Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.

Identification of fetal liver stroma in spectral cytometry using the parameter autofluorescence.

The fetal liver (FL) is the main hematopoietic organ during embryonic development. The FL is also the unique anatomical site where hematopoietic stem cells expand before colonizing the bone marrow, where they ensure life-long blood cell production and become mostly resting. The identification of the different cell types that comprise the hematopoietic stroma in the FL is essential to understand the signals required for the expansion and differentiation of the hematopoietic stem cells. We used a panel of monoclonal antibodies to identify FL stromal cells in a 5-laser equipped spectral flow cytometry (FCM) analyzer. The "Autofluorescence Finder" of SONY ID7000 software identified two distinct autofluorescence emission spectra. Using autofluorescence as a fluorescence parameter we could assign the two autofluorescent signals to three distinct cell types and identified surface markers that characterize these populations. We found that one autofluorescent population corresponds to hepatoblast-like cells and cholangiocytes whereas the other expresses mesenchymal transcripts and was identified as stellate cells. Importantly, after birth, autofluorescence becomes the unique identifying property of hepatoblast-like cells because mature cholangiocytes are no longer autofluorescent. These results show that autofluorescence used as a parameter in spectral FCM is a useful tool to identify new cell subsets that are difficult to analyze in conventional FCM.