Table 2.

Comparison of bioanalytical aspects and strategies between qPCR and flow cytometry

	qPCR	Flow cytometry
Assay sensitivity 9	Highly sensitive: LLOQ ≤ 50 copies of transgene/µg DNA	Sensitivity depends on the relative affinity of the CAR/TCR detection agent, i.e., anti‐ID vs. dextramer
Sample matrix and storage 46 , 47	Frozen whole blood	Whole blood at room temperature, run 24–72 hours for absolute cell quantitation
Sample batching 46 , 47	Yes	No, must be analyzed within 1–2 days post collection to enable absolute cell quantitation
Detection 47	Measures copies of transgene in the cell	Measures cells with surface expression of transgenic CAR/TCR, multiplexed with other markers of T‐cell phenotype/subset
Post‐treatment detection duration 8	Can often detect transgene in circulation for years depending on cell persistence	Generally, can detect engineered T‐cells in circulation for months to years, but may fall below assay sensitivity at later time points depending on cell persistence
Other considerations	The extent of transgene incorporation into genome is generally unknown. Does not measure expression level of the transgenic CAR/TCR on the cell surface. Applying newly developed qPCR method with volume‐based unit enables more accurate evaluation of in vivo ACT kinetics due to reduced bias from processes such as lymphodepletion 11	Does not provide direct quantitation of the extent/density of CAR/TCR expression on the cell surface. CAR/TCR downregulation upon T cell activation may lower the mean fluorescence intensity without affecting the cell count

ACT, adoptive cell therapy; anti‐ID, anti‐idiotype; CAR, chimeric antigen receptor; LLOQ, lower limit of quantification; qPCR, quantitative polymerase chain reaction; TCR, T cell receptor.