Conceived and designed the experiments: JD AL I-BA-G TL SS. Performed the experiments: JD AL IA. Analyzed the data: JD AL I-BA-G SS. Contributed reagents/materials/analysis tools: TL SS. Wrote the paper: JD AL. Critically reviewed the manuscript and approved the final version to be published: I-BA-G TL SS. Fabrication of the acoustophoresis chip: AL.
The authors have read the journal's policy and have the following conflicts: One of the authors, Thomas Laurell, is engaged in Acousort AB, a start-up company from Lund University, which develops applications of acoustophoresis. The work presented in the current paper is not biased by commercial interest from Acousort AB.
Excessive collection of platelets is an unwanted side effect in current centrifugation-based peripheral blood progenitor cell (PBPC) apheresis. We investigated a novel microchip-based acoustophoresis technique, utilizing ultrasonic standing wave forces for the removal of platelets from PBPC products. By applying an acoustic standing wave field onto a continuously flowing cell suspension in a micro channel, cells can be separated from the surrounding media depending on their physical properties.
PBPC samples were obtained from patients (n = 15) and healthy donors (n = 6) and sorted on an acoustophoresis-chip. The acoustic force was set to separate leukocytes from platelets into a target fraction and a waste fraction, respectively. The PBPC samples, the target and the waste fractions were analysed for cell recovery, purity and functionality.
The median separation efficiency of leukocytes to the target fraction was 98% whereas platelets were effectively depleted by 89%. PBPC samples and corresponding target fractions were similar in the percentage of CD34+ hematopoetic progenitor/stem cells as well as leukocyte/lymphocyte subset distributions. Median viability was 98%, 98% and 97% in the PBPC samples, the target and the waste fractions, respectively. Results from hematopoietic progenitor cell assays indicated a preserved colony-forming ability post-sorting. Evaluation of platelet activation by P-selectin (CD62P) expression revealed a significant increase of CD62P+ platelets in the target (19%) and waste fractions (20%), respectively, compared to the PBPC input samples (9%). However, activation was lower when compared to stored blood bank platelet concentrates (48%).
Acoustophoresis can be utilized to efficiently deplete PBPC samples of platelets, whilst preserving the target stem/progenitor cell and leukocyte cell populations, cell viability and progenitor cell colony-forming ability. Acoustophoresis is, thus, an interesting technology to improve current cell processing methods.
Hematopoietic stem cell transplantation is a well-established therapy for haematological malignancies and other diseases
Administration of hematopoietic growth factors, which is required for effective progenitor cell mobilization from the bone marrow into the blood, has been reported to decrease platelet counts in healthy donors before apheresis
Microfluidic devices have shown great potential in the field of complex biofluids such as blood
The use of acoustic forces has recently emerged as a non contact and label free method of cell manipulation
The laminar flow also enables medium switching as the acoustic force can be used to move particles from one suspension into a medium flowing in parallel
The acoustic force on a particle is also size-dependent as the force is proportional to the volume. This can be utilized as a fractionation step as larger particles are transported to the pressure node faster than smaller ones. By tuning the acoustic power and the flow rate it becomes possible to sort out the larger particles
A combination of the medium switching and the size dependent fractionation mentioned above is used in the current study in which we investigated the performance of the acoustophoresis technique for platelet removal, aiming to efficiently deplete PBPC samples of intact platelets whilst preserving the composition and functionality of the collected target cells.
PBPC samples were obtained from 2 healthy donors and 8 patients for evaluation of acoustophoretic cell separation efficiency. For each PBPC sample, 6–12 sample portions of the separated target and waste fraction, respectively, were sequentially collected for analysis. (
PBPC sample | Target | Waste | |
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WBC (×103/µl) | 13.4 (7.2–23.3) | 6.1 (1.9–15) | 0.1 (0–0.4) |
PLT (×103/µl) | 86.5 (16–314) | 8.0 (0–61) | 31.0 (0–128) |
RBC (×106/µl) |
<0.1 | <0.1 | <0.1 |
Lymph (×103/µl) | 3.1 (1.9–13.7) | 1.5 (0.2–9.1) | - |
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WBC (×105) | 13.4 (7.2–23.3) | 7.8 (2.4–18.7) | 0.2 (0–0.8) |
PLT (×105) | 86.5 (16–314) | 10.2 (0–78) | 62.0 (0–256) |
RBC (×108) | - | - | - |
Lymph (×105) | 3.1 (1.9–13.7) | 1.9 (0.3–11.6) | - |
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WBC (%) | 97.8 (80–100) | 2.9 (0–22) | |
PLT (%) | 12.5 (0–54.6) | 89.0 (56.7–100) |
Abbreviations: WBC, white blood cells; PLT, platelets; RBC, red blood cells; Lymph, lymphocytes.
Data are presented as the median (range) of 10 samples.
The PBPC samples were diluted 1∶5 before cell count.
The RBC count was below the detection limit of 0.1 (×106/µl) given in the instrument manual
The addition of wash buffer to the acoustophoresis-chip and the difference in flow rate between the central outlet (20 µl/min) and the side oultlet (2×20 µl/min), gave a sample dilution so that the cells of 100 µl PBPC sample were distributed to 128 µl target sample (central outlet) and 200 µl waste sample (side outlet). Cell numbers were calculated accordingly.
The separation of WBC was not influenced by storage, as reflected by a WBC separation efficiency of ≥98% to the target fractions of the stored samples.
Cytospins from PBPC samples (n = 4) were prepared before and after acoustophoresis separation for evaluation of cell morphology (
PBPC sample | Target | Waste | |
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Granulo-/monocytes (%) | 70 (60–78) | 68 (50–73) | 12 (0–81) |
Lymphocytes (%) | 17 (16–28) | 20 (15–39) | 51 (0–91) |
Promyelocytes (%) | 11 (1–12) | 9 (6–20) | 1 (0–1) |
Blast cells (%) | 2 (1–2) | 2 (1–9) | 3 (0–8) |
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CD34 (%) | 2.5 (1.5–3.4) | 2.3 (1.0–3.1) | 1.6 (1.1–2.8) |
CD3 (%) | 16 (7.2–24) | 17 (9.7–24) | 38 (8.3–68) |
CD3/4 (%) | 9 (3.8–11) | 10 (4.1–12) | 20 (6.5–35) |
CD3/8 (%) | 6 (4.9–7.6) | 7 (5.0–8.1) | 17 (4.9–29) |
CD14 (%) | 43 (39–47) | 45 (40–47) | 23 (1.9–40) |
CD56 (%) | 1.9 (1.6–6) | 2.3 (1.5–5.7) | 1.4 (1.6–18) |
CD19 (%) | 2.8 (0.5–5.2) | 1.3 (0.3–2.2) | 1.0 (0.3–1.6) |
Viability (%) |
98 (97–99) | 98 (97–99) | 97 (95–98) |
Data are presented as the median (range) of 4 samples (sample no. 2–4 and 7).
Data are presented as the median (range) of 3 samples (sample no. 2–4).
Viability was determined using Propidium Iodide (PI) in flow cytometry analysis.
The median relative number of granulocytes/monocytes was 70% (range 60–78%) versus 68% (50–73%) and the median relative number of lymphocytes was 17% (range 16–28%) versus 20% (range 15–39%) in the PBPC samples and target fractions, respectively. The very small total number of leukocytes which were lost in the waste fraction comprised mainly lymphocytes (median 50%, range 0–91%).
Flow cytometry analysis of PBPC samples (n = 3) before and after acoustophoretic separation revealed a similar (p>0.1) distribution of leukocyte populations in the PBSC samples and target fractions. The median relative number of CD14 + cells was 43% (range 39–47%) versus 45% (range 40–47%), the median relative number of CD3+ cells was 16% (range 7.2–24%) versus 17% (range 9.7–24%) and the median relative number of CD34+ cells was 2.5% (range 1.5–3.4%) versus 2.3% (range 1.0–3.1%), in the PBSC samples and target fractions respectively. The few leukocytes that were detected in the waste fraction were mainly lymphocytes (median 38%, range 8.3–68%). The median viability, as measured by PI-negativity, was 98% (range 97–99%) and 98% (range 97–99%) in the PBPC samples and target fractions, respectively.
Cells from PBSC samples (n = 9) were plated in methylcellulose before and after acoustophoretic separation and evaluated for colony-forming ability (
The number of colonies/1,000 plated cells from 9 different PBPC samples and their corresponding target fractions are presented. The numbers of colonies are given for each single PBPC sample and as median (range) of 4–6 sequentially collected sample portions from each target fraction, respectively.
Analysis of platelet activation was performed on cells from PBPC samples (n = 5) before and after acoustophoretic separation, using the α-granule membrane protein CD62P (P-selectin) as an activation marker (
Analysis of platelet activation is based on the surface expression of P-selectin (CD62P). Representative dot plots and histograms from one of five samples are shown.
Platelet activation based on the surface expression of P-selectin (CD62P). The median relative numbers of single platelets expressing CD62P (
The excessive collection of platelets using current centrifugation-based PBPC apheresis presents a problem in PBPC processing, with a negative impact on the performance (yield and purity) of cell product manipulation, such as selection or depletion of specific cell subsets
Based on recent developments in the use of acoustic forces in cell separation
A possible negative influence of acoustophoresis on cell viability could be excluded as cell viability post sorting was not different from pre-sorting values. Furthermore, colony-forming ability, which is a sensitive assay for functional capacity of early hematopoietic progenitor cells, was not affected by the acoustophoresis procedure.
As previously described, RBC (7 µm in diameter, density 1.100 g/mL) and platelets (2–4 µm in diameter, density 1.058 g/mL)
The alpha-granule membrane protein P-selectin (CD62P) is a widely used marker for detection of platelet activation
Our findings of platelet activation induced by acoustophoretic cell separation were well in agreement with previous reports on platelet activation in standard centrifuge-based cell apheresis technology, where activation levels ranging from 10–30% of the collected platelets are seen with different cell separators. Also in PBPC apheresis, significant platelet activation may be induced as shown by the detection of CD62P positive platelets in the circulation of donors
The possible adverse effects, infusing PBPC products containing activated platelets has to be considered. Adhesion of CD62P expressing platelets to circulating leukocytes has been suggested to play an important role in the pathogenesis of inflammatory events
Currently used methods for platelet depletion of PBPC products are based on low speed, successive manual
In applications demanding high throughput, several separation channels can be operated in parallel, similar to the approach previously used in microchip-based blood washing where a device comprising eight separation channels in a bifurcation tree was used to increase the flow rate to 0.5 ml/min
In summary, we conclude that the acoustophoresis technique as described herein can be utilized to efficiently deplete PBPC samples of platelets, whilst preserving the target leukocyte fraction, cell viability and progenitor cell colony-forming ability. Acoustophoresis is, thus, an interesting technology to improve current cell processing methods, with the potential to efficiently deplete PBPC products of intact platelets for re-transfusion to the donor, while preserving the number and function of the collected PBPC in a future clinical apheresis setting.
Sampling of patient and donor PBPC products for use in the current study was approved by the Regional Ethical Review Board at Lund University. Written informed consent was obtained from all participants involved in the study.
Samples were obtained from PBPC products collected after standard mobilization treatment of healthy donors (G-CSF, Neupogen; Amgen, Thousand Oaks, CA, USA) and patients (protocol specific chemotherapy + G-CSF). Large volume leukapheresis was performed with a Cobe Spectra (Cobe, Lakewood, CO, USA), using the MNC program, version 7.0. On the day of the leukapheresis, or after a maximum of 24 hours storage, 1 mL of PBPC sample was removed from the collection bag, analysed using a standard automated hematology analyzer (Sysmex KX-21N, Sysmex, Kungsbacka, Sweden), and surplus cells were used for further experimentation.
The chip was designed to consist of a single acoustophoresis channel dividing at each end of the chip into a trifurcation with one central orifice and the laterally dividing branches joining to form a single side orifice, thus resulting in a chip with two inlets at one end and two outlets at the other (
The chip was equipped with three syringe pumps (WPI SP210iwz, World Precision Instruments, Sarasota, FL, USA) connected to 5 mL plastic syringes (BD Plastipak Luer-Lok Tip, Franklin Lakes, NJ, USA) to control the flow through the chip (
Two pumps connected to the chip outlets and set in withdrawal mode, and one pump connected to the chip central inlet, infusing wash buffer were responsible for flow control. The PBPC sample was connected to the chip side inlet and entered into the separation channel at a net flow rate set by the three pumps. A switching valve injector was inserted between the chip outlets and the outlet syringe pumps enabling the collection of outlet samples. The ultrasonic standing wave was generated by a piezoelectric transducer operated via an amplifier by a waveform generator setting the resonance frequency to 2 MHz.
The two pumps in withdrawal mode were set to give a flow rate through each of the three exit branches of 20 µl/min. The wash buffer was infused at a flow rate of 40 µl/min, leaving a net flow rate of 20 µl/min (10 µl/min in each side inlet branch) for the PBPC sample. The sampling loops were pre-filled with PBS in order to eliminate air flowing into the system when the loops were set to collection mode. The PBPC samples were diluted 1∶5 in PBS and connected to the side inlet of the chip. The operational parameters were tuned by visual inspection of the outlet trifurcation and set when leukocytes were optimally focused into the central outlet and platelets were distributed to the lateral branches (
From each PBPC sample, cytospin slides were prepared from target and waste fraction by centrifugation of 5,000–10,000 cells onto glass slides (5 minutes, 750 rpm. Labex Instruments AB, Göteborg, Sweden) onto glass slides. The cells were stained for 5 minutes with May-Grünwald solution (May-Grünwald, Eosin; Histolab Products, Västra Frölunda, Sweden), washed, fixed for 20 minutes in Giemsa (Histolab Products), washed and blotted dry. The cells were evaluated using a Nikon H 55OL microscope.
PBPC samples, target and the waste fractions were analysed for CD34−, CD3−, CD3/4−, CD3/8−, CD14−, CD56− and CD19-positive cells, using a four-colour flow cytometer (FACSCalibur; BD, Becton Dickinson, San Jose, CA, USA). Flurochrome-labelled monoclonal antibodies were used in different combinations as follows: anti-CD34 phycoerythrin (PE), anti-CD45 fluorescein isothiocyanate (FITC), peridinin chlorophyll protein complex (PerCP) or allophycocyanin (APC), anti-CD3 FITC, anti-CD4 APC, anti-CD8 PE, anti-CD56 PE, anti-CD19 PE or PerCPCy5, anti-CD14 PE or PerCP (all BD) and anti-CD15 PE (Becton Dickinson, Pharmingen, San Diego, CA, USA). For isotype controls, IgG1 (FITC) and IgG1 (PE) were used (BD). Propidium Iodine (PI, 100 ug/ml, Sigma-Aldrich) was used for dead cell exclusion. 50,000 events were acquired and data were analyzed with CellQuest software (BD).
PBPC samples, target and waste fractions were evaluated for their colony-forming ability in hematopoietic progenitor cell assays using standard methylcellulose culture (MACS HSC-CFU media complete with EPO, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Cells were plated at a concentration of 1,000 cells/mL and incubated for 14 days (Thermo Forma Steri incubator, 37°C, 5% CO2). Hematopoietic progenitor cell colony forming units (CFU) were enumerated based on standard criteria using an Olympus IX70 microscope.
Control samples were obtained from stored (7 days), blood bank platelet concentrates, which were prepared from pooled buffy-coats, suspended in platelet additive solution T-SOL™ (Baxter Healthcare corp., Deerfield, IL, USA) and depleted of leukocytes by filtration according to standard blood bank procedures. Samples were analyzed for platelet cell count (Sysmex KX-21N, Sysmex, Kungsbacka, Sweden). A number of 20×106 platelets from the control samples, the PBPC input samples, the target and the waste fractions were directly suspended in cold Cellfix (0.5%, BD biosciences). A number of 20×106 platelets from the control samples and the waste fractions were incubated with 20 µM TRAP (Thrombine Receptor Activator Peptide, stock solution 200 µM, Sigma-Aldrich), for 20 minutes in room temperature, after which they were suspended in Cellfix as described above. Control and acoustophoresis samples were washed twice (centrifuged at 1200×g, for 5 minutes) in PBS and re-suspended in 1 ml of DBA (PBS+0.2% BSA+0.1% Sodium Azid, Mallinckrodt Baker, Phillipsburg, NJ, USA). Flowcytometric analysis of CD61 (fibrinogen receptor, platelet specific), CD62P (P-selectin, α-granule membrane protein expressed on the surface of activated platelets) and CD45 (leukocytes) was performed. Fluorochrome-labelled monoclonal antibodies (all BD, Biosciences) were used as follows: anti-CD61 FITC, anti-CD62P PE and anti CD45 PerCP. Samples were analysed using a three-color flow cytometer (FACS-Scan, BD Biosciences). 10 000 events were acquired from each sample and data were analyzed with CellQuest software (BD) (
Data are presented as median (range) or as mean ± standard deviation (SD). Statistical analysis was performed using the Wilcoxon signed rank test for paired observations and the Wilcoxon log rank sum test for comparison of two sample groups. For the latter, a two-sided p-value of <0.05 was considered significant.
(AVI)