Quantification of solution-free red blood cell staining by sorption kinetics of Romanowsky stains to agarose gels.

The imaging and quantification of stained red blood cells (RBCs) are important for identifying RBCs in hematology and for diagnosing diseased RBCs or parasites in cytopathology. Romanowsky staining has been used traditionally to produce hues in blood cells using a mixture of anionic eosin Y and cationic methylene blue and azure B. While Romanowsky stains have been widely used in cytopathology, end-users have experienced problems with varying results in staining due to the premature precipitation or evaporation of methanol, leading to the inherent inconsistency of solution-based Romanowsky staining. Herein, we demonstrate that the staining and destaining of blood smears are controllable by the contact time of agarose gel stamps. While the extent of staining and destaining is discernable by the hue values of stamped red blood cells in micrographs, the quantification of adsorbed and desorbed Romanowsky dye molecules (in particular, eosin Y, methylene blue and azure B) from and to the agarose gel stamps needs a model that can explain the sorption process. We found predictable sorption of the Romanowsky dye molecules from the pseudo-second-order kinetic model for adsorption and the one phase decay model for desorption. Thus, the method of agarose gel stamping demonstrated here could be an alternative to solution-based Romanowsky staining with the predictable quantity of sorption and timing of contact.

Hydrogel Stamping for Rapid, Multiplexed, Point-of-Care Immunostaining of Cells and Tissues.

In the era of precision oncology, multicolor fluorescence imaging has become a core technology for multiplexed molecular analysis of cellular and tissue specimens. However, conventional solution-based staining is labor-intensive and time-consuming and requires considerable expertise to yield optimal results, which creates difficulties for employing this technology in resource-limited settings. Here, we report a new immunostaining method based on hydrogel stamping, which is simple, fast, easy to use, and reproducible. We showed that a hydrophilic hydrogel stamp could effectively transfer fluorescent antibodies to targets and withdraw an excess solution when the reaction is completed, obviating the need for extra washing. This unique property allows for quality immunostaining in 5 min for cells using one-eighth of antibody consumption compared to the conventional solution-based method. Furthermore, we implemented fluorescence quenching and immunocycling with hydrogel staining for multiplexed analysis of 9 protein markers at a single cell level. Finally, we applied the immunocycling method to human breast cancer tissue samples and showed quality immunostaining over a large area (∼2 cm2) in 30 min for molecular subtyping of breast cancer. The hydrogel immunostaining could open new opportunities for rapid, automated, and multiplexed profiling in compact point-of-care systems for molecular cancer diagnosis.

Temporal changes in immune cell composition and cytokines in response to chemoradiation in rectal cancer.

We measured systemic changes in the immune response in 92 patients receiving preoperative chemoradiation therapy (CRT) and subsequent surgery for rectal cancer. The peripheral blood was sampled five times from the onset of CRT until surgery. Lymphocytes decreased continuously during CRT but increased after CRT. The increased lymphocyte population was predominantly CD8+ T lymphocytes, which accounted for a significantly larger proportion in patients without residual lymph node metastasis than in those with residual lymph node metastasis. Neutrophils and monocytes decreased during the initial two weeks of CRT but were maintained or increased afterwards. Neutrophil and monocyte counts were significantly lower in patients with a pCR (pathologic complete response) than in those without a pCR two weeks after CRT began but not at the initiation of CRT. All cytokines showed dramatic changes one month after the termination of CRT. Cytokines related to the antitumour immune response increased, and those related to tumour progression decreased. The predictive value of cytokines was not clear. In short, we observed that immune components in peripheral blood are affected by CRT and show dynamic changes over time. We identified biomarker candidates to predict the pathologic response in the future.