To determine the range of reference clone IC50 values measured by different
labs for a core set of antimalarials. Intended participants. All laboratories ...
WWARN In Vitro Pilot QA/QC Project Determining the global range of IC50 values for reference clones The project at a glance: Aim
To determine the range of reference clone IC50 values measured by different labs for a core set of antimalarials
Intended participants
All laboratories conducting in vitro assessments on field isolates
Reference clones
Plasmodium falciparum strains 3D7 and W2
Molecular validation
WWARN’s Molecular Module - microsatellites, Pfmdr1 copy number
Drugs
Chloroquine, mefloquine, desethylamodiaquine, dihydroartemisinin
Drug source
WWARN’s QA/QC module – reference material programme
Experimental set-up
Lab’s standard, existing methodology
Drug wells
≥ 7 different drug concentrations
Drug-free wells
≥ 2 per plate (ideally more)
Number of repeats
3 or more
Data submission
To WWARN
Data analysis
Intra- and inter-laboratory, using WWARN’s In Vitro Analysis Tool
WWARN contacts
Sabina Dahlström
[email protected] Charles Woodrow
[email protected]
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Notes A. Reference clones The study will examine the in vitro sensitivity of Plasmodium falciparum strains 3D7 and W2. Laboratories may submit data relating to either of these reference clones, or both, according to what is currently used within each laboratory. WWARN can provide assistance to laboratories wishing to join the study but not currently in possession of a stock of either reference clone. B. Molecular validation of reference clones To ensure that all laboratories use valid cultured lines, each will prepare a blood spot of their reference culture at both the start and completion of the culture period. Blood spots sent to the WWARN In Vitro Module (Paris) will be analysed by the WWARN Molecular Module (Baltimore). The spot taken at the start of the culture period will be tested retrospectively to confirm that the culture remained unchanged during the project. Molecular testing is based on multiple microsatellites to confirm strain as well as Pfmdr1 copy number. If for any reason (e.g. contamination) a separate frozen vial or live culture reference clone is used, new validation samples should be taken immediately and at the end of the culture. C. Drugs to be studied Four drugs will be tested: chloroquine - because of the wide range of IC50 values obtained across reference clones; and mefloquine, desethylamodiaquine and dihydroartemisinin given their global importance as components of artemisinin-combination therapy. D. Source of drug It is critical that laboratories participating in this study use drugs from WWARN’s QA/QC module in Bangkok http://www.wwarn.org/research/tools/qaqc. Drugs will be provided at no cost to laboratories that complete a short registration form and material transfer agreement. Suggested drug solvents are provided in Appendix 1. E. Experimental set-up Laboratories should use their own existing methodology for parasite culture, synchronization and assay readout ; suggested protocols are also available on the WWARN website. F. Drug wells To produce appropriate data for calculation of IC50, at least seven different drug concentrations should be applied in duplicate or triplicate. A suggested plate layout with drug concentrations is shown in Appendix 2. G. Drug-free controls WWARN uses data from drug-free wells as positive controls to set the top constraint for IC50 calculations. Consequently every plate needs to have at least 2 drug-free wells, and preferably 8-12 given the importance of control data in calculating IC50. WWARN uses data from all drug-free wells on a given plate irrespective of location. H. Number of repeats To obtain estimates of both intra- and inter-laboratory variation, three separate assessments for each drug / reference clone pair should be undertaken at least one week apart. I. Data submission Data files uploaded to the Data Contribution portal on the WWARN website should hold assay results in the form of a 96-well plate, along with a schematic of the drug applied in each well (if any) and its concentration in nM.
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J. Data analysis - individual laboratory Each laboratory will receive a standard report generated by WWARN’s In Vitro Analysis and Reporting Tool (IVART) detailing individual sample drug sensitivity curves and derived IC50 values. For an example from the IVART beta version, see Appendix 3. K. Data analysis – overall Individual drug sensitivity constants produced by IVART will be combined into a pooled analysis. An analytical plan for this pooled analysis has yet to be defined precisely. However,each laboratory will receive an aggregated set of results indicating the spread of IC50 data among the participating laboratories and summarizing the results of the reference clone molecular tests. Individual laboratories will not be identified in any reports.
Questions What if my reference clone turns out not to be what I think it is – will my work be wasted? Molecular validation is a foundation for all in vitro assessments involving reference clones, so molecular data will be a highly relevant output for this project, forming the first aspect of any presentation of the pooled data. In an earlier pilot study, WWARN noted that four of eleven 3D7 reference clones submitted for testing by microsatellite markers and Pfmdr1 copy number were not 3D7. This is an important finding which confirms that reference clones should be validated regularly. I already have these drugs – why should I go to the trouble of using those from WWARN QA/QC? WWARN’s QA/QC programme acquires all antimalarial drug standards from one independent company which specializes in the synthesis of drug standards. All reference materials and standards are accompanied by certificates of analysis (CoA). Each reference standard is re-certified one month prior to expiry date. Reference standards are stored and continuously monitored at the QA/QC unit under optimal conditions for humidity, temperature, and stability data to maximize their useful lives. By utilizing the same source of standards and same weighing procedures for all laboratories, it is possible to minimize bias arising from poor quality standards and weighing inaccuracies. Laboratories are encouraged to order exactly-measured 0.5-1.0 mg drug aliquots (n=4) to use in this study as well as larger 20.0 mg samples for longer term use. Laboratories may request other drugs for other in vitro projects linked to WWARN. What if my results differ from everyone else’s? There are many valid reasons why laboratories will obtain different results from the same drug-clone combination. These include intrinsic differences in assay methodology and parasite culture. The aim of this project is simply to measure the variability remaining when three parameters have been standardized: drug supply, reference clone identity and analytical method. It should be stressed that there are no ‘right’ answers. The overall goal is to understand the range of variability as a step towards standardization of in vitro assessment across all in vitro laboratories.
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Appendix 1: Suggested drug solvents Drug
Solvent to dissolve powder
Solvent for dilutions
Chloroquine
water
water
Mefloquine
methanol
water
Desethylamodiaquine
methanol
water
Dihydroartemisinin
methanol
water
Appendix 2: 96-well plate assay layout Concentrations in nM
A B C D E F G H
CQ MQ DQ DHA
1
2
3
4
5
6
7
8
9
10
11
12
0
6.25
12,5
25
50
100
200
400
800
1600
3200
0
0
6.25
12,5
25
50
100
200
400
800
1600
3200
0
0
2
4
8
16
32
64
128
256
512
1024
0
0
2
4
8
16
32
64
128
256
512
1024
0
0
3.75
7.5
15
30
60
120
240
480
960
1920
0
0
3.75
7.5
15
30
60
120
240
480
960
1920
0
0
0.125
0.25
0.5
1
2
4
8
16
32
64
0
0
0.125
0.25
0.5
1
2
4
8
16
32
64
0
4
Appendix 3: Extract from In Vitro Analysis and Reporting Tool
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