using mass spectrometry in diagnosis &...

Using Mass Spectrometry in Diagnosis &

Management

James C. Ritchie, Ph.D, DABCC, FACB

Director

Core & Special Chemistry LaboratoriesCore & Special Chemistry Laboratories

2011 ASCP Annual Meeting1700 – 1800

October 20th, 2011Veronese 2503

Disclosures

James C Ritchie, Ph.D.

• Research / Educational Grants

– BeckmanCoulter, Inc

– Abaxis, Inc

– Abbott Diagnostics

– Roche Diagnostics

– Waters, Inc.

• Federal Grants:

– R01-71531- Collaborative R01, BPD in Pregnancy

– P50-77083 - Predictors of Antidepressant Response (CIDAR)

– P50-77928 - Perinatal Stress: Pathways to Vulnerability (TRCBS)

– MH-69056 - Emory / GSK/ NIMH Collaborative Mood Disorders Initiative

– MH-078105 - Early Experience, Stress and Neurobehavioral Development

Center

Discussion Today

• MS Technology Primer

• Problems unique to LC-MS/MS

• One Lab’s Initial Experience

• New tools for old problems (new assays, etc)• New tools for old problems (new assays, etc)

• What’s Coming

• Summary

Fear Factors in the Clinical Lab

• 1970’s – Immunoassay

• 1980’s – PCR

• 1990’s – PGx • 1990’s – PGx

• 2000’s -Mass Spectrometry

Introduction to Mass Spectrometry

CH3COCH3CH3COCH3 CH3+COCH3CH3+COCH3 CH3C

+OCH3CH3C+OCH3

+CH3+CH3

+COH+COH

Mass Spectroscopy

Sample

Inlet

Sample

Inlet

Ionization

& Adsorption

of Excess Energy

Ionization

& Adsorption

of Excess Energy

Mass AnalysisMass Analysis

+COCH3+COCH3

Fragmentation

(Dissociation)

Fragmentation

(Dissociation)

DetectionDetection

Electron Ionisation Electron Ionisation

Electron

Trap

Ion Repeller Mass AnalyserIon Repeller

Sample

MoleculesElectrons

Ions

Chemical Ionization

• Reagent gas enters before samples (methane)

• An electron beam ionizes the reagent gas creating gas ions

• When sample molecules enter the source they collide with the reagent gas resulting in a collide with the reagent gas resulting in a charge transfer from the gas ions to the sample molecules.

• This is a valuable as it creates a “soft, non-fragmenting” ionization.

+

+

+

+

+ +

Matrix Assisted Laser Dissorption Ionization (MALDI)Matrix Assisted Laser Dissorption Ionization (MALDI)

Laser

e.g. N2 (354nM)

Nd-YAG (266nM), etcDesorbed

sample and

matrix ions

UV-adsorbing matrix

mixed with sample

Surface Enhanced Laser Desorption Ionization (SELDI) = a form of MALDI employing selective surfaces to pre-select compounds of interest

Mass spectrometry

CH3COCH3CH3COCH3 CH3+COCH3CH3+COCH3 CH3C

+OCH3CH3C+OCH3

+CH3+CH3

+COH+COH

Sample

Inlet

Sample

Inlet

Ionization

& Adsorption

of Excess Energy

Ionization

& Adsorption

of Excess Energy

Mass AnalysisMass Analysis

+COCH3+COCH3

Fragmentation

(Dissociation)

Fragmentation

(Dissociation)

DetectionDetection

Mass Analyzers

Two Classes:

• Beam-type

– Ions make one trip through the instrument and then strike the detector where they are destructively strike the detector where they are destructively detected (micro to milliseconds).

• Trapping–type

– Ions are held in a confined space by a combination of magnetic, electrostatic, and /or RF electrical fields. Fields are then manipulated to allow ions to be released (seconds to minutes)

Quadrupole Theory

In a quadrupole instrument, only electric fields are used to separate

ions according to mass, as they pass along the central axis of four

parallel, equidistant rods (poles) which have fixed (DC) and

alternating (RF) voltages applied to them.

Quadrupole TheoryPre-filter Quadrupole Mass Filter Stable Trajectory

Unstable Trajectories

•Only ions with the correct m/z values have stable trajectories within

an RF/DC quadrupole field.

• Ions with unstable trajectories collide with the rods, or the walls of the

vacuum chamber, and are neutralised.

•These stabilities are governed by the Mathieu equations.

Principles of Time-of-Flight

BAtm +=

m

eVv

acc2

=

2

2

1mveV

acc= mass

time

Source Detector

Time-of-Flight Mass Spectrometry

Basic TOF Instrument

Q-TOF Illustration

Ion Trap Mass AnalyzerIon Trap Mass Analyzer

• Ion traps are ion trapping devices that make use of a three-dimensional quadrupole field to trap and mass-analyze ionsmass-analyze ions

• invented by Wolfgang Paul (Nobel Prize1989)

• Offer good mass resolving power

FTFT--ICRICRFourierFourier--transform ion cyclotron resonancetransform ion cyclotron resonance

• Uses powerful magnet (5-10 Tesla) to create a miniature cyclotron

• Originally developed in Canada (UBC) by A.G. Marshal in 1974Marshal in 1974

• FT approach allows many ion masses to be determined simultaneously (efficient)

• Has higher mass resolution than any other MS analyzer available

LTQ Orbitrap Mass Spectrometer

Olsen, JV, et al, Molecular & Cellular Prot, 2009,8; 2759

IonizationIonizationMass

analysis 2Mass

analysis 2FragmentationFragmentation

Massanalysis 1Mass

analysis 1

MSMSMSMS

MS/MS vs GC-MS

InjectionInjectionMass

analysisMass

analysisIonizationIonizationGC

SeparationGC

Separation

INLETINLET SEPARATESEPARATE IDENTIFYIDENTIFY

GCGC MSMS

INLETINLET SEPARATESEPARATE IDENTIFYIDENTIFY

Tandem QuadrupoleInstrument Design

MS1 MS2Collision

Cell

Argon gasArgon gas

Precursor(s)Precursor(s)Product(s)Product(s)

Multiple Reaction MonitoringMRM

Both the first and second quadrupole mass analyzers are

held Static at the mass-to-charge ratios (m/z) of the

precursor ion and the most intense product ion,

respectively.

Both the first and second quadrupole mass analyzers are

held Static at the mass-to-charge ratios (m/z) of the

precursor ion and the most intense product ion,

respectively.

Static (m/z 315.1) Static (m/z 109.0)

Precursor(s)Precursor(s)

Capillary

~3kV

Capillary

~3kV

Ions evaporate

from the

Ions evaporate

from the

Electrospray Ionisation Electrospray Ionisation

~3kV~3kV

As droplet evaporates, the

electric field increases and ions

move towards the surface.

As droplet evaporates, the

electric field increases and ions

move towards the surface.

from the

surface

from the

surface

Electrospray or Atmospheric Pressure Chemical Ionization (APCI)

Issues Unique to LC-MS/MS Analyses

• Matrix Effects

• Differential ionization of Internal Standards or Calibrators

• In Source Transformation (fragmentation)

• Isobaric Compounds and Isomers• Isobaric Compounds and Isomers

• Cross-Talk effects

• Chromatographic Resolution

• Carry-Over

Vogeser & Seger, 2010, Clin Chemhttp://www.clinchem.org/cgi/doi/10.1373/clinchem.2009.138602

Sample Preparation

• Matrix: Serum, whole blood, urine

• Pretreatment process

– Protein Precipitation Protocols: Fast & Easy. However

associated with longer periods of ion suppression due associated with longer periods of ion suppression due

to early eluting, low molecular weight matrix

constituents

– Solid-Phase Extraction & Liquid-Liquid Extraction:

Slower & more chance for error. Do have shorter

periods of ion suppression.

Vogeser & Seger, 2010, Clin Chemhttp://www.clinchem.org/cgi/doi/10.1373/clinchem.2009.138602

In Source Transformation:

• Fragmentation occurring after column separation but before collision cell– MAP-G > MPA > MPA Fragment

– MPA-BE > MPA > MPA Fragment

• Can be a problem with endogenous drug metabolites– Check real patient samples in extended

chromatographic runs

Isobaric Compounds & Isomers

• Extremely important when measuring

endogenous analytes

• Often mandates complete chromatographic

resolution

Cross-Talk•Occurs when several mass transitionswith identical product ions are acquiredwith identical product ions are acquiredover a short time interval.

•If collision cell does not empty completely spurious signals can be recorded in a subsequent trace

•Common when several metabolites of a single drug are detected with identical fragment ions

•Solution: Increase interscan delay

“Analyte co-elution is generally

feasible due to the high

selectivity of SRM/MRM

experiments, if co-elution of

isobaric analyte isomers or ion

source fragmentation of the

analyte metabolites can be

ruled out”ruled out”

Vogeser & Seger, 2008, Clin Biochem, 41:649-662

Carryover

The AOI at 500 ng/mL causes a 331% Carry-Over.

10 ng/mL 100 ng/mL 500 ng/mL

Plasma 1 537 451 517

Plasma 2 539 481 410

Analyte 108000 1010000 4700000

Plasma 3 529 737 1660

Plasma 4 495 460 481

%Carryover = (Aavg – Bavg)/Bavg x 100

A = plasma pool 3, 5 ,7 B = plasma pool 2, 4, 6, 8.

Plasma 4 495 460 481

Analyte 107000 978000 4660000

Plasma 5 464 831 1980

Plasma 6 522 581 528

Analyte 105000 985000 4550000

Plasma 7 445 916 2900

Plasma 8 425 539 575

Analytical Sensitivity

• Limit of Absence (LOA)

– 20 replicates of zero calibrator or specimens without analyte run across multiple days

• LOA = mean + 2SD or + 3 SD

• Limit of Detection

– Measure specimens with levels (naturally, diluted or spiked) that – Measure specimens with levels (naturally, diluted or spiked) that approximate the LOA, but are consistently detectable

• LOD = mean of detectable concentration + 2SD or +3 SD of specimens

• Also, noise x 3

• Limit of Quantification (Functional Sensitivity)

– Minimum concentration where concentration can be measured reliably

• Imprecision CV > 20%

• Noise x 10

• Goals: Must be below clinical needs

ThroughputSelectivity

Requirements of Clinical Laboratory Methodology

Automation

Ease of Use

Sensitivity

Accuracy

Emory Transplant Center

�Region’s largest and only comprehensive organ and tissue transplant program.

In 2010 the Emory Center performed a total of 453 transplants:transplants:

160 Kidney22 Heart80 Liver26 Pancreas24 Lung128 Stem Cell13 Islet Cell

Choosing if Mass Spectrometry is Right for Your Lab

� Define your assay requirements

� Define your analytes and understand any alternative approaches

� Identify a mass spectrometer

� Test compounds

� Visit other labs

Can You Measure Your Analyte of Interest Using

Mass Spectrometry?

• Do you have access to the AOI?

• Have others done this already?

20

30

40

50

# o

f P

ub

lica

tio

ns

• Does it “fly” (ionization mode)?

• Are the calibrators/IS available?

• Is it financially viable?

• What are the clinical needs?

0

10

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

Year

# o

f P

ub

lica

tio

ns

2003: What Were Other Labs Doing for IS Drugs?

Reference Laboratories Methodology

Quest HPLC and TDx (by request)

Mayo Labs HPLC + LC- MS/MS

Lab Corp LC- MS/MS

ARUP LC - MS/MS

Academic Centers Methodology

MUSC LC - MS/MS

UNC LC - MS/MS

Univ. of Mich. LC - MS/MS

Univ. Penn HPLC (LC - MS for C2 protocols)

Univ. Washington (Seattle) Children’s

TDX

Duke LC - MS/MS

2003

Immunosuppressant Testing

Measurand Volume / yr Cost / Analysis Cost / yr

Cyclosporine 7588 $10.80 $81,950

Tacrolimus 13120 $12.00 $157,440

Sirolimus 744 $ 57.45* $42,743

TOTALS 21,452 $282,133

• Rapamycin TAT 1 to 4 days

• Expecting volume to increase substantially in future

• Each drug is a separate analysis, no opportunity for multiplexing

• Total cost to system over 7 years = $1,974,931

TOTALS 21,452 $282,133

*Sendout-Mayo Labs

LC-MS/MS

Proposal

Measurand Volume / yr Cost* / Analysis Cost / yr

Cyclosporine 7588 $6.35 $48,184

Tacrolimus 13120 $6.35 $83,312

Rapamycin 744 $6.35 $4,724Rapamycin 744 $6.35 $4,724

TOTALS 21,452 $136,220

* Cost includes reagents, labor, & instr. depreciation

• All drug TATs within 1 day• Capable of expanding to meet increased need• Runs are multiplexed • Total cost to system over 7 years = $953,540• Saving to system over 7 years = $1,021,391• Provides opportunities to do other assays

Process for Developing a Clinical Method

• Method Selection

• Selection of Key Operator• Outline/plan methods• Acquisition of materials• Set quality goals

Make a Implementation Plan Validate the Method

• Imprecision• Recovery• Linearity• Sensitivity• Specificity• Set quality goals

• Validation• Monitoring and statistics• SOP preparation, staff training

• Specificity• Interferences• Specimen• Method Comparison• Assay Calibration

LC-MS/MS Analyses

• Most current methods are home-brews

– Resources:

• Develop from scratch

• Previously validated method from colleagues

• Literature

• Vendors

• Most variation between labs related to differences in methods &

standards

• CLIA – High Complexity

Guidance Documents

• EU Directive 2002/657/EC

• FDA Guidance 118

• FDA Special Controls Guidance for Particular Drugs

• CAP Chemistry & Toxicology Checklist

• CLSI Guidance EP10-A3E

• SOFT and AAFS Guidelines

• WADA Identification Criteria

• New York State – Clinical Laboratory Standards of Practice

Useful ResourcesCLSI Guidelines

– EP10 Preliminary Evaluation of Quantitative Clinical Laboratory

measurement Procedures

– EP15 User Verification of Performance for Precision and Trueness

– EP05 Evaluation of Precision Performance of Quantitative – EP05 Evaluation of Precision Performance of Quantitative

Measurement Methods

– EP06 Evaluation of the Linearity of Quantitative Measurement

– EP09 Method Comparison and Bias Estimation

– GP31 Laboratory Instrument Implementation, Verification, and

Maintenance

– EP50 Mass Spectrometry in the Clinical Laboratory

EP50 Mass Spectrometry in the Clinical

Laboratory

• General overview of mass spectrometry and clinical applications

• Guidelines on analytical method development and validationand validation

• Seeks to harmonize some of the international documents

• http://www.clsi.org/source/orders/free/C50-A.pdf

Immunosuppressant Drugs by LC/MS/MS –

Protocol (circa 2004)

Microfuge tube protocol.

1. In a 1.5mL Eppendorf tube accurately pipette:50µL Lysis Solution A (0.4M ZnSO4)

200µL whole blood (Calibrators, QCs or patient samples)

2. Briefly vortex mix samples (5 – 10sec)

3. Add 500µL of Precipitating Solution (25ng/mL Ascomycin + 100 3. Add 500µL of Precipitating Solution (25ng/mL Ascomycin + 100 ng/mL CycloD in acetonitrile)

4. Vortex mix for approximately 1 minute or until the entire sample is thoroughly mixed

5. Centrifuge for 5 minutes at 14,500rpm

6. Cut top off of micro-centrifuge vial. Place vial into HPLC autosampler

7. Inject 10µL on the LC-MS/MS system. Use C-18 column (4X3mm –Phenemonex)

Immunosuppressant Drugs by LC/MS/MS -

Protocol

Compound

Precursor Ion

Daughter Ion

Cyclosporine A 1220 1203

Tacrolimus 821.5 768.5Tacrolimus 821.5 768.5

Rapamycin 931.6 864.5

Ascomycin 809.5 756.4

Cyclosporine D 1234.1 1217.2

Cyclosporine A & D

Immunosuppressant Standard (20/20/500 ng/mL)

Ascomycin, FK-506, Rapamycin

Immunosuppressant Standard (20/20/500 ng/mL)

Cyclosporine D

Cyclosporine A

Rapamycin

FK-506

Ascomycin


Limits

Compound

LOD

(ng/mL)

LOQ

(ng/mL)

Linearity

(ng/mL)

Tacrolimus 0.1 0.6 40

Cyclosporine A 3.0 10 1000

Rapamycin 0.3 0.6 40


Imprecision

Interassay

Tacrolimus Cyclosporine A Rapamycin

Concentration 4 90 4Concentration

(ng/mL)

4 90 4

%C.V. 7 10 13

Intra-assay

%C.V. 2 7 9

Matrix effects

1. Prepare extracts on 5 random blood samples from patients not receiving an IS drug.

2. Prepare 2 extracts using water as matrix.

3. To 500 uL of each extract add 150 ng Cyclo A, 75 ng Cylo D, 15 ng FK-506, 15 ng Rapa, and 20 ng Asco (all in 50ul).

4. Compare whole blood recoveries from water.

FK-506(Area)

Cyclo A(Area)

Rapa(Area)

Asco(Area)

Cyclo D(Area)

Water 3651 75885 1267 8578 36918

#1 4241 77293 1580 8650 38890

#2 4834 71217 1317 8787 34821

#3 4486 73005 1359 8218 27974

#4 4836 71500 1360 9023 36014

#5 4394 75963 1402 8225 32160

Blood mean 4559 73796 1404 8581 33972

Recovery 125% 97% 111% 100% 92%

AMR & CRR

AMR(ng/mL)

CRR(ng/mL)

Cyclosporine A 100 - 1000 10 - 2000Cyclosporine A 100 - 1000 10 - 2000

FK-506 0.3 - 40 0.3 - 80

Rapamycin 0.6 - 40 0.6 - 80

LC/MS/MS - Throughput

• Run time = 2 mins/sample

• Cycle time = 3.5 mins.

• One run contains 18 samples + 4 stds + 3 • One run contains 18 samples + 4 stds + 3 controls.

• A run takes 87.5 mins.

• 5 runs can be performed in 7.3 hours.

• This means a maximum of 90 specimens can be analyzed per shift per instrument.

Emory Medical Laboratories

Immunosuppressant Monitoring

• Average monthly workload for 2004:

» Tacrolimus – 1322

» Cyclosporine A – 732

» Sirolimus – 426» Sirolimus – 426

This equals 2480 samples a month or 83 samples per day

• Analysis performed in the Special Chemistry

Section, 7 days per week, dayshift only.

Billboards You’ll Never See:Billboards You’ll Never See:

RAPA – Assay Comparison

(both LC/MS/MS)

Demming Regression:Emory = 1.006 Mayo + 0.05R = 0.9903N = 24Average Bias = 0.10

FK-506 Assay Comparisons

Demming Regression:LC/MS/MS = 1.022 IMx-0.117R = 0.9380N = 39Average Bias = 0.064

400

500

600

HPLC (Mayo)

(ng/mL)

CyclosporineLC/MS/MS vs HPLC

n=19 random patient specimens

LCMS HPLC100 114200 215400 416

HPLC = 1.0056 LCMS + 13.548R2 = 0.9574

0

100

200

300

0 100 200 300 400 500 600

HPLC (Mayo)

(ng/mL)

LC/MS/MS (Emory)(ng/mL)

How does immunoassay compare to

LC-MS/MS?

Pure Compound:Pure Compound:

1. LC/MS/MS = 0.912Abbott -2.071, r = 0.9122. On average the LC/MS/MS value is 9.7% lower than the Immunoassay .3. Immunoassay Therapeutic Range was 50 to 400 ng/mL4. From this comparison, LC/MS/MS Therapeutic Range is 43 to 363 ng/mL

Patient Sample Comparison

Emory Cyclosporine Assay Comparison

(using patient samples)

1500

2000

2500

LC/MS/MS

n=185 patient samples w ith complete data as of 12/16/03

LC/MS/MS = 0.789TDx - 46.752

R2 = 0.9011

0

500

1000

0 500 1000 1500 2000 2500

Immunoassay (Abbott Monoclonal)

LC/MS/MS

n=185 patient samples with complete data as of 12/15/03 Therapeutic Range comparable to TDx assay = -7 to 269ng/mL

On average the LC/MS/MS values are 44% lower than the immunoassay values

• Cyclosporine (CsA) is a cyclic undecapetide of MW 1203.6 Da.

• It is used primarily in solid organ and bone marrow transplantation.

Cyclosporine

transplantation.

• CsA exerts its immunosuppressive effect by binding to cyclophilin, inhibiting calcineurin, and halting T-cell receptor transcription of the IL-2 gene.

• CsA is metabolized by Cytochrome P450 -3A4 and 3A9, in the liver, small intestine, and kidney .

Cyclosporine

• At least 30 metabolites (hydroxylated, demethylated, cyclized, and oxidative by-products) have been identified.

• Metabolites AM1 and AM9 have approx. 14 to 16% of the activity of the parent drug (in the MLC assay). Little is known about the activity of other metabolites.

• The above two metabolites are seen at 150 and 75% concentrations relative to the parent drug at steady state in kidney transplant patients.

Cyclosporine

• Therapeutic drug monitoring is mandated as bioavailability, metabolism, and excretion can differ markedly among individuals.

• Additionally, nephrotoxicity, hepatoxicity, and neurotoxicity can occur with overdosage whereas graft rejection can result from underdosage.

• The current recommendation for CsA monitoring is to analyze trough concentrations of the drug in EDTA whole blood using a method specific for the parent compound. Monitoring of the active metabolites is not currently recommended. (Oellerich et al, Consensus Conference

on CsA Monitoring in Organ Transplantation:report of the consensus panel. Ther. Drug Monit. 1995, 17:642-654.)

Metabolic Pathway of Cyclosporine A

AM1, AM9, AM4n are the primary metabolites found in human blood and urineOther metabolites result from further metabolism of the primary metabolites.

Metabolite Toxicity ?

• Toxic mechanisms of CsA and its metabolites are not

necessarily identical. No good models of organ specific

toxicity exist for CsA.

• CsA metabolites are in general less toxic than parent CsA in

rat models or in the renal epithelial cell line (LLC-PK ).rat models or in the renal epithelial cell line (LLC-PK1).

• Clinical reports have not shown a clear association between

blood concentrations of CsA metabolites and neuro- /

nephrotoxicity.

Update Memo

Moving CsA Testing to LC-MS/MS Assay

• “Methodology will change as of 11/24/03”

• “New Therapeutic Range:

43 to 363ng/mL (Specific for parent drug)”

• “Individual patient levels may vary by as much as 70%”

• “Recommend if see a drop of 30 to 40% and patient is stable, no change. If more than that proceed to increase dose slowly.”

Laboratory Update Memo:

Moving CsA Testing to LC-MS/MS Assay

Advantages:Advantages:– Measures 3 immunosuppressive drugs

(CsA, Tacrolimus, Sirolimus) simultaneously.

– Specific for only parent compound of each drug.– Specific for only parent compound of each drug.

– Corresponds very well to the HPLC method which has been

regarded as the “gold standard” method for CsA

determinations. (Holt, et al, Ther. Drug Monitor., 24:59-67, 2002)

– Increased lab efficiency, decreased costs, decreased reliance

on single vendor of immunoassay.

11/24/03

• Due to a distribution problem memo not

distributed until 11/26/03

Duplicate Patient Testing

• To help transition to the new methodology lab agreed to perform duplicate cyclosporine testing.

Transplant Type

# Patients Specimens

Heart 82 128

Kidney 231 247

Liver 23 41testing.

• 833 patient specimens were analyzed (12/15/03

thru 01/15/04).

Liver 23 41

Lung 23 243

Stem Cell & Bone Marrow

8 52

Pancreas 5 42

C2 Protocols

13 45

Total Data SetCyclosporine Patient Sample Comparisons

12/15/03 thru 01/15/04

1200

1400

1600

1800

2000

LC/MS Immuno

40 100

102 200

227 400

601 1000

LC/MS/MS = 0.6232TDx - 22.659

R2 = 0.9629

0

200

400

600

800

1000

0 500 1000 1500 2000 2500 3000

TDx - Immuno Assay

(ng/mL)

LC/MS/MS

(ng/mL)

Heart Transplant Specimens

Heart Transplant Specimens

n=128

350

400

450

500

LC/MS Immuno

44 100

97 200

203 400

522 1 000

LC/MS/MS = 0.5313TDx - 9.3919

R2 = 0.9157

0

50

100

150

200

250

300

0 100 200 300 400 500 600 700

TDx - Immuno Assay

(ng/mL)

LC/MS/MS

(ng/mL)

Kidney Transplant Specimens

Kidney Transplants

n=247

1000

1200

1400

LC/MS/MS (ng/mL)

LC/MS Immuno

41 100

104 200

231 400

613 1000

LC/MS/MS = 0.6362TDx - 23.124

R2 = 0.9669

0

200

400

600

800

0 200 400 600 800 1000 1200 1400 1600 1800 2000

TDx - Immuno Assay (ng/mL)

LC/MS/MS (ng/mL)

Individual Patient Data

0

200

400

600

800

1000

1200

1400

12/22/03

12/23/03

12/24/03

12/25/03

12/26/03

12/27/03

12/28/03

12/29/03

12/30/03

12/31/03

1/1/04

1/2/04

1/3/04

1/4/04

1/5/04

1/6/04

1/7/04

1/8/04

1/9/04

1/10/04

1/11/04

1/12/04

Cyclosporine (ng/mL)

TDx-Immunoassay

LC/MS/MS

Kidney Transplant Patient: PI (10/16/03)

12/22/03

12/23/03

12/24/03

12/25/03

12/26/03

12/27/03

12/28/03

12/29/03

12/30/03

12/31/03

1/10/04

1/11/04

1/12/04

Date

Individual Patient Data

0

50

100

150

200

250

300

350

12/26/03

12/27/03

12/28/03

12/29/03

12/30/03

12/31/03

1/1/04

1/2/04

1/3/04

1/4/04

1/5/04

1/6/04

1/7/04

1/8/04

1/9/04

1/10/04

1/11/04

1/12/04

Date

Cyclosporine (ng/ml)

TDx-Immunoassay

LC/MS/MS

Kidney Transplant Pat: AL (5/1/92)

New Transplant Specific

Ranges ?

Post Heart Transplant

Immunoassay

CsA

LC/MS/MS

CsA

(Based on sample

comparison)

LC/MS/MS

CsA

(Heart transplant group)

1 to 180 days 300 150 150

6 to 18 months 250 123 125

18 mos to 2 yrs 200 97 100

>2yrs 150 70 75

Conclusions

1. Developing an LC-MS/MS assay for clinical use is just like setting up any other assay in the lab.

2. Defining the process and selecting a good chief operator are key.

3. Today’s medical technologists are capable of understanding 3. Today’s medical technologists are capable of understanding and using LC-MS/MS for routine analyses

4. Standardizing the analytical process and system maintenance are crucial for success.

5. LC-MS/MS can be a cost effective tool in the clinical laboratory

Conclusions

6. Looking at the Emory transplant population LC-MS/MS

cyclosporine concentrations are 40 to 60% lower than the

corresponding immunoassay values (TDx)

7. Individual patients “track” consistently in the LC-MS/MS

assay

8. Analysis of cyclosporine A metabolites does not seem

warranted for the vast majority of patients.

9. It is not enough to just have better technology

… the best resources when switching assays are data and

good communication.

Can LC-MS/MS analysis of IS drugs be

considered a routine test?

1. Authors describe the analysis of tacrolims in their lab over a 4.5

yr period by four laboratory scientists.

2. During this period they ran 4029 batches which included 61,027

patient samples.

3. Accuracy was 97.6 to 98.5% and Imprecision was <8.0%3. Accuracy was 97.6 to 98.5% and Imprecision was <8.0%

4. Only four batch failures occurred out of the 4029 batches (0.1%

failure rate).

5. Of the 4 batch failures, 3 were due to errors attributed to the

scientist and 1 due to instrument malfunction.

These data document the ruggedness of the method and suggest it is

ideally suited as a routine test for tacrolimus in the clinical setting

P.J. Taylor, et al., J. Chromatogr. B (2011), doi:10.1016/j.jchromb.2011.06.024

Survey CSM-A 2011

CAP Proficiency Testing

• 480 labs participated:

Cyclo = 485, Tacro = 479, Rapa = 247

• 68 Labs used LC-MS/MS:• 68 Labs used LC-MS/MS:

Cyclo = 66, Tacro = 64, Rapa = 70

• Immunoassays:

Cyclo = 8, Tacro = 4, Rapa = 2

Results

Cyclosporine Mean

Immunoassay

% CV

LC-MS/MS

%CV

CS-01 199.65 ng/mL 9.2 9.1

CS-02 351.50 8.6 9.7

CS-03 78.93 12.9 9.0

TacrolimusTacrolimus

CS-01 8.49 ng/mL 13.4 8.6

CS-02 16.06 10.1 9.3

CS-03 4.00 22.3 9.6

Sirolimus

CS-01 10.82 ng/mL 7.4 15.9

CS-02 22.64 7.9 13.9

CS-03 4.28 11.6 16.3

K/DOQI – Classification of CKD based on GFR stratification

Renal Impairment

STAGE 1Kidney DamageNormal or

STAGE 2Kidney DamageMild Kidney

STAGE 3ModerateKidney

STAGE 4Severe Kidney

STAGE 5Kidney

Measuring Renal Function Using Iothalamate

Glomerular filtration rate (GFR) is the best estimate

of kidney function and determines your stage of

kidney disease (NKDEP).

Normal or Kidney Function

Mild Kidney Function

Kidney Function

Kidney Function

Kidney Failure

015306090120

Adapted from K/DOQIGFR

• Not bound by protein

• Freely filtered by glomerulus

• Ease of measuring

• Assay interferences

• Secreted by renal tubule

• 24 hr: creatinine creatine

• Possible incomplete and inaccurate 24 hr urine collection

Creatinine

Advantages Disadvantages

Endogenous

Analytes Used For Calculating GFR

inaccurate 24 hr urine collection

• Gold standard for measurement of GFR

• Cumbersome

• Expensive

• Limited availability

• Usually limited to clinical research

• Close correlation with inulinclearance

• Widespread availability

• Non-radiolabeled analyte

• Timed clearance

Inulin

Iothalamate

EndogenousExogenous

Emory Transplant Center GFR Assessment

Pt Void (UO) Pt Void (U1)/Plasma (P2)Pt Void (UE)/Plasma (P1)

Clinical Relevance

0 min

300mg Iothalamate SubQ

110 min

Iothalamate U1 (Urine) (µg/mL) X flow (mL/min)Iothalamate Avg P1 & P2 (Plasma) (µg/mL)

GFR =

65 min5 min

Iothalamate MRM

Iothalamate

Iohexol

Iohexol

Product Ion #2

Product Ion #1

Time (min) Time (min)

IohexolIothalamate

Product Ion #2

Product Ion #1

H+

NH4+

Na+

Ion Suppression

Methanol Supernatant Dried Ext in Mobile Phase A

SampleIothalamate

Area SampleIothalamate

Area

Exp #1 Exp #2

Sample Area Sample Area

Mobile Phase 101905 Mobile Phase 69508

Plasma 1 31941 Plasma 1 67397



Suppression 69% Suppression 8%

Deming Regression Bland-Altman

Accuracy

y = 1.07x + 1.80R2 = 0.96

y = 1.09x + 0.19R2 = 0.73

Linear Regression Bland-Altman

y = 1.06x – 11.25R2 = 0.80

GFR

Total Error = Bias + (1.96 X SD)

Total Error = 5.85 + (1.96 X 6.2)

Total Error = 18%

BiasMDL = (mx + b) – xMDL

BiasMDL = (mx + b) – 90 mL/min

BiasMDL = 5.85

A UPLC LC-MS/MS Assays for the Newer Antidepressants,

Antipsychotics, and their Active MetabolitesMetabolites

Why Monitor?

• Check Adherence

• Differential Metabolism

• Rough Therapeutic Ranges• Rough Therapeutic Ranges

• To Check Clearance of Drug When Switching to

Another or in Special Populations

• Suspect Serotonin Syndrome

• Suspect Overdose

Types of “Newer” Antidepressants

• Selective Serotonin Reuptake Inhibitors (SSRIs)

– Citalopram (Celexa)

– Escitalopram (Lexapro)

– Fluoxetine (Prozac)

– Fluvoxamine (Luvox)

– Paroxetine (Paxil)

– Sertraline (Zoloft)

• Serotonin-norepinephrine reuptake inhibitors (SNRIs)

– Duloxetine (Cymbalta)

– Milnacipram (Ixel)

– Venlafaxine (Effexor)

– des-Venlafexine (Pristiq)

Types of “Newer” Antidepressants

• Noradrenergic & specific serotonergic antidepressants (NaSSAs)

– Mianserin (Tolvon)

– Mirtazapine (Remeron, Avanza, Zispin)

• Norepinephrine reuptake inhibitors (NRIs) • Norepinephrine reuptake inhibitors (NRIs)

– Atomoxetine (Strattera)

– Mazindol (Mazanor, Sanorex)

– Reboxetine (Edronax)

– Viloxazine (Vivalan)

• Norepinephrine-dopamine reuptake inhibitors (NDRIs)

– Bupropion (Wellbutrin, Zyban)

2nd Generation Antipsychotics

Antipsychotic Drugs Trade Name

Olanzapine (+active metab) Zyprexa

Risperidone Risperdal

9-OH Risperidone Paliperidone9-OH Risperidone Paliperidone

Ziprasadone Geodon

Clozapine(+active metab) Clozaril

Haloperidol Haldol

Quetiapine Seroquel

Aripiperazole (+active metab) Abilify

Atomoxetine(+active metab) Strattera

Objectives

• To develop LC-MS/MS methods to measure serum levels of the newer ADs and APs using “off the shelf reagents”

• Methods must also measure active • Methods must also measure active metabolites of these compounds

• Method must be robust and cost effective versus sending to a reference laboratory

Sample Extraction

• 100 µL Sample, QC, or Standard

• (AD) = 10 µL Mobile Phase A

(AP) = 50 uL 0.4M ZnSO4 , sit 2 mins

• 200 µL Internal Standard (in MEOH)

• Vortex 1 min, let stand 5 mins

• Centrifuge at 14,000xG for 5 mins

• Transfer supernatant to sample vial

• Inject 5 µL of extract

Compound Parent Ion Daughter Ion Cone (V) Collision (eV) Dwell RT(min)

des-Mirtazapine 252.13 195.10 43.00 18.00 0.01 1.17

ODV 264.00 106.70 30.00 37.00 0.01 0.95

Mirtazapine 266.00 194.70 39.00 23.00 0.01 1.15

d3-Mirt 269.00 194.70 39.00 23.00 0.01 01.14

Venlafaxine 278.20 120.80 30.00 35.00 0.01 1.74

d6-Ven 284.00 120.80 30.00 35.00 0.01 1.72

des-Sertraline 291.80 158.50 17.00 25.00 0.01 2.75

des-Fluoxetine 295.90 133.90 20.00 5.00 0.01 2.58

MRM - ADs

des-Fluoxetine 295.90 133.90 20.00 5.00 0.01 2.58

Douloxetine 298.00 153.70 18.00 6.00 0.01 2.43

d6-des-Fluox 301.80 139.80 20.00 5.00 0.01 2.57

Sertraline 305.90 158.50 20.00 25.00 0.01 2.69

d3-Sert 308.90 158.50 20.00 25.00 0.01 2.69

Fluoxetine 310.20 148.10 30.00 10.00 0.01 2.56

Reboxetine 314.00 175.60 31.00 13.00 0.01 2.18

Fluvoxamine 319.20 258.20 30.00 10.00 0.01 2.52

Citalopram 325.10 108.90 40.00 25.00 0.01 1.94

Paroxetine 330.00 69.80 30.00 30.00 0.01 2.34

d6-Parox 336.00 75.80 35.00 30.00 0.01 2.34

Tuned to Max Sensitivity for des-Sertraline

Compound Parent Ion Daughter Ion

20

ionCone (V) Collision (eV) Dwell RT

(min)

d8-N-desmethyl-Olanzapine

307.00 260.20 212.70 40.00 35.00 0.01 1.53

N-desmethyl-Olanzapine 299.00 255.80 212.70 38.00 26.00 0.01 1.54

Olanzapine 313.00 255.80 197.70 34.00 25.00 0.01 1.57

4-OH Atomoxetine 272.00 44.00 21.00 14.00 0.01 2.15

9-OH-Ripseridone 427.00 206.90 109.90 45.00 31.00 0.01 2.14

d3,13C2-Risperidone 415.50 192.90 109.90 47.00 31.00 0.01 2.20

Risperidone 411.10 190.90 109.90 47.00 31.00 0.01 2.20

d8-Ziprasidone 421.00 193.80 46.00 32.00 0.01 2.49

Ziprasidone 413.00 193.80 46.00 32.00 0.01 2.50

MRM MRM -- APsAPs

Ziprasidone 413.00 193.80 46.00 32.00 0.01 2.50

nor-Clozapine 313.00 269.90 40.00 25.00 0.01 2.55

d8-Clozapine 335.00 274.70 40.00 25.00 0.01 2.58

Clozapine 327.00 270.00 38.00 25.00 0.01 2.59

d4-Haloperidol 380.00 168.90 126.90 40.00 26.00 0.01 2.64

Haloperidol 375.90 164.90 122.80 40.00 26.00 0.01 2.65

Quetiapine 384.00 252.80 45.00 27.00 0.01 2.64

d7-Atomoxetine 263.10 43.90 19.00 13.00 0.01 2.72

Atomoxetine 256.00 43.90 19.00 13.00 0.01 2.72

dehydro-Aripiperazole 446.00 284.80 36.00 25.00 0.01 2.70

d8-Aripiperazole 456.00 292.80 38.00 38.00 0.01 2.71

Aripiperazole 448.00 284.80 175.90 38.00 30.00 0.01 2.72

Tuned to Max Sensitivity for n--desmethyl-Olanzapine

ADsADs -- 100 100 ngng//mLmL Std (TIC)Std (TIC)

MIRTs Paroxs

Fluv & Fluox

ODV

Vs+C

Rebox

Duolox

Fluv & Fluox

des-Fluoxs

Serts

APsAPs -- 100 100 ngng//mLmL STD (TIC)STD (TIC)

Risp

Olanz’s

OH-Atomox + OH-Risp

Zip

Cloz’s

Hal + Quet

Atomox + Aripip’s

ImprecisionIntra-assay:ADs ADs - 5 to 10% at 75 & 300 ng/mL (n=10)APs APs - 4 to 11% at 5/100 & 15/350ng/ml (n=10)

Interassay:

ADsADs - 7 to 12% at 75 & 300 ng/mL (n=10)APs APs - 3 to 12% at 5/100 & 15/350 ng/mL (n=13)

Linearity

Assays PerformanceAssays Performance

Linearity ADsADs – 0.2 to 2000 ng/mL (LOD=0.05, des-Sert = 0.1)APs APs – 0.2 to 2000 ng/mL (LOD=0.05 ng/mL)

Matrix Effects (Matuszewski, et al 2003)ADs ADs - 11 (reboxetine) to -3% (Venlafexine)APsAPs - <4% for all compounds

Absolute RecoveryADsADs – 75 to 114% from 5 to 550 ng/mLAPsAPs – 84 to 108% from 2.3 to 228 ng/mL

Citalopram Assay Comparison

y = 0.7883x - 0.3088

R2 = 0.7537

50

100

150

LC/MS/MS

(ng/m

L)

Assay ComparisonsAssay ComparisonsEmory HPLCEmory HPLC--UV vs LC/MS/MSUV vs LC/MS/MS

0

0 50 100 150HPLC-UV

(ng/mL)

Paroxetine Comparison

y = 0.9341x - 21.943

R2 = 0.9689

0

100

200

300

400

500

600

700

800

0 200 400 600 800

HPLC-UV

LC/MS/MS

Flouxetine

y = 0.7021x + 13.284

R2 = 0.7917

0

100

200

300

0 50 100 150 200 250 300

UK - All Methods Mean

Emory-LC/MS/MS

des-Fluoxetine

y = 0.6895x + 7.9303

R2 = 0. 6832

0

50

100

150

200

250

300

0 50 100 150 200 250 300

U K - A l l M e t h o d s M e a n

Sertraline

y = 1. 18 9 8 x - 4 . 0 8 8 2

R2 = 0 . 9 9 3 7

9 0

12 0

15 0

des-Sertraline

y = 1.0415x + 0.3831

R2 = 0. 9312

200

250

300

Assay Comparisons Assay Comparisons -- UKEQASUKEQAS

0

3 0

6 0

0 3 0 6 0 9 0 12 0 15 0

U K - A ll M e t hods M e a n

0

50

100

150

0 50 100 150 200 250 300


Venlafexine

y = 1.1114x + 50.656

R2 = 0.8056

0

100

200

300

400

500

0 100 200 300 400 500


ODV

y = 0.9163x + 28. 79

R2 = 0.7573

0

100

200

300

400

500

0 100 200 300 400 500

U K - A l l M e t h od s M e a n

Clinical Use

• 2010 VolumesParoxetine – 15

Fluoxetine – 20

Citalopam – 18

Considerations:

1. AD and AP assays are essentially

the same for the technologist

2. Reagents, Standards , and

controls are very stable at -80CCitalopam – 18

Venlafaxine - 12

Sertraline – 14

Olanzapine – 15

Risperidone - 18

Clozapine – 52

Quetiapine – 11

Aripiperazole - 5

• Total = 180/yr

controls are very stable at -80C

3. No stat turnaround requirements

4. Can be used to fill down time on

LC-MS

Small runs can be cost effective !

Lab Test of the Decade

800

1000

1200

Vitamin D deficiency

0

200

400

600

20

00

20

01

20

02

20

03

20

04

20

05

20

06

20

07

20

08

20

09

20

10

PubMed Search

Lab Test of the DecadeEmory Medical Laboratories

Financial Justification

• EML:

– Vitamin D biggest send-out test

– A new LC–MS/MS instrument was purchased for

vitamin D testing:

• Waters Xevo TQ MS• Waters Xevo TQ MS

• 28716 tests @ a cost of $13.05

• Estimated cost by mass spec:

– Reagents/calibrators/ standards/ labor : $4.50

• Estimated savings: $8.55/test

– ~ $200K/yr (after factoring in maintenance contract)

Acquisition of Materials

• Endogenous compounds:

– Stripped sera vs standards prepared in BSA

• Golden West Biologics

– Vitamin D 25-Hydroxy, D2 & D3 Total < 1 ng/ml Trigylcerides &

Cholesterol < 10 mg/dlCholesterol < 10 mg/dl

– Vitamin D 25-Hydroxy, D2 & D3 Total < 1 ng/ml, Triglycerides

& Cholesterol normal range

• 4% BSA

• Calibrators & QC materials were

purchased from Chromsystems

• Internal Standard: D6 25-OH D3 (Cerillient)

Sample Extraction

• To 300 µL of serum or plasma, add: – 20 µL of internal std (D6 25-OH D3, 250 ng/mL in

EtOH),

– 150 µL 0.2 mol/L ZnSO4, and vortex.

– Add 300 µL MeOH, – Add 300 µL MeOH,

– 750 µL hexane, vortex and centrifuge.

• Transfer 400 µL of the hexane layer to a glass tube and dry under a stream of N2.

• Reconstitute with 75 µL mobile phase A and transfer to LC vial.

• Inject 20 µL.

Chromatographic Separation

D3: 3.18 min

401.3 > 159.2

D6: 3.18 min

407.2 > 159.2

D2: 3.26 min

413.4 > 355.3

What’s Next ??

Proteomics

The systematic analysis and documentation of

proteins in biological samples with a focus on

state-related expression of proteins.”state-related expression of proteins.”

Proteomics is the study of multiprotein systems, in which the focus is on the interplay of multiple, distinct proteins in their roles as part of a larger system or network (systems biology).

Challenges

Lancet, 2002, 359:572-577

Technical ChallengesAnalytical:

– Must span up to 10 orders of magnitude in concentration

– Be able to detect multiple forms of the same analyte (PTMs, isoforms, etc)

– Lack of a generally applicable technique for – Lack of a generally applicable technique for protein quantitation

– Limited throughput of todays proteomic platforms

– Problems with high MW, basic, or hydrophobic proteins

Technical Challenges

Bioinformatics:

• Analyses produce a huge amount of data

• Must have comprehensive databases to get accurate sequence information and accurate sequence information and identification.

• No agreed upon algorithms for standardization of analyses

Challenges of Biomarker Discovery in

Plasma

• Currently there is no single comprehensive proteomics platform for plasma.

• Multi-dimensional fractionation is crucial to penetrate deeper into the abundance distributionpenetrate deeper into the abundance distribution

• Where possible discovery efforts should center on tissues or non-plasma fluids where they occur at higher concentrations

• “Ultimately however we need to measure biomarkers in plasma to be most effective”

L. Anderson, Plasma Protein Institute

The way forward

• Proteomics currently is a biomarker discovery tool which promises to revolutionize our thinking about health and disease.

• This technology may someday provide disease specific panels to aid diagnosis and treatment.panels to aid diagnosis and treatment.

• Many barriers must be overcome before these assays reach the clinical laboratory.

• However, just as with small molecules, these new techniques can be employed incrementally to solve some of our knotty clinical problems.

Thyroglobulin (Tg)

• A 660 Kd dimeric thyroid protein which forms the backbone for thyroid hormone production

• Serum Tg is used in conjunction with imaging to manage patient treatment after removal of thyroid carcinoma.thyroid carcinoma.

• Approximately 10 to 25% of patients have anti-Tg antibodies which potentially can interfere with the immunoassays commonly used to measure Tg

• Commercial Tg immunoassays lack concordance across platforms most likely due to posttranslational modifications in vivo.

1.1.1.1. Method uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by anti----1.1.1.1. Method uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by anti----peptide antibodies (SISCAPA)peptide antibodies (SISCAPA)peptide antibodies (SISCAPA)peptide antibodies (SISCAPA)

2.2.2.2. Three digest peptides are used to assure specificityThree digest peptides are used to assure specificityThree digest peptides are used to assure specificityThree digest peptides are used to assure specificity3.3.3.3. Analytical sensitivity of 2.6 Analytical sensitivity of 2.6 Analytical sensitivity of 2.6 Analytical sensitivity of 2.6 ngngngng/ml approaches that of /ml approaches that of /ml approaches that of /ml approaches that of

immunoassay (1.0ng/immunoassay (1.0ng/immunoassay (1.0ng/immunoassay (1.0ng/mLmLmLmL))))4.4.4.4. No interference from antiNo interference from antiNo interference from antiNo interference from anti----TgTgTgTg antibodiesantibodiesantibodiesantibodies

Comparison to ImmunoassayComparison to Immunoassay

Other Targets of Opportunity• Β-hCG (Beta human chorionic gonadotropin)

• Sex Hormone Binding Globulin

• Lipoproteins (LPa. Apo-B, B-100, Apo A1, etc)

• C-reactive protein (CRP)

• D-dimers

• α -acid glycoprotein • α1-acid glycoprotein

• ACTH

• Glycated albumin

• Βrain-Natriuretic peptide (BNP)

• Ferritin

• α-Fetoprotein

• And many many more……

1. Enables an unbiased identification of microorganisms.

2. Can be used for gram-positive and gram-negative bacteria, yeast and multicellular fungi without presumptions or pretesting.

3. Applications from clinical microbiology, food, feed safety and analysis

MALDI Biotyper: The next generation microbial identification system for the 21st century

Microbiology ?

Microbial identification system based on bench top microflexMALDI-TOF mass spectrometer

•Unbiased identification of bacteria, yeasts and multicellularfungi•Cost-effective sample preparation•High resolution identification down to species level in one step•Analysis of mixed cultures•Interfaces for LIMS integration

J.Banoub (ed.) Detection of Biological Agents for the Prevention of Bioterrorism,2010, Springer Sci

J.Banoub (ed.) Detection of Biological Agents for the Prevention of Bioterrorism, 2010, Springer Sci

Status of LC-MS/MS Assays at EML

Today:

Clinically in Use

CyclosporineRapamycinTacrolimusMycophenolic acidBusulfanAntidepressants (14)

In Development

25 (OH) Vitamin DAntidepressants (14)Antipsychotics (12)

Research

ArgatrobanLenalidomideLevamisoleBile AcidsIodothalamateFlumazenil

25 (OH) Vitamin DEverolimusGlucocorticoidsTestosteroneMetanephrines

� High specificity

� Multiplexing possible

� High sensitivity (femtomolar LODs) and accuracy

� Wide linear range

Throughput

AutomationAccuracy

Selectivity

Advantages of MS in the Clinical Advantages of MS in the Clinical LaboratoryLaboratory

�

� Limited sample prep with short chromatographic run-times

� Low sample volumes –pediatrics

� Alternative Matrices – blood spots, saliva, hair

Automation

Ease of UseSensitivity

Accuracy


� Automation (including sample prep)

� Ease of Use - (autotunes & autocals), smart systems

� LIS connectivity

Throughput

AutomationAccuracy

Selectivity

Requirements for MS to gain wide acceptance Requirements for MS to gain wide acceptance in Clinical Laboratoriesin Clinical Laboratories

� Low carryover

� 24 hr service

� Manufacturer supported procedures and reference ranges

� Cost Effectiveness

Automation

Ease of UseSensitivity

Accuracy


Today I’ve shown you:• A new instrument to

understand….

• A few new concepts……

• A few new things to watch

out for……….

• A glimpse of the future……

Ultimately to provide more accurate analyses And better patient care

Remember all instruments are simply tools

AcknowledgementsAcknowledgements

ECTRL Laboratory Staff Clinical Chemistry FellowsPatricia Scott-Harrell, B.S. Marion Snyder, Ph.D.Bailey Glover, M.D. Ross Molinaro, Ph.D.Clay Ramsey, B.A. Charbel AbuDiwan, Ph.DLisa Maxwell, RN

EML Special Chemistry StaffWilla Zhang, MSCora Tomblin, MT (ASCP)Tom Tuten, MS

http://www.pathology.emory.edu/ECTRL/

Tom Tuten, MS

QUESTIONS ???

Methodology

• EML had analyzed CsA previously using the Abbott monoclonal immunoassay on the TDx/Flx in our Core Laboratory (“in by 10am, results by 3pm”).

• Therapeutic Range: 50 to 400 ng/mL.

• A semi-automated FPIA assay.

• Cross-reactivity with metabolites: AM1 = 9.3%, AM9 = 23.5%.(Steimer

W, 1999, Clin Chem, 45:371-381)

• The cross-reactivity of the CsA metabolites in the Abbott monoclonal assay matches closely with their pharmacological potency in the MLC assay. (Murthy et al, 1998, Clin Biochem, 31:159-163)

Hamwi, et al, Cyclosporine Metabolism in Patients after

Kidney, BoneMarrow, Heart-Lung, and Liver Transplants in Early and late

Posttransplant Periods, Am J Clin Pathol, 114:536-543, 2000

Transplant Type

White bars = Early period (≤ 3 months)

Black bars = Late period (>3 months)

Transplant TypeA=KidneyB=Bone MarrowC=Heart-LungD=Liver

Hamwi, et al, Cyclosporine Metabolism in Patients after

Kidney, BoneMarrow, Heart-Lung, and Liver Transplants in Early and late

Posttransplant Periods, Am J Clin Pathol, 114:536-543, 2000

Transplant TypeA=KidneyB=Bone MarrowC=Heart-LungD=Liver

White bars = Early period (≤ 3 months)

Black bars = Late period (>3 months)