(P-CB-15) Quantitation of Peripheral Blood Mononuclear Cells (PBMCs) in Leukocyte Reduction System (LRS) Chambers

The most common source of human white blood cells (WBCs) for laboratory use had been buffy coats, whereby WBCs are separated from red blood cells (RBCs) by centrifugation and expression from whole blood (WB) donor units. The introduction and widespread adoption of leukoreduction blood filters in WB and apheresis collection sets by blood collection establishments have helped minimize HLA alloimmunization, CMV transmission, and febrile nonhemolytic transfusion reactions in transfusion recipients. These filters, usually considered a waste product of the blood or component collection, are a useful source of peripheral blood mononuclear cells (PBMCs) for research. Many researchers have switched from buffy coats to apheresis leukocyte reduction system chambers (LRSC) to obtain PBMCs for their research. This study determined the count and viability of PBMCs in LRSCs based on product collection type (single, double, and triple) platelet products.

Study

Design/Methods:

Residual LRSCs (under IRB approved protocol) from healthy allogeneic platelet donors were obtained from an apheresis collection system (Trima Accel, Gambro BCT, Lakewood, CO). Post collection, LRSCs were stored at 2-8°C overnight to imitate storage conditions prior to distribution for research.

In this study 10 single, 10 double, and 10 triple platelet products (collection target values were 4.0 x 10¹¹, 6.2 – 6.8 x 10¹¹, and 9.5 – 10.3 x 10¹¹, respectively) LRSCs were obtained and PBMCs isolated utilizing a polysucrose and sodium diatrizoate density medium (Sigma-Aldrich, St. Louis, MO). Cells were quantified by an automated cell counter (DeNovix CellDrop BF Automated Cell Counter, Wilmington, DE) using 0.4% Trypan Blue staining for viability, total cell, and live cell count. Acetic acid cell counts were treated with 3% acetic acid prior to enumeration to rule out red cell carry over.

Results/Findings:

On average, single platelet derived LRSCs contained 1.46 x 10⁹ live PBMCs, approximately 1.50 x 10⁹ fewer PBMCs than double platelet LRSCs. Triple platelet LRSCs contained 2.40 x 10⁹ PBMCs compared to double derived LRSCs that contained 2.96 x 10⁹ PBMCs. PBMC viability decreased minimally as the product split increased. Figure 1.

Conclusions:

PBMCs isolated from LRSCs are viable starting materials for research. Depending upon the end user’s application, selection of single platelet derived product yields fewer PBMCs when compared to a double or triple derived product. The non-linear recovery of PBMCs from platelet derived LRSCs is attributable to the WBC saturation of the LRSC. In longer apheresis procedures clearing of LRSC on the apheresis collection system returns WBCs to the donor, facilitating further collection of platelet products, explaining the decrease in PBMCs in triple vs double derived LRSCs.