Dataset Information

Establishment of the reference intervals of whole blood neutrophil phagocytosis by flow cytometry.

ABSTRACT:

Objective

To investigate the reference intervals (RIs) of the whole blood neutrophil phagocytosis by flow cytometry (FCM) and to study the application value of neutrophil phagocytosis in infectious diseases.

Methods

Pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923) cultured for 18-24 h were labeled by fluorescence probe carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), and then incubated with whole blood at 37℃. The phagocytosis of pathogens by neutrophils was detected by flow cytometry, and a reference interval was established.

Results

In the healthy adults, the reference interval for the neutrophil phagocytosis to Escherichia coli was 46.91%-83.09% and to Staphylococcus aureus was 33.92%-69.48%. This method showed good reproducibility. Neutrophil phagocytosis was negatively correlated with the neutrophil count, neutrophil percentage, and neutrophil-to-lymphocyte ratio (NLR, p < 0.05).

Conclusion

We have successfully established the RIs of neutrophil phagocytosis in whole blood in healthy adults by flow cytometry (FCM), which might be of important clinical value in the diagnosis, treatment, and prognosis of infectious diseases.

SUBMITTER: Yang F

PROVIDER: S-EPMC8373350 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Severe falciparum malaria anaemia (SMA) is a frequent cause of mortality in children and pregnant women. The most important determinant of SMA appears to be the loss of non-parasitized red blood cells (np-RBCs) in excess of loss of parasitized (p-) RBCs at schizogony. Based on data from acute SMA where excretion of haemoglobin in urine and increased plasma haemoglobin represented respectively less than 1% and 0.5% of total Hb loss, phagocytosis appears to be the predominant mechanism of removal of np- and p-RBC.Estimates indicate that np-RBCs are cleared in approximately 10-fold excess compared to p-RBCs. An even larger removal of np-RBCs has been described in vivax malaria anaemia. Estimates were based on two single studies both performed on neurosyphilitic patients who underwent malaria therapy. As the share of np-RBC removal is likely to vary between wide limits, it is important to assess the contribution of both np- and p-RBC populations to overall RBC loss, and disclose the mechanism of such variability. As available methods do not discriminate between the removal of np- vs p-RBCs, the purpose of this study was to set up a system allowing the simultaneous determination of phagocytosis of p- and np-RBC in the same sample. METHODS AND RESULTS: Phagocytosis of p- and np-RBCs was quantified in the same sample using double-labelled target cells and the human phagocytic cell-line THP-1, pre-activated by TNF and IFN? to enhance their phagocytic activity. Target RBCs were double-labelled with fluorescent carboxyfluorescein-succinimidyl ester (CF-SE) and the DNA label ethidium bromide (EB). EB, a DNA label, allowed to discriminate p-RBCs that contain parasitic DNA from the np-RBCs devoid of DNA. FACS analysis of THP-1 cells fed with double-labelled RBCs showed that p- and np-RBCs were phagocytosed in different proportions in relation to parasitaemia. CONCLUSIONS: The assay allowed the analysis of phagocytosis rapidly and with low subjective error, and the differentiation between phagocytosed p- and np-RBCs in the same sample. The presented method may help to analyse the factors or conditions that modulate the share of np-RBC removal in vitro and in vivo and lead to a better understanding of the pathogenesis of SMA.