www.elsevier.com/locate/jim
Journal of Immunological Me
Research paper
Evaluation of leukocyte stabilisation in TransFixR-treated blood
samples by flow cytometry and transmission electron microscopy
B. Canonicoa,b,*, L. Zamaia,c, S. Burattinib, V. Grangerd, F. Mannelloc, P. Gobbib,
C. Felicia, E. Falcieria,b,e, J.T. Reillyd, D. Barnettd, S. Papaa,b
aCentro di Citometria e Citomorfologia, Universita di Urbino bCarlo BoQ, Urbino, ItalybIstituto di Scienze Morfologiche, Universita di Urbino bCarlo BoQ, Urbino, Italy
cIstituto di Istologia ed Analisi di Laboratorio, Universita di Urbino bCarlo BoQ, Urbino, ItalydDepartment of Haematology, Royal Hallamshire Hospital, Sheffield, UK
eITOI-CNR, Istituti Ortopedici Rizzoli, Bologna, Italy
Received 5 January 2004; received in revised form 8 July 2004; accepted 9 September 2004
Available online 12 October 2004
Abstract
In this report, we have evaluated the effects of a TransFixR-based stabilisation technique on leukocyte scatter characteristics,immunophenotyping, membrane permeability, absolute cell counting and morphology to extend previously reported flow
cytometric data focused on the lymphocyte population. We show that scatter characteristics, immunophenotyping and absolute
cell counting are well preserved, particularly in the lymphocyte population. Nevertheless, a general increase in membrane
permeability, evaluated by propidium iodide (PI) uptake, was observed in TransFixR-treated leukocyte subsets. Ultrastructural
observations show selective morphological preservation (up to 10 days of storage) of lymphocytes and, to a lesser extent, of
monocytes. In contrast, granulocytes have necrosis-like features, although the plasma membrane seems well preserved.
Therefore, electron microscopy observations reflect modifications induced in different cell populations as evidenced by flow
cytometry (FC). The data indicate that this short-term stabilisation method is particularly suitable for the analysis of human
lymphocytes and it is a good procedure for quality control programmes for inter- and intra-laboratory performance evaluation;
good results are obtained with respect to antigen definition and absolute cell counting procedures. Any apoptotic pathways in
leukocyte subsets are blocked for at least 10 days.
D 2004 Elsevier B.V. All rights reserved.
Keywords: TransFixR; Flow cytometry; Phenotype; Cell permeability; Apoptosis; Ultrastructure
0022-1759/$ - s
doi:10.1016/j.jim
Abbreviation
electron microsc
phosphate-buffe
* Correspon
Italy. Tel.: +39 7
E-mail addr
thods 295 (2004) 67–78
ee front matter D 2004 Elsevier B.V. All rights reserved.
.2004.09.013
s: FI, fluorescence intensity; UK NEQAS, United Kingdom National External Quality Assessment Scheme; TEM, transmission
opy; PI, propidium iodide; SSC, side scatter; FSC, forward scatter; PBCs, peripheral blood cells; FC, flow cytometry; PBS,
red saline.
ding author. Centro di Citometria e Citomorfologia, University of Urbino bCarlo BoQ, via dell’Annunziata 4, 61029 Urbino,
22 329032; fax: +39 722 329198.
ess: [email protected] (B. Canonico).
Table 1
Monoclonal antibodies used for specific immunofluorescence
evaluation
Fluorochrome Clone Manufacturer
Anti-CD45 FITC HI30 Caltag Laboratories
Anti-CD14 R-PE TUK4 Caltag Laboratories
Anti-CD19 PE 4G7 Becton Dickinson
Anti-CD16 PeCy5 3G8 Caltag Laboratories
Anti-CD4 PE SK3 Becton Dickinson
Anti-CD8 PerCP SK1 Becton Dickinson
Anti-CD3 FITC S4.1 Caltag Laboratories
Anti-CD38 R-PE HIT2 Caltag Laboratories
B. Canonico et al. / Journal of Immunological Methods 295 (2004) 67–7868
1. Introduction
Stabilisation protocols were developed initially to
create biological standards (long-term stabilised cells)
for the evaluation of cytometer performance in
different immunofluorescence assays and to train
technicians (Barnett et al., 1996, 1998a,b, 2000a;
Whitby et al., 2002). Long-term stabilised cells have
also been used to compare and check other immuno-
fluorescence techniques such as the Quantum Simply
Cellular and the QuantiBRITE methods showing that,
apart from cytometer standardisation, biological
standards such as CD45 ABC/lymphocytes are
essential to check the performance of antigen quanti-
tation methods (Barnett et al., 2000b; Bikoue et al.,
2002). Thus, these potential reference preparations act
as a full process control and provide a valuable means
for checking quality control systems, important steps
when evaluating immunofluorescence staining pat-
terns in a qualitative and quantitative manner (Barnett
et al., 1996, 1998a,b, 2000a,b; Bikoue et al., 2002;
Jani et al., 2001; Reilly and Barnett, 2001; Whitby
et al., 2002).
Recently, a new stabilisation product has been
developed, called TransFixR, which has been
shown to maintain cell integrity for at least 7–10
days (Barnett et al., 1998a,b; Shafaie et al.,
2000a,b; Jani et al., 2001), without affecting the
accuracy of human lymphocyte subset definition
and their absolute count (Shafaie et al., 2000b; Jani
et al., 2001). Furthermore, the increased cost burden
of transportation is removed as this novel fixative
permits the transportation of whole blood specimens
for prolonged periods without inducing the pro-
found morphological and phenotypic changes
appearing in fresh blood (Jani et al., 2001).
However, no study has been undertaken to examine
the morphological alterations, if any, induced by
both the long- and short-term TransFixR stabilisa-
tion technique. In the present study, we report the
effects of TransFixR on the morphology of the
different human peripheral blood cell (PBC) line-
ages and show, through multidisciplinary approaches,
the selective effectiveness of this stabilisation
method on lymphoid populations, as well as the
differences in response to TransFixR treatment of
other leukocyte subpopulations (i.e., monocytes and
granulocytes).
2. Materials and methods
2.1. Samples and reagents
Donor peripheral blood samples from 10 healthy
subjects (aged 38F5 years, equally distributed
between both sexes) were collected into K2 EDTA
tubes following venipuncture at the Transfusion
Centre of Urbino Hospital. Informed consent was
obtained according to the 1975 Helsinki Declaration
of Human Rights. No patient had taken any hormonal
medication or pharmacological drug for at least 6
months prior to the study. Subjects reporting clinical
symptoms or altered laboratory markers in the
previous year, as well as those who had been
medically treated for haematological diseases, were
excluded from the study.
TransFixR (Barnett et al., 1998a,b) reagent was
prepared in Sheffield (United Kingdom National
External Quality Assessment Scheme [UK NEQAS]
for Leukocyte Immunophenotyping) and supplied to
the Centre of Cytometry and Cytomorphology, Uni-
versity of Urbino, where the study was conducted.
To lyse erythrocytes, a NH4Cl-based lysing sol-
ution (PharMingen, Becton Dickinson Biosciences,
San Diego, CA, USA) was used. Propidium iodide
(PI) purchased from Calbiochem (La Jolla, CA,
USA) was dissolved in phosphate-buffered saline
(PBS; stock solution 1 mg/ml). The monoclonal
antibodies employed are shown in Table 1. We used
TruCOUNTk tubes (Becton Dickinson Biosciences,
Palo Alto, CA, USA) for absolute count evaluation.
Glutaraldehyde, OsO4, epoxy resin, uranyl acetate and
lead citrate were obtained from TAAB Laboratories
Equipment Ltd, Aldermaston, Berkshire, UK.
B. Canonico et al. / Journal of Immunological Methods 295 (2004) 67–78 69
2.2. TransFixR treatment
Each peripheral blood sample was divided into
two: TransFixR reagent was added in a 1:10 ratio
within 5 h after venisection of donor, whilst 0.1 M
PBS (pH 7.3) was added at the same ratio to an
untreated blood sample. Samples were then main-
tained at 4 8C for 10 days. Before flow cytometry
(FC) and transmission electron microscopy (TEM)
analyses, diluted samples were allowed to reach room
temperature for at least 30 min before sample
preparation. Red blood cells were lysed with 2 ml of
NH4Cl lysing solution prior to analysis of the white
peripheral blood cells by FC and TEM.
2.3. Flow cytometry
Scatter characteristics, immunophenotyping, mem-
brane permeability and absolute cell count were
evaluated by FC using a FACScan flow cytometer
equipped with an argon ion laser tuned at 488 nm. For
all FC measurements, at least 2500 events in the
lymphocyte area were collected from each sample
tube using CellQuestk Software (Becton Dickinson,
San Jose, CA, USA). Instrument set-up and calibra-
tion was performed prior to data acquisition according
to well-established procedures (Schwartz, 1989;
Stewart and Stewart, 1990).
2.3.1. Scatter signal analyses
Analyses of forward and side scatter (FSC and
SSC, respectively), associated with morphological
aspects, were performed to distinguish lymphocytes,
monocytes, granulocytes and to detect the changes in
FSC and SSC over time in treated and untreated
samples. In particular, it is known that FSC analysis
detects apoptotic cell shrinkage (Luchetti et al., 2003;
Zamai et al., 1993, 2000).
2.3.2. Monoclonal antibody labelling
To evaluate antigen expression on TransFixR-treated and untreated samples, we added fluoro-
chrome-conjugated monoclonal antibodies as appro-
priate to 50 Al of donor whole blood. mAb volumes
used were as follows: 5 Al for anti-CD45 FITC, anti-
CD14 R-PE, anti-CD3 FITC, anti-CD16 Pe-Cy5 and
anti-CD38 R-PE; 10 Al for anti-CD19 PE, anti-CD4
PE and anti-CD8 PerCP. Samples were gently mixed
and incubated for 15 min at room temperature in the
dark. After a lysing step, they were analysed by FC.
FACScompk software was routinely applied for
mAbs labelling evaluation.
2.3.3. Propidium iodide supravital staining
To evaluate membrane integrity using PI uptake,
50 Al of blood sample were pipetted carefully into the
bottom of a test tube, then PI was added to a final
concentration of 50 Ag/ml. Samples were gently
resuspended and incubated for 20 min at room
temperature in the dark. After a lysing step, the
samples were analysed by FC. Supravital staining
using PI (Zamai et al., 1996, 2001) can distinguish
between PI brightly stained (necrotic), PI dimly
stained (apoptotic) and PI negative (viable) cells. A
separate setting of the FL3 PMT, necessary for PI
stained sample analysis, was performed on PI stained,
UV-treated fresh leukocytes.
2.3.4. Absolute cell counting
TruCOUNTk tubes were used for absolute count
evaluation. Briefly, 50 Al of TransFixR-treated and
untreated blood samples were carefully dispensed by
means of a reverse pipetting technique at the bottom
of the tube. Five microlitres of monoclonal antibody
anti-CD45 FITC conjugated was added. After 15 min
of incubation at room temperature, red cell lysis was
carried out. Samples were acquired on the FACScan
within 60 min. A minimum of 2500 lymphocytes
were collected. Set-up and calibration procedures
were optimised for the absolute counting protocols
(Brando et al., 2000; Gratama et al., 2000).
2.4. TEM observations
Untreated and TransFixR-treated peripheral blood
cells were pelleted by centrifugation at 1800 rpm for
10 min, immediately fixed with 2.5% glutaraldehyde
in 0.1 M PBS (pH 7.3), post-fixed with 1% OsO4 in
the same buffer, dehydrated with alcohol and
embedded in araldite (Falcieri et al., 2000). To
obtain better direct ultrastructural observations, semi-
thin sections were stained with toluidine blue at 60
8C. Thin sections were collected on nickel grids,
stained with uranyl acetate and lead citrate, and
analysed with a Philips CM 10 electron microscope
at 80 kV.
B. Canonico et al. / Journal of Immunological Methods 295 (2004) 67–7870
2.5. Statistical analysis
Mean values and their standard deviations were
calculated. In order to explore the existence of
statistically significant differences between the differ-
Fig. 1. FSC vs. SSC dot plots of untreated (left panel) and TransfixR-treate4; E,F: day 10. Ten-day-old blood cells (E) show a subpopulation of apo
observed for granulocytes (FSC reduction). Ten-day-old TransfixR-treatevalues, without comparing any of the apoptotic subpopulation (F).
ent samples, the paired Student’s t-test was used. P
values b0.05 were considered statistically significant.
To calculate differences in fluorescence intensity (FI)
values (as channel values) over time, we considered
the original input on fresh untreated cells as 100%.
d (right panel) peripheral blood cells over time: A,B day 0; C,D day
ptotic lymphocytes in the area close to the viable cells; the same is
d myelomonocytic cells show a reduction both for FSC and SSC
B. Canonico et al. / Journal of Immunological Methods 295 (2004) 67–78 71
3. Results
3.1. Flow cytometry
FC analysis was performed at days 0, 1, 2, 3, 4, 7
and 10 in order to check the timing of changes in
scatter characteristics, immunophenotyping, mem-
brane permeability and absolute count.
3.1.1. Scatter signal analyses
TransFixR treatment generated slightly lower
forward and side scatter granulocytes with respect to
the fresh ones, just 1–2 days after the treatment (data
not shown). At day 4, lower SSC and FSC values
were also observed on TransFixR-treated peripheral
Fig. 2. CD45 positivity evaluation of lymphocytes (A –C), monocytes (D
incubation time. The filled peak represents labelled fresh untreated cells,
overlaid. D = day.
blood monocytes (Fig. 1D), suggesting that myelo-
monocytic cells show not only the highest sensitivity
to the treatment but also that this sensitivity is time-
dependent (i.e., rapid in granulocytes, slower in
monocytes).
Starting at day 4, untreated leukocytes (granulo-
cytes, monocytes and lymphocytes) show two distinct
clusters: viable and dead cells with reduced FSC
values (due to cell shrinkage). In contrast, treated
leukocytes (Fig. 1B,D,F) lack the typical reduction of
FSC present on dead untreated leukocytes (Fig. 1E vs.
F). Therefore, a unique cluster of the different
leukocyte populations with homogeneous scatter
characteristics was maintained over time only in the
TransFixR-treated samples, allowing appropriate
– F) and granulocytes (G – I) from the same donor during 10 days
peaks from TransfixR-treated cells at day 0, day 4 and day 10 are
B. Canonico et al. / Journal of Immunological Methods 295 (2004) 67–7872
gates to be drawn and correct immunophenotyping
evaluations to be obtained only on treated samples.
3.1.2. Monoclonal labelling analysis
As expected, untreated cells tend to lose their
antigenic pattern over time. This phenomenon is
consistently evident on apoptotic cells (at lower FSC
values) (Frey, 1997 and data not shown).
Fig. 2A–C shows histograms expressing the FI of
the monoclonal antibody anti-CD45 FITC conjugated
on untreated (fresh) and TransFixR-treated samples,
Fig. 3. Dot plots CD45 vs. SSC of fresh (A) and transfixed leukocytes at
monocytes and, particularly for granulocytes, is observed in (C) (day
characteristics allow optimal detection of the different leukocyte subsets.
revealing stability in surface labelling of treated
lymphocytes over all time periods. As expected,
similar results were obtained with anti-CD3 and the
important marker of leukocyte activation and differ-
entiation, CD38, which remained stable up to day 10
(data not shown). These results were obtained on
treated samples and the reference FI value was
represented by FI of fresh untreated lymphocytes.
FI relating to the same monoclonal antibody anti-
CD45 on TransFixR-treated monocytes (Fig. 2D–F)
revealed a slight decrease from day 0 to day 4
day 0 (B), day 4 (C) and day 10 (D). Reduction in SSC values for
4) and (D) (day 10). However, both CD45 expression and SSC
L, lymphocytes; G, granulocytes.
B. Canonico et al. / Journal of Immunological Methods 295 (2004) 67–78 73
(15F4% decrease in FI with respect to FI of fresh
cells, meanFS.D.) (Pb0.05); while, from day 4 to day
10, FI values remained almost stable (decrease in FI of
17F3% with respect to FI of fresh cells). The analyses
were also carried out on granulocytes (Fig. 2G–I),
Fig. 4. Effects of both TransFixR cell stabilisation and incubation timing o
untreated and TransfixR-treated granulocytes and lymphocytes. The upper
10, with progressively increasing percentages of PI positive events, especia
treated granulocytes (day 0, day 4, day 10), revealing the cluster of PI brigh
untreated lymphocytes at day 0, day 4 and day 10, revealing the cluster of P
lymphocytes.
showing a decrease in FI values with respect to fresh
cells of about 20% at day 4 and about 30% at day 10.
Although myelomonocytic cells show a decrease in
FI values with respect to fresh cells, this fact does not
interfere with detection of different leukocyte subsets
n granulocyte and lymphocyte membrane: FL1 vs. FL3 dot plots of
panels (A –C) shows untreated granulocytes at day 0, day 4 and day
lly of PI dim events (apoptotic). Cytograms (D –F) show TransfixR-t events as the unique population (day 10) (F). Dot plots G – I show
I positive events. The bottom panels (J – L) shows TransfixR-treated
Fig. 5. Variations in absolute counts during the 10 days of storage of
both untreated and TransfixR-treated lymphocytes, granulocytes
and monocytes (A, B and C, respectively). Values are reported as
percentages compared to the absolute number of cells at day 0
representing 100%. (TransfixR-treated and untreated leukocytes are
shown as empty and dotted columns, respectively.)
B. Canonico et al. / Journal of Immunological Methods 295 (2004) 67–7874
(Fig. 3A–D). The distinction between positive and
negative events can still be observed for the full
duration of storage and typical differences in CD45
expression between different leukocyte subsets are
also well detected. These conditions allow gates to be
drawn around different clusters showing virtually
identical percentage values from day 0 to day 10.
3.1.3. PI fluorescence analysis
Supravital PI uptake was used to detect both cell
death in untreated cells (Zamai et al., 2001) and the
possible alterations of membrane permeability
induced by TransFixR. Our results confirm that
untreated granulocytes are the leukocyte subpopula-
tion most sensitive to cell death, probably due to their
bcl-2 negative peculiarity (Zamai et al., 2002). Fig.
4A–C shows an increased percentage (from 15% to
88%) of PI positive cells (Pb0.02) (especially of PIdim
cells), demonstrating progressive and significant cell
death in untreated samples. In contrast, in TransFixR-treated granulocytes (Fig. 4D–F), it is possible to
detect an early increase in PIbright events when
compared to the untreated sample; such an increase
is apparent in almost all the population starting from
day 4 with all granulocytes exhibiting a PIbright pattern
at day 10 (day 0 vs. day 10, Fig. 4D vs. F, respectively;
Pb0.01). Interestingly, granulocytes went straight
from negative to bright, without presenting a PIdim
pattern, typical of apoptotic cells.
In contrast, lymphocytes are more resistant to cell
death and less sensitive to TransFixR treatment (Fig.
4G–L). At day 0, TransFixR-treated blood samples, as
well as untreated cells, are PI negative (Fig. 4G vs. J,
respectively).
At day 4, PI+ events are slightly more represented
in the dot plot of TransFixR-treated lymphocytes
compared to the untreated control (Fig. 4H vs. K,
respectively), whereas at day 10, there are no
significant differences in PI+ events in untreated
and TransFixR-treated lymphocytes (Fig. 4I vs. L,
respectively).
3.1.4. Absolute cell count
Absolute cell counting was performed at day 0, day
4 and day 10, on both TransFixR-treated and
untreated samples. Gates on granulocytes, monocytes
and lymphocytes were established based on SSC/
CD45 dot plots (see Fig. 3). Following this gating
,
Fig. 6. TEM of peripheral blood cells at day 0 (A, B), day 4 (C–F) and day 10 (G, H) from fresh (A, C, D, G) and TransFixR-treated (B, E, F, H)blood. Up to 4 days, good preservation of lymphocytes can be observed after TransFixR treatment, while granulocytes at 4 days undergo a
progressive necrotic-like feature. At 10 days, several granulocytes with typical apoptotic chromatin changes appear in fresh blood, but this is not
present in TransFixR-treated cells. A progressive necrotic-like feature of lymphocytes can be seen at this time, nevertheless, apparent membrane
integrity is maintained (a). *Apoptotic granulocyte. (A, B, D, E, F, H) bar=2 Am; (C, G) bar=1 Am.
B. Canonico et al. / Journal of Immunological Methods 295 (2004) 67–78 75
B. Canonico et al. / Journal of Immunological Methods 295 (2004) 67–7876
strategy, we counted both dead and living cells, only
excluding debris. Counting test tubes allowed us to
evaluate the stability in lymphocyte number and
antigen density and to provide more details on cells
collected for ultrastructural analysis. There was no
significant difference at day 0 between absolute
numbers of lymphocytes, monocytes and granulocytes
in treated and untreated samples, while during the 10
days of storage we found significant differences
between TransFixR-treated (with cell count similar
to fresh count) and untreated samples (Pb0.05) (Fig.
5A–C). It is well known that untreated cells tend to
die over time, finally becoming debris, excluded by
gates.
3.2. TEM observations
At day 0 (Fig. 6A,B), morphological analysis
showed good maintenance of cell morphology for
all the different cell types present in peripheral blood
samples both in fresh (Fig. 6A) and TransFixR-treated(Fig. 6B) specimens, demonstrating that the stabilisa-
tion procedure does not interfere with the post-
fixation for TEM. At day 4, the morphology of
lymphocytes showed no significant changes in both
untreated (Fig. 6C) and TransFixR-treated blood
samples (Fig. 6E), whereas the ultrastructure of the
granulocytic population showed the initial features of
apoptosis in the untreated sample (Fig. 6D) and clear
necrosis-like patterns in the treated sample (Fig. 6F).
Finally, at day 10, while the majority of untreated
granulocytes had entered apoptosis (Fig. 6G), the
TransFixR-treated samples showed, besides an evi-
dent granulocyte necrotic-like feature, a progressive
modification of some lymphocytes towards a necrotic-
like pattern even when the integrity of the cell
membrane seemed to be maintained (Fig. 6H).
4. Discussion
The chemical or physical stabilisation (fixation) of
biological samples represents a fundamental step in
terms of standardisation of protocol analysis. Unfortu-
nately, such procedures often produce artefacts in the
samples to various degrees (Dubochet and Sartori
Blanc, 2001). Protocols able to maintain cell integrity
for several days after collection are needed. First, in
order to reduce costs and efficiency, centralisation of
sample analysis is becoming a goal for health systems
in Europe as well as in developing countries. More-
over, this is useful for setting up the cytometers
employed in routine clinical activity. Finally, central-
isation reduces the variable delay between the
collection and analysis of the specimens for better
in-house organisation of routine sample analysis. For
example, the fact that over time, untreated cells tend
to undergo apoptosis (finally becoming debris) and to
lose surface antigen density (Frey, 1997), means that
the correct evaluation of immunophenotyping a long
time after blood collection (N72 h) is not possible
(Mandy et al., 2003). A protocol and reagent able to
maintain lymphocyte integrity for several days is
essential to achieve these goals.
To date, FC is the most widely used and reliable
analytical approach in the clinical analysis of blood
cells, particularly for the diagnosis of haematological
neoplasias (Hrusak and Porwit-MacDonald, 2002)
and in the monitoring of HIV progression and
therapeutic effectiveness (Storie et al., 2004).
Recently, advances have been made by the develop-
ment by UK NEQAS for Leukocyte Immunopheno-
typing of a unique whole blood stabilising process
that ensures the maintenance of both the light scatter
characteristics and antigenic profile required to
provide successful cellular immunophenotyping for
both European Quality Assessment studies and
clinical samples (Barnett et al., 1996; Jani et al.,
2001; Reilly and Barnett, 2001). For flow cytometric
evaluation of whole blood, one of these fixing
methods uses a stabilising reagent, called TransFixR(Barnett et al., 1998a,b). Here, we highlight the fact
that the TransFixR methodology stabilises leukocytes
for at least 10 days. However, the present study,
examining cell permeability and ultrastructural mor-
phology, demonstrates that this stabilising procedure
preserves the biological integrity of lymphocytes,
although it has a mild effect on the granulocytic
population and a lesser effect on the monocytic
population. The different effects on granulocytes and
monocytes could be explained by recent findings
indicating that, although neutrophils and monocytes
are derived from a common progenitor, up-regulation
of XIAP and bcl-XL contributes to the increased
longevity of cells in the monocytic lineage (Miranda
et al., 2003).
B. Canonico et al. / Journal of Immunological Methods 295 (2004) 67–78 77
The paired analyses of ultrastructural morphology
and FC demonstrate that during the 10 days of storage
after TransFixR treatment, both lymphocyte morphol-
ogy and membrane antigenicity are maintained;
although at the end of the storage period investigated,
cell morphology is slightly altered, the TransFixRstabilisation does not affect absolute counts and CD
antigen density. In particular, the results of our
multidisciplinary study show that the lymphocyte
profile in FSC and SSC is maintained by means of
TransFixR reagent, thus enabling discrimination of
the different cell populations in human peripheral
blood up to 10 days after collection.
The ultrastructural analysis has shown substantial
preservation of TransFixR-treated blood cell mor-
phology, particularly evident in lymphocytes in the
early treatment stage. Successively, granulocytes
undergo a progressive necrosis-like feature, while
lymphocytes appear well preserved. Surprisingly,
necrosis-like behaviour of granulocytes seems to
occur with an apparent membrane preservation,
while permeability to PI is increased. This unex-
pected and interesting behaviour, very unusual in
necrotic death and widely reported in apoptosis, can
be undoubtedly correlated to the TransFixR mem-
brane stabilisation process. Although a general
increase in membrane permeability was observed in
TransFixR-treated leukocyte subsets, we nevertheless
show that scatter characteristics, immunophenotyping
and absolute cell counting are well preserved
particularly in the lymphocyte population on Trans-
FixR-treated samples.
Cytofluorimetric and ultrastructural data together
indicate: (i) maintenance of physical characteristics
identical to fresh cells, especially for peripheral
blood lymphocytes for at least 10 days; (ii) the
absence of any apoptotic pattern in TransFixR-treated leukocytes; (iii) the TransFixR-inducedbnecrogenic-like effectQ on granulocytes; (iv) sub-
stantial preservation of the lymphocyte CD antigenic
profile; (v) structural preservation of the membrane;
(vi) alterations in membrane permeability, particu-
larly evident in TransFixR-treated granulocytes and
detectable progressively also in lymphocytes,
towards the end of the storage time and not affecting
antigen density and absolute number assessment. The
maintenance of light scatter characteristics and
antigenic pattern of the peripheral blood cells is the
main goal for use as a reference standard in routine
laboratory practice.
In conclusion, our data indicate that TransFixRstabilising reagent can reliably be used in flow
cytometric procedures; good results are obtained in
antigen definition and absolute counting procedures,
blocking any apoptotic pathways in leukocyte subsets.
Moreover, although at day 0, TransFixR-treated and
untreated samples did not show any ultrastructural and
morphological changes, the intrinsic biological heter-
ogeneity of leukocytes may determine differences in
the final cellular response of different leukocyte
subsets to the TransFixR stabilisation. The biological
significance of these findings is not completely
understood and further biochemical and molecular
investigations are needed and are currently in progress
in our laboratories.
Acknowledgements
We are thankful to the entire staff of the
Transfusion Centre of the Urbino Hospital. We are
grateful to Mr. Federico Bastianelli and to Dr.
Mario D’Atri for their skillful assistance with image
processing. We also want to thank Dr. Francesca
Luchetti, Dr. Massimo Della Felice and Dr. Claudia
Masoni for their useful collaboration. This work
was supported by EU FP5 grants bEurostandardsQQLRI-CT-2000-00436 and bEuromeetQ QLG-1-CT-
2002-30275 (SP), COFIN 2002 and 2003 (LZ),
MIT (Marche Innovation Training) contract number
IRC4.27/ADD2.
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