Evaluation of leukocyte stabilisation in TransFixR-treated ... · 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

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.


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


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


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.


(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.)


(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.



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).


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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