Analysis of lymphocytes and its correlation to rejection in transplant patients

Kidney transplantation is the treatment of choice for patients suffering from Chronic Kidney Disease and is associated with longer survival and better quality of life compared to those patients who have not received a transplant and remain on dialysis.

In recent decades, thanks to advances in medicine, short- and medium-term renal graft survival has improved remarkably; however, long-term graft survival has not shown significant improvements, rejection being mediated by antibodies (AMR) the most probable cause of graft loss after the first year post-transplant.

The role of cTfh cells in the production of antibodies

Follicular helper T cells (Tfh) play a fundamental role in the production of antibodies, since they induce the differentiation of B cells into antibody-producing cells. Tfh are generally located in secondary lymphoid organs (SLOs), even though, in 2011 circulating Tfh cells (cTfh) were described as Tfh cells that are located in peripheral blood .

Thus, given the already explained role of cTfh cells in the production of antibodies, it could be interesting, from a clinical and prevention point of view, to analyze cTfh levels in peripheral blood of kidney transplant patients. Hence, patients with higher cTfh levels in peripheral blood would be more prone to suffering acute antibody-mediated rejection than those patients with low levels of these cells.

Flow cytometry to perform analysis of lymphocytes

The analysis of cTfh lymphocytes can be performed by flow cytometry, requiring first the isolation of peripheral blood mononuclear cells (PBMCs) by density gradient with LymphoprepTM.

For this method, 15mL of whole blood is diluted with 15mL of buffered saline supplemented with fetal calf serum. The 30mL of diluted blood is carefully dispensed into a 50mL tube containing 15mL of LymphoprepTM. Then, the 50mL tube is centrifuged at 800 G for 20 minutes at room temperature (15-25ºC). This centrifugation step can be carried out with universal, ventilated equipment such as the Unicen 21 that has a control that allows defining the way of working beyond the operating values.

As a result of this centrifugation, the cells are divided into 4 layers based on their density, which from bottom to top are: layer with erythrocytes, layer with LymphoprepTM, layer with PBMCs, and layer with plasma. After centrifugation, the serum layer is discarded to gain access to the PBMCs layer, which is the one we are interested in. Finally, the layer of PBMCs is collected, and the cells are labeled with the optimal antibodies to analyze the cTfh in the flow cytometer.

The analysis of cTfh cells by flow cytometry is an interesting parameter to prevent antibody-mediated rejection in kidney transplant patients, and for this, PBMCs must be previously isolated by density gradient centrifugation.