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Light transmission aggregometry using pre-coated microtiter plates and a Victor X5 plate reader
Pernille Just Vinholt1, Mads Nybo1, Camilla Brødsgaard Nielsen1
1Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark.
Plos One
|October 13, 2017
Summary
This study presents a simplified 96-well light transmission aggregometry (LTA) protocol using pre-coated microtiter plates. This method allows for the simultaneous testing of multiple agonist concentrations and can be stored at -80°C for up to 15 days without affecting results.
Area of Science:
- Hematology
- Biotechnology
- Assay Development
Background:
- Classical light transmission aggregometry (LTA) is cumbersome for high-throughput analysis.
- Microtiter plates offer a solution for concomitant testing of multiple samples in LTA.
- Pre-coating plates with agonists could further simplify LTA procedures.
Purpose of the Study:
- To establish a simplified 96-well LTA protocol.
- To enable pre-coating of microtiter plates with agonists for storage.
- To validate the use of pre-coated plates for platelet aggregation testing.
Main Methods:
- Developed and validated a 96-well LTA protocol using a Victor X5 plate reader.
- Utilized pre-coated microtiter plates stored at -80°C.
- Tested a range of agonist concentrations (collagen, arachidonic acid, ADP, TRAP, PAR-4) and shaking times.
Main Results:
- Minimum platelet-rich plasma requirement of 45 μL and platelet count of 100 x10^9/L.
- Optimal absorbance reading at 595 nm; higher aggregation at 37°C.
- Platelet aggregation results remained stable after storage at -80°C for 15 days.
Conclusions:
- The developed 96-well LTA protocol is suitable for platelet aggregation testing.
- Pre-coated microtiter plates can be stored at -80°C for at least 15 days.
- The protocol allows for concomitant testing of multiple agonist concentrations.