At ESCCA 2026 in Salzburg, Sven introduced an epigenetic qPCR approach that could bring several CAR-T monitoring questions into one standardized workflow.
Last month, I traveled to Salzburg for the 2026 conference of the European Society for Clinical Cell Analysis, or ESCCA. The annual meeting brings together scientists working across basic, translational, and clinical applications of cytometry, particularly flow cytometry, hematology, immunology, cancer, and cell biology. Its theme played on Salzburg’s most famous musical association, the Sound of Music, with the theme “The Cells are alive… with ESCCA.”
My presentation offered a deliberately contrary thought…the cells do not have to be alive.
In clinical trials, shipping fresh blood samples is an expensive challenge which can be an insurmountable barrier for global trials. While ddPCR is forgiving of cell quality, for applications such as flow cytometry the cells must remain alive. Typically, cell preservation requires complex or expensive methods and collection of blood into vacutainers. Then, a sample leaves a clinical site and immediately enters a race against time, carried at controlled ambient temperature through couriers, customs, weekends, and everything else that can intervene between a patient and a laboratory. For flow cytometry, this urgency is understandable. More than that it measures physical characteristics, it is only when the cellular matrix is intact that the cell can be seen as a cell and thus "counted" by flow, so those cells must reach the laboratory in suitable condition. But every time-sensitive shipment adds another place where an otherwise well-designed study can become operationally fragile.
That statement sounds simple, perhaps even obvious once someone has said it. Yet one of the things that continues to surprise me is how few scientists know that immune cells can be measured epigenetically. This is especially striking at a conference devoted to clinical cell analysis. At Epiontis, now part of Precision for Medicine, we have spent decades developing epigenetic approaches to immune-cell quantification. Yet the underlying idea has still not entered the general scientific vocabulary. Even people who spend their careers measuring cells may instinctively assume that the cell itself must remain intact and viable. Epigenetic immune monitoring begins from a different premise.
Cells develop a characteristic DNA methylation pattern. Some loci become demethylated only when the corresponding gene is expressed, and some of these epigenetic marks are highly specific to particular cell types. Because the number of gene copies per cell is known, we can use quantitative PCR to measure the demethylated DNA and calculate the proportion of a selected cell population. The early work focused on the Treg-specific demethylated region within FOXP3. The broader principle proved applicable to many leukocyte populations, enabling separate qPCR assays for T cells, B cells, NK cells, monocytes, and other immune-cell types. Normalization methods account for differences in PCR efficiency, supporting quantitative comparison across assays.
This approach is powerful but it does not make flow cytometry obsolete. Flow cytometry examines many markers on the same cell and identifies detailed subpopulations. Epigenetic analysis measures defined markers separately and cannot always show whether two markers occur on the same individual cell. That makes flow cytometry particularly valuable in exploratory studies where the relevant biology is still being mapped. Once the markers of interest are established, however, the stability and standardization of epigenetic monitoring become powerful. The method can work across matrices including frozen whole blood, anticoagulated blood, dried blood spots, and tissue, and it is supported by long-term assay monitoring and quality controls within an ISO 15189-accredited medical laboratory environment.
CAR-T cell therapy creates an interesting test case. Monitoring commonly involves several technologies. PCR/ddPCR can assess vector-copy-number integration as well as distribution and persistence of CAR-Ts cells in body fluids and tissues. Once CAR-T cell numbers are high enough, and assuming specific antibodies are available, PCR can determine activation and senescence status of the CAR-T cells. Microscopy, flow cytometry, or molecular methods may then help assess therapeutic response. In autologous therapies, the drug is already patient-specific. The number of changing parameters increases further when adding several analytical technologies, each with its own samples, reagents, workflows, and interpretation, which makes comparison scientifically and operationally complex.
Our question was whether the major measurements could be brought into one epigenetic qPCR workflow. Lentiviral vectors used in CAR-T therapies contain relatively conserved elements, including WPRE and RRE, which integrate with the transgene. We developed bisulfite-specific qPCR assays for these regions and included the targets in a normalization plasmid. This creates a potential route to measuring vector integration alongside endogenous T and NK cells for pharmacokinetic monitoring and B-cell content as one indicator of response. Theoretically, these measurements could come from one sample and one PCR plate, with samples provided as frozen blood or dried blood spots.
We evaluated the concept using frozen whole-blood samples from CAR-T recipients at University Hospital Leipzig in a blinded setting, with flow cytometry used for comparison. The assays detected true-positive signals in the relevant lentiviral CAR-T samples, supported estimation of vector copies per cell, and enabled calculation of CAR T cells as a proportion of all T cells. B-cell monitoring could be conducted alongside those analyses. These results show feasibility, not the final answer. More data are required to establish where the approach is strongest, where its limitations matter, and how it should be applied in clinical programs.
The practical consequence returns us to Salzburg and to those living cells. If the analytical question can be answered from DNA, the sample no longer has to survive the journey in the same way. Smaller volumes may be possible. Samples may be frozen, stored, batched, or collected as dried blood spots rather than dispatched individually under time-critical conditions. For global studies, that can simplify a chain of activity reaching from the clinical site to the central laboratory and finally to the testing laboratory. The scientific proposition is important, but its operational meaning may be equally valuable: sometimes the best way to measure cells is to stop requiring them to remain alive.
What could your CAR-T monitoring strategy look like if the cells did not have to remain alive? Talk with Precision for Medicine about integrating epigenetic analysis into your clinical development plan.