HA Convention 2007 Service Priorities & Programmes 8

(1)

Dr. Michael H. M. Chan

MBChB FRCPA FHKCPath FHKAM(Pathology) Specialist in Chemical Pathology

Department of Chemical Pathology Prince of Wales Hospital

New Territories East Cluster

HA Convention 2007

Service Priorities & Programmes 8

An Expenditure

-

saving Cluster

-

wide Cyclosporin

A Service with Improved Analytical Performance

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Cyclosporin A (CysA) Service Provision in PWH

• Therapeutic drug monitoring for transplanted patients

• Methodology & Instrumentation – Monoclonal antibody

– Fluorescence polarization immunoassay (FPIA) – Abbott TDx analyser

– Measurement range: 25 – 1500 ug/L

– Trough level (C₀) and 2-hour post-dose (C₂) monitoring • Service provision to NTEC (PWH, AHNH, NDH)

• Service provision to outside NTEC (CMC, KWH, UCH) • ~5,000 requests received annually

(3)

Laboratory Procedure for Cyclosporin Analysis

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Tracer

High [CysA]

Trace bound to antibody inhibiting

analyte binding

Low rotation of tracer

Principle of Fluorescence Polarization Immunoassay

Analyte bound to antibody inhibiting

tracer binding

High rotation of tracer molecule emits fluorescence under polarized light Tracer CysA CysA

(5)

Problems with Traditional Immunoassay Method

• Antibody cross-reacts with inactive metabolites

ÆFalsely high results

• Small number of reagent suppliers available Æ High consumable cost (HK$110 per sample)

CysA

CysA inactive metabolite

(6)

New Gold Standard Method

• High performance liquid chromatography tandem mass spectrometry (LC-MS/MS)

– Highly sensitive with better detection limit

– Highly specific for detection of the parent drug only – Better analytical performance over TDx

– Wider assay range: 5 – 4000 ug/L – Much lower consumable cost

(7)

• Separation of molecules by their differential polarities between the stationary phase and the mobile phase

• Stationary phase: silica particles (2-5 um)

• Mobile phase: aqueous buffers, organic solvents, etc

Principle of Liquid Chromatography

Column

(stationary phase)

Analytes mixed with haemolysed blood in mobile phase

MS/MS Detector Analytes are separated

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MS1 Collision

Cell MS2 Detector

Principle of Tandem Mass Spectrometry Detector

• Selective, accurate and precise method

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800 1000 1200 1400m/z 0 100 % 1202.8 1007908 800 1000 1200 1400m/z 0 100 % 1220.1 39105120 1225.1 5145552 Selected parent ion, [M+NH₄] Selected daughter ion Protonated parent ion, [M.H]+

Mass Spectra for Cyclosporin A

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Expenditure Saving Initiative

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Cashflow Analysis

1,260,000 (440,000) (1,000,000) 2,700,000 Saving 2,100,000 500,000 1,000,000 600,000 ---Grand Total 200,000 6th_{– 100,000} 6th_{– 100,000} 200,000 5th_{– 100,000} 5th_{– 100,000} 200,000 4th_{– 100,000} 4th_{– 100,000} 200,000 3rd _{– 100,000} 3rd _{– 100,000} 200,000 2nd_{– 100,000} 2nd_{– 100,000} 1,100,000 1st_{– Warranty} 1,000,000 1st_{– 100,000} 5,000 HK$20 LC-MS/MS 3,360,000 60,000 ---3,300,000 ---Grand Total 560,000 6th _{– 10,000} 6th_{– 550,000} 560,000 5th _{– 10,000} 5th_{– 550,000} 560,000 4th _{– 10,000} 4th_{– 550,000} 560,000 3rd_{– 10,000} 3rd _{– 550,000} 560,000 2nd _{– 10,000} 2nd_{– 550,000} 560,000 1st_{– 10,000} 0 1st_{– 550,000} 5,000 HK$110 Abbott TDx Overall Maintenance Instrument Reagent / Yr CysA / Yr Cost / CysA

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10 ul whole blood + 50 ul water + 50 ul 0.2M ZnSO₄ 100 ul precipitating reagent

(Internal standard CsA-d4)

Centrifugation

Procedure for LC

(13)

• Clinicians can have results of both methods

–

TDx measurement

–

LC-MS/MS measurement

–

Results authorized only when both results were available

–

Result reporting delay: 2-3 days

• N = 352

• Separate analyses for C

₀

and C

₂

–

Different in drug / metabolites ratio

Parallel Run for 6 Months

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0 100 200 300 400 400 350 300 250 200 150 100 50 0 TDX LCMS LCMS = 0.81 * TDX - 7 r=0.9795

Passing

-

Bablok Regression & Bland

-

Altman Plot (C

₀₀

)

0 100 200 300 400 500 20 0 -20 -40 -60 -80 -100 -120 AVERAGE of LCMS and TDX LCMS - TDX Mean -37.4 -1.96 SD -76.8 +1.96 SD 1.9

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0 500 1000 1500 2000 2500 2500 2000 1500 1000 500 0 TDX LCMS LCMS = 0.99 * TDX - 97 r=0.9867 p<0.0001

Passing

-

Bablok Regression & Bland

-

Altman Plot (C

₂₂

)

0 500 1000 1500 2000 2500 3000 150 50 -50 -150 -250 -350 AVERAGE of LCMS and TDX LCMS - TDX Mean -108.1 -1.96 SD -228.4 +1.96 SD 12.1

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Quality Control Precision Performance

140-163 3.2 152.8 178 LC-MS/MS 129-183 6.4 155.6 224 TDx Range (ug/L) CV (%) Mean (ug/L) N Low Level QC 390-450 2.6 424.9 179 LC-MS/MS 389-496 3.7 432.6 215 TDx Range (ug/L) CV (%) Mean (ug/L) N Mid Level QC 749-867 2.8 820 177 LC-MS/MS 788-942 3.6 858.4 213 TDx Range (ug/L) CV (%) Mean (ug/L) N High Level QC

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Pharmacokinetic Study

• 49 Chinese kidney-transplanted subjects with stable renal

function were recruited

• Trough cyclosporin A levels (C₀) were collected under nurse

supervision

• Neoral® were taken under nurse supervision

• One-hour (C₁), 2-hour (C₂), 4-hour (C₄), and 6-hour (C₆ )post-dose cyclosporin A levels were collected under nurse

supervision Time (hours) 0 1 2 3 4 5 6 W hol e bl ood cy cl os por in lev e l ( µg/L ) 0 400 800 1200 1600 area-under-curve (AUC) = 145.2 ? 45.6 mg.hr/L

Area-under-curve (AUC) for each subjects using both TDx and LC-MS/MS methods were calculated

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Prediction of AUC with C

₀₀

and C

₂₂

by TDx and LC

-

MS/MS

0.642 162 ± 65 (59-413) 0.721 238 ± 77 (124-488) 0.488 265 ± 109 (108-569) 0.535 382 ± 130 (184-726) C₄ 0.904 550 ± 257 (105-1353) 0.933 797 ± 266 (226-1614) C₂ 0.804 747 ± 366 (60-1708) 0.773 1023 ± 460 (98-1806) C₁ 0.604 98 ± 40 (18-218) 0.607 133 ± 46 (52-258) C₀ r LC-MS/MS [CysA] in ug/L Mean ± SD (Range) r TDx [CysA] in ug/L Mean ± SD (Range)

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Reference Range Study

• 6, 13 and 20 November 2005

• 176 patients were recruited to perform C₀ and C₂ under nurse supervision

• Whole blood CysA concentrations were measured by TDx and LC-MS/MS methods

• Stable renal function: MDRD >30 ml/min/1.73m2

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• 158 patients satisfied the stable renal function criteria – 41 patients on Diltiazem

– 117 patients not on Diltiazem

TDx

• 117 patients without Diltiazem – C₀: 50 – 229 ug/L – C₂: 409 – 1103 ug/L • 41 patients on Diltiazem

– C : 55 – 259 ug/L

LC-MS/MS

• 117 patients without Diltiazem – C₀ : 35 - 179 ug/L – C₂ : 336 - 966 ug/L • 41 patients on Diltiazem – C : 55 – 204 ug/L

Results

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Summary

• LC-MS/MS assay generally yields lower numerical results due to its high specificity in measuring only the parent drug instead of additional cross-reactivity metabolites

• Accordingly, new reference intervals were developed for trough (C₀) and 2-hour post-dose (C₂) monitoring in which C₂ correlates with AUC much better than C₀

• The analytical performance has been improved as indicated by the reduction of coefficient of variation, better limit of detection, and extended measurement range

• The reagent cost per test has been greatly reduced from HK$110- to

HK$20-• With the significant reduction in reagent cost per test, the HK$1.2M expenditure saving initiative can be targeted after the 6th year of service

• This pioneering initiative should be applicable to other clusters as well as for monitoring newer and even more expensive immunosuppressants such as Tacrolimus, Sirolimus, and Everolimus

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Acknowledgement

Department of Chemical Pathology PWH

• Dr C S HO

• Prof Christopher W K LAM

Department of Pathology NDH • Mr Y K LUK • Mr Daniel C W LEUNG • Dr Michael W M SUEN NTEC Nephrologists • Dr C B LEUNG • Dr K M CHOW • Dr Y L CHENG • Dr Alex W Y YU • Prof Philip K T LI NTEC Administration • Prof Philip K T LI • Dr K K LAI