Advances in Bioscience and Biotechnology, 2012, 3, 630-642 ABB
http://dx.doi.org/10.4236/abb.2012.35082 Published Online September 2012 (http://www.SciRP.org/journal/abb/)
A simple and efficient method for the preparation of live
leukocytes from peripheral blood using the LeukoCatchTM
system
Ayumi Okamoto1*, Kosuke Torigata1*, Minami A. Sakurai1, Daisuke Okuzaki1,2, Hodaka Fujii3,
Toshinari Ohmine4, Daisaku Miura5, Shoic hi Kimura6, Norikazu Yabuta1, Hiroshi Nojima1,2#
1Department of Molecular Genetics, Osaka University, Osaka, Japan
2DNA-Chip Development Center for Infectious Diseases, Osaka University, Osaka, Japan
3Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
4Laboratory for Clinical Investigation, Osaka University Hospital, Osaka University, Osaka, Japan
5Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe, Japan
6Fukae Kasei Co., Ltd., Kobe, Japan
Received 16 June 2012; revised 27 July 2012; accepted 14 August 2012
ABSTRACT
Leukocytes from peripheral blood (PB) are of great
value for diagnosis as well as basic and clinical re-
search. However, no easy, centrifugation-free method
is available for the isolation of live leukocytes from
blood. We here develop a simple and quick method
for the purification of viable leukocytes from whole
blood using novel tools, named tLeukoCatch (tip-type)
or sLeukoCatch (syringe-type), which is equipped
with three Pall filter layers and captures leukocytes
but not red blood cells (RBCs) in whole blood. Indeed,
we showed that several million leukocytes per mL
(~35% of the recovery rate) were captured and eluted
from whole blood. The number of contaminant RBCs
decreased from several million to several thousand.
When mouse blood was hemolysed, almost all of the
lysed RBC fragments were removed by passage
through sLeukoCatch. Optical microscopic observa-
tion confirmed that the recovered leukocytes were
sufficiently healthy to respond to growth stimuli. Ef-
ficient leukocyte recovery was also confirmed for
hemolysed human blood. These results suggest that
the LeukoCatchTM system is useful for bedside diag-
nosis and basic research with blood samples.
Keywords: Novel Tool; Leukocyte Purification;
Centrifugation-Free; Diagnosis; PBMC
1. INTRODUCTION
Leukocytes play important roles in maintaining the de-
fense system of the body by regulating inflammatory
processes and initial responses to microbial infections.
Peripheral blood (PB) mononuclear cells (PBMCs) or
peripheral blood leukocytes (PBLs) is ideal cells for di-
agnosis as well as basic and clinical research. Cultured
leukocytes from patients have been useful materials in
diagnostic and therapeutic applications for various auto-
immune diseases and cancers. Indeed, the characteriza-
tion of PBLs from animal disease models and patients
has aided in the elucidation of the pathological mecha-
nisms of rheumatoid arthritis and other autoimmune dis-
orders [1-3].
With the rapid development of immunology in the past
decade, many new immunotherapeutic strategies have
been proposed. Immunotherapy based on antitumor im-
mune memory is a new modality for cancer treatment
that holds great promise for improving patient survival
with minimal toxicity. Other cancer therapies that have
drawn recent interest include cancer vaccination, which
improves the antitumor activity of T cells from cancer
patients; cell transfer therapy, which uses chimeric anti-
gen receptors to activate human T cells to attack tumor
cells; and the stimulation of other leukocyte types [4,5].
The isolation and characterization of PBLs and circulat-
ing tumor cells (CTCs) also could contribute to the early
detection of cancer, selection of effective treatment, and
estimation of prognosis [6]. Accordingly, effective tech-
niques for handling PBLs and CTCs are needed.
Leukocytes can be separated from whole blood by
rapidly sedimenting the red blood cells (RBCs) by cen-
trifugation. The resulting supernatant contains mainly
leukocytes, platelets, and a few RBCs. The most com-
mon and classical method for this process, known as
Ficoll 400 (or Percoll) gradient centrifugation, is based
*These two authors contributed equally to this work.
#Corresponding author.

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642 631
on methodology established through the pioneering work
of Bøyum [7]. A routine one-step centrifugation proce-
dure splits the blood cells into two major fractions: after
centrifugation, mononuclear cells are located on the top
of the separation fluid, whereas erythrocytes and granu-
locytes have sedimented to the bottom [8].
Modifications of the density gradient centrifugation
method and medium formulation have improved the
quality of isolation and analysis of highly purified leu-
kocytes from PB [9].
Some of these techniques are now available as com-
mercialized kits, such as Ficoll-Hypaque [10]. Neverthe-
less, these leukocyte separation procedures require labo-
ratory-based professional and time-consuming proce-
dures using instrumentation that is not routinely available
at the bedside or in a common clinical facility. Moreover,
their protocols are too complex to apply to an automated
machine to treat hundreds of samples simultaneously.
Novel methods to replace the gradient centrifugation
method have been reported, which are based on immuno-
magnetic separation techniques. These methods are char-
acterized by their specificity of targeting PBLs with a
simple procedure [11,12]. However, they yield a biased
population of leukocytes because only a limited number
of leukocytes associate with the antibodies that are at-
tached to the magnetic beads. Thus, a quick, easy, inex-
pensive, nonbiased, and efficient method for the purifica-
tion of PBLs from RBC extracts is required to expand
the utility of leukocytes as a diagnostic target.
Serum immunoglobulin and RBC hemoglobin com-
prise most of the protein in blood cells and must be re-
moved before the protein components in leukocytes can
be analyzed. New devices for trapping and removing
PBLs, such as leukocytopheresis [13,14], granulocyte-
monocyte-apheresis [15] and Adacolumn [16], have been
developed and are used in clinical practice for the treat-
ment of inflammatory bowel disease and rheumatoid
arthritis [17]. A less expensive filtration device, called
LeukoTrap (Pall, Leukosorb B Medium, LKB-3R), is
available that efficiently captures leukocytes but not
RBCs or platelets. This tool has been successfully used at
the bedside [18] to prepare leukocyte-depleted platelet
concentrates from whole blood [19-21]. These devices
are mainly intended to remove PBLs and purify RBCs,
and the captured PBLs are discarded.
By utilizing previous filtration systems (such as Leu-
koTrap) in an opposite manner, we assumed that we
could obtain an efficient device to capture and recover
sufficiently pure PBLs, with minimum contamination by
RBCs. We present a novel technique, called the tLeuko-
Catch (tip-type) or sLeukoCatch (syringe-type) system,
which allows the one-step separation of PBLs from
RBCs in blood samples. The basic design of this system
is similar to that of our original LeukoCatchTM tool [22],
which consisted of a layer of filters held at the bottom of
a tip or a syringe that captured leukocytes but not RBCs
from a blood sample. Notably, the recovered PBLs are
viable and can be efficiently expanded in a culture dish;
this feature may expand the use of PBLs as research and
diagnostic targets. The results from the present study
indicate that tLeukoCatch and sLeukoCatch provide a
readily available, easily processed, and convenient source
of functional human PBLs for use not only in basic re-
search of the human immune system but also in clinical
research for the diagnosis of PBL-related diseases.
2. MATERIALS AND METHODS
2.1. Cell Culture
Human prostate cancer PC-3 cells (CRL-1435: ATCC,
Manassas, VA) and mouse prostate cancer TRAMP-C1
cells (CRL-2730: ATCC, Manassas, VA) were main-
tained in Dulbecco’s modified Eagle’s medium (DMEM,
Sigma-Aldrich Co., Milwaukee, WI) with 5% or 10%
fetal bovine serum (FBS, HyClone, Logan, UT) supple-
mented with 100 U/mL penicillin, 100 μg/mL strepto-
mycin, 10% Nu-Serum IV (BD Biosciences), and 10 nM
dihydrotestosterone (DHT) in a CO2 (5%) incubator at
37˚C. Cells in the logarithmic growth phase were used
for experiments.
2.2. Application of sLeukoCatch to PC-3
Samples
PC-3 cells (1.7 × 106 cells/experiment) suspended in 2
mL of medium were passed through (sucked-and-poured)
the sLeukoCatch five times. The captured PC-3 cells
were eluted forcefully each time by fresh medium for
five times. The five fractions of eluted PC-3 cells (first to
fifth eluted fractions) were assessed for their viability by
the trypan blue exclusion test. An equal number (9.0 ×
104 cells/mL) of nontreated PC-3 cells and the first frac-
tion of eluted PC-3 cells were seeded separately in each
plate with DMEM. The cell growth rates over 6 days
were measured by the trypan blue exclusion test.
2.3. Flowcytometry
Leukocytes (2.0 × 106 cells/experiment) eluted from
sLeukoCatch were stained by the CycleTESTTM PLUS
DNA Reagent Kit (BD Biosciences) according to the
manufacturer’s instructions. Samples were divided into
four tubes. Cells were counted four times for each sam-
ple to obtain the mean value with a FACS Calibur and
CellQuest software. This experiment was independently
repeated twice.
To examine the viability of mouse peripheral lympho-
cytes eluted from sLeukoCatch, eluates were stained with
propidium iodide (PI) and analyzed by flowcytometry.

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642
632
To examine the function of eluted mouse peripheral
lymphocytes, eluted cells were stimulated with 5 µg/mL
anti-CD3 antibody (145-2C11, BD Biosciences), 5 µg/mL
anti-CD28 antibody (Clone 37.51, BD Biosciences), and
5 ng/mL mouse IL-2 (R&D Systems) for 24 hours. Cells
were stained with fluorescein isothiocyanate-conjugated
anti-CD3 and phycoerythrin-conjugated anti-CD69 (H1.2
F3, BD Biosciences) antibodies and analyzed with FACS
Calibur. To confirm that there was no difference in size
and granularity between nontreated diluted mouse pe-
ripheral blood (mPB) and the first fraction of mouse
lymphocytes eluted from sLeukoCatch, both samples
were stained with PI and analyzed with FACS Calibur.
2.4. Application of sLeukoCatch to
Hemolysed Mouse Blood
To prevent blood clotting, a heparin-coated 1 mL syringe
was prepared by passing ~0.05 mL of heparin sodium
(1000 U/mL Novo-Heparin; Mochida Pharmaceutical
Co., Ltd., Tokyo, Japan) through a syringe several times.
A mouse was sacrificed by the injection of pentobarbital
sodium (Somnopentyl, Kyoritsu Seiyaku, Tokyo, Japan),
and its blood (0.5 mL) was obtained with the heap-
rin-coated 1 mL syringe. This experiment was performed
with the lowest degree of neuropsychological sensitivity,
pain, suffering and distress, which have been ensured by
the employment of approved methods of anaesthesia and
animal handling and by the application of specific anti-
pain drugs whenever required. The collected blood was
transferred into a collection tube with the heparin-coated
syringe and diluted with 1 mL of phosphate-buffered
saline (PBS) without Ca2+/Mg2+ ions [PBS(-)]. Next, 3.5
mL of RBC lysing buffer (Sigma-Aldrich Co., Milwau-
kee, WI) was added to the tube, for a total volume of 5
mL. The blood mixture was drawn into a 10 mL sLeu-
koCatch syringe. To encourage hemolysis, the tube was
gently turned upside-down by hand once per second for 5
minutes.
Lysed RBC ghosts were evacuated through gentle and
slow movement (over 90 seconds) of the syringe piston.
It was anticipated that most of the leukocytes would be
captured by the filters during this process. Finally, 1 mL
of PBS(-) or RPMI + 10% FBS was sucked into the
sLeukoCatch syringe, incubated for 5 seconds, and then
forcefully flushed back out into a plastic collection tube
for subsequent analysis.
2.5. Application of sLeukoCatch to Human
Blood Samples without Hemolysis
Blood samples (2 mL per tube) drawn from one of the
authors (H.N, male, age 60) into vacuum blood collec-
tion tubes containing 1 mM EDTA were sucked into the
sLeukoCatch and poured back into the tube. After re-
peating this process 5 times, supplemented DMEM (2
mL) in a fresh container was sucked into the sLeuko-
Catch and poured back into the container to evacuate the
captured cells. This process was repeated various times.
The collected cells were incubated in 5% CO2 at 37˚C.
2.6. Application of sLeukoCatch to
Hemolysed Human Blood Sa mples
Blood samples collected from one of the authors (H.N,
male, age 60) into heparin vacuum blood collection tubes
(5 mL per tube) were passed through the sLeukoCatch
syringe 5 times. Then, 5 mL of RBC lysing buffer in a 15
mL plastic tube was sucked into the sLeukoCatch sy-
ringe to induce hemolysis. The tubes were gently turned
upside-down by hand once per second for 5 minutes at
room temperature. The hemolysed sample was gently
flushed into the same tube through a slow movement of
the syringe piston over 2 minutes. Subsequently, the
captured leukocytes were rinsed once with 5 mL of PBS(-)
to remove the RBC ghosts. Fresh culture medium (1 mL)
in a 15 mL plastic tube was sucked into the sLeukoCatch
syringe. The captured leukocytes were eluted out into the
same tube by strongly pushing the syringe piston.
2.7. Hematologic Analysis of Blood Samples
The numbers of leukocytes and RBCs were counted with
an LH 750 hematology analyzer (Beckman Coulter,
Fullerton, CA) immediately after the fresh blood sample
was hemolysed and treated with or without the sLeuko-
Catch. Triplicate samples were counted.
2.8. Ethical Permission
All animal experiments were approved by the animal
care and use committee of the Research Institute for Mi-
crobial Diseases, Osaka University (#BikenA-H19-36-0).
All experiments respected the welfare of animals and
have been conducted strictly according to the legal and
ethical requirements demanded by law. Details of animal
welfare and steps taken to ameliorate suffering are in-
cluded in the methods section of the manuscript.
All of the experiments utilizing human blood samples
were performed after obtaining a written informed con-
sent with the approval of the ethics committee of Osaka
University (#303).
3. RESULTS
3.1. Structure and Use of the tLeukoCatch
and sLeukoCatch Systems
We previously reported that LeukoCatchTM, equipped
with five layers of filters (Pall, Leukosorb B Medium,
LKB-3R), could be used to isolate whole protein extracts

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642 633
from leukocytes in human blood with minimum con-
tamination by hemoglobin from RBCs [22]. We sur-
mised that this system would also be useful for the
preparation of live leukocytes with similar purity. For
this purpose, we fabricated a tool containing three layers
of Pall filters at the bottom of a plastic container, such as
a pipette tip (tip-type; tLeukoCatch) or medical syringe
(syringe-type; sLeukoCatch) (Figure 1A). We limited
the number of filters to three because there was no dif-
ference in the efficiency of leukocyte capture when more
than three filters were used in a similar LeukoCatchTM
instrument [22]. Also, a three-filtered tool is easier to
handle because blood movement through the filters is
less sticky.
The handling process of tLeukoCatch or sLeukoCatch
was simple and easy (Figure 1B). First, the lid of a vac-
uum collection tube (2 mL of blood per tube) was opened.
The blood sample inside was sucked up through the fil-
ters by moving the Pipetman’s plunger button (for tLeu-
koCatch) or syringe piston (for sLeukoCatch) upward
(Figure 1B(i); steps a and c) and downward (steps b and
d) five times by hand. Then, the tip or syringe needle
was immersed into a fresh container (e.g., a 15 mL plas-
tic tube) containing supplemented DMEM, and 2 mL of
the DMEM was sucked up into the upper-side space of
the filter (Figure 1B(ii); step e) to wash the filters. The
plunger button or piston was pushed down to evacuate
the captured leukocytes into a fresh tissue culture plate
(Figure 1B(ii); step f). This elution process may be re-
peated many times to increase the recovery rate of the
captured leukocytes. The collected leukocytes may be
stored frozen in the presence of 7% dimethyl sulfoxide
(DMSO) or transferred to a tissue culture plate and
maintained at 37˚C in a CO2 (5%) incubator.
Figure 1. Schematic drawings of the LeukoCatchTM system and its usage. (A) Structure of the
LeukoCatchTM system, in which the three-layered stacked filters are installed between the stoppers
at the bottom of a syringe. An enlarged view of the stacked configuration of the filtering system is
shown in an inset; (B) Schematic drawing of a protocol for the preparation of live leukocytes (or
PBMCs) from a 2 mL blood sample. The protocol consists of two procedures: capture of leuko-
cytes by the LeukoCatchTM filters (i) and leukocyte elution by a cell culture medium (e.g., DMEM)
(ii). Thin horizontal arrows show the flow of the procedure (from step a to step j). Thick vertical
arrows indicate the multiple movements of the piston to allow the blood (i) or elution medium (ii)
to pass through the stacked filters in the LeukoCatchTM system. In each step, the piston was moved
upwards (steps a, c, e, g, and i) or downwards (steps b, d, f, h, and j) by hand. The whole procedure
may be completed within 3 minutes.

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642
634
3.2. sLeukoCatch Enabled the Capture and
Recovery of Live Tissue Cultured Cells
Before applying the LeukoCatchTM system to blood sam-
ples, we first tested its capture rate and recovery effi-
ciency using tissue cultured cells. We tested the ability of
the sLeukoCatch to collect human prostate cancer PC-3
cells. Briefly, PC-3 cells were passed through the filters
by gently pushing and pulling the syringe piston five
times. After passing the PC-3 cells through the sLeuko-
Catch five times, about 82% of PC-3 cells were captured
(Figure 2A). PC-3 cells that were captured on the filters
were eluted forcefully by passing the DMEM medium
into a test tube (1 - 5 times by serial passage). This pro-
cedure resulted in an elution recovery rate of 23% of
captured PC-3 cells at the first passage (first fraction)
(Figure 2B). The cells in the first eluted fraction were as
healthy as nontreated PC-3 cells, as shown by their simi-
lar cell growth rates (Figure 2C) and images under a
microscope (Figure 2D).
Next, we used the tLeukoCatch to collect mouse pros-
tate cancer TRAMP-C1 cells using a Gilson Pipetman.
The capture of TRAMP-C1 cells by tLeukoCatch was
more efficient (86%) than that by sLeukoCatch (Figure
S1A); however, the elution rate (11% at 1st elution) was
much lower than that of sLeukoCatch even after the 5th
elution (Figure S1B), which can be probably attributed
to the weak pipetting force of the Gilson Pipetman. Be-
cause tLeukoCatch was originally designed to treat large
numbers of blood samples, each with a volume of less
than 0.5 mL on an automated machine (see Discussion),
we refrained from using tLeukoCatch for manual ma-
ipulations and used sLeukoCatch hereafter. n
Figure 2. sLeukoCatch allow the capture and recovery of live PC-3 cells. (A) PC-3 cells were
passed through an sLeukoCatch five times. NT, nontreated. Bar graphs represent the number of
PC-3 cells (flow-through) that were not captured after being passed through the sLeukoCatch sys-
tem five times; (B) PC-3 cells were passed through the sLeukoCatch system five times. Captured
PC-3 cells were eluted by fresh medium (sLeukoCatch system); (C) Equal numbers of nontreated
PC-3 cells and PC-3 cells from the first eluted fraction (1st) were seeded in separate plates with
DMEM. Bar graphs represent the numbers of nontreated PC-3 cells and PC-3 cells in the 1st eluted
fraction that were assessed by the trypan blue exclusion test over 6 days. Each data point represents
an average of three independent experiments; (D) Cell images of nontreated PC-3 cells and PC-3
cells in first eluted fraction (1st) on day 4. Bar = 50 μm.

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642 635
3.3. sLeukoCatch Allows the Capture of
Leukocytes from Human Peripheral Blood
To isolate viable cells from human peripheral blood
(hPB) by sLeukoCatch, a blood sample (2 mL) in a vac-
uum blood collection tube containing 1 mM EDTA was
aspirated into the sLeukoCatch by hand and blown out
gently five times to capture the leukocytes (see Figure
1B(i)). Cells that were captured on the filters were eluted
forcefully into a test tube containing 2 mL of fresh
DMEM and 10% inactivated FBS. This process was re-
peated five times to maximize the number of collected
cells (Figure 1A(ii)). Because the movement of the sy-
ringe piston was smooth even when whole blood was
used, we did not dilute the blood sample by PBS(-).
Nearly 35% of the leukocytes or white blood cells
(WBCs) were captured in the filters of the sLeukoCatch,
as judged by the difference between the values of the
nontreated (NT) and pass through (PS) samples (Figure
3A). The captured leukocytes were eluted efficiently by
successive elution steps (Figures 3A and B; see columns
1st to 5th). Almost all of the leukocytes were eluted into
the medium by repeating the three elution processes
(Figure 3B). Because the fourth and fifth eluted samples
contained very small numbers of residual leukocytes that
were hard to detect precisely, the ratio of eluted leuko-
cytes from the filters in Figure 3B shows a value of
>100%. From a practical perspective, the fourth and fifth
elution steps may be skipped in future uses of the tech-
nique.
Similar distributions of neutrophils, lymphocytes, and
monocytes, as detected by a hematology analyzer, were
observed during the nontreated and elution processes,
which suggested the nonbiased capture of these leuko-
cytes (Figure 3C). The proportion of platelets included
in the first to third eluted fractions were largely reduced,
which suggested an efficient separation of platelets from
the eluted leukocytes (Figure 3D). The number of RBCs
decreased by two orders of magnitude, from 5.2 × 106
cells (nontreated hPB) to 68,000 cells (third eluted frac-
tion) in 1 μL of hPB (Figure 3E). Thus, the decreasing
number of RBCs in the eluted samples increased the ratio
of leukocytes to RBCs (Figure 3F). Microscopic obser-
vation clearly showed the successful separation of RBCs
from leukocytes in the eluted fraction (Figure 3G).
Taken together, these results show that intact leukocytes
in hPB samples can be captured and eluted without any
osmotic stresses in a procedure that requires handling for
only a few minutes.
3.4. sLeukoCatch Provides RBC-Free
Leukocytes When Used for
Hemolysed Mouse Blood
The hemolysis of RBCs can be a convenient process to
remove RBCs efficiently. However, leukocytes are also
hemolysed, although much less efficiently, when they are
immersed in hemolysis buffer for a long time. Thus, it is
important to determine the proper timing at which RBCs
but not leukocytes are hemolysed. We prepared mouse
peripheral blood (mPB) and assessed the timing of
hemolysis using various ratios of blood to hemolysis
buffer. A ratio of 1:9 yielded almost complete hemolysis,
as judged by the clear blood solution obtained after gently
turning the tube upside-down once per second by hand at
room temperature for 5 minutes (data not shown).
After 5 minutes, when almost all of the RBCs were
hemolysed, we pushed down on the sLeukoCatch syringe
piston slowly over 90 seconds to promote the capture of
leukocytes but not RBC ghosts by the sLeukoCatch fil-
ters (Figure 4A). This process removed the serum and
hemoglobin. We sucked 1 mL of PBS(-) or RPMI + 10%
FBS into the sLeukoCatch syringe, incubated it for 5
seconds, and then flushed the solution back out by
forcefully pushing the syringe piston to retrieve the cap-
tured leukocytes. Almost all of the lysed RBC fragments
were removed through this process.
We tested whether the eluted leukocytes were viable.
As shown in Figure 4B, a sizable population of lym-
phocytes was easily detected. The viability of the eluted
cell population was more than 95% and was comparable
to that of the hemolysed (but not eluted) cell population.
These results show that treatment with sLeukoCatch did
not markedly affect viability. To assess the functionality
of the eluted mouse lymphocytes, the eluted cells were
stimulated with anti-CD3 antibody and anti-CD28 anti-
body plus recombinant mouse IL-2 for 24 hours. The
stimulated lymphocytes became larger (Figure 4C), sug-
gesting their activated phenotype. Moreover, CD3 (+)
cells showed markedly increased expression of CD69, an
activation marker of T cells (Figure 4D). These results
show that the lymphocytes purified with sLeukoCatch
were functional.
We tested whether there was a difference in size and
granularity between nontreated mPB cells and cells in
the eluted fraction. A sizable population of lymphocytes
was easily detected in both samples by flowcytometry
(Figure 4E). No change in the size and granularity dis-
tribution of the lymphocytes was found after using the
LeukoCatchTM system. These results suggest that the
eluted fraction contained the whole population of leuko-
cytes.
3.5. sLeukoCatch Retrieves RBC-Free
Leukocytes from Hemolysed Human Blood
We applied the hemolysis technique to hPB samples.
Human blood was more resistant to the hemolysis buffer
than mouse blood when it was diluted as described above

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642
636
Figure 3. sLeukoCatch allows the efficient capture of RBC-free leukocytes from human peripheral
blood (PB). (A) Numbers of leukocytes in the nontreated (NT), pass-through (PS), and first (1st) to
fifth (5th) eluted fractions, respectively, are shown. Data were obtained with an LH1511 cell counter;
(B) Ratio of eluted versus captured leukocytes before (NT) and after (PS and fractions 1st to 5th) pas-
sage through the sLeukoCatch. Accumulated percentages of leukocytes eluted from sLeukoCatch at
each elution step (1st to 5th) are shown, revealing an almost complete recovery; (C) Percentage of
neutrophils (NE), lymphocytes (LY), monocytes (MO), and others in the NT, PS, and eluted fractions
1st + 2nd + 3rd after use of the sLeukoCatch; (D) and (E) Numbers of platelets (PLTs); (D) and red
blood cells (RBCs); (E) in the PB, PS, or eluted fractions 1st to 3rd are shown. Cell numbers were
counted with an LH1511 cell counter; (F) Relative RBC versus leukocyte content in the PB, PS, and
eluted fractions 1st to 3rd. Bar graphs for eluted fractions 4th and 5th are not shown because few RBCs
were found; (G) Microscopic images of PS (left panels) and eluted fraction 1st (right panels) at 20×
and 40× magnifications. Regions displayed as enlarged images (40×) are encircled in the 20× images.
Arrowheads indicate putative leukocytes. Bar = 25 μm.

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642 637
Figure 4. sLeukoCatch obtains live and functional leukocytes without RBC debris from hemolysed
mouse peripheral blood (mPB). (A) Schematic protocol for the preparation of hemolysed mouse blood
from 10 mL of diluted blood sample: (i) hemolysis of mouse blood, (ii) capture of leukocytes by the
sLeukoCatch filters, (iii) rinse, and (iv) leukocyte elution by cell culture medium. Thin horizontal ar-
rows show the flow of the process (from step a to h). Thick vertical arrows indicate the movements of
the syringe piston to allow the blood (ii) or RPMI (iv) to pass through the stacked filters in the sLeuko-
Catch system. In each step, the piston was moved upwards (steps a, c, e, and g) or downwards (steps b,
d, f, and h) by hand; (B) Flowcytometric analysis of an eluted sample. The eluted fraction from mPB
was stained with propidium iodide (PI) and analyzed by flowcytometry, which identified leukocytes by
their FSC and SSC properties as basic “lymphocyte gating”; (C) Microscopic images of eluted leuko-
cytes after 24 hours with or without stimulation by anti-CD3 antibody, anti-CD28 antibody, and IL-2; (D)
CD69 expression on nonstimulated or stimulated T cells was analyzed by flowcytometry. CD3 (+) cells
were gated for analysis of CD69 expression; (E) Histogram representing the lymphocyte size and
granularity. Non-treated mPB and sLeukoCatch-mediated eluted fraction from mPB were analyzed by
flowcytometry, which identified leukocytes by their FSC and SSC properties as lymphocyte gating.

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642
638
(data not shown). Thus, we performed hemolysis directly
in the sLeukoCatch syringe (Figure 5A). Briefly, blood
samples collected into heparin (5 mL per tube) were
passed through the sLeukoCatch syringe five times to
remove most of the RBCs. Then, 5 mL of hemolysis
buffer in a 15 mL plastic tube was sucked into the sLeu-
koCatch syringe. Hemolysis of the residual RBCs was
induced by gently turning the tube upside-down by hand
every second for 5 minutes at room temperature. The
hemolysed blood sample was gently flushed into the
same tube through a slow movement of a syringe piston
over 90 seconds. Subsequently, the captured leukocytes
were gently rinsed once by using 5 mL of PBS(-) to re-
move the RBC ghosts. Finally, fresh culture medium (1
mL) in a 15 mL plastic tube was sucked into the sLeuko-
Catch syringe, and the captured leukocytes were eluted out
into the same tube by forcefully pushing the syringe piston.
We found that the approximately 77% of the nontreated
hPB sample (~6000/μL) was in the PS fraction. The loss
of leukocytes during the PBS(-) rinse process was small
(<17%), as judged by the hematologic analysis that was
performed immediately after elution (Figure 5B). Nota-
bly, the captured leukocytes were almost completely
recovered in the first and second eluted fractions (Figure
5B). Interestingly, the eluted leukocytes were almost free
of RBCs and their ghosts, according to the hematologic
analysis (Figure 5C) and microscopic observation (data
not shown). Indeed, the ratio of leukocytes to RBCs in-
creased markedly in the first (76×) and second (65×)
eluted fractions (Figure 5D). These results indicate that
sLeukoCatch retrieved about 10% RBC-free leukocytes
in 2 mL from 5 mL of hemolysed hPB. This number may
be large enough to perform further analysis for diagnos-
tics as well as basic and clinical research.
4. DISCUSSION
The successful identification of biomarkers for PBLs in
infectious disease, autoimmunity, and cancer is critical
not only for diagnosis but also for tracking drug effects at
early time points in clinical trials, which may prevent
costly late trial failures. Although popular, the Ficoll
density centrifugation method is laborious and inaccurate.
Therefore, a simple technique for the preparation of
PBLs from whole blood has been eagerly awaited.
In the present study, we report a simple, rapid and ef-
ficient preparation technique called the LeukoCatchTM
system that enables the recovery of live leukocytes from
mouse and human blood samples through blood handling
for only a few minutes (Figure 1). Tissue cultured cells
were effectively captured and recovered with this system,
yielding healthy cells with a normal growth rate (Figure
2). When applied to human blood samples, sLeukoCatch
effectively captured and successfully separated leuko-
cytes from RBCs (Figure 3). A dramatic removal rate of
RBC debris was observed in the recovered leukocytes
from mouse blood samples when hemolysis was per-
formed before elution (Figure 4). This procedure will be
useful for samples in which hemolysis is not toxic to the
required leukocytes.
We prepared two kinds of modified LeukoCatchTM
systems: the tLeukoCatch and the sLeukoCatch. Because
Gilson’s Pipetman is manually handled, its evacuation
ability is not strong enough to pass through all blood
samples smoothly. Thus, the primary utility of the tLeu-
koCatch system is as an automated system, in which the
evacuation power can be mechanically controlled, with
large numbers of blood samples. In contrast, sLeuko-
Catch is useful when used for a small number of samples
at the bedside. The tLeukoCatch and sLeukoCatch sys-
tems will contribute to the preparation of leukocytes or
immune-related cells that can subsequently be analyzed
by various means. It is particularly convenient for bed-
side use because it can be easily handled by those who
are not familiar with medical sciences.
As one potential application, LeukoCatchTM may be
helpful for improving the efficiency of cancer immuno-
therapies [4]. The basic strategy for tumor immunother-
apy involves the stimulation of patient leukocytes, such
as the cytotoxic T lymphocytes (CTLs), natural killer
cells, or lymphokine-activated killer cells (LAKs), and
their subsequent activation by putative tumor-specific
antigens. Tumor-specific antigen peptides have been ex-
plored by mass spectrometry (MS) with clinical tumor
samples, and identified peptide sequences have been
registered in databanks after extensive research [23].
However, mainly due to economic reasons, only the top-
ranking peptides in the list are applied to patients, with-
out considering the differences in efficacy of the differ-
ent peptides among individual tumors. The Leuko-
CatchTM system would enable clinicians to collect and
isolate, in a simple and rapid manner at the bedside, live
leukocytes and circulating malignant cells, which can
subsequently be cultured, expanded, and applied to vari-
ous detailed examinations.
We propose a novel strategy for tumor immunotherapy
using tumor cells and leukocytes that are obtained
freshly from each patient with the LeukoCatchTM sys-
tem (Figure 6). This work flow consists of two parts: (A)
the profiling and identification of tumor-specific peptides,
and (B) the screening and evaluation of peptides for the
stimulation of patient leukocytes. Both parts start from a
culture step after use of the LeukoCatchTM system (Fig-
ures 6A(i) and B(i)).
Tumor cells and leukocytes are cultured until a suffi-
cient number of cells for analysis are obtained. The
membrane fraction purified from tumor cells is applied
to MS analysis to identify tumor-specific antigens. This

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642 639
Figure 5. sLeukoCatch is useful for the isolation of RBC-free leukocytes from hemolysed human peripheral
blood (hPB). (A) Schematic protocol for the purification of leukocytes from 5 mL of hemolysed hPB: (i) cap-
ture of leukocytes by the sLeukoCatch filters, (ii) hemolysis of captured cells, (iii) PBS rinse, and (iv) leukocyte
elution by DMEM. Thin horizontal arrows show the flow of procedure (from step a to l). In each step, the sy-
ringe piston was moved upwards (steps a, c, e, g, i, and k) or downwards (steps b, d, h, j, and l) by hand; (B and
C) Bar graphs indicate the number of leukocytes (B) or RBCs (C) in nontreated (NT), passed-through (PS), or
eluted fractions 1st and 2nd. Data were obtained with an LH1511 cell counter; (D) Bar graphs show relative
amounts of leukocytes and RBCs that were included in the NT, PS, and eluted fractions 1st and 2nd.

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642
640
Figure 6. Schematic for the novel use of the LeukoCatchTM system for tumor im-
munotherapy. The LeukoCatchTM system enables the collection of CTCs and leu-
kocytes simultaneously from PB samples at the bedside. (A) Collected tumor cells
can be used for cell culture (i), antigen profiling (ii), and peptide ranking (iii) for
the identification of suitable antigens from a preconstructed antigen library; (B)
sLeukoCatch can also be used for cell culture (i), the screening of effective pep-
tides (ii), and peptide ranking (iii) to pursue more effective stimulations by anti-
gens. See the main text for details.
analysis is performed for several hundred patients to
construct an archive of tumor-specific antigens. In this
archive, the prevalent antigens for specific tumors are
ranked according to their frequency of detection among
tested patients (Figure 6A(ii)). The absence of proteins
harboring the peptide sequences of these putative tu-
mor-specific antigens in the membrane fractions of nor-
mal cells should be confirmed before the antigens are
listed in the archive.
The existence of a preconstructed antigen archive plays
an important role in this proposal because the peptide
sequences obtained from the MS analysis of the patient
tumor cells are screened against this archive to confirm
their tumor specificity. Analysis of novel patients may
lead to the identification of new candidate peptides,
which may help to enrich the archive content (Figure
6A(ii)). For more effective therapy, in vitro stimulation of
the patient leukocytes with these antigens may also be
important (Figure 6B(ii)). In the final step, the most ef-
fective immunotherapeutic protocol is determined through
the integration of the data (Figures 6A(iii) and B(iii)).
Our findings showing that recovered PC-3 cells grow nor-
mally may support the practical applicability of this pro-
tocol at the bedside, which will be tested in our future
works.
Taken together, the results of this study indicate that the
LeukoCatchTM system allows the rapid and simple prepa-
ration of leukocytes, which may be useful not only for
bedside diagnosis but also for basic research using blood
samples. This technique will contribute to the preparation
of immune cells that can be reliably analyzed by various
means.
5. AUTHORS’ CONTRIBUTIONS
H.N. made contributions to the conception and design of
this study and drafted the manuscript. S.K. constructed

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642 641
the LeukoCatchTM device. A.O., K.T. and D.M. per-
formed the mouse study. A.O., K.T. and N.Y. contrib-
uted to the tissue culture experiments. T.O., A.O., M.A.S.,
D.O., and K.T. participated in the blood fractionation and
data analysis. A.O., K.T. and H.F. contributed to the
flowcytometry analysis of eluted leukocytes. All authors
read and approved the final manuscript.
6. FUNDING
This work was supported in part by grants-in-aid for
Scientific Research S (#15101006), Scientific Research
B (#20370081) and Exploratory Research (#1651085)
from the Ministry of Education, Culture, Sports, Science
and Technology of Japan (http://www.mext.go.jp/eng
lish/) to HN., and Support Projects (#H22-0910-KD77Go)
from the New Energy and Industrial Technology Devel-
opment Organization (NEDO), the Ministry of Economy,
Trade and Industry of Japan to SK. No additional exter-
nal funding was received for this study. The funders had
no role in study design, data collection and analysis, de-
cision to publish, or preparation of the manuscript.
7. ACKNOWLEDGEMENTS
We thank Ms. Kana Ao-Ooi of our laboratory for technical assistance
and Mr. Shin Moto and Ms. Saki Terashita of Fukae Kasei Co., Ltd. for
technical advice. We also thank Dr. Kate Edmondson and Dr. Patrick
Hughes of Bioedit, Ltd. for their critical reading of the manuscript.
REFERENCES
[1] Chen, D.Y., Hsieh, T.Y., Hsieh, C.W., Lin, F.J. and Lan,
J.L. (2007) Increased apoptosis of peripheral blood lym-
phocytes and its association with interleukin-18 in pa-
tients with active untreated adult-onset Still’s disease.
Arthritis & Rheumatism, 57, 1530-1538.
doi:10.1002/art.23088
[2] Kitamura, T., Kabuyama, Y., Kamataki, A., Homma,
M.K., Kobayashi, H., et al. (2008) Enhancement of lym-
phocyte migration and cytokine production by ephrinB1
system in rheumatoid arthritis. American Journal of Physi-
ology-Cell Physiology, 294, 189-196.
doi:10.1152/ajpcell.00314.2007
[3] Attur, M., Belitskaya-Lévy, I., Oh, C., Krasnokutsky, S.,
Greenberg, J., et al. (2011) Increased interleukin-1β gene
expression in peripheral blood leukocytes is associated
with increased pain and predicts risk for progression of
symptomatic knee osteoarthritis. Arthritis & Rheumatism,
63, 1908-1917. doi:10.1002/art.30360
[4] Mellman, I., Coukos, G. and Dranoff, G. (2011) Cancer
immunotherapy comes of age. Nature, 480, 480-489.
doi:10.1038/nature10673
[5] Curran, K.J., Pegram, H.J. and Brentjens, R.J. (2012) Chi-
meric antigen receptors for T cell immunotherapy: Cur-
rent understanding and future direction. The Journal of
Gene Medicine, 6, 405-415. doi:10.1002/jgm.2604
[6] Yu, M., Stott, S., Toner, M., Maheswaran, S. and Haber,
D.A. (2011) Circulating tumor cells: Approaches to isola-
tion and characterization. The Journal of Cell Biology,
192, 373-382. doi:10.1083/jcb.201010021
[7] Bøyum, A. (1968) Isolation of mononuclear cells and
granulocytes from human blood (Paper IV). Scandinavian
Journal of Clinical and Laboratory Investigation Sup-
plement, 97, 77-89.
[8] Bøyum, A., Brincker Fjerdingstad, H., Martinsen, I., Lea,
T. and Løvhaug, D. (2002) Separation of human lym-
phocytes from citrated blood by density gradient (Nyco-
Prep) centrifugation: Monocyte depletion depending upon
activation of membrane potassium channels. Scandina-
vian Journal of Immunology, 56, 76-84.
doi:10.1046/j.1365-3083.2002.01102.x
[9] Tomana, M., Parton, A. and Barnes, D.W. (2008) An
improved method for separation of leucocytes from pe-
ripheral blood of the little skate (Leucoraja erinacea).
Fish and Shellfish Immunology, 25, 188-190.
doi:10.1016/j.fsi.2007.12.006
[10] http://www.pathology.unibe.ch/Lehre/downloads/Ficoll.pdf
[11] Cotter, M.J., Norman, K.E., Hellewell, P.G. and Ridger,
V.C. (2001) A novel method for isolation of neutrophils
from murine blood using negative immunomagnetic sepa-
ration. American Journal of Pathology, 159, 473-481.
doi:10.1016/S0002-9440(10)61719-1
[12] Burger, D.E., Wang, L., Ban, L., Okubo, Y., Kühtreiber,
W.M., et al. (2011) Novel automated blood separations
validate whole cell biomarkers. PLoS One, 6, e22430.
doi:10.1371/journal.pone.0022430
[13] Ward, D.M. (2011) Conventional apheresis therapies: A
review. Journal of Clinical Apheresis, 26, 230-238.
doi:10.1002/jca.20302
[14] http://www.asahi-kasei.co.jp/medical/en/list/
[15] Ghosh, S., Kaplan, G. and Panaccione, R. (2009) Granu-
locyte-monocyte apheresis for the treatment of ulcerative
colitis—Is this the end of the road? Nature Clinical Prac-
tice Gastroenterology & Hepatology, 6, 6-7.
doi:10.1038/ncpgasthep1299
[16] Adacolumn. http://www.adacolumn.com/
[17] Danese, S., Angelucci, E., Marconi, S., Repici, A. and
Malesci, A. (2008) Oral beclomethasone: A review of its
use in inflammatory bowel disease. Mini Reviews in
Medicine Chemistry, 8, 1459-1463.
doi:10.2174/138955708786786444
[18] http://www.pall.com/medical_39479.asp
[19] Dzik, S. (1993) Leukodepletion blood filters: Filter de-
sign and mechanisms of leukocyte removal. Transfusion
Medicine Reviews, 7, 65-77.
doi:10.1016/S0887-7963(93)70125-X
[20] Ferrer, F., Rivera, J., Corral, J., González-Conejero, R.
and Vicente, V. (2000) Evaluation of leukocyte-depleted
platelet concentrates obtained by in-line filtration. Vox
Sanguinis, 78, 235-241.
doi:10.1046/j.1423-0410.2000.7840235.x
[21] Mascaretti, L., Baggi, L., Riva, M., Proserpio, P., Dassi,
M., et al. (2002) Lymphocyte subsets in inline filtered
packed red blood cell units: Comparison between low and

A. Okamoto, K. Torigata et al. / Advances in Bioscience and Biotechnology 3 (2012) 630-642
642
high spin procedures. Transfusion and Apheresis Science,
26, 167-174. doi:10.1016/S1473-0502(02)00009-5
[22] Okuzaki, D., Kimura, S., Yabuta, N., Ohmine, T. and
Nojima, H. (2011) LeukoCatch, a quick and efficient tool
for the preparation of leukocyte extracts from blood.
BMC Clinical Pathology, 11, 9.
doi:10.1186/1472-6890-11-9
[23] http://www.cancerimmunity.org/peptidedatabase/Tcellepi
topes.htm
LIST OF ABBREVIATIONS
CTC: circulating tumor cells; CTC: cytotoxic T lym-
phocyte; LEK: lymphokine-activated killer cell, LY:
lymphocyte; MO: monocyte; NE: neutrophil; NT:
nontreated, PB: peripheral blood; PBL: peripheral blood
leukocyte; PBMC: peripheral blood mononuclear cells;
PBS: phosphate-buffered saline; PS: pass through; PTL:
platelet; RBC: red blood cell.
SUPPORTING INFORMATION
Figure S1. tLeukoCatch efficiently captures TRAMP-C1 cells.
(A) TRAMP-C1 cells were passed through a tLeukoCatch five
times. Bar graphs show the number of TRAMP-C1 cells before
tLeukoCatch treatment (NT, nontreated) and after five passages
through tLeukoCatch filters; (B) TRAMP-C1 cells were passed
through tLeukoCatch five times. The captured TRAMP-C1
cells were eluted five times (1st to 5th) with fresh DMEM.