daily Data Evaluation

i. Data Reductionii. Evaluation of QC resultsiii. Evaluation of abnormal samples

Standard Operating Procedure

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 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

• 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

• 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 Preparation

• In-house Prepared Internal Standards– Available from Dr. H.J. ten Brink, Cambridge

Isotope Labs and CDN Isotopes Inc. – 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

• 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 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

• 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 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

• 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

1. Add Internal Standard (critical step) we use 12-channel pipetter. 2. Extract 20 minutes on a shaker (room temperature). 3. Transfer extract to a fresh plate. (We keep original plate). 4. Evaporate Solvent using warm air and gentle warming. 5. Add Butanolic HCl, cover (we use Teflon plates) and heat to

approximately 60 C for 20 minutes (ovens corrode over time). 6. Evaporate Butanolic HCl using warm air. 7. Add reconstitution solvent. 8. Cover with Aluminum foil and analyze by MS/MS.

Documentation -- Preparation

Documentation -- MS/MS Analysis

Derivatized Method, Challenges or Hints

0102030405060708090

1stQtr

2ndQtr

3rdQtr

4thQtr

EastWestNorth

High Quality Control C4

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0 50 100 150 200

Day

Con

c (µ

mol

e/L)

9413

9412

9405

9406

LCL

UCL

• 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

• 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 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.

• Quality Control Plan• Documents quality control decisions

– Acceptable plate quality control• Control results within ± 3 sd• Allow some number outside ± 3 sd

• Latitude in decision making• Document decisions• Checked and Reviewed Daily• Reviewed for trends

Evaluation of Quality Control Samples.

Analysis of TrendsSample Average Phenylalanine

0.25

0.45

0.65

0.85

1.05

1.25

1.45

1.65

1.85

0 50 100 150 200 250 300 350

Day

Co

nc (

mg

/dL

)

Average High Quality Control Phenylalanine

2

3

4

5

6

7

8

9

10

0 50 100 150 200 250 300 350

Day

Co

nc (

mg

/dL

)

9413

9412

9405

9406

LCL

UCL

Evaluation of Abnormal Results

Evaluation of Results

References

• Morbidity and Mortality Weekly Report, April 13, 2001

• Clinical Laboratory Improvement Amendments• Clinical Laboratory Evaluation Program• Basic QC Practices, 2nd Edition, James O.

Westgard, Ph.D.

Summary

• Plan Ahead• Train & Educate• Rigorous Method Validation is Required• Start with well defined rules and written Standard

Operating Procedures• Be consistent• Follow long-term trends as well as the daily

results.