WP4: Enumeration methods for cells

The aim of this workpackage is to develop and evaluate reference measurement procedures for the enumeration of macroscopic entities and determination of their concentrations. The accuracy of correct identification and enumeration of cells is essential to a wide range of clinical and industrial applications, such as the production of biopharmaceuticals, cellular therapies, tissue engineering and clinical diagnosis. Therefore, a range of samples will be developed which replicate the complexity of real life samples. The quantitative characterisation of these samples will allow for the development, testing and validation of primary and secondary enumeration techniques.

For selected blood cells relevant for haematological and immunological diagnosis and therapy control, a primary reference method will be established. A simple model enumeration of red blood cells (RBC) will first be investigated and the protocols adapted to other target cells and within this project.

To allow manufacturers and end users to assess the trueness of their results, a secondary reference method and a selected method will be developed. It is intended that both, the secondary and the selected method can be applied using standard instrumentation available in (manufacturers) bioresearch laboratories and routine laboratories. In contrast to the primary reference measurement method where the particles per volume are directly enumerated, the secondary and selected methods rely on relative measurements with respect to a calibrator of defined particle number or particle concentration. Correlated with the reduced requirements, corresponding higher uncertainties will be obtained.

To establish traceability and to validate the secondary and selected methods, calibrators are needed and hence the investigation of artificial as well as biological material will also be investigated.

Besides particle detection and enumerated in flow (flow cytometry) at rates of 100 Hz to 5 kHz, microscopy will be used with respect to its potential as primary and/or secondary reference measurement method.

In the later stages of the JRP, the measurement procedures for accurate enumeration will be applied to address specific critical applications for cell characterisation, transfusion and transplantation as well as rare cell detection and enumeration. Accurate enumeration of rare cells is crucial in particular because of their unambiguous identification. To improve rare cell enumeration, surface staining, cell sorting and subsequent genetic classification by qPCR or dPCR will be exploited as a link between this workpackage and WP2. For genotyping, a specific target gene will be detected and the copy number measured using the enumeration methods developed in WP2.