SUPPLEMENTARY MATERIALS, PART I Questionnairedownload.lww.com/wolterskluwer_vitalstream_com/... · # Minimal Experience Level Select One 1 0 – 3 months 2 3 – 6 months 3 6 –

1

The Impact of Laboratory Practices on Inter-laboratory Variability in Therapeutic Drug Monitoring of Immunosuppressive Drugs. Christians U, MD, PhD1, Vinks S, PharmD, PhD2, Langman L, PhD3, Clarke W, PhD4, Wallemacq P, PhD6, Van Gelder T, MD, PhD7, Renjen V, MD8, Marquet P, MD, PhD5, Meyer EJ, BS9 1iC42 Clinical Research and Development, University of Colorado, Aurora, CO, USA; 2Cincinnati Children’s Hospital and Medical Center; 3Mayo Clinic, Rochester MN, USA; 4John Hopkins School of Medicine, Baltimore, Maryland, USA; 5Cliniques Universitaires St. Luc UCL, Brussels, Belgium; 6Erasmus Medical Center, Rotterdam, The Netherlands; 7Navigant Consulting Inc., New York, NY, USA; 8University Hospital of Limoges, Limoges, France; 9Novartis Pharmaceutical Corp., East Hannover, NJ, USA

SUPPLEMENTARY MATERIALS, PART I Questionnaire

2

PROJECT: PRECISION

Assessment of Inter-lab Variability of TDM Analytical Assays for Immunosuppressant Drugs

QUESTION FLOW OF THE SURVEY:

A. SCREENER – RESPONDENT & LAB PROFILE:

B. PROCESS OF CONDUCTING TDM ANALYTICAL ASSAYS:

C. TDM ANALYTICAL ASSAY DEVELOPMENT:

D. TDM ANALYTICAL ASSAY VALIDATION:

E. TDM ANALYTICAL ASSAY IMPLEMENTATION:

F. TDM ANALYTICAL ASSAY QC:

G. PROFICIENCY TEST:

H. HYPOTHESIS FOR INTER-LAB VARIABILITY OF TDM ANALYTICAL ASSAYS:

TABLE COLOR KEY:

Questions relevant to all TDM analytical assay methods

Questions related to immunoassays only

Questions related to mass spectrometry only

Questions related to HPLC-UV only

Respondent Profile Lab Profile

Sam. Acq. & Samp. Prep. & Ext. Conduct of Assays QA / QC

Assay Development

Assay Validation

Assay Implement.

Control / QC

Proficiency Test

Hypotheses

3

Sam. Acq. & Receipt Samp. Prep. & Ext. Conduct of Assays QA / QC

A. SCREENER – RESPONDENT & LAB PROFILE:

1. Please indicate the name of your laboratory:

(This question is for sample control purpose only. Your answer will not be shared with

an outside party.)

____________________

2. Please provide your current job title:

# Title Select all that apply

1 Lab Director / Medical Director

2 Lab Manager / Supervisor

3 Lab technician

4 Other, please specify: __________

3. Please indicate your educational background:

# Education Select all that apply

1 Ph.D.

2 M.D.

3 Masters

4 Bachelors


4. How many years in total have you been performing or supervising TDM analytical

assays for immunosuppressant drugs?

# Years Select One

1 < 1 year

2 1 – 2 years

3 2 – 5 years

4 > 5 years

Lab Profile

Respondent Profile

Respondent Profile

Respondent Profile

4


5. How many years have you been in this lab?

# Years Select One

1 < 1 year

2 1 – 2 years

3 2 – 5 years

4 > 5 years

6. Approximately, how many employees work at your laboratory site / facility?

# Number of Employees Select One

1 < 20

2 20 – 100

3 100 – 500

4 500 – 2,000

5 > 2,000

7. How many people perform TDM analytical assays in your lab?

# # of People Performing TDM

Assays

Select One

1 1 – 3

2 4 – 6

3 7 – 10

4 11 – 20

5 More than 20

8. In your lab, which type of TDM analytical assays is the primary one performed for the

following immunosuppressant drugs?

Select One

# TDM Analytical Assay Method cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Immunoassay

Respondent Profile

Lab Profile

Lab Profile

Lab Profile

5


2 Mass spectrometry

3 HPLC-UV

4 Others, please specify:

__________

5 Currently not performing TDM

analytical assays for the

indicated immunosuppressant

9. What are all of the TDM analytical assay methods, instrumentation systems, and/or kits

that are available in your lab ,or are in the process of being implemented, for the testing

of the following immunosuppressant drugs (please select all)?

Select all that apply

# Immunoassay Methods cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 ACMIA

2 ADVIA

3 CEDIA

4 CMIA

5 EMIT

6 FPIA

7 MEIA

8 QMS

9 Manual ELISA


__________


# Immunoassay Instrumentation

Systems

cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Abbott Architect c Series

2 Abbott Architect i Series

3 Abbott AxSYM

4 Abbott IMx

5 Abbott TDx

6 Beckman Access

7 Beckman UniCel DxC Access

Series

Lab Profile

6


8 Beckman UniCel Dxl Access

Series

9 Olympus AU Series

10 Ortho VITROS Series

11 Roche COBAS Series

12 Roche Elecsys

13 Roche Hitachi 917

14 Roche Modular

15 Siemens ADVIA Centaur

16 Siemens ADVIA 1250



19 Siemens Dimension EXL

20 Siemens Dimension RxL

21 Siemens Dimension Vista

22 Siemens Dimension Xpand

23 Siemens IMMULITE

24 Thermo Scientific Indiko

25 Thermo CDx 90

26 Thermo Scientific MGC 240

27 Manual ELISA

28 Other, please specify:

__________

Please select all that apply & specify model numbers

# Mass Spectrometer

Manufacturer


1 Agilent

2 AB Sciex

3 Bruker

4 Thermo Scientific

5 Waters


__________


# Mass Spectrometer Kit cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Waters MassTrak

2 Chromsystems MassTox

3 Recipe ClinMass

7


4 Developed in house


__________

10. What is the primary TDM assay instrumentation system and/or kit that your lab uses for

the following immunosuppressant drugs?


# Immunoassay Methods cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 ACMIA

2 ADVIA

3 CEDIA

4 CMIA

5 EMIT

6 FPIA

7 MEIA

8 QMS

9 Manual ELISA


__________


# Immunoassay Instrumentation

Systems


1 Abbott Architect c Series

2 Abbott Architect i Series

3 Abbott AxSYM

4 Abbott IMx

5 Abbott TDx

6 Beckman Access

7 Beckman UniCel DxC Access

Series

8 Beckman UniCel Dxl Access

Series

9 Olympus AU Series

10 Ortho VITROS Series

11 Roche COBAS Series

Lab Offering

8


12 Roche Elecsys

13 Roche Hitachi 917

14 Roche Modular

15 Siemens ADVIA Centaur




19 Siemens Dimension EXL

20 Siemens Dimension RxL

21 Siemens Dimension Vista

22 Siemens Dimension Xpand

23 Siemens IMMULITE

24 Thermo Scientific Indiko

25 Thermo CDx 90

26 Thermo Scientific MGC 240

27 Manual ELISA

28 Other, please specify:

__________

Please select all that apply & specify model numbers

# Mass Spectrometer

Manufacturer


1 Agilent

2 AB Sciex

3 Bruker

4 Thermo Scientific

5 Waters


__________


# Mass Spectrometer Kit cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Waters MassTrak

2 Chromsystems MassTox

3 Recipe ClinMass

4 Developed in house


__________

9


11. Does your lab test the following immunosuppressant drugs using TDM analytical assays

for clinical or research purpose?

Select One

# Clinical or Research? cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 For clinical purpose

2 For research purpose

3 Both

12. Your laboratory is affiliated with:

# Affiliation Select all that apply

1 A university

2 A research center

3 A hospital

4 A transplant center

5 A commercial lab

13. In which country does your lab reside?

____________________ (SCROLL DOWN / DROP DOWN MENU)

COUNTRY LIST:

U.S.

Germany

France

Belgium

Italy

Spain

UK

Netherlands

Denmark

Norway

Sweden

Brazil

Mexico

Lab Profile

Lab Profile

Lab Profile

10


Australia

South Africa

India

Russia

South Korea

Taiwan

Argentina

14. Please provide the zip code or postal code of your laboratory’s location:

____________________

15. From where does your lab typically receive samples for TDM analytical assays of the

following immunosuppressant drugs?

Select the most representative area for each compound

# Geographic Span cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Local (within hospital or

community)

2 Regional

3 National

4 International

5 Not sure

16. Approximately how many patient samples does your laboratory run each month using

the primary TDM analytical method for each of the following immunosuppressant

drugs?

Select One

# Monthly Volume of TDM Assays cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 < 500

2 500 - 1,000

3 1,000 – 2,000

Lab Profile

Lab Profile

Lab Profile

11


4 2,000 – 4000

5 > 4,000

17. Does your lab have a formal training procedure for people who perform TDM analytical

assays for immunosuppressant drugs?

# Training Procedure Select One

1 No

2 Yes

3 Not sure

18. What is the minimal experience level of the people performing TDM analytical assays in

your lab?

# Minimal Experience Level Select One

1 0 – 3 months

2 3 – 6 months

3 6 – 12 months

4 More than 1 year

19. What is the maximal experience level of the people performing TDM analytical assays in

your lab?

# Maximal Experience Level Select One

1 < 6 months

2 6 – 12 months

3 1 – 3 years

4 > 3 years

20. Please indicate what percentage of your professional time is spent in the lab or managing

the lab (vs. teaching, conducting research, etc.)

Lab Profile

Lab Profile

Lab Profile

Respondent Profile

12


# % Time Spent Select One

1 < 25%

2 25 – 50%

3 50 – 75%

4 75 – 100%

21. On a scale of 1 to 7, how experienced are you with the following activities?

(1 means “no experience” and 7 means “very experienced”)

Experience 1 2 3 4 5 6 7

Knowledge of available platforms to conduct

TDM analytical assays in your lab

Laboratory requirements for accepting,

receiving and preparing samples

Laboratory requirements for conducting TDM

analytical assays

Evaluation and implementation of

commercial TDM analytical assays

Processes of designing and developing TDM

analytical assays

Processes of validating TDM analytical assays

Processes of controlling TDM analytical

assays, assay QC, maintenance, and training

Definition of detection and/or quantitation

ranges for TDM analytical assays

Laboratory standards for reporting TDM

analytic assay results

Technical supervision / lab management

Training & professional development of staff

members

Research to improve laboratory processes and

results

Fiscal operations of laboratory / laboratory

division

22. On a scale of 1 to 7, how experienced are you with immunoassays, mass spectrometry,

and HPLC-UV for TDM analytical assays?

(1 means “no experience” and 7 means “very experienced”)

Respondent Profile

13


Experience with IA, Mass Spec., and HPLC-

UV

1 2 3 4 5 6 7

Immunoassays

Mass spectrometry

HPLC-UV

B. PROCESS OF CONDUCTING TDM ANALYTICAL ASSAYS:

1. Does your lab specify a standard procedure for sample handling prior to arrival in the

laboratory?

# Standard Procedure Select One

1 No

2 Yes

3 Not sure

2. Which anticoagulant does your lab use or accept during sample acquisition?


# Anticoagulant Cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 EDTA

2 Heparin

3 Citrate

4 None, use serum instead


__________

3. If your lab receives samples from outside entities for TDM analytical assays, what are the

acceptable shipping conditions?

Drop-down menu: “Acceptable,” “Not acceptable”

Respondent Profile

Sam. Acq. &

Sam. Acq. &

Sam. Acq. &

14


# Shipping Condition Cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Samples shipped at ambient

temperature, unlimited time

2 Samples can remain at ambient

temperature for up to 24 hours

3 Samples shipped refrigerated or

frozen

4 Ship at ambient temperature,

refrigerate or freeze upon receipt

5 No special conditions specified

4. What percentage of all samples received is rejected for TDM analytical assays for the

following reasons?

Select One

# Rejection Reason 0% 1-2% 3-5% > 5%

1 Requirements of minimal

sample volumes not met

2 Incorrect anticoagulant used

during sample acquisition

3 Samples clotted

4 Requirements of storage /

shipping temperatures not met

5 Incorrect matrix

6 Samples mislabeled

7 Incomplete documentation /

missing paperwork

8 Sample age specification not met

9 Others (please specify):

__________

Sam. Acq. &

15


5. What is the average elapsed time from sample receipt to sample preparation?

Select One

# Elapsed Time from Receipt to

Preparation


1 Within 4 hours

2 Within 24 hours

3 Within 1 week

4 Within 2 weeks

5 More than 2 weeks

6. How are patient samples for TDM analytical assays stored upon receipt prior to

preparation?

Select One

# Storage Condition cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Ambient temperature

2 Refrigerated (0 – 4°C)

3 Frozen at -20°C

4 Frozen at -80°C


__________

7. Does your lab have a standard sample preparation procedure?

# Standard Procedure Select One

1 No

2 Yes, specified by manufacturers

3 Yes, developed by the lab

4 Not sure

Sam. Acq. &

Sam. Acq. &

Samp. Prep. & Ext.

16


8. In your lab, what is the typical batch size for patient samples for TDM analytical assays?

Select One

# Batch Size cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 1 – 10 samples

2 10 – 25

3 26 – 50

4 50 – 100

5 > 100

9. If applicable, what percentage of samples is resuspended using the following methods

prior to sample pretreatment or extraction?

# Resuspension Method Please assign percentage

(%)

1 Manual tube inverting

2 Vortexing

4 Mechanical tube rocker

5 Ultrasound

6 No resuspension performed


__________

10. Is there a standard dilution procedure in your lab?

Select One

# Standard Procedure cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 No standard dilution procedure

2 Yes, standard procedure for all

samples

3 Standard procedure exists for

samples that are outside the

linear range of assays

Samp. Prep. & Ext.

Samp. Prep. & Ext.

Samp. Prep. & Ext.

17


11. How does your lab perform sample pretreatment / extraction?

Select One

# Sample Pretreatment / Extraction cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Manual

2 Automated

3 Using online column extraction

(for mass spectrometry)

12. What sample precipitation / extraction solutions does your lab use?

Select One

# Pretreatment / Extraction cyclosporine tacrolimus sirolimus everolimus mycophenolate

Immunoassay

1 Methanol

2 Acetonitrile

3 Diethyl ether

4 Manufacturer proprietary


__________

Mass Spec. & HPLC-UV

6 ZnSO4 + methanol precipitation

7 ZnSO4 + acetonitrile

precipitation

8 Solid phase extraction

9 Liquid phase extraction


__________

Samp. Prep. & Ext.

Samp. Prep. & Ext.

18


13. Does your lab concentrate samples during sample precipitation / extraction? If so , what

method does your lab use?

Select One

# Concentration Method cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Never concentrate samples

2 Speed vacuum

3 Evaporation under nitrogen

4 Temperature elevation

5 Concentration on column


__________

14. What is the elapsed time from sample preparation to the start of the run?

Select One

# Elapsed Time from Preparation

to Run Start


1 < 1 hour

2 1 – 4 hours

3 4 – 8 hours

4 8 – 12 hours

5 12 – 24 hours

6 > 24 hours

Samp. Prep. & Ext.

Samp. Prep. & Ext.

19


15. What is the average run time for every batch?

Select One

# Average Run Time for Every

Batch


1 Less than 1 hour

2 1 – 2 hours

3 2 – 4 hours

4 4 – 8 hours

5 8 – 24 hours

6 More than 24 hours

16. If samples are not tested immediately (< 1 hour until batch start) after pretreatment, are

samples stored in a temperature controlled manner?

Select One

# Samples Temperature

Controlled?


1 No

2 Yes, refrigerated (0 – 4°C)

3 Yes, frozen at -20°C

4 Yes, frozen at -80°C

17. How long does your lab retain the original blood sample?

Select One

# Retaining of Blood Sample cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 1 – 2 days

2 1 week

3 2 weeks

4 1 month

5 > 1 month

Samp. Prep. & Ext.

Samp. Prep. & Ext.

Samp. Prep. & Ext.

20


18. At what temperature does your lab retain the original blood sample?

Select One

# Retaining Temperature cyclosporine tacrolimus sirolimus everolimus mycophenolate



3 Frozen at -20°C

4 Frozen at -80°C


__________

19. Are the following equipment and materials used during TDM analytical assays regulated

by any standard procedure in your lab?

# Regulated by Standard Procedures? Yes No

1 Centrifuges (e.g. specifying temperature

and speed)

2 Types of pipettes

3 Types of pipette tips

4 Sample containers

5 Stock solutions

6 Chemical grade

20. Does your lab consistently use the same centrifuge each time an assay is performed for

an immunosuppressant drug?

# Same Centrifuge Each Time? Select One

1 No

2 Yes

Samp. Prep. & Ext.

Samp. Prep. & Ext.

Samp. Prep. & Ext.

21


21. If multiple centrifuges are used, on a scale of 1 to 7, how well are centrifuges controlled

for the following parameters during sample preparation?

(1 indicates “not at all controlled” and 7 indicates “extremely well controlled”)

# Parameter Not

Sure

1 2 3 4 5 6 7

1 Temperature

2 Speed (e.g. rpm)

3 Rotor diameter

4 g force

5 Others, please specify: __________

22. Are centrifuges serviced and verified on a regular basis?

On a regular basis?

# Centrifuge Yes No

1 Service

2 Verification

23. What types of pipettes does your lab use during the following steps of sample

preparation?


# Type of Pipettes Transferring

blood sample

Adding protein

precipitation

solutions

Adding

internal

standards

Transfer

extracts to

instruments

1 Manual single channel fixed

volumes pipettes

2 Manual single channel variable

volume pipettes (also known as

mechanical, adjustable or digital)

3 Manual multichannel variable

volume pipettes

4 Manual single channel positive

displacement pipettes

Samp. Prep. & Ext.

Samp. Prep. & Ext.

Samp. Prep. & Ext.

22


5 Electronic single channel

variable volume pipettes

6 Electronic multichannel variable

volume pipettes

7 Robotic pipettes


__________

24. How often does your lab calibrate pipettes?

# Frequency Select One

1 Every 3 months or more frequent

2 Every 6 months

3 Every 12 months

4 Once in > 1 year

5 Never

25. Does your lab use barrier pipette tips for TDM analytical assays?

# Use of Barrier Pipette Tips Select One

1 No

2 Yes, for some samples

3 Yes, for most samples

4 Yes, for all samples

26. Does your lab assign expiration dates to stock solutions?

# Expiration Date Select One

1 No

2 Yes

3 Not sure

Samp. Prep. & Ext.

Samp. Prep. & Ext.

Samp. Prep. & Ext.

23


27. What grade of chemicals does your lab use for TDM analytical assays?

Select One

# Chemical Grade For Solvents for

Sample

Preparation

For Reference

Materials

(Mass Spec. &

HPLC-UV) For

HPLC

1 Reagent grade

2 HPLC grade

3 Spectroscopy grade


__________

28. What quality of water does your lab use for TDM analytical assays?

Select One

# Water Quality For Solvents for

Sample

Preparation

For Reference

Materials

(Mass Spec. &

HPLC-UV) For

HPLC

1 Filtered only

2 Filtered and de-ionized

in house

3 Milli-Q (filtered and de-

ionized)

4 HPLC-grade

5 Double-distilled


__________

Samp. Prep. & Ext.

Samp. Prep. & Ext.

24


29. How are patient samples introduced to mass spectrometers?

Mass Spec.

# Introduction of Samples Select One

1 Flow injection

2 Standard HPLC

3 Ultra-performance HPLC (U-

HPLC)


__________

30. What type of columns in chromatography is used in your lab for TDM analytical assays?


# Column Please select one or specify

1 Size (e.g. length &

diameter)

Please specify length: __________

Please specify diameter: __________

2 Material C8 C18 Others, please

specify:

__________

3 Extended pH Yes No

4 Composition Silica gel Synthetic

materials

Others, please

specify:

__________

5 End-capped Yes No

6 Particle size Please specify: __________

Conduct of Assays

Conduct of Assays

25


31. What is the composition of the mobile phase in chromatography used in your lab for

TDM analytical assays?

Mass Spec. & HPLC-UV Select all that apply

# Composition of the Mobile

Phase


1 Methanol

2 Acetonitrile

3 Water, pH not adjusted

4 Water, pH adjusted to 3 – 5



7 Water, pH adjusted to > 8


__________

32. At what (column) temperature does your lab run the mobile phase in chromatography?

Mass Spec. & HPLC-UV Select One

# Column Temperature cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Below room temperature

2 Room temperature, uncontrolled

3 Room temperature, controlled

4 Between room temperature and

40°C

5 40 – 65°C


__________

Conduct of Assays

Conduct of Assays

26


33. What elution method in chromatography does your lab use for TDM analytical assays?

Mass Spec. & HPLC-UV Select One

# Elution Method cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Isocratic

2 Gradient

3 No chromatography used

34. What ion source does your lab use for mass spectrometry (for the primary / prevalent

instrument / system in your lab)?

Mass Spec. Select One

# Ion Source cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Electrospray ionization

2 Atmospheric pressure chemical

ionization (APCI)


__________

35. What type of mass spectrometer does your lab use (for the primary / prevalent

instrument / system in your lab)?

Mass Spec. Select One

# Mass Analyzer cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Single stage quadrupole

2 Triple stage quadrupole

3 Ion trap


__________

Conduct of Assays

Conduct of Assays

Conduct of Assays

27


36. (MASS SPEC & HPLC-UV) What internal reference standards are used for each

immunosuppressant drug?

Mass Spectrometry Select One

# Internal Standard cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Ascomycin

2 Cyclosporin D (CsD)

3 Isotope labeled cyclosporin A

(CsA)

4 Isotope labeled tacrolimus

5 Isotope labeled sirolimus

6 Isotope labeled everolimus

7 Isotope labeled mycophenolate


__________

37. What ion or ion transitions are monitored to quantify each immunosuppressant drug?

Mass Spectrometry Please specify

# Ion or Ion Transitions cyclosporine tacrolimus sirolimus everolimus mycophenolate

Examples: 1224.9/1112.6 826.6/616.2 936.8/409.2 980.8/389.4 343.3/229.1

38. How many ion transitions (quantifier and qualifier ions) are monitored for each

immunosuppressant drug?

Mass Spectrometry Please specify

# # of Ion Transitions Monitored cyclosporine tacrolimus sirolimus everolimus mycophenolate

Conduct of Assays

Conduct of Assays

Conduct of Assays

28


39. When weighing reference materials (e.g. internal standards, calibrators, and controls),

does your lab compensate for drug impurities or water content (e.g. sirolimus is almost

never > 98% pure)?

Select One

# Impurity Compensation cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 No

2 Yes

40. How does your lab store stock solutions containing internal standards?


# Storage Temperature cyclosporine tacrolimus sirolimus everolimus mycophenolate



3 Frozen at -20°C

4 Frozen at -80°C

41. What is the shelf life of the stock solutions containing internal standards under the above

storage condition in your lab?

Mass Spec.

# Storage Time Select One

1 < 6 hours

2 < 12 hours

3 < 24 hours

4 < 1 week

5 Batched and stored in

refrigerators


__________

Conduct of Assays

Conduct of Assays

Conduct of Assays

29


42. Does your lab use the same stock solutions when preparing controls and calibrators?

# Same Stock Solutions Select One

1 No

2 Yes

43. When testing multiple immunosuppressant drugs, does your lab test them separately or

run multi-analyte assays?


# Testing of Multiple Analytes cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Test individually

2 Multi-analyte assay

44. Does your lab run immunoassays for patient samples in batch or random access mode?

Immunoassay

# Batch or Random Access Select all that apply

1 Batch, please specify how many

times per day: __________

2 Upon receipt (random access

mode)

Conduct of Assays

Conduct of Assays

Conduct of Assays

30


45. Does your lab purchase commercial calibrators (e.g. from assay kits) or prepare them in

house? If prepared in house, are those calibrators made from whole blood?


# Source of Calibrators cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Commercial calibrators

2 Prepared in house and from

whole blood

3 Prepared in house, but not from

whole blood


__________

46. Does your lab purchase commercial controls (e.g. from assay kits) or prepare them in

house? If prepared in house, are those controls made from whole blood?


# Source of Controls cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Commercial controls

2 Prepared in house and from

whole blood

3 Prepared in house, but not from

whole blood


__________

47. How are calibrators and controls prepared in your lab?

# Preparation of Calibrators &

Controls

Select One

1 From ready-to-use calibrator and

control sets

2 Dilution from stock solutions

QA / QC

QA / QC

QA / QC

31


48. How is dilution performed for calibrators?

# Dilution Select One

1 Serial dilution

2 Direct or individual dilution

49. Are new lots of quality control samples, calibrators, and internal standards tested against

previous lots (qualified)?

# Qualification of Controls,

Calibrators, & Internal Standards

Select One

1 No qualification

2 Yes, we qualify

3 Not sure

50. How many different calibrators does your lab run for a typical calibration curve?

Select One

# Calibrators cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 1

2 2 – 3

3 4 – 6

4 7 – 8

5 > 8

QA / QC

QA / QC

QA / QC

32


51. How often does your lab run calibrators?

Select One

# Calibrator Frequency cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Within each batch

2 2 – 3 times a day

3 Once per day

4 Once per week


__________

52. How many different quality control samples does your lab run?

# QC Samples Select One

1 0

2 1

3 2

4 3

5 > 3

53. How often does your lab run quality control samples?

Select One

# QC sample Frequency cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Within each batch

2 2 – 3 times a day

3 Once per day

4 Once per week


__________

QA / QC

QA / QC

QA / QC

33


54. How does your lab run controls and calibrators?

Select One

# Manner cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 In singlicate

2 In duplicate

3 In triplicate

55. What type of quality control samples is used for each immunosuppressant drug?

Select One

# Type of QC Samples cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Artificial plasma, serum or

whole blood

2 Commercial plasma or whole

blood

3 Prepared in house


__________

56. Are calibrators and controls stored on or off board on the instrument in your lab?

Immunoassay Select One

# Storage of Calibrators &

Controls


1 On board

2 Off board

QA / QC

QA / QC

QA / QC

34


57. Are reagents stored on or off board on the instrument in your lab?

Immunoassay Select One

# Storage of Reagents cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 On board

2 Off board

58. What are the lower limits of detection (ng / mL) for the assays used in your lab?

Please specify

# LLOD cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 ng / mL

59. What are the quantitation ranges (ng / mL) for the five immunosuppressant drugs?

Please specify

# Quantitation Range cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Upper limit of quantitation (ng /

mL)

2 Lower limit of quantitation (ng /

mL)

QA / QC

QA / QC

QA / QC

35

Assay Development

C. TDM ANALYTICAL ASSAY DEVELOPMENT:

1. Who in your lab is responsible for determining the detection and quantitation ranges of

the assays?

# Assay Designer Select all that apply

1 Lab Director / Medical Director

2 Lab Manager / Supervisor

3 Lab technician

4 Research scientist

5 Clinical physician


2. How is the lower limit of detection determined?

# Determination of LLOD Select all that apply

1 Signal to noise: 3:1

2 Others, please specify: __________

3. During initial assay validation, how are the lower and upper limits of the quantitation

range determined?

# Limit of Quantitation Please specify

1 Lower limit of quantitation (LLOQ)

2 Upper limit of quantitation

Assay Development

Assay Development

Assay Development

36

Assay Development

4. How are the analytical ranges determined for the five immunosuppressant drugs?

Select One

# Determination of Analytical

Ranges


1 LLOQ and upper limit of

quantitation

2 Clinical considerations


__________

5. Based on the ranges, where does your lab place calibrators?

# Placement of Calibrators Select all that apply

1 At the extremes of the detection

range

2 At the extremes of the

quantitation range

3 Between the extremes of the

detection & quantitation ranges

6. What are the acceptance criteria for calibration curve?

# Acceptance Criteria for

Calibration Equation


1 Number of outliers

2 Correlation co-efficient ≥ 0.999

3 Intercept

4 Deviation from nominal value

has to be within ±15% for at least

2/3 of the calibrators

7. How does your lab establish therapeutic target ranges for immunosuppressant drugs?

Assay Development

Assay Development

Assay Development

37

Assay Development

Select One

# Therapeutic Target Ranges cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Regulatory agency (e.g. FDA)

approved reference ranges

2 Published reference ranges

3 Established by the lab


__________

Assay Development

38

Assay Validation

D. TDM ANALYTICAL ASSAY VALIDATION:

1. Which regulatory guidance does your lab primarily follow for TDM analytical assay

validation?

# Guidance for Assay Validation Select One

1 College of American Pathologists

(CAP)

2 Regulatory agency (e.g. FDA)

guidance

3 Clinical Laboratory & Standards

Institute

4 Applicable ISO Standard


__________

2. What parameters or analyses does your lab validate for commercial and lab developed

assays?


# Parameters or Analyses Commercial

Assays

Lab

Developed

Assays

1 Specificity

2 Lower limit of detection (LLOD)

3 Upper limit of detection

4 Lower limit of quantitation (LLOQ)

5 Upper limit of quantitation

6 Lower limit of blank (LLOB)

7 Range of linear response

8 Within-run imprecision

9 Within-day imprecision

10 Day-to-day imprecision

11 Accuracy

12 Linearity

13 Matrix interferences

14 Matrix effects (e.g. ion suppression)

15 Extraction recovery

Assay Validation

Assay Validation

39

Assay Validation

16 Carry over

17 Dilution integrity

18 Partial volume verification

19 Stock solution stability

20 Extracted sample (auto-sampler) stability

21 Freeze thaw cycle stability

22 Long-term stability

23 Cross-validation with other laboratories

3. How is instrument performance (operating qualification) ensured?

# Instrument Performance Select all that apply

1 Not performed

2 OQ repeated after major repair

3 Scheduled requalification

4 Not re-qualified

4. When determining imprecision, how many replicates per run does your lab run? How

many runs per day? How many days in total?

Please specify

# For Imprecision Within-run

Imprecision

Within-day

Imprecision

Day-to-day

Imprecision

1 # of replicates per run

2 # of runs per day

3 # of days for imprecision runs in

total

Assay Validation

Assay Validation

40

Assay Validation

5. What level of within-run imprecision (% CV) is considered to be acceptable for quality

control samples with concentrations near the middle of the standard curve?

Select One

# Level of Within-run Imprecision

(% CV)


1 < 5%

2 5-10%

3 10-15%

4 15-20%

5 > 20%

6. What level of within-day imprecision (% CV) is considered to be acceptable for quality


Select One

# Level of Within-day Imprecision

(% CV)


1 < 5%

2 5-10%

3 10-15%

4 15-20%

5 > 20%

Assay Validation

Assay Validation

41

Assay Validation

7. What level of day-to-day imprecision (% CV) is considered to be acceptable for quality


Select One

# Level of Day-to-day Imprecision

(% CV)


1 < 5%

2 5-10%

3 10-15%

4 15-20%

5 > 20%

8. How many patient samples does your lab use during method comparison (i.e. cross-

validation)?

# # of Patient Samples Select One

1 None

2 1-25

3 25-50

4 50-100

5 > 100

9. How are method comparisons conducted in your lab?

# Cross-validation Select all that apply

1 No method comparison

conducted

2 Cross-validate within the lab

3 Cross-validate across centers

4 Cross-validate against other

methods (e.g. immunoassays

cross-validated against mass

spectrometry, or vice versa)

Assay Validation

Assay Validation

Assay Validation

42

Assay Validation

10. Under what situations would your lab conduct partial validation for TDM analytical

assays?

# Partial Validation Situation Select all that apply

1 Addition of new types of

transplant samples (e.g.

measuring kidney transplant

samples using assays validated

for liver transplant samples)

2 Method transfer to new

instruments

3 Instrument repair

4 Method modification (e.g.

reagent changes, parameter

optimization)

5 Proficiency test failure

6 In response to corrective action


__________

11. Under what situations would your lab conduct re-validation for TDM analytical assays?

# Re-validation Situation Select all that apply

1 Method modification

2 Acquisition of new instruments

3 Proficiency test failure

4 In response to other corrective

measures


__________

Assay Validation

Assay Validation

43

Assay Validation

12. How often does your lab conduct full re-validation for TDM analytical assays?

# Frequency of Re-validation Select One

1 Once in a year

2 Once in 2 years

3 Once in 5 years

4 Once in 10 years

5 Never conducted re-validation

13. When was the last time your lab conducted re-validation for TDM analytical assays?

# Last Time Select One

1 3 months ago

2 6 months ago

3 1 year ago

4 More than 1 year ago

5 More than 1 year ago, when the

assay was first implemented

6 Never validated

Assay Validation

Assay Validation

44

Assay Implement.

E. TDM ANALYTICAL ASSAY IMPLEMENTATION:

1. What level of training does lab personnel receive before conducting TDM analytical

assays?

# Level of Training Select all that apply

1 Direct observation of patient test

performance/employee duties

2 Direct observation of

performance of instrument

maintenance and function checks

3 Observation for compliance with

safety protocols

4 Assessment/evaluation of

problem solving skills

5 Assessment of test performance

6 Monitoring, recording and

reporting of test results

7 Review of intermediate test

results and work product records

for compliance with standard

operating procedures and

applicable work load limits

2. Who provides training for TDM analytical assays?

# Trainer Select all that apply

1 Assay developer

2 Assay conductor

3 Assay supervisor

4 Lab director

5 Training technologists /

education specialists


__________

Assay Implement.

Assay Implement.

45

Assay Implement.

3. How many years of TDM analytical assay experience does the trainer have?

# Years of TDM Assay Experience Select One

1 < 2 years

2 3 – 5 years

3 6 – 10 years

4 > 10 years

4. How often does your lab provide refresher training or re-training?

# Frequency of Re-training Select One

1 Once in a month

2 Once in a year

3 As necessary

4 In response to corrective actions

only

5 No policy in place

Assay Implement.

Assay Implement.

46

Proficiency Test

F. TDM ANALYTICAL ASSAY CONTROL / QC:

1. By whom are the target values (means) and acceptable ranges of QC samples determined

(e.g. by manufacturer vs. by laboratory)?

# Determined By Select One

1 Manufacturer

2 Lab

2. When quality control samples are outside the acceptable ranges, what actions are taken

prior to re-running of assays?

# Actions Taken Select all that apply

1 Perform maintenance

2 Test system suitability

3 Recalibrate

4 Pass internal QCs

5 Pass external QCs

6 Pass stored proficiency testing

samples

7 Successful cross-validation with a

reference laboratory

8 Partial validation of the assays as

deemed fit-for-purpose

3. What samples does your lab use to monitor imprecision?


# Samples Used cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Internal QC samples

2 External QC samples

3 Pooled patient samples

4 Proficiency test samples


__________

Control / QC

Control / QC

Control / QC

47

Proficiency Test

4. What samples does your lab use to monitor long-term quality of assay performance?

# Samples Used Select all that apply

1 Internal QC samples

2 External QC samples

3 Pooled patient samples

4 Proficiency test samples


__________

5. What criteria are used to confirm and monitor long-term stability and validity of assays

in your lab?

# QC Criteria Select all that apply

1 Linearity

2 Precision

3 Accuracy

4 Sensitivity

5 Specificity

6. How does your lab test system suitability?

(System suitability: check for readiness of mass spectrometry system)

Mass Spec.

# Procedure of Testing System

Suitability


1 Neat blank (e.g. methanol)

2 Neat standard at LLOQ

3 Neat standard at upper limit of

quantitation

4 Additional neat standard

between LLOQ and upper limit

of quantitation

5 Carry over control

Control / QC

Control / QC

Control / QC

48

Proficiency Test

7. How often does your lab test system suitability?

Mass Spec.

# Frequency of System Suitability

Testing


1 Every batch

2 Daily

3 Weekly

4 Monthly

5 Twice in a year

6 Annually

7 After each maintenance

8 After repeated calibrator / control

failures

9 Not done at all

8. What is the preventive maintenance schedule of instruments in your lab?

# Preventive Maintenance Schedule Select One

Water filtration system

1 Weekly

2 Monthly

3 Twice in a year

4 Annually

5 No scheduled preventive

maintenance

Immunoassay


Immunoassay analyzers

1 Weekly

2 Monthly

3 Twice in a year

4 Annually


maintenance

Control / QC

Control / QC

49

Proficiency Test



HPLC valves and filters

1 Weekly

2 Monthly

3 Not at all

Mass Spec.


Mass spectrometer

1 Twice in a year

2 Annually

3 As required

4 As recommended by

manufacturer


maintenance

9. Who carries out preventive maintenance in your lab?

# Preventive Maintenance Select One

1 By laboratory personnel

2 Through service contract with

manufacturer or equally qualified

outside provider

10. Does your lab use multiple instruments (e.g. due to high-volume demand or as a

backup)? If yes, are these instruments cross-validated?

# Multiple Instruments Select One

1 Never use

2 Yes, but NOT cross-validated

3 Yes, and cross-validated

Control / QC

Control / QC

50

Proficiency Test

11. How often are internal QA audits (including laboratory, in-process, assay performance

and documentation) conducted?

# QC Audits Select One

1 Every month

2 Every 3 months

3 Every 6 months

4 Every year

5 Never

12. Is there a long-term tracking procedure for assay performance (e.g. based on QC

samples) in place in your lab?

# Long-Term Tracking Procedure Select One

1 No

2 Yes

13. Is there any other policy for quality control / quality improvement that your lab adheres

to?

# Policy for QC Select One

1 No other policy

2 Additional policy exists (please

specify: __________)

Control / QC

Control / QC

Control / QC

51

Proficiency Test

G. PROFICIENCY TEST:

1. What TDM analytical assay proficiency test program or cross-validation scheme does

your lab participate in for the following immunosuppressant drugs?


# Proficiency Tests cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 College of American

Pathologists (CAP) proficiency

programs

2 Analytical Services International

(ASI) proficiency programs

3 Other proficiency programs,

please specify: __________

4 External cross-validation

5 No proficiency program or

external cross-validation

2. How often does your lab conduct periodic cross-validation / proficiency sample runs?

Select One

# Frequency of Proficiency Runs cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 Every month

2 Every 3 months

3 Every 6 months

4 Once in a year

5 Once in a couple of years

6 Never participated in cross-

validation or proficiency tests

Proficiency Test

Proficiency Test

52

Proficiency Test

3. Does your lab run proficiency test samples on all of your instruments?

# Proficiency Tests Select One

1 No

2 Yes

4. What corrective measures does your lab take when proficiency tests are out of range?

# Actions Taken Select all that apply

1 Perform maintenance

2 Test system suitability

3 Recalibrate

4 Pass internal QCs

5 Pass external QCs

6 Pass stored proficiency testing

samples

7 Successful cross-validation with a

reference laboratory

8 Partial validation of the assays as

deemed fit-for-purpose

5. Is there a re-test policy for aberrant samples (e.g. samples that fall out of the reference

ranges, or the ones identified by clinicians)?

# Re-test Select One

1 No

2 Yes

3 Not sure

Proficiency Test

Proficiency Test

Proficiency Test

53

Hypotheses

H. HYPOTHESIS FOR INTER-LAB VARIABILITY OF TDM ANALYTICAL ASSAYS:

1. How much do you agree with the following statements?

(1 means “not at all agree” and 7 means “very much agree”)

Statement 1 2 3 4 5 6 7

Current inter-lab (i.e. among labs) variability

of TDM analytical assays is a huge issue.

Current intra-lab (i.e. within a lab) variability

of TDM analytical assays is a huge issue.

A commercially available assay is more

reliable than a lab developed test.

I feel our quantitation ranges are adequate at

addressing clinical needs.

I feel able to address clinicians’ concerns

around interpretation of TDM analytical assay

results and associated dosing / titration of

immunosuppressant drugs.

2. Please rank immunoassay, mass spectrometry, and HPLC-UV as a TDM analytical assay

method for immunosuppressant drugs across the following parameters.

Parameter Immunoassay Mass Spectrometry HPLC-UV

Sensitivity

Drop-down menu for each column: “Best,” “Good,” “Not as

good as the other two.”

Specificity

Linearity

Accuracy

Precision

Technical difficulty Drop-down menu for each column: “Most difficult,”

“Difficult,” “Not as difficult as the other two.”

Cost effectiveness Drop-down menu for each column: “Best,” “Good,” “Not as

good as the other two.” Clinical decisions

Result turnaround time

Impact on lab work flow Drop-down menu for each column: “Strong impact,” “Some

impact,” “Not as impactful as the other two.” Impact on lab infrastructure

Hypotheses

Hypotheses

54

Hypotheses

3. Do you think there is a significant bias problem across laboratories?

Select One

# Significant Bias cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 No

2 Yes

3 Not sure

4. Does your lab plan to switch to another TDM analytical assay method for the following

immunosuppressant drugs?

Select One

# Switching Decision cyclosporine tacrolimus sirolimus everolimus mycophenolate

1 No switch planned

2 Switch to immunoassay

3 Switch to mass spectrometry

4 Switch to HPLC-UV

5 Not sure

5. To what extent do you believe the following factors contribute to inter-lab variability of

TDM analytical assays for immunosuppressant drugs?

(1 means “not at all” and 7 means “very much”)

# Contributor 1 2 3 4 5 6 7

1 Sample collection

2 Sample shipping & storage

3 Suboptimal sample preparation

(e.g. extraction)

4 Suboptimal assay design

5 Technician error

6 Inappropriate controls,

calibrators, internal reference

standards

7 Reagent stability

Hypotheses

Hypotheses

Hypotheses

55

Hypotheses

8 Inappropriate internal standard

9 Instrument maintenance

10 Multiple instruments

11 Use of lab developed tests vs.

commercially available ones

12 Insufficient assay performance

tracking

13 Insufficient personnel

qualification or training


__________

Documents

SUPPLEMENTARY MATERIALS, PART I Questionnairedownload.lww.com/wolterskluwer_vitalstream_com/... · # Minimal Experience Level Select One 1 0 – 3 months 2 3 – 6 months 3 6 –