Background: Hereditary spherocytosis (HS) is the most common cause of inherited chronic hemolysis. The various tests developed for HS screening have many shortcomings. The purpose of this study was to develop a new, simple, reliable test using flow cytometry.Methods: In this new test, deionized water, a hemolysis‐inducing agent, is spiked to a red cell suspension during acquisition, and the count of red cells is measured sequentially in real‐time using flow cytometry. In this study, the optimal parameters for this test were established, and the discriminatory power of the adopted protocol was verified among several groups with various degrees of osmotic fragility (OF).Results: The established protocol successfully discriminated between blood analyzed immediately and blood stored for 24 h (P< 0.00001). With healthy individuals as a reference, the OF measured by this protocol was significantly decreased in patients with iron deficiency anemia (P= 0.030) and was significantly increased in HS patients (P= 1.1 × 10⁻⁹). This new test found all 11 HS patients to have increased OF.Conclusions: This new test was verified to be simple, quantitative, objective, and cost‐effective for the measurement of OF. We suggest this test as another effective approach for HS screening, although its assay performance will require further verification through more clinical trials. © 2008 Clinical Cytometry Society

The aim of this study was to investigate the feasibility of nuclear molecular imaging using the human sodium iodide symporter (hNIS) as a reporter gene to monitor macrophage migration toward the inflammatory foci. Methods: A stable macrophage cell line coexpressing hNIS and green fluorescent protein (GFP) genes (RAW264.7/hNIS-GFP and R_NIS cell) was established from an immortalized macrophage cell line (RAW264.7 cells). ¹²⁵I uptake was determined (for hNIS protein functional activity), and flow cytometry analysis (to examine GFP gene expression), a cell proliferation assay, a cytokine assay, and a phagocytic activity assay were performed. ^99mTc-pertechnetate images were acquired at 1 d after subcutaneous inoculation of R_NIS cells in nude mice. Chemical inflammation was induced for in vivo imaging in the thigh of nude mice by turpentine oil injection. Small-animal PET with ¹⁸F-FDG and ¹²⁴I was performed with an intravenous administration of RAW264.7 or R_NIS cells in inflammation-induced animals. Results: The expression of hNIS and GFP genes was confirmed in R_NIS cells by flow cytometry and immunofluorescent staining. ¹²⁵I uptake was about 67 times higher in R_NIS cells than in RAW264.7 cells. No significant difference was observed in cell proliferation, cytokine production, and phagocytic activity between RAW264.7 and R_NIS cells. ^99mTc-pertechnetate imaging revealed increased tracer uptake at the inoculation site. PET with ¹²⁴I demonstrated a donut-shaped uptake, correlating with uptake shown by the ¹⁸F-FDG PET images, at the inflammation site of mice administered R_NIS cells. ¹²⁴I uptake (percentage injected dose per gram) was about 2.12 times higher at the inflammation site in the R_NIS mice than in RAW264.7 mice. By immunohistochemistry, the migration of macrophages was further confirmed by positive staining for GFP and hNIS at the inflammation site of R_NIS mice. Conclusion: These data support the feasibility of hNIS reporter gene imaging to monitor the macrophage migration toward an inflammatory lesion. Macrophages expressing hNIS may provide a new strategy to investigate the cellular behavior seen with inflammatory response in a preclinical model.

Kim Z, et al. Ann Lab Med. 2016 Jan;36(1):28-35. https://doi.org/10.3343/alm.2016.36.1.28

The effect of the transplant dose of each cell subset on engraftment kinetics and transplantation outcomes was evaluated in HLA-identical allogeneic peripheral blood stem cell transplantation (PBSCT). Sixty-nine patients were included in this retrospective study. Engraftment kinetics, transplantation outcomes, and immune reconstitution up to 1 year after transplantation were analyzed according to the transplant dose of CD34+ and non-CD34+ cells, including natural killer (NK) cells and CD8+ cytotoxic T (Tc) cells. An accelerated neutrophil engraftment was strongly associated with a higher transplant dose of NK cells (12 versus 16 days, P < .001) and Tc cells (13 versus 16 days, P < .001) but not CD34+ cells (P = .442). Survival analyses revealed a favorable prognosis for patients who received a higher dose of non-CD34+ cell subsets, rather than CD34+ cells, in terms of overall survival (OS; P = .024 for NK cells and .050 for Tc cells) and nonrelapse mortality (NRM; P = .005 for NK cells, .060 for Tc cells). In addition, a higher transplant dose of NK and Tc cells was correlated with a faster lymphoid reconstitution. In multivariate analyses, rapid neutrophil engraftment was correlated with a higher transplant dose of NK cells (P = .001) and Tc cells (P = .004). Moreover, an increased OS was associated with the NK cell dose (P = .007) and chronic graft-versus-host disease (P = .009), whereas a decreased NRM was associated with the NK dose (P = .024). In conclusion, in a PBSCT setting, a higher transplant dose of NK and Tc cells accelerated neutrophil engraftment, improved the immune reconstitution, and decreased NRM, thereby increasing OS after allogeneic PBSCT.