clinical application of lc-ms - massep.org · (thermo fisher scientific) for online cleanup...

Mark Kellogg Children’s Hospital Boston 27 September 2011

Clinical Application of LC-MS

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This presentation discusses the issues faced in translating a biomarker of clinical utility to a validated clinical assay

Regulatory Requirements

Preanalytical Issues

Analytical Issues

Post-Analytical Issues

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Clinical Laboratory = A facility for the examination of materials derived from the human body for the diagnosis, prevention or treatment of any disease or the assessment of health

Children’s Hospital Boston

Department of Laboratory Medicine

Assay Performance

• Precision – How close together the measured values are…

– The “good” for most endocrine assays

• Accuracy – How close is our “answer” to the actual quantity of

analyte present…..

– The “bad” for most endocrine assays

– Requires standardized reference material



• Analytical Sensitivity – The slope of the analytical function…

• Change in signal versus analyte concentration

– How small of a difference in concentration can be detected • Often used to describe limits of detection

– Particularly important with hormones • Often nmol or pmol levels

• AND….Pediatric samples start with less material…....



• Analytical Specificity (Selectivity)

– Free from interference from

• Endogenous substances – Including isoforms, pre-hormones…

• Exogenous substances – Steroid drugs…

– Nutritional supplements….



• “Diagnostic sensitivity”:

– percentage of persons who have a given disorder who are identified by the assay as positive for the disorder.

• High analytical sensitivity does not guarantee acceptable diagnostic sensitivity.



• “Diagnostic specificity” – is the percentage of persons who do not have a given condition who

are identified by the assay as negative for the condition.

– False-positive reactions occur:

» because of sample contamination

» Improper sample collection, handling, processing

• E.g. glucose on cells (5-8% decrease/hr)

» Subclinical disease in the “healthy” study population(s)

All diminish the diagnostic specificity of the assay



Reference Intervals…

• Required by Clinical Laboratory Improvement Amendments of 1988 – US Food and Drug Administration-cleared or approved nonwaived test

system "verify that the manufacturer's reference intervals (normal values) are appropriate for the laboratory's patient population.“

– Laboratories that modify US Food and Drug Administration-approved tests or develop their own assays are required to establish their own reference intervals for their assays.

– Regulations also specify that reference intervals be included in laboratory reports or made available upon request to individuals who order tests.



Reference Interval….

• an interval that, when applied to the population serviced by the laboratory correctly includes most of the subjects with characteristics similar to the reference group and excludes the others.

– No RI is completely "right" or "wrong."

– The majority of RIs in use refer to the central 95%

– Dependent on test method!



How to…..

• Select a statistically sufficient group

– a minimum of 120

– healthy reference subjects. • However…..Health is a relative condition lacking a universal

definition.

• Difficult to “detect” subclinical disease….



How to…..

• Recruiting is costly, time-intensive, and virtually an impossible task for most laboratories.

– further magnified in establishing RIs for different age groups (eg, pediatric patients and geriatric patients), uncommon sample types (eg, cerebrospinal fluid and aspirations), timed collections, challenge tests, and serial measurements.



How to…..

• Option 1: to verify RIs that have been reported by the manufacturer or as established by another laboratory.

– requires only 20 healthy subjects to recruit.



How to…..

• Option 2: "transfer" the RI to a new method.

– demonstrate that the 2 methods produce comparable results.



Decision Values

• defines specific medical decision limits that clinicians use to diagnose or manage patients.

• “Reference intervals” that incorporate medical decision limits are often defined with clinical trials and adopted by laboratories from the medical literature.

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Regulatory and Accrediting agencies include CMS, FDA, CDC, Joint Commission, CAP…….

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Imprecise, Inaccurate immunoassays

18yo female reference int: 20-75 ng/dL

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Imprecise, Inaccurate immunoassays…..

PTH: Reference Interval 15-65 pg/mL

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Imprecise, Inaccurate immunoassays or lack of immunoassays are driving mass spectrometry into the clinical laboratory

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Cost, Lack of Knowledge, Unfriendly user interface and regulatory fear are impeding use of mass spectrometry in the clinical laboratory

© Can Stock Photo Inc. / photomak

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Cost is significantly more than the usual clinical instrumentation

$150,000 for automated random access clinical chemistry analyzer with:

25 tests onboard

100 samples/hr throughput

$75,000 for automated ELISA system with: 2 plates on board “unlimited” menu

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Cost is significantly more than the usual clinical instrumentation

A minimum of $400,000 for chromatography and mass spectrometer:

One analyte at a time

2-20 samples/hour throughput

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LDT’s – Laboratory Developed Tests are “High Complexity Tests”

Staffing Requirements

(A) At least 60 semester hours, or equivalent, from an accredited institution that, at a minimum, include either–

(1) 24 semester hours of medical laboratory technology courses; or (2) 24 semester hours of science courses that include-- (i) Six semester hours of chemistry; (ii) Six semester hours of biology; and (iii) Twelve semester hours of chemistry, biology, or medical laboratory technology in any combination; and

(B) Have laboratory training that includes either of the following:

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Fewer people with hands-on chemistry skills

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LDT’s – Laboratory Developed Tests are “High Complexity Tests”

Validation Requirements

(2) Each laboratory that modifies an FDA-cleared or approved test system, or introduces a test system not subject to FDA clearance or approval (including methods developed in-house and standardized methods such as text book procedures, Gram stain, or potassium hydroxide preparations), …. must, before reporting patient test results, establish for each test system the performance specifications for the following

performance characteristics, as applicable: (i) Accuracy. (ii) Precision. (iii) Analytical sensitivity. (iv) Analytical specificity to include interfering substances. (v) Reportable range of test results for the test system. (vi) Reference intervals (normal values). (vii) Any other performance characteristic required for test performance.

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Instrument “up-time” is critical as clinicians will not accept….”sorry the instrument is down”

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Routine LC-MS in the clinical lab are relatively simple types of analyses

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Clinical Applications in LC-MS need front end automation and computerization on the back end…..

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Immunosuppressant use requires therapeutic drug monitoring to prevent acute transplant rejection due to wide inter- and intra-individual variation in absorbance and metabolism.

• Cyclosporine A: calcineurin inhibitor…decreased interleukin-2 production

• Tacrolimus: calcineurin inhibitor…prevents T cells for transitioning from

Go to G1 of cell cycle

• Sirolimus: mTOR inhibitor…prevents T cells from transitioning from G1 to S phase of the cell cycle

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Previous ISD method….in use for 8 years

• 1.5mL deionized H2O + 100uL Internal Std: 32-desmethoxyrapamycin & Ascomycin

• 500uL EDTA whole blood • 500uL Acetonitrile • 4mL zinc sulfate solution (7.5 g zinc sulfate, 400 mL ddH2O, 100 mL MeOH, and 300 mL ACN)

• 1 minute vortex, centrifuged 3 min • Supernate extracted on 3mL Oasis HLB columns

• Two 3mL ddH2O washes and 1 3mL 75% MeOH Wash • Column dried, ISD eluated with 2mL 90% MeOH • Eluate dried under N2, reconstitued in 40uL MeOH • 15uL injected • C18 column w/ 30:45:25 MeOH:ACN:H2O mobile phase • 8 minute run time

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Vitamin D has been “linked” to decreased risk for kidney disease, heart disease, diabetes, high blood pressure, cancer……..the “miracle” vitamin!!

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• 50uL serum or plasma

• 100uL 1N NaOH + d6D3 as IS

• 30 min incubation at RT

• 3mL hexane, vortex, 8min centrifugation

• Top layer dried down under N2

• Batches of 24 samples

Previous Vitamin D method

D2

D3

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Mobile A: 0.1% formic acid

Mobile B: 100% MeOH

C18-DB 15cmx4.6mm x3um

LC Gradient Time (minutes) Percent Methanol

0.01-1.70 50

1.71-3.50 80-95

3.51-4.70 95

4.71-5.00 50

Previous Vitamin D method

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Goal…combine immunosuppressant and Vitamin D analysis into one bench, one tech, one day….

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Vitamin D new method

• 50ul of serum, standard and control

• 200ul acetonitrile (with IS)

• Centrifuged for 5 minutes at 13000 rpm (1.57rcf)

• Fifty µL of the extract was injected onto the TLX-2 for online cleanup (Cyclone-P 0.5x50mm column)

• 1.5ml per min with 0.05% formic acid:methanol=80:20

• Eluted to a Phenomenex Kinetex 50x4.6mm, 2.6um column at flow rate rate of 0.7ml per min 0.05% formic acid: methanol=7:93 connected to the API-5000.

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ISD new method..

• 50ul of whole blood sample, standard or control

• 50ul of 25mM ZnSO4 briefly then mixed vigorously with 200ul methanol:acetonitrile=70:30 (with IS’s)

• Centrifuged for 10 minutes at 13000 rpm (1.57 rcf).

• Thirty µL of the extract was injected onto the Aria TLX-2 (Thermo Fisher Scientific) for online cleanup (Cyclone-P 0.5x50mm column) at flow rate 3ml per min with 15mM ammonium acetate/0.1% formic acid:methanol=80:20

• Eluted onto a Thermo Hypersil Gold 50x3mm column at flow rate of 0.75ml per min with 100% methanol/15mM ammonium acetate to the API 5000

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Challenge with ZnSo4 not extracting all sirolimus and forming precipitates….

Several ZnSO4 solutions tried:

1.2mM in 80% MeOH

1:1:1 ZnSO4:Acetone:ACN

Final ZnSO4 extraction:

25mM ZnSO4

Followed by 70:30 MeOH:ACN

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Quantitation of ISD and D analytes were carried out in 5 min by sharing the same mass spectrometer with atmospheric pressure ionization in the positive-ion mode for multiple reaction monitoring (MRM).

• Total acquisition time for each method is 0.8 minutes

• with dwell times 25-75 msec

• Sir: 931.7/864.4 m/z; Tac: 821.6/768.3 m/z;

• CyA: 1202.9/1184.9 m/z Des: 901.7/834.4 m/z;

• Asc: 809.6/756.3 m/z; CyD: 1216.9/1198.9 m/z

• OHD2: 395.3/209.1 m/z; OHD3: 383.2/365.4 m/z;

• d6D3: 389.3/211.2 m/z • APCI; Temperature: 480 C; Curtain gas: 15 psi;

• Ion source Gas 1: 35 psi, Ion source Gas 2: 15 psi

• Collision gas: 9 psi; Nebulizing current: 4 u

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Similar performance..

Linear Range:

2 – 100 for Sir and Tac; 2 – 2000 ng/mL for CyA

2 - 100 ng/mL for OHD2 and OHD3

Lower limit of quantitation (LOQ):

0.5 ng/mL for Siro and Tac; 10 ng/mL for CyA

1.0 ng/mL for OHD2 and OHD3

Within day imprecision < 5%

Between day impresion <8%

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y = 0.9025x + 12.061 R² = 0.9665

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y = 0.9613x + 0.1444 R² = 0.9087

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Sirolimus

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y = 0.9788x + 0.3931 R² = 0.9621

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Tacrolimus

y = 0.9489x + 0.8783 R² = 0.9717

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25OHD3

y = 1.0591x + 0.7196 R² = 0.9837

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Improved performance that allowed a reduction of 1.5FTE, same day reporting of Vitamin D and a 60 minute buffer to our required ISD reporting time

• 48 samples of each “method” prepped in 30 min.

• Versus 12 /hr / method

• 20 sample each reported every 60 minutes

• Versus 7.5/h for ISD and 20/h for Vit D

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The combination of automated sample processing, Turboflow sample cleanup and “multiplexed” sample presentation to a single mass spectrometer brings LC-MSMS much closer to the needs of a clinical laboratory operation

Acknowledgements: Terry Law, Jim Dunn Sam Polito, Kellie Parent

clinical application of lc-ms - massep.org · (thermo fisher scientific) for online cleanup...

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