In blood banks, leukoreduction filters (LRFs) are used to remove leukocytes from blood components in order to prevent some complications related with transfusion.
Several groups have shown that LRF could be a good source of normal human cells (1, 2). Leukoflex LST-1 (Maco Pharma,Tourcoing, France) is used by the Iranian Blood Transfusion Organization (IBTO) to prepare leukoreduced products. Indeed, a blood bag (450 mL with 1.6 × 109 to 4 × 109 white blood cells), represents a potential source of large numbers of granulocytes, such as neutrophils. Since LRFs are discarded after the preparation of red blood cells (RBC), they represent an interesting source for purifying white blood cells (WBC). The present study is our experience in isolation of neutrophils from whole-blood leukoreduction filters.
Blood bags from healthy donors were collected after written consent. All donors were screened for infectious diseases (hepatitis B, hepatitis C and HIV) and negative samples were included in the study. Blood bags (with a volume of 450 ml) with a half-life of less than 24 hours filter by LRF at 22° C by gravity flow. The ends of the Leukotrap filters were sealed, and instead of being discarded, were stored at 4 °C prior to our use. The cells were extracted from filters by back-flushing with a 60-mL syringe filled with Phosphate buffered saline (PBS), pH 7.4, without MgCl2 and CaCl2, containing 5 mM EDTA and 2.
5% sucrose. The PBS was homogenized with the filter content and then collected in a sterile tube. Neutrophils can survive in a LRF up to 24 hours after collection of blood, so back-flushing of LRF may preferably take place during this time. Granulocytes were separated from mononuclear cells by centrifugation through Ficoll (Lymphodex, Inno-Train, Germany) at 400×g for 30 min.
After aspirating mononuclear cells along with the supernatant, granulocytes were collected. To increase the number of cells, three filters were simultaneously washed. The wash fluid was then aliquoted to six 15 ml tubes and the granulocytes layer from each tube was combined into one tube. The cells were twice washed by low speed centrifugation (300 g for 10 min) in PBS 1X to remove contaminating erythrocytes. This left a pellet containing leukocytes, which consisting mostly of neutrophils. RBC, PLT, and WBC counts were performed with an automatic hematology analyzer (Figure 1).
Over 90% of the cells were viable by trypan blue exclusion.Our experience showed LRF is a major source of leukocytes for research, as they are available in large quantities, easy to handle and they are destroyed after blood processing.