Modified Leukocyte Filter Removes Tumor Cells from the Salvaged Blood

Background Intraoperative blood salvage, an effective blood conservation strategy, has not been applied in onco-surgery, because of potential malignant cell contamination. In this study we tested effectiveness of a modified leukocyte depletion filter (M-LDF) for removal of tumor cells. Materials and Methods The effects of M-LDF and regular LDF on removal of cells (HepG2 cell line) were compared. The safety of M-LDF was tested with blood (collected and washed during onco-surgery), the salvaged blood mixed with tumor cells from the solid tumor of the same patient, or mixed with HepG2 cells (n=30 in each protocol). Cancer cells were identified by flow cytometry, culture and bioassay with and without filtration. Results M-LDF removed 5-log of HepG2 and nucleated cells, which was much higher than regular LDF, and cells were destroyed when they passed through M-LDF. Cytokeratin-positive cells in all samples were removed by M-LDF. Invasive growth adherent cells were found in most of unfiltered samples and 67% of the inoculated nude mice developed tumors in LDF-treated sample. Neither adherent cells nor nude mice developed tumors were found in M-LDF-treated samples. Discussion and Conclusion Since M-LDF can effectively remove and destroy cancer cells in the salvaged blood, it has great potential for clinical application.


Introduction
Surgical resection is still a preferred treatment for the solid tumors. One third of the banked blood is used during onco-surgery [1]. Unfortunately, blood transfusion may increase the risk of recurrence and mortality in patients with malignancies [2][3][4] with a shorter survival time [5]. Thus, an effective blood conservation strategy to minimize the adverse effects of allogeneic blood is needed during onco-surgery. Intraoperative blood salvage (IBS) has been used for 3 decades as an effective blood conservation strategy. However, it does not prevail in onco-surgery, because of malignant cell contamination in the salvaged blood [6]. Therefore, 50 Gy gamma irradiation has been applied to eliminate these malignant cells in Germany [7], but with limited use elsewhere [8]. A leukocyte depletion filter (LDF) can also remove tumor cells from the salvaged blood [9,10]. Some clinical trials [11] suggest LDF used for IBS does not increase the risk of tumor recurrence, however, the potential metastasis still exists [12,13], because not all nucleated cells can be removed. Therefore, its safety in application during onco-surgery is still questioned [1] and to further improve malignant cell removal is required. Our pilot studies indicate that pore size of the filter membrane and the wash solution are two key elements for cell depletion. Regular LDFs with a pore size of 25 μm remove 2-3 log of leukocytes from banked blood. However, salvaged blood, with the scarcity of plasma and platelets, is quite different from banked blood. Regular LDFs remove only 1-2 log of leukocytes and are ineffective for removal of malignant cells from the salvaged blood, which is consistent with another report [14]. With advances in materials and production processes, modified LDF (M-LDF) with a pore size as small as 12-18 μm is now available, which can remove 3-4 log of leukocytes in salvaged blood [15,16]. Mannitol-adenine-phosphate (MAP) solution not only helps preserve red blood cell (RBC) morphology and function and reduces hemolysis by improving energy production [17], but also improves tumor cell removal from 2-3 log to 4-5 log in IBS during onco-surgery [15,16]. In the present study, therefore, the effectiveness of this M-LDF on malignant cell removal was tested.  respectively. Studies were conducted according to the Declaration of Helsinki. All patients signed informed consents before participation. Animal studies were approved by the Institutional Animal Ethical Committee at Affiliated No. 2 Hospital of Zhejiang University. Immunocompromised BALB/c Foxn1 nu mice were from the Animal Center of Zhejiang University, and animals were euthanized by overdose of phenobarbital sodium at the end of the experiment. M-LDF was produced by Separator Haemo-Technology Beijing Co Ltd, Beijing, China, and regular LDF was produced by the Institute of Blood Transfusion, Chinese Academy of Medical Sciences. The salvaged blood was not transfused to patients in Protocol 2, 3 and 4. MAP solution (1.5g/L sodium citrate, 0.2g/L citric Acid, 7.93g/L glucose, 0.94g/L sodium biphosphate, 0.14g/L adenine, 4.97g/L sodium chloride, 14.57g/L mannitol) was produced by Sichuan Nigale Biomedical Co., Ltd., Chengdu, China.

Protocol 1: Effect of M-LDF and LDF on removal of malignant cells in media
The remaining was filtered with M-LDF, and cell count, viability (100 ml) and culture (100 ml) were assessed again. HepG2 cells were mixed with normal saline (NS 0.9 g/L sodium chloride) and filtered with a regular LDF as control (n = 8).

Protocol 2: Effect of M-LDF on removal of malignant cells in shed blood
From April 1 to December 31, 2010, thirty patients (hepatic carcinoma or pancreatic cancer, Stage I) scheduled for tumorectomy were recruited. Shed blood was collected from the skin incision to tumor removal, as described before [15]. Blood was anti-coagulated (heparin 25 u/ ml in MAP solution), and washed with an automated salvaging device (Jingjing Autologous Blood Recovery System, Model 3000P; Jingjing Medical Equipment Co., Ltd, Beijing, China) by MAP solution (1500-2000 ml for 250 ml RBC). Only salvaged blood, with hematocrit at 30-60% and greater than 250 ml of volume, was eligible for use.
Fourteen ml of the salvaged blood was sampled for cell count (2 ml), identification (4 ml) and culture (8 ml). The remaining blood was filtered with M-LDF, and reassessed for cell count (10 ml), identification (20 ml), and culture (40 ml). Filter membranes were also examined by light microscopy (BX51, Olympus, Japan) and transmission electron microscope (H-600IV, Hitachi, Osaka, Japan) for cell and filter interaction.

Protocol 3: Performance of M-LDF on the tumor cell overload
From August to November, 2010, thirty patients (hepatic carcinoma, gallbladder cancer, bile duct cancer or pancreatic cancer, Stage I) were recruited in this protocol. Blood collection and washing procedures were the same as for Protocol 2. To mimic the overload of tumor cells in the salvaged blood, freshly removed tumors (about 2×2×2 cm 3 ) were treated for single-tumor cell suspension [16]. Briefly, the tumor was mechanically and enzymatically dissected. The cells were isolated by centrifugation at 81× g for 3 min, and then suspended in phosphate-buffered saline (PBS). The isolated tumor cells were mixed with the salvaged blood and used for M-LDF assessment as described in Protocol 2.

Protocol 4: Safety assessment of M-LDF
From August to November, 2010, a total of 30 patients were recruited in this protocol. Exclusion criteria, blood collection and washing procedure were the same as for Protocol 2. The HepG2 cell (about 10 6 −10 7 ) at exponential growth phase was mixed with 250 ml of salvaged blood. Samples were taken for cell count, identification and culture as in Protocol 2, both before and after M-LDF treatment. In addition, nucleated cells were isolated from the salvaged blood (50ml before and 120 ml after M-LDF treatment, respectively), and injected subcutaneously into the left axilla of BALB/c Foxn1 nu mice for bioassay.
Cellular viability. HepG2 cells were concentrated before and after LDF treatment in Protocol 1. Cellular viability was determined by flow cytometry analysis. Cells were incubated with 15μM propidium iodide (PI, red, Sigma, USA) and 10 μM calcein acetoxy-methyl ester (calcein-AM, green, Dojindo, Japan) for 5 min and 30 min respectively with no light exposure, and then washed by PBS. Data acquisition was performed on Calibur (BD-FACS Aria, BD, USA) with FACS Diva 5.0 software and analyzed by FCS Express (De Novo Software, Thornhill, ON). PI-negative and calcein-AM-positive cells were considered as viable. After filtration, HepG2 cells stained with PI and calcein-AM were also examined with a laser confocal microscope (Nikon A1, Nikon, Japan) for morphology.
Blood cell count and morphology. The RBC count was determined by an automated blood analyzer (MINDRAY, BC-3000, China). The salvaged blood was lysed with ammonium chloride solution at room temperature, and centrifuged at 300×g for 5 min. Nucleated cells were counted under a light microscope. Morphology and bare nuclei of nucleated cells were identified with Wright's stain [15,16]. At least 100 cells per slide were counted.
To test the sensitivity of this cell separation method, 5 HepG2 cells labeled with AE1/ AE3-PE (Anti-pan cytokeratin, clone AE1 /AE3, Cat: 53-9003, eBioscience, USA) were added into 50 ml of the salvaged blood. The blood was lysed, and nucleated cells were isolated (see above) and smeared on a slide. Labeled cells were counted under a fluorescence microscope (DM4000B Leica-Ernst-Leitz, St. Wetzlar, Germany). After repeating counts 5 times, 24 cells in total of 25 cells (96%) were found in 5 samples.
Cell culture and identification. For cell culture, the nucleated cells were isolated by centrifugation as in Protocol 1, and by density gradient centrifugation as in protocols 2, 3 and 4 [15,16]. The salvaged blood was diluted 1:1 with sterile PBS. Then lymphocyte separation medium (LTS1077-1 TBD, China) was slowly added to the blood, and centrifuged at 300×g for 15 min at room temperature. The intermediate white layer was extracted and washed 3 times in PBS to isolate nucleated cells. The cells were cultured in the glucose-rich DMEM media (SH30022, Hyclone, USA) supplemented with 10% fetal bovine serum (SV30087.02 Hyclone, USA) at 37°C. The medium was refreshed every 24 hrs. After 15 days of incubation, the medium was removed, and the cells were fixed in 4% paraformaldehyde for 10 min, and stained with CK19-PE or AE1/AE3-PE in the last 3 protocols.
To assess proliferative capacity of malignant cells, nucleated cells from samples before (×10 7 cells) and after M-LDF treatment (×10 3 cells) in protocol 4 were injected respectively into the left axilla of immunocompromised BALB/c Foxn1 nu mice (4 week old, n = 60). Tumors that were 2-3 cm in diameter at inoculation site were removed for pathological and immunofluorescence examination. If no tumor was observed after one year, tissues at injection sites were then obtained and examined.

Statistical analysis
All data were analyzed by SPSS18.0 (Chicago, IL). All the quantitative data were examined for their distribution. Normal distribution data were expressed by the mean ± standard deviation, and one-way ANOVA was used to compare the difference before and after treatment. Nonnormal distribution data were expressed by a median (min, max) and the Kruskal-Wallis test was applied to compare differences among groups. Rates of tumor development in nude mice were expressed as a percentage, and examined with the Fisher exact test (protocol 4). P<0.05 was considered statistically significant. No viable cells or cell colonies were found in M-LDF-treated samples, except fragmented debris (Fig 1), suggesting cells were damaged when they passed through the filter. However, both viable cells (100%) (Fig 1) and cell colonies (37.5%)(S1 Fig) were found in LDF samples.

Protocol 3: Performance of M-LDF during tumor cell overload
The filtrate contained as high as 8.74×10 7 tumor cells (range from 0.26 to 294.00 ×10 7 ) / 250 ml of salvaged blood, M-LDF removed up to 4 log of nucleated cells. AE1/AE3 + cells were found in all untreated-samples, and invasive growth cells were found in 85% of untreated-samples (n = 7, Fig 6). They were absent after M-LDF filtration.   Fig 7A1), which was confirmed by pathological and immunofluorescent examinations ( Fig  7A2 and 7A3). Neither clones were found in culture and nor tumors developed in mice in the M-LDF-treated group (Fig 7B).

Discussion and Conclusion
For the first time we demonstrated that M-LDF is very effective in removing cells. It removed 4 log of nucleated cells and more malignant cells (protocol 1), with 96% RBC recovery. Furthermore, nucleated cells were destroyed as they passed through the M-LDF. No viable proliferative or epithelial cells were found after M-LDF filtration. Therefore, M-LDF may have potential for use during onco-surgery.
As reported, cancer cells were in salvaged blood [6] and regular LDF can only remove 2-3 log of malignant cells. Our results confirmed the previous studies [14,22]. Therefore, there is a risk of metastasis for using salvaged blood [23] and it is not safe for onco-surgery patients. Interestingly, meta-analysis [24] plus a clinical trial [25] show that IBS decreased the risk of metastasis. In our study, we found cancer cells and invasive growth cells in salvaged blood (Protocol 3), which caused malignant tumors in 67% of nude mice (Protocol 4). This discrepancy may result for two reasons. First, the seed requires the right microenvironment at the right time for survival [26]. Therefore, less than 0.01% of seeds formed metastatic lesions [26]. Second, invasive growth cells in hepatoma samples were identified as CD133 + , suggesting metastasis is likely caused by a specific subtype of cancer cells, which are scarce in the salvaged blood.
Whether malignant cells in IBS will affect prognosis remains unknown, however, it is prudent to remove malignant cells before transfusion. Our results show that the M-LDF is effective in eliminating the malignant cells (5 log) even with cell overloading (10 8 /L). We did not find viable malignant cells by flow cytometry, immunofluorescence or cell culture in vitro and in vivo after M-LDF filtration as reported [11,14,15,16]. Cells may be destroyed or lose   their adhesive characteristics, which is critical for metastasis formation, as they pass through the filter.
In conclusion, our results demonstrate that the M-LDF can effectively remove a variety of cells. Since no viable malignant cells were found in salvaged blood after M-LDF filtration, IBS has significant potential for clinical application in onco-surgery.