Challenges of Operating a High Throughput Clinical Mass

Spectrometry Lab

Alan L. Rockwood, Tiffany A. Bradshaw, and Jason C.C. Sawyer

ARUP Laboratories, Dept. of Pathology, University of Utah School of Medicine

Asian Pacific Conference on Chromatography & Mass Spectrometry 2010

15 January 2010

rockwoal@aruplab.com

An Individual Behind Every Sample

Outline

• Introduction– Reference laboratory industry– Our institution– Mass spectrometry in this industry– Challenges

• Case studies• Additional points

Reference Laboratory Industry

Why adopt MS technology?

• Lower operating cost

• Freedom from supply disruptions (analytespecific reagents)

• Higher quality assays (better patient care)

Clinical LC-MS/MS trends and drivers

• Early– Immunosuppressants– Misc. analytes (e.g. methylmalonic acid)

• Current– Small molecule hormones (e.g. testosterone)

• Future– Proteins and peptides

Mass spectrometry: a disruptive technology?

LC-MS/MS technologyapplied here

New technology

Disrupt or Sustain?

New technology

Disrupt

Disrupt or Sustain?

New technology

Sustain

Challenges• Capital Cost

• Staffing– Recruitment– Training– retention

• Service/maintenance

• Backup capabilities

• R&D issues

• Assay troubleshooting

• Growth

• Supplier disruptions

• Proficiency testing

Case #1: The great acetonitriledrought

• Starting Oct 2008: worldwide acetonitrileshortage– Olympics – production shutdowns in China– Hurricane Ike – production shutdowns in

Texas– Worldwide economic downturn – decreased

production of industrial resins and plastics (acetonitrile is a byproduct of acrylonitrile)

• Supply disruptions and increased costs

Actions taken

• Vendor commitments

• Stockpile extra supplies– At premium price

• Altered and validated methods to decrease usage or use other solvents

Lessons learned

• Economic conditions outside of medical sector can impact clinical laboratory

• Be flexible to modify and validate methods to respond to changing conditions

Case #2: Mysterious contamination

• New high sensitivity assay successfully transitioned from R&D to production lab

• Several weeks later– Runs started to fail– Negative controls failed

• peak at retention time of interest– Ion ratios “out” indicating interferences

Investigation

• Many factors investigated (big picture down)– MS parameters– HPLC setup– Water quality– Solvents– Columns– Extraction Procedures– 96 well plates– Mats

Diagnosis

• Contaminated mats– Not a new product– Was used in validation– No previous interference issues – An unknown (new) manufacturing process

appeared to be the source.

Lessons learned

• Monitoring ion ratios crucial to maintaining quality

• Quality of supplies crucial to maintaining quality

• Supplies and suppliers not necessarily uniform in quality– Lot to lot variation can be serious

Case #3: Importance of SOP

• High sensitivity assay for certain steroids began to fail

• High background in chromatograms

• Column fouling, contamination, background, and column life

Investigation

• Investigated many factors

• SOP specified freezing of liq.-liq. extraction samples prior to phase separation

• Partial thawing of aqueous phase was occurring

• Slushy transition layer must be excluded

Solution

• Modify SOP to assure that no thawing occurs prior to phase separation– Time out-of-freezer-on-bench– Careful pipetting technique

• Train staff to better understand this aspect of SOP

Lessons learned

• Clarity and completeness of SOP is crucial• Training of staff to understand principles

underlying SOP is crucial• Diligence in following SOP is crucial• SOP issues can be subtle and difficult to

troubleshoot• R&D validation is fine, but production

experience is the acid test of any assay

Case #4: The sick Instrument

• Early adoption of new MS model• Instrument passed installation specifications• Erratic behavior of instrument

– Erratic sensitivity– Need for frequent cleaning– Unreliable in routine production– Altered instrument specifications

• Frequent service calls• Fingerpointing

Resolution

• Many months of troubleshooting and many service calls

• Several instrument deficiencies found

• Personnel learned to better operate within limitations of instrument

Lessons learned

• Ordering early in product cycle is risky

• Don’t assume an instrument is faultless

• Don’t assume your own laboratory is faultless

Case #5: Damaged stationary phase

• High sensitivity 2D LC-MS/MS assay for certain analytes was working well

• Method transferred to a commercial column multiplexing system

• Erratic assay performance– Inconsistent– High background– Decreasing sensitivity– Failed runs

Investigation

• Weeks of in-house and vendor-aided troubleshooting ensued investigating a wide variety of potential issues

Valve switching arrangement

Diagnosis

• Pressure fluctuations damage stationary phase

• Problem unfamiliar to the vendor– Our 2D LC setup was slightly different from

the typical setup used in their system

Solution

• Balancing flow resistance on waste line to the resistance of the analytical flow path minimized pressure fluctuations, etc.

• Reliability improved markedly

Lessons learned

• Operating instrument outside vendor’s experience can uncover unexpected problems– When using new applications of technology, a

collaborative relationship works best to understand and troubleshoot the new assay.

• High volume production uncovers problems not evident during R&D validation

Case #6: Turn around time

• An analyte is measured in either serum or urine

• Turn around times for serum consistently met

• Turn around times for urine difficult to meet

Investigation

• Serum sample assay performed entirely within MS lab

• Urine sample assay required result from secondary lab (creatinine)– Samples forwarded to other lab after being

run in MS lab

Problem resolution

• What would you do to resolve this problem?

Case #7: Staffing shortage

• Position for medical technologist open for 6 months.

• Medical Technologists (MT) or equivalent degree/experience needed for laboratory accreditation in US.

• Career awareness is low

Medical Technologists Wanted

Kibak, Phil. The Worsening Shortage of Lab Staff. AACC Clin Lab News 2008; 34:5

Response

• Support existing MT programs – Provide training slots to support MT programs

• Educational benefit exists for employees to upgrade education and credentials

• Rely on more people with “categorical”certification

Compensation: a fundamental issue?

• Median income of occupations in US:– medical technologist: $53,000– food scientist: $60,000– biologist (other): $65,000– chemist: $66,000 – chemical engineer: $85,000

Source: www.bls.gov/oes/current/...

Training issues

• Medical technology training:– Little or no mass spectrometry

• Months of additional training and experience required in MS lab

Case #8: Staff retention

• First employee in MS production lab served several years, but had additional career aspirations

• Gained an acceptance to law school

• (Many other MS lab personnel have left to attend professional schools)

Appropriate response

• Realize that MS lab attracts many high quality and ambitious people

• Realize that the net effect is a better lab• Accept fact that some will leave to pursue

other career goals• Encourage rather than discourage staff to

reach their full potential

Case #9: Know when to retire

• Reliable ~10 year old tandem MS– First instrument at ARUP– Producing ~8000 routine patient sample

results per month– Developed frequent breakdowns– Eventually “unrepairable”

Actions taken

• Secondary instrument takes on work of two (8000 samples/month)

• Attempts to repair instrument over many months

• Recently decommissioned instrument• Ordered replacement instrument

Lessons learned

• Instruments don’t last forever

• Lab personnel and management need to recognize when to decommission an instrument

• Be adaptable and have a plan for downtime– Reserve capacity

Case #10: No backup instrument

• Specialized hormone test run on single instrument

• Risk: instrument may go down

• Potential consequences– Sendout cost– Turn around time– Patient care

Response

• Large labs: Order and validate backup instrument(s)

• Small labs: Be sure you have a good sendout lab for backup

Issue: Interfacing with vendor

• Mass spectrometers are not designed as “clinical analyzers”

• Service support– Response time– Cost– Multiple vendor involvement and the blame

game

Response

• Internal service group– First and second line service

• Vendor-supplied service– as backup

• Not appropriate for smaller labs

Issue: Growth

• Can become buried in growth– Staffing

• Recruitment• Training• Retention

– Instrument shortages– Consumables, etc.– R&D targets

Growth of an assay

Assay sample volume

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2005 2006 2007 2008 2009

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Response and Lesson

• Get ahead of the curve– Staffing– Instrumentation– Management - split labs– Etc.

• Dangers– Too far ahead of the curve– Everolimus example

R&D challenges and issues

• Structure– Dedicated R&D personnel-based– Production personnel-based

• Staffing– Difficulty in recruiting R&D professionals

• Throughput• Coordination with routine testing lab

Case #11: MS improves patient care

• 17 yr blond female. – Male pattern hair growth

• obscured by long hair along jaw and light hair color

– Recalcitrant acne– Slightly overweight– Athletic build– No menses to date– Mom: history of delayed menarche

Differential Diagnosis

• Ethnic or family traits• Oversensitivity of hair follicles to

androgens• Androgen excess

– Polycystic ovary disease– Tumors (adrenal, ovary)– Congenital adrenal hyperplasia– Insulin resistance– Obesity

Laboratory results

• Testosterone by immunoassay– Twice upper reference interval limit

• Is this result useful?

Immunoassays for Testosterone in Women: Better than a Guess?

• Are assays that miss target values by 200–500% meaningful? Guessing would be more accurate and additionally could provide cheaper and faster testosteroneresults for females—without even having to draw the patient’s blood.

David A. Herold and Robert L. Fitzgerald, Clin Chem 49: 1250-1251, 2003;

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IA V

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Low-level Testosterone Method Comparison:LC-MS/MS to other methods

LC-MS/MS to Immunoassay

MM Kushnir…, Clin Chem, 52:120-128(2006).

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0 20 40 60 80Comparative methods, ng/dL

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LC-MS/MSGC-MS

Mass Spec Comparisons

Testosterone Standard of Care

• Immunoassay adequate for high level measurements (normal adult males)

• LC-Tandem MS for low level analysis (women, children, hypogonadal men)

Lesson

• Immunoassays unreliable for low level testosterone

• LC-MS/MS is more reliable for low level testosterone

• LC-MS/MS often improves patient care

Predicting the Future

Summary

• MS advantages and opportunities– Lower cost (sometimes)– Freedom from ASR suppliers– Improved patient care (often)

• MS challenges– Challenges are many– Can be met by good planning and operations

Acknowledgments

• ARUP Laboratories

• Staff in the mass spec lab