Abstract: SA-PO711
B and T Cell Subsets in Patients with Membranous Nephropathy: Comparison of Frozen Stored vs. Freshly Collected Blood Samples
Session Information
- Pathology and Lab Medicine: Clinical
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Pathology and Lab Medicine
- 1602 Pathology and Lab Medicine: Clinical
Authors
- Vink- van Setten, Coralien, Radboud University Medical Center, Nijmegen, Netherlands
- van de Logt, Anne-Els, Radboud University Medical Center, Nijmegen, Netherlands
- Wetzels, Jack F., Radboud University Medical Center, Nijmegen, Netherlands
Background
It is suggested that characterisation of B and T cell subsets may provide relevant information in auto-immune diseases. Rosenzwajg et al (Kidney Int 2017; 92,277-237) studied B and T cell subsets in Rituximab treated patients with primary membranous nephropathy (pMN). They observed a decrease of regulatory T cells as well as NK cells in active disease, whereas numbers of naive B cells were increased. The increase in Treg after Rituximab predicted treatment response. In the literature B and T cell subsets are evaluated using frozen peripheral blood mononuclear cells (PBMCs) or freshly isolated blood samples. We compared these conditions, a comparison which is lacking in the literature.
Methods
In 18 patients with pMN, we collected 20 mL of EDTA plasma. 10mL was used for fresh whole blood analysis and 10mL was used for isolation of PBMCs, and samples were stored in liquid nitrogen for 6 – 12 months. We characterised immune cell subsets with flowcytometry with fluorescently labelled cell surface markers: CD3, CD4, CD5, CD8, CD14, CD16, CD19, CD20, CD24, CD25, CD27, CD38, CD45, CD45 RA, CD45RO, CD56, CD127, HLA-DR, IgD, IgM.
Results
The distribution of the various B and T cell subsets when comparing both conditions showed a good correlation(table). The figure shows the correlation for NK cells and Tregs respectively. Our data show a weak correlation specifically within the Treg subset.
Conclusion
Our study suggests that frozen PBMC samples can be used for the characterisation of T and B lymphocytes and NK-cells, except for the Treg population. This may be improved by intracellular staining techniques. Further validation is needed.
Comparison of fresh and frozen immune cell subsets
| Cell type | Fresh whole blood | Frozen PBMCs | Spearman correlation coefficient (P value) |
| Monocytes | 20.3% | 31% | 0.631 (0.005) |
| Lymphocytes | 79.7% | 69% | 0.631 (0.005) |
| total T-cells (within lymphocytes) CD4 (within T-cells) Treg (within T-cells) CD8 (within T-cells) | 73.4% 69.2% 3.1% 26.6% | 67.9% 68.1% 4.4% 27.6% | 0.811 (0.000) 0.934 (0.000) 0.421 (0.082) 0.935 (0.000) |
| total B-cells (within lymphocytes) | 3.8% | 6.5% | 0.824 (0.000) |
| NK-cells (within lymphocytes) | 10.9% | 9.0% | 0.939 (0.000) |