Dr. Andrea Döscher (Oldenburg/ DE), Claudia Grote (Oldenburg/ DE), Prof. Thomas Müller (Braunschweig/ DE)
Background
Recovery and survival of transfused platelets can be assessed by digital PCR of mitochondrial markers. Therefore, we investigated the key analytical qualities of the droplet digital PCR of seven mitochondrial markers.
Methods
The SPEF1 gene served as internal control for the entire process and was tested for intra-and inter-assay variability with 20 replicates and 10 independent PCR runs, respectively. Limit of blank (LoB: mean copy number NTC + 1.645 x SD copy number NTC), limit of detection (LoD: LOB + 1.645 x SD copy number non-blank material) and limit of quantification (LoQ: lowest platelet concentration with a coefficient of variation ≤ 25%) were determined with 10different PCR runs and samples measured in duplicate. The linearity was evaluated by dilution of platelets (0.07 to 10 G/L) tested in quadruples. Determination of the assays" validity was done by comparison with flow-cytometry and hematology analyzers (Symex XS 800i and Advia 2120i).
Results
Assay linearity was demonstrated by R^2-values above 0.99, the slope varied in respect to the different markers between 0.738 and 1.382 and intercept ranged from -0.03 to 0.16. DNA was prepared from EDTA anticoagulated samples (n=10) on a daily basis and tested in the ddPCR assay. The maximal difference in platelet count between day one and day five of sample preparation was 0.5 G/L. DNA samples stored at -30°C showed mean differences to the initial results of 0.131± 0.5 (one month), 0.121 ± 0.5 (three months), 0.130 ± 0.2 (six month) and 0.126 ± 0.180 G/L platelets after 12 months" storage. Validity of ddPCR results was in the range of R^2 = 9.3 – 9.5. The calculated recovery of the internal control was 79.9%±7.93.
Conclusion
Quantification of endogenous and transfused platelets with ddPCR offers a sensitive and reliable tool to monitor platelets in patients.
Offenlegung Interessenkonflikt:
Kein Interessenkonflikt
Invited talks abstract/summary