Evaluation of a New Method of Leukocyte Extractions from the Leukoreduction Filter

Abstract

This study’s purpose was to optimize the leukocyte extraction protocol and evaluate the efficacy of this new protocol. 12BioR blood filters were collected from Tehran Blood Transfusion Center. A twosyringe system and Multi-step rinsing were designed for cell extraction. The final purpose of this optimization was: (1) removed the residual RBCs, (2) reversed the leukocyte trapping process, and (3) remove the microparticles to obtain the high yield of target cells. Finally, Extracted cells were evaluated by Automated Cell count; Samples smear differential cell count, Trypan blue, and Annexin-PI staining. The results showed that on average 11.88 × 10⁸ ± 3.32 leukocytes recovered after indirect washing and that the mean count of granulocytes, lymphocytes, and Monocyte in this sample was 5.24 ± 2.18 × 10⁸, 5.57 ± 1.74 × 10⁸, and 0.56 ± 0.38 × 10⁸ respectively. Also, the mean percent of manual differential cell count after concentration was 42.81%, 41.80%, and 15.82% for granulocytes, lymphocytes, and monocytes respectively. Moreover, viability and apoptosis assay showed > 95% viability in mononuclear cells recovered from LRFs. It is concluded that the use of a double-syringe system and RBC and microparticles removal from leukoreduction filters lead to acceptable viable leukocyte count that can be used in in vitro and in vivo studies.

Introduction

In recent years, blood transfusion organizations have focused on implementing technological advances to increase the safety of the blood component. Leukoreduction is a processing step to improve safety in the routine preparation of blood components [1–6]. Leukocyte removal from the blood component typically prevents the possible side effects of these cells and their cytokines in the potential recipient [7–11].

Conventionally human blood is broadly used as a reliable source of primary cells for academic research and standard therapy. This source is typically obtained by venipuncture of volunteer participants [12–15]. Recently Leukocyte reduction filters (LRFs) are introduced as a reliable source of alternative human cells. Leukocyte reduction filters offer several advantages as a source of various cells for research: (1) Since 400–450 ml of blood is a normal donation (1 unit) therefore the particular advantage of LRFs is that they typically obtain many more cells from a volunteer participant in comparison to conventional methods. (2) Another advantage is that the filter cells are safe because the blood donated to blood banks is properly screened for the presence of several viral infections (e.g., HIV, HBV, and HCV). (3) In notable addition, LRFs are waste products and can be adequately provided by blood banks to researchers at little to no direct cost [16–30].

Recently, the last version of leukoreduction filters with a pore diameter of about 2–6 microns are developed with the efficacy of > 99% to remove leukocytes [2]. Nanofiber’s structure in leukoreduction filters is based on biodegradable polymers and Polybutylene terephthalate nanofiber is mostly used in the last version of leukoreduction filters. The cell isolation mechanism is a combination of physical, chemical, and biological processes. The physical construction of nanofiber-based materials results in increasing non-deformable cell retention [31–33]. In physical process separation, cells are separated from the blood by blocking, bridging, adhesion, and interception mechanisms [31].

Different subtypes of leukocytes for special purposes were extracted in different studies. Anti-tumor activity of extracted NK cells [34], allograft transplantation of CD34 + stem cells [35], defensin purification from extracted neutrophils [36], and dendritic cells (DC) obtained from extracted monocytes [37] were investigated in related studies. These studies used different human sources for cell extraction and the main limitation of them is the low number of cells extracted. The usage of some LRFs as a supply has been previously described and various methods have been used to reverse the trapping mechanisms and release cells with high yield [16–18]. Since LRFs are designed to leukocytes trap, releasing and extracting these cells is a challenging process. Moreover, Due to the short half-life of leukocytes, rapid extraction with the highest efficiency is critical to achieving the expected clinical and research benefits of these cells. This study focuses on evaluating a facilitate fast method for extraction of trapped leukocytes in LRFs as a valuable source.

Method and Material

Filter Supply

12 BioR blood filters (FRESENIUS KABI, AUSTRIA) were collected from Tehran Blood Transfusion Center (TBTC, Iran) after leukodepletion of 450 ml of whole blood from normal healthy donors. Each donated blood unit was screened according to the TBTC screening tests guideline. Cell extraction was started less than 4 h after the blood filtration.

Elusion Buffer Preparation

Chemical materials were obtained from Merck (Merck, Germany). Filter elution buffer was prepared according to Table 1. Buffer sterilization by 0.22 um filter (Membrane Solution, China) was performed before use. The pH of the filter elution medium was adjusted from pH 7.2 to 7.4 [16].

Table 1.

Filter elution medium preparation

Components		Concentration (%)	Molarity (mM)
D-PBS (Without MgCl2 and CaCl2)	Potassium Chloride (KCl)	7.5	0.20
	Potassium Phosphate Monobasic (KH2PO4)	13.6	0.20
	Sodium Chloride (NaCl)	5.84	8.00
	Sodium Phosphate Dibasic (Na2HPO4)	14.2	1.15
Na2–EDTA		0.16	5
Sucrose		2.5	73

Filter Washing

Briefly, a two-syringe system and Multi-step rinsing were designed to wash the filter. The goal was: (1) removed the residual RBCs, (2) reversed the leukocyte trapping process, and (3) remove the microparticles to obtain a high yield of target cells.

Direct Washing (RBC Removal)

In the first step, the exterior surfaces of the filters and tubing were wiped with 70% ethanol (Merck, Germany), and the following procedures were performed in a biosafety cabinet (Jal Tajhiz, Iran). Sterile scissors were used to cut the sealed ends of the tubing. RBCs are removed by rinsing the filter in the direction of blood filtration to minimum interference in the leukocyte extraction process. Tow 50 ml sterile syringe (Mina, Iran) Connected to both sides of the filter, and then pouch NaCl (0.9%) solution to direct position of filtration with slow and steady state. Each time pouched 25 ml solution blocked the input port, and wait for the filter plunging then suction the NaCl slowly with another sterile syringe. Each step was repeated four times. This sample is named Direct Sample Fig. 1A.

Fig. 1.

Filter washing schematic. Direct washing (A), Indirect washing (B)

Indirection Washing (Leukocyte Extraction)

In this step, we tried to release the leukocyte by reversing the direction of rinsing, mechanical forces to release the white blood cells, and suction. For this purpose, the two 50 ml sterile syringes were connected to both sides of the filter, then pouch cold elution Buffer to the opposite direction of filtration with slight pressure. Each time pouched 25 ml solution blocked the input port, waited for the filter to plunge, create a mechanical force for changing the pore size of the filter, and suctioned the elution buffer with another syringe with slight pressure for the release of semi-adherent cells. Each step was repeated four times. This sample was named Indirect Sample and saved for the next steps. Figure 1B

Microparticle Removal and Concentrating

To remove the platelets particles trapped in the filter as well as the cells microparticles created during the washing process, the solution obtained from indirect washing was centrifuged (Eppendorf, Germany) for 15 min at 800 g for 3 times, and a superior solution containing microparticles gently remove (this sampled was named Microparticle Sample) then added 50 ml elution buffer to precipitate and was centrifuged in 2000 g for 15 min, discarded the superior solution then added 20 ml elution buffer to precipitate. [38] This sample was named Concentrate Sample Fig. 2A.

Fig. 2.

Separation process. Concentrate sample (A), Ficoll up and Ficoll down samples (B)

Density Gradient Cell Separation

Cell suspension obtained from step 4 was added to density gradient medium (Inno-train, Germany) in a 2:1 volume ratio in a slow manner in order not to mix the layers. The tube was Centrifuged at 800×g for 20–30 min with the brake OFF. The mononuclear cells are harvested carefully by inserting the pipette directly through the upper layer. The harvested cells were washed twice with PBS and named Ficoll up Samples Fig. 2B. The precipitate cells resulting from this process were also named Ficoll down Samples Fig. 2B

Automated Cell Count

White blood cells were counted by an automatic multi-parameter XN-550 hematology analyzer (Sysmex Partec, Germany) based on light scattering to determine simultaneously absolute cell count of total white blood cells (WBC) and their subtypes. All reagents for the hematology analyzers (e.g., cell lysing reagents, sheath reagents) were supplied by Sysmex Company. Yields and purities of the leukocytes were analyzed and measured using the output results.

Differential Cell Count

Smear preparation and Giemsa staining were performed for each sample according to standard protocol. Briefly, a small drop of sample is placed on the glass slide, Second slide is pulled back into the blood drop and then pushed in another direction to the end of the smear slide, Slides fixed in methanol (Merck, Germany) for 5–7 min, Stained with Diluted Giemsa for 15–60 min, and evaluated using a light microscope (Olympus, USA) as follows: A drop of oil was placed on the stained slide and used the 100 X objective for counting. Each white cell seen was counted and recorded on a differential cell counter until 100 white cells were counted. Results are expressed as a percentage of the total leukocytes counted.

Apoptosis Assay by Flow cytometry

To measure apoptosis and necrosis of mononuclear cells which are mainly used for research, cells obtained from Ficoll up samples were incubated with FITC AnnexinV (Biolegend, USA) and propidium iodide (PI) (Biolegend, USA), and analyzed by flow cytometry within 1 h. Briefly, cells were suspended in Annexin V Binding Buffer at a concentration of ~ 2 × 10⁶ cells, 100 μl of suspension were transferred to a test tube and 5 μl of FITC Annexin V [1 mg/ml] was added, then 5 μl of propidium iodide [1 mg/ml] Viability Staining Solution was added and incubated for 15 min at room temperature (25 °C) in the dark finally 400 μl of Annexin V Binding Buffer was added to each tube. Cells that were propidium iodide (PI) negative and Annexin V negative are considered viable, PI negative and Annexin V positive cells are considered apoptotic, and cells that are positive to both PI and Annexin V are considered necrotic.

Viability Assay by Trypan Blue

Mononuclear cell viability was evaluated to confirm the apoptosis assay results. A sample of cells was mixed with an equal volume of trypan blue 0.25% (Biowest, France) and incubated at room temperature for 5 min. Then ten microliters from each sample were transferred to the hemocytometer slide (HBG, Germany). Stained and unstained cells were counted as dead and viable cells, respectively, using a light microscope (Olympus, USA).

Percent of viable cells was calculated using the following formula:

$$\text{No}.\text{of}\text{Viable}\text{Cells}\text{Counted}/\text{Total}\text{Cells}\text{Counted}\left(\text{viable}\text{and}\text{dead}\right)\times 100=\%\text{viable}\text{cells}$$

Statistical Analysis

Statistical analyses were carried out with SPSS software [SPSS 22, SPSS Inc, Chicago, IL]. Descriptive statistics together with graphics analysis were used to evaluate quantitative data of the sample and the measures. Data with normal distribution were analyzed by parametric test and data with abnormal distribution were analyzed by nonparametric test. A p-value of less than 0.05 indicates that a difference is significant.

Ethical Consideration

The project was approved by the Ethics Committee of the High Institute for Research and Education in Transfusion Medicine. (IR.TMI.REC.1399.010).

Results

Cells Extraction Efficiency

Automated Cell Count

Direct, Indirect, concentrated, Ficoll Up, Ficoll Down and Microparticle samples were counted and compared in terms of cell number and distribution of cell populations. The results showed that on average 11.88 × 10⁸ ± 3.32 leukocytes recovered after indirect washing and that the mean count of granulocytes, lymphocytes, and Monocyte in this sample was 5.24 ± 2.18 × 10⁸, 5.57 ± 1.74 × 10⁸, and 0.56 ± 0.38 × 10⁸ respectively. Also, data showed that on average 2.14 × 10¹¹ ± 0.98 RBC was obtained after direct washing while on average 0.04 × 10¹¹ RBC remained in the indirect sample, that this reduction is significant (P value = 0.001). Also, the result of platelet count showed that after microparticles removal platelet count significantly decreased in the concentrated sample compared to the indirect sample (94.662 ± 23.39 × 10⁸ versus 356.68 ± 102.54 respectively) (P = 0.031). The details were shown in Table 2 and the distribution graph of the different leukocyte populations was shown in Fig. 3.

Table 2.

Automated and differential cell count

Cell count		Automated	Differential
		Mean (No) ± SD	Mean (%) ± SD
Direct Volume: 100 cc	WBC (× 108)	0.00
	RBC (× 1011)	2.14 ± 0.98	Not diff
	Platelet (× 108)	23.11 ± 10.42
In Direct Volume: 100 cc	WBC (× 108)	11.880 ± 3.32
	RBC (× 1011)	0.04 ± 0.01
	Neutrophil (× 108)	5.24 ± 2.18	Not diff
	Lymphocyte (× 108)	5.57 ± 1.74
	Monocyte (× 108)	0.56 ± 0.38
	Platelet (× 108)	356.68 ± 102.54
Concentrate Volume: 20 cc	WBC (× 108)	10.97 ± 2.85
	Neutrophil (× 108)	2.778 ± 1.71	42.81 ± 2.53
	Lymphocyte (× 108)	5.208 ± 1.36	41.80 ± 2.41
	Monocyte (× 108)	0.678 ± 0.25	10.85 ± 1.76
	Platelet (× 108)	94.662 ± 23.39
Ficoll up Volume: 5 cc	WBC (× 108)	6.8105 ± 1.36
	Neutrophil (× 108)	0.6085 ± 0.27	17.47 ± 4.96
	Lymphocyte (× 108)	3.28 ± 1.02	72.21 ± 6.33
	Monocyte (× 108)	1. 35 ± 0.78	12.79 ± 1.23
	Platelet (× 108)	53.7115 ± 12.02
Ficoll down Volume: 5 cc	WBC(× 108)	1.858 ± 0.67
	Neutrophil (× 108)	0.958 ± 0.39	76.72 ± 5.35
	Lymphocyte (× 108)	0.3495 ± 0.16	23.72 ± 5.81
	Monocyte (× 108)	0.024 ± 0.011	1.50 ± .58
	Platelet (× 108)	4.3455 ± 2.56
Microparticles Volume: 100	WBC (× 108)	0.6 ± 0.17
	Platelet (× 108)	208.126 ± 129.23	Not diff

Fig. 3.

Automated cell counter Graph. Indirect (A), Concentrate (B), Ficoll UP (C), Ficoll down (D), Control (E)

Differential Cell Count

To confirm the results of automated cell counting the Direct, Concentrate, Ficoll Up, Ficoll Down and microparticle samples smear was stained (Fig. 4), and the percentage of different leukocyte populations was manually counted. Also, the mean percent of neutrophils, lymphocytes, and Monocyte was 42.81%,41.80%,and10.82% respectively in Concentrate samples, the mean percent of neutrophils, lymphocytes, and Monocyte was 17.47%, 72.21%, and 12.79% respectively in Ficoll Up samples, and mean percent of neutrophil, lymphocyte and Monocyte was 76.72%, 23.72%, and 1.5% respectively in ficoll down samples (Table 2).

Fig. 4.

Samples Smear. Concentrate (A) Ficoll up (B), Ficoll down (C), Direct (D), Microparticles (E)

Viability and Apoptosis Assay

Trypan blue staining showed that the mean viability of recovered mononuclear cells was 95.60% ± 2.88. Also, the flow cytometry assay showed that on average 95.84% ± 3.17 cells were viable (Annexin-/PI-), 3.45% ± 2.24 Cells were apoptotic (Anexin + /PI-), and 2.27% ± 1.14 cells was necrotic (Anexin + /PI +) Fig. 5.

Fig. 5.

Viability assay. Flow cytometry assay of one sample shown > 97% Annexin/PI negative cells (A) Mean percent of viability in Trypan blue, and flowcytometry assay (B)

Discussion

The results of this study showed that understanding the mechanism of cell entrapment and reversing this process can be useful in extracting leukocytes from leukoreduction filters. The results showed that the RBCs removal leads to effective filter cleaning and facilitates leukocyte subtype extraction during the density-gradient separation process. Also, this study tried to reverse the entrapment mechanism therefore adhesive cells were first isolated using cold elution buffer. Then, to overcome the blocking, bridging, and interception mechanisms the filter pore sizes and the position of the cells were changed by inducing mechanical force. Finally, the syringe suction leads to the collection of semi-adherent cells as well as released cells following mechanical force. moreover, the study shows that mechanical force and suction can leads to shear stress and cell microparticles creation that can interfere with the subtype leukocyte extraction in the next step. For this reason, microparticle removal was performed (Table 3).

Table 3.

Time comparison between current method and reference method

Washing method	Filter number	Time (Minute)
Current method	1	8.80
	2	9.20
	3	8.10
	4	11.30
	5	9.30
	6	7.20
	7	10.12
	8	8.20
	9	7.30
	10	7.90
	N	10
	Mean	8.7420
	SD	1.28404
Reference method [16]	1	24.50
	2	27.30
	3	25.50
	4	21.20
	5	23.90
	6	22.10
	7	26.20
	8	31.40
	9	25.10
	10	27.40
	N	10
	Mean	25.4600
	SD	2.90410

The EDTA-containing elution buffer for leukocyte extraction was introduced first time by Meyer [16]. Also, Abby K. Wegehaupt showed that the use of a single syringe system can lead to the effective extraction of leukocytes. [17] These two extraction methods are not repeatable and are a very time-consuming process. The present study is a modified method of the two mentioned studies, actually in this study, a two-syringe system was created for push and suction of elution buffer therefore balancing the pressure applied to the filters prevents the filter from bulking and re-trapped cells in the filter.

The results of automated cell counting showed that on average 11.88 × 10⁸ ± 3.32 leukocytes are recovered by a double syringe system used in this study. In this regard, Meyer investigated the 4 different types of filters and the average number of extracted leukocytes was 11.4 × 10⁸ which is comparable to the present study. However, there is a significant reduction in time spent by double syringe systems extraction compared to the Meyer method (8.74 min ± 1.28 versus 25.46 min ± 2.9 P value = 0.0001) (data not shown) [16].

Abby K. Wegehaupt’s study also showed that an average of 3.56 × 10⁸ leukocytes is obtained after washing with a single syringe system, which is significantly less than the current study, However differences in the type of filter and the different elution buffer can also be influential factors. [17].

In our study, a reduction in the percentage of granulocytes was observed. This result was similar to Meyer and Abby K. Wegehaupt's study. In this regard as Meyer pointed out: “The reduction of granulocytes in those filters is due to disintegration of parts of the granulocytes after adhesion to the filters, which in consequence can no longer be isolated from the filters”.

However, Sassani obtained a higher percentage of granulocytes in his study, which seems to be using dextran40 solution and uniform pressure using a poly static pump is more efficient in the neutrophil recovery. However, the cell count in the Sassani study is significantly less than in the present study, which indicates a higher efficiency of the two-syringe system in total leukocyte extraction. [18].

As Sassani and Abby K. Wegehaupt showed in their study our Sysmex graphs analyses revealed that the extracted leukocytes exhibited light scatter properties similar to those of lysed whole blood in other studies [21, 22], evidencing the presence of granulocytes, monocytes, and lymphocytes. Also, other graphs related to the Ficoll Up and Ficoll Down samples consistent with the percentage of cells obtained in manual and automated cell counting [18] Fig. 3.

Also, the analyses of stained smears reveal that all populations of leukocytes are identifiable in differential counting. However, because neutrophils were slightly deformed it seems that the study of neutrophil function (Chemotactic Activity, Phagocytic Capacity, and Bactericidal Capacity) following the extraction process can be useful [39].

Most studies used the method of density-gradient separation to remove RBCs and platelets from the filter leukocyte extracted mixture [16–30]. In this study, we used direction washing and light centrifuge speed methods for RBCs and microparticle removal respectively. The results of automated counting of RBCs showed a significant reduction of RBCs in the indirect sample, while the leukocyte count in the direct sample remains zero, which indicates the effectiveness of this method in removing RBCs. On the other hand, the platelet count after the light centrifuge speed process in the concentrated sample is significantly reduced compared to indirect samples. Because the filters used in this study filtered the whole blood these particles due released the attached platelets from the filter fibers and created microparticles during the extraction process. Also, the results of Giemsa staining of light centrifuge speed samples show micro-particles with different sizes and giant shapes. (Fig. 4) However, the purified RBCs and microparticles from leukocyte populations can use as a source for research work [17]. The results of survival and apoptosis tests indicate high survival of cells extracted by this method and it seems that the light centrifuge cycle can be useful in removing excess particles and apoptotic bodies.

Conclusion

It is concluded that the use of a double-syringe system, as well as RBC and microparticles removal from leukoreduction filters, leads to obtaining an acceptable viable leukocyte count that can be used in various in vitro and ex vivo studies. The main limitation of this study was the evaluation of one brand of filters and it is suggested that the comparison of different brands of filters be done in future studies.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.