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Purification and properties of peritoneal eosinophils from pediatric dialysis patients
R L Roberts1, B J Ank, I B Salusky
1Department of Pediatric Immunology/Allergy, UCLA School of Medicine 90024.
Insights
A new method isolates pure peritoneal eosinophils from pediatric patients on dialysis. This technique yields large quantities of eosinophils for studying their function and composition.
Area of Science:
- Immunology
- Cell Biology
- Nephrology
Background:
- Peritoneal eosinophilia is common in patients on peritoneal dialysis.
- Pediatric patients on continuous peritoneal dialysis often exhibit high peritoneal eosinophil counts.
Purpose of the Study:
- To develop a method for isolating large numbers of pure peritoneal eosinophils from pediatric patients undergoing peritoneal dialysis.
- To enable further study of eosinophil function, subpopulations, and granule constituents.
Main Methods:
- Collected peritoneal fluid from pediatric patients with high eosinophil counts.
- Concentrated cells via centrifugation.
- Utilized a discontinuous Percoll gradient (45%, 55%, 65%, 75%) for cell separation.
- Analyzed cell purity and characteristics at different gradient interfaces.
Main Results:
- Isolated peritoneal fluid contained approximately 77% eosinophils.
- The densest cell band (above 75% Percoll) achieved 94.7% eosinophil purity.
- Isolated eosinophils exhibited normal morphology and comparable function to blood eosinophils in assays.
- Monocyte/macrophage counts increased at lower Percoll concentrations.
Conclusions:
- A Percoll gradient centrifugation method effectively isolates highly pure peritoneal eosinophils from pediatric patients on peritoneal dialysis.
- This method provides a valuable tool for research into eosinophil biology and function in the context of peritoneal dialysis.
Abstract:
Peritoneal eosinophilia frequently occurs in patients undergoing peritoneal dialysis. We have devised a method for isolating large numbers of these peritoneal eosinophils from pediatric patients on continuous peritoneal dialysis. Patients were selected on the basis of previous high peritoneal eosinophil counts and had an age range of 1.5-11 years. The unfractionated peritoneal fluid contained 7.9 +/- 3.7% neutrophils, 3.8 +/- 1.0% lymphocytes, 11.0 +/- 3.7% monocytes/macrophages, and 77.3 +/- 6.3% eosinophils (based on Wright stain) and up to 2 x 10(9) cells could be recovered from 1 liter of peritoneal dialysate. The cells were concentrated by centrifugation and the cell suspension then layered over a discontinuous Percoll gradient consisting of layers of 45%, 55%, 65%, and 75% Percoll. The gradients were centrifuged resulting in the formation of bands of cells at the interfaces of the layers. The densest band of cells (above 75% Percoll) contained 94.7 +/- 1.8% eosinophils (mean with median of 98%) and 4.3 +/- 16% neutrophils. The eosinophil counts were 72.2 +/- 7.1% above the 65% layer, 57.1 +/- 8.7 above 55%, and 40.9 +/- 10.9% above 45%. The monocyte/macrophage count increased from 0.1% above the 75% layer to 42.9% above the 45%. The denser eosinophils (above 75% and 65%) had the appearance of normal blood eosinophils and comparable function to blood eosinophils in cytotoxic and oxidative assays. This method provides a means of obtaining large numbers of very pure eosinophils for study of eosinophil function, eosinophil subpopulations, or eosinophil granule constituents.