Leukocytes are used for a variety of research purposes. In recent years, the demand for primary human cells has been increased. The gold standard to obtain these cells is leukapheresis.18,19 However, the use of buffy coats or leukapheresis products for research purposes is highly expensive and is not available at every blood bank. Several groups have examined the phenotype and function of immune cells isolated from LRFs, compared to standard buffy coats. They observed that the percentages of T cells, B cells, monocytes and dendritic cells (DCs) were largely similar between peripheral blood mononuclear cells (PBMCs) isolated from LRFs and buffy coats.8,18,20

The number of cells recovered from different LFRs is dependent on the volume of phosphate buffered saline (PBS) used to flush back them from the filter. In a study by Izquierdo et al.,21 blood bags from healthy donors were less than 24h old when processed through an LRF by gravity flow at room temperature (22°C). Cells were recovered from filters at high numbers (about 5×108 PBMCs), with a viability >95%, and the phenotypic and functional experiments confirmed that they were similar to those cells obtained from buffy coats. In a study by Peytour et al.,22 discarded LRFs were processed through a rapid (<5h) and easy 4-step procedure (WBC elution, platelet depletion, MNC separation and CD34+ cell selection) less than 24h after blood donation. They obtained 0.4±0.05×105 highly enriched CD34+ cells per LRF. The functional properties, such as cell cycle status, growth rate and myeloid differentiation in liquid culture, were rather similar to steady state peripheral blood (SSPB) and umbilical cord blood (CB) CD34+ cells. Ebner et al.,23 conducted a study to isolate CD14+ precursors from LRFs to generate DCs and compared them with isolated cells from buffy coats. They revealed that the yield of PBMCs obtained from LRFs is higher than that from buffy coats. The generated DCs had short culture and maturation periods, similar to the DCs obtained from the leukapheresis procedure. Thus, they concluded that LRFs have the potential to become a standard procedure for preparation of large quantities of DCs. Weidinger et al.,19 showed that PBMCs obtained from LRFs have a lower fraction of apoptotic and necrotic cells than PBMCs from leukapheresis. Furthermore, in a study by Pfeiffer et al.,24 PBMCs and especially high-quality monocyte-derived dendritic cells (moDCs) were generated from LRFs, comparable to those derived from leukapheresis products or whole blood. Néron et al.,25 demonstrated that cells recovered from WBF2 filters represent a good source of viable PBMCs because each unit can provide 2×108 to 3×108 PBMCs that can be used to prepare cryopreserved samples for phenotypic and functional investigations. Recently, we performed a study26 on the isolation of cells from Leukoflex LST-1 filters and approximately, 4.4×108 leukocytes (mononuclear cells+granulocytes) were obtained from each filter. Teleron et al.,17 designed a study to evaluate the capacity of LRFs as a source of peripheral blood-derived endothelial progenitor cells (EPCs). They displayed that the mean yield of EPCs from one LRF is 14-fold higher than that from fresh blood, probably due to the greater numbers of PBMCs obtained from each LRF. Accordingly, they concluded that LRFs can provide a safe and cheap source of EPCs.

Another application of LRFs is using them in genetic analysis. Cook et al.,27 reported that the LRF is a source of large quantities of DNA for the study of genetic variations in human populations. A mean of 1520μg (ranging from 450 to 3750μg) of DNA per sample was obtained. Also, they indicated that the LRF-recovered DNA remains suitable for polymerase chain reaction (PCR) and sequencing methods after 2 years of storage at −40°C to 4°C. They concluded that this procedure allows for the attainment of sufficient quantities of genomic DNA for many experiments. However, these genetic experiments require the specific consent of the donor and ethical approval.

Peytour et al22 introduced a procedure to obtain 0.4±0.05×105 highly enriched CD34+ cells per LRF. They showed that CD34+ cells trapped in filters are functionally similar to those harvested from other sources, such as bone marrow (BM), cord blood or peripheral blood. Therefore, LRFs can be an alternative source of stem cells for hematopoietic transplantation. Ivanovic et al28 conducted a study to investigate the viability and the functional properties of mononuclear cells (MNCs) and CD34+ cells recovered from LRFs. The results displayed that the functionality of LRF-recovered CD34+ cells was well preserved with a significant ability of ex vivo expansion. They also confirmed the ability of these cells to differentiate into erythroid, megakaryocytic, and myelomonocytic lineages, upon the addition of certain cytokines in their liquid cultures. The problem of the relatively low amount of CD34+ cells in each LRF can be resolved by pooling cells recovered from several LRFs utilized for a continuous donor. Hence, LRF-recovered CD34+ cells can be applied to clinical procedures to generate mature ex vivo cells. Some studies disclosed that peripheral blood MNCs can differentiate into the CD34+ progenitors and they may differentiate into the various non-hematopoietic cell lineages.29,30 Therefore, it is possible that LRF-recovered MNCs become a main source of cells for cell-based regenerative therapy in future. In addition, it is suggested that LRF CD34+ cells are good candidates for cell/tissue engineering, such as cardiac cell therapy,31,32 and the attainment of pluripotent stem cells (iPS cells).33

The PBMNs are frequently used for the DC culture in adoptive immunotherapies. Today the gold standard to obtain monocytes for DC therapies is leukapheresis. In a study by Izquierdo et al.,21 monocytes recovered from WBF3 filters differentiated into in vitro DCs. The main feature of these cells is the capacity to stimulate native T cells and initiate primary immune responses.34 This characteristic has led to the use of DCs in cancer immunotherapy.35,36

Human α-defensins, also called human neutrophil peptides 1–3 (HNP 1–3), are cationic antimicrobial peptides with three disulfide bridges.37–40 These peptides are effector molecules of the innate immunity. They also show chemotactic activity for monocytes and T cells. Defensins are mainly produced by neutrophils, but their production by other leukocytes, such as NK cells, B cells, T cells, monocytes and macrophages, has been reported.41–43 Previous studies have also demonstrated that defensins display anticancer activity.44–46 Synthesis of α-defensins using chemical and recombinant approaches is challenging due to the essential role of the disulfide bonds for its function. Recently LRFs have been introduced as a major production source for natural defensins.26,47 We performed a study48 at the Iranian Blood Transfusion Organization (IBTO) and found that α-defensins purified from neutrophils trapped in LST-1 filters have anticancer activity that can be used for clinical applications in the future. Vossier L et al49 have also found that human defensins extracted from LRFs have high antimicrobial activity with minimal inhibitory concentration (MIC) values between 0.12 and 1μg/mL. In addition, other polypeptides with antimicrobial activity have been found in neutrophils, such as calprotectin and LL-37,50 that can be extracted from the neutrophils trapped in leukofilters.

The preclinical studies for drug screening involve the use of animals, which is expensive and leads to the suffering of these animals. A number of alternative systems have been developed for use in research, such as tissue culture, stem cells, microbiological systems, DNA chips and computer analysis models. The benefits of these alternatives include the decrease in the number of animals used, reduction in the costs and control of the variables of the experiment.51,52 In addition, it is reported that mouse models may poorly mimic human disorders, such as inflammatory disease.53,54 The LRFs can be used as a culture medium with a valuable source of primary human cells. Therefore, it can be used as an alternative to animal models.