The quantification of immune cell subpopulations in blood is important for the diagnosis, prognosis and management of various diseases and medical conditions. Flow cytometry is currently the gold standard technique for cell quantification; however, it is laborious, time-consuming and relies on bulky/expensive instrumentation, limiting its use to laboratories in high-resource settings. Microfluidic cytometers offering enhanced portability have been developed that are capable of rapid cell quantification; however, these platforms involve tedious sample preparation and processing protocols and/or require the use of specialized/expensive instrumentation for flow control and cell detection. Here, we report an artificial intelligence-enabled microfluidic cytometer for rapid CD4+ T cell quantification in whole blood requiring minimal sample preparation and instrumentation. CD4+ T cells in blood are labeled with anti-CD4 antibody-coated microbeads, which are driven through a microfluidic chip via gravity-driven slug flow, enabling pump-free operation. A video of the sample flowing in the chip is recorded using a microscope camera, which is analyzed using a convolutional neural network-based model that is trained to detect bead-labeled cells in the blood flow. The functionality of this platform was evaluated by analyzing fingerprick blood samples obtained from healthy donors, which revealed its ability to quantify CD4+ T cells with similar accuracy as flow cytometry (<10% deviation between both methods) while being at least 4× faster, less expensive, and simpler to operate. We envision that this platform can be readily modified to quantify other cell subpopulations in blood by using beads coated with different antibodies, making it a promising tool for performing cell count measurements outside of laboratories and in low-resource settings.
© 2025. The Author(s).

The immunological characteristics that could protect children with coronavirus disease 2019 (COVID-19) from severe or fatal illnesses have not been fully understood yet.
Here, we performed single-cell RNA sequencing (scRNA-seq) analysis on peripheral blood samples of 15 children (8 with COVID-19) and compared them to 18 adults (13 with COVID-19).
The child-adult integrated single cell data indicated that children with the disease presented a restrained response to type I interferon in most of the major immune cell types, along with suppression of upstream interferon regulatory factor and toll-like receptor expression in monocytes, which was confirmed by in vitro interferon stimulation assays. Unlike adult patients, children with COVID-19 showed lower frequencies of activated proinflammatory CD14+ monocytes, possibly explaining the rareness of cytokine storm in them. Notably, natural killer (NK) cells in pediatric patients displayed potent cytotoxicity with a rich expression of cytotoxic molecules and upregulated cytotoxic pathways, whereas the cellular senescence, along with the Notch signaling pathway, was significantly downregulated in NK cells, all suggesting more robust cytotoxicity in NK cells of children than adult patients that was further confirmed by CD107a degranulation assays. Lastly, a modest adaptive immune response was evident with more naïve T cells but less activated and proliferated T cells while less naïve B cells but more activated B cells in children over adult patients.
Conclusively, this preliminary study revealed distinct cell frequency and activation status of major immune cell types, particularly more robust NK cell cytotoxicity in PBMC that might help protect children from severe COVID-19.
Copyright © 2024 Jia, Li, Hu, Chang, Zeng, Liu, Lu, Xu, Zhai, Qian and Xu.

Here, we present a protocol for collecting, dissociating, isolating, staining, and analyzing immune cells from pancreatic cancer tissues for flow cytometry. The isolated cells can also be used for single-cell RNA sequencing and other related procedures. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2023).1.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

We previously identified the recombinant (r) macrophage (M) infectivity (I) potentiator (P) of the protozoan parasite Trypanosoma cruzi (Tc) (rTcMIP) as an immuno-stimulatory protein that induces the release of IFN-γ, CCL2 and CCL3 by human cord blood cells. These cytokines and chemokines are important to direct a type 1 adaptive immune response. rTcMIP also increased the Ab response and favored the production of the Th1-related isotype IgG2a in mouse models of neonatal vaccination, indicating that rTcMIP could be used as a vaccine adjuvant to enhance T and B cell responses. In the present study, we used cord and adult blood cells, and isolated NK cells and human monocytes to investigate the pathways and to decipher the mechanism of action of the recombinant rTcMIP. We found that rTcMIP engaged TLR1/2 and TLR4 independently of CD14 and activated the MyD88, but not the TRIF, pathway to induce IFN-γ production by IL-15-primed NK cells, and TNF-α secretion by monocytes and myeloid dendritic cells. Our results also indicated that TNF-α boosted IFN-γ expression. Though cord blood cells displayed lower responses than adult cells, our results allow to consider rTcMIP as a potential pro-type 1 adjuvant that might be associated to vaccines administered in early life or later.
Copyright © 2023 Ait Djebbara, Mcheik, Percier, Segueni, Poncelet and Truyens.

The role of innate lymphoid cells (ILCs)-including natural killer cells, helper-like ILC1s, ILC2s, ILC3s, and lymphoid tissue inducers-in human cancer is still poorly understood due to the scarcity of cell number. To address this, we present a protocol to analyze or purify ILCs from human blood, adjacent intestine, and colorectal tumor tissue. We describe steps for tissue and blood treatment, density centrifugation, antibody staining, and cell sorting. For complete details on the use and execution of this protocol, please refer to Qi et al. (2021).1.
Copyright © 2023. Published by Elsevier Inc.