Transfusion and Apheresis Science 30 (2004) 2328

www.elsevier.com/locate/transci

Blood component fractionation: manual versus

automatic procedures
Daniela Pasqualetti a,*, Alessandro Ghirardini b, Maria Cristina Arista a,
Stefania Vaglio a, Azis Fakeri a, Alan A. Waldman c, Gabriella Girelli a
a
Department of Cell Biotechnology and Hematology, University La Sapienza, Blood Bank, Via Chieti 7, Rome 00161, Italy
b
Istituto Superiore di Sanita, Laboratory of Epidemiology and Biostatistics, Rome, Italy
c
Waldman Biomedical Consultancy, Oceaside, New York, USA

Abstract
Over the last few years, quality system requirements have been introduced for blood components. The necessary
compliance with standard productions will have a considerable impact on Blood Banks. The introduction of automated
methods is the most satisfactory means to meet these requirements for blood component preparation.
A new device has been developed to automate the fractionation of blood into components. We evaluated the ecacy
of this instrument as compared to manual methods. A total of 218 units of blood have been collected, into several
dierent commercial blood bag systems (77 into standard quadruple bag systems, 141 into bag systems with integrated
in line lters), and used to evaluate the universality of the instrument. Whole blood units were processed using the Top/
Top system and the Compomat G4 (Fresenius HemoCare). A separate program protocol was developed for each kind
of bag.
Use of the Compomat G4 resulted in a statistically signicant (p < 0:001) increase of the hemoglobin in ltered red
cell concentrates (RCC) in comparison with the manual procedure, and a similar trend, even not statistically signicant,
has been observed for ltered RCC. Regardless of bag systems, we were able to observe a statistically signicant in-
crease of platelets in the platelet concentrates (PCs), when comparing automatic versus manual procedure.
The automated procedure has been shown to be fast, and easy for the operators. This device reliably produces
acceptable blood components, and has been shown adaptable to use with dierent blood bag systems.

2003 Elsevier Ltd. All rights reserved.

1. Introduction cussed over the last few years, and bringing stan-
dard production into compliance will have a
The implementation of a quality system for considerable impact on Blood Banks. In actual
blood component production has been much dis- practice, it often has been necessary to change
working methods in order to guarantee repro-
ducible production of the blood components,
* introducing careful controls of the processing
Corresponding author. Tel.: +39-06-85795-524; fax: +39-
06-85795-501. procedures involved and performing quality tests
E-mail address: pasqualetti@bce.uniroma1.it (D. Pasqua- of the blood products on a routine basis. While in
letti). the past the procedures for preparing fractions of
1473-0502/$ - see front matter
2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.transci.2003.07.002
24 D. Pasqualetti et al. / Transfusion and Apheresis Science 30 (2004) 2328
blood components were based completely on
manual operations, automated methods have re-
cently been introduced as the most ecient and
eective way to full the standards for blood
components production and conform with the
European guidelines [1].
Using collection bags which allow for removal
of materials only through a port on the top (Top/
Top system), standard blood fractionation in the
United States begins with the separation, by low-
speed centrifugation, of platelet-rich plasma
(PRP), red cell concentrates (RCC) and buy-coat
(BC) from whole blood (WB). In a subsequent
high-speed centrifugation, platelet concentrates
(PCs) and platelet poor plasma (PPP) are derived
from the PRP [2]. In Europe, in the 1990s, a dif-
ferent approach was introduced, one designed to
decrease development of the platelet storage lesion
and in vitro activation due to the second centri-
fugation [3,4]. Using collection bags which allow
for removal of materials through ports on both the
top and the bottom (Top/Bottom system), it is
possible, following an initial high-speed centrifu-
gation of the WB, to produce PPP on the top, with
the platelets concentrating into a BC on a cushion
of RCC at the bottom [5]. After collection of this
BC, the platelets can be separated by a gentle low-
speed centrifugation, a process that presumably
produces less damage than high-speed packing,
which causes formation of tight pellets of plate-
lets [6].
In actual practice, however, examination of
platelets stored for up to ve days indicated no
signicant dierences in platelet survival and
function with the two preparative techniques [7,8],
and it is still being debated whether or not platelets
from PRP or from BC are better [8].
While there have been several studies performed
to validate the performance of an automatic
instrument with the Top & Bottom system [911],
the results of using an automatic extractor for the
Top/Top system for blood component preparation
have not been reported.
As part of our eorts to improve product
quality and to reduce manufacturing problems, we
have tested the Compomat G4 (Fresenius Hemo-
Care) equipment developed to automate Top/Top
fractionation, and compared the quality of the
components produced with those obtained by the
standard manual method. As part of this study, we
evaluated Top/Top blood bag systems from dif-
ferent manufacturers.
2. Materials and methods
To check on the universality of the automatic
instrument Compomat G4 (Fresenius HemoCare),
we collected from blood donors 218 blood units,
each of 450 ml 10%, into 63 ml of CPD antico-
agulant solution, using blood bag collection sys-
tems from dierent manufacturers.
Standard quadruple bag systems were used for
77 of the blood collections, while blood bag
collection systems with integrated in-line lters
were used for 141 of the blood collections. In the
rst group of bag systems, manual procedures
were used to manufacture components from 13
units collected into Maco Pharma bags, 12 units
collected into Terumo bags, and 10 units col-
lected into Fresenius bags, while the automatic
device was used to manufacture components
from 8 units collected into Maco Pharma bags,
20 units collected into Terumo bags, and 14
units collected into Fresenius bags. In the second
group of bags system with in-line lters, manual
procedures were used to manufacture compo-
nents from 80 units collected into Fresenius bags
and from 18 units collected into bags manufac-
tured by Baxter, while the automated device was
used to manufacture components from 31 units
collected into Fresenius bags and 12 units col-
lected into Baxter bags.
The quadruple bag systems were processed
immediately after blood collection, while all the in-
line bag systems were processed after 2 h since
blood collection and after cooling at a conditioned
temperature between 18 and 22 C. All the WB
units were centrifuged at 550g for 8 min at 20 C,
with an acceleration rate of 2 and deceleration 1 in
a Beckman J-6M/E centrifuge, to produce RCC.
PC were prepared from PRP by a second centri-
fugation at 2200g for 10 min.
The automated fractionation was performed
using Compomat G4 (Fresenius-HemoCare).
 D. Pasqualetti et al. / Transfusion and Apheresis Science 30 (2004) 2328
Following each of the centrifugations, the blood
packs are taken out of the bucket, the primary
bag hung on the pins on the front of the machine,
and the satellite bags placed on the top of the
Compomat G4, with the integrated tubing being
placed in built-in automated clamper/sealers. After
the rst centrifugation of the WB, as directed
by the software, three presses combined with
the integrated detectors express out PRP and,
if desired, BC, into satellite bags, leaving the
RCC. After the second centrifugation, the pre-
sses and integrated detectors express out the PPP
into a satellite bag, leaving the PCs. The detectors,
the sensitivity of which were set to meet our stan-
dards, allow control over the blood component
separation, and standardization for dierent bags.
The separation of the 141 blood units with in-
line lters was performed with the same procedure
as described above, placing the lter upside down
on the right hand side panel of the Compomat. In
addition, this equipment has been used on Frese-
nius lters with the partial squeezing of Sag-M
through the lter by means of the top press. The
ltration of the 141 RCC with in-line lters was
carried out by gravity.
By using Compomaster, a software running on
Windows NT that manages the Compomat, the
Compomat G4 is able to process the blood units
with a programmed and personalized protocol for
each kind of bag. The Compomaster/Compomat
programs every procedure, from the start to end of
separation, and registers all the data. At our site,
in order to obtain the best recovery results for the
RCC, dened as lowest hemoglobin loss into the
small BC volume and a low level of WBC con-
tamination, we developed specic parameters for
the processing programs for the dierent bag sys-
tems, as follows:
(a) Fresenius and Maco Pharma blood bag sys-
tems:
The upper press is pushed out to 54.0 mm and
the red cell level reaches rst the DET A2 by
means of both presses, then the DET A6 + B using
only the lower press with a speed 20% to reduce
the squeezing ow. The combination of these
parameters mainly denes the features of the BC,
in terms of volume and hematocrit.
25
(b) Terumo and Baxter blood bag systems:
Upper press out to 53.0 mm, both presses out
DET A1 and lower press out DET A8 + B with a
speed 25%.
(c) Fresenius and Baxter blood bag systems with
in-line lters:
Upper press out 50.0 mm, lower press out DET
A6 with speed 15% and then lower press out DET
A8 + B with speed 20%.
(d) PRP derived from all systems:
Both presses to 45.0 mm, upper press to 56.0
mm and lower press to 58.0 mm.
After standardization of the method for stain-
ing of leukocytes with a commercial kit (Leuco-
Count, Becton Dickinson, San Jose, Costa
Mesa-CA, USA), this kit and calibrated ow cyto-
metry (FACScan, Becton Dickinson, San Jose,
Costa Mesa-CA, USA) were used to count the
absolute number of the residual [12]. Other
hematological parameters were measured with an
automated analyser (AcT.5DiCP-Coulter, Instru-
mentation Laboratory, Milan, Italy).
Statistical analysis was performed by comparing
mean value of the relevant variables, by using the
Epi Info software (version 6) produced by the
Centers for Disease Control and Prevention (CDC).
3. Results
The average gain in hemoglobin recovery in
RCC was equal to or more than 3 g/unit for both
ltered and not ltered RCC. As shown in Tables
1 and 2, the automatic procedure is associated with
a statistically (p < 0:001) signicant increase in
hemoglobin recovery in ltered RCC, while for
RCC the dierence does not reach the signicant
level (p 0:12). The apparent greater increase in
red cell recovery for the Fresenius bags (Table 3) is
felt, in part, to be due to the large standard devi-
ation in the volumes of the individual collections.
With regard to the higher values for hemoglo-
bin in the ltered RCC (Table 2), this is apparently
due to a decreased loss in the lters, a loss that, for
the Baxter bags, falls to 11.17% (Table 4).
It should be noted that the Fresenius lter
was hard-housing and the Baxter lter was
26 D. Pasqualetti et al. / Transfusion and Apheresis Science 30 (2004) 2328

Table 1 Table 4
Comparison of results between manual and automatic proce- Comparison of the results produced by Compomat G4 of l-
dure in standard bag systems from all dierent manufactures tered red cell concentrates with bag systems with in-line lters
Overall summary from the two dierent manufacturers used
Manual procedure Compomat G4
of standard bag (N 35) (N 42) Results with the two Fresenius Baxter
systems Mean (SD) Mean (SD) bag systems with in-line (N 31) (N 12)
lters manufacturers Mean (SD) Mean (SD)
RCC
Volume (ml) 335.7 (22.6) 351.6 (24.6) Filtered RCC
Hb (g/unit) 61.0 (7.7) 64.6 (5.7) Hb (g/unit) 58.1 (7.8) 62.09 (5.24)
Ht (%) 52.9 (5.3) 53.2 (2.2) Hb loss (%) 10.4 (4.4) 11.17 (8.6)
WBC Log depletion 4.14 (0.30) 4.08 (0.27)
PCs
Plts (10e11) 0.62 (0.17) 0.81 (0.2) PCs
Recovery (%) 73.8 (14.1) 92.6 (14.5) Plts (10e11) 0.70 (0.3) 0.68 (0.7)
Recovery (%) 84 (22) 76 (30)

Table 2
Comparison of results between manual and automatic proce- priming and ltration was carried out by gravity.
dure in in-line lter systems from all dierent manufactures There was no statistically signicant dierence
Overall Summary of Manual procedure Compomat G4 (p 0:52) in residual WBC (Table 4) for both
in-line lter systems (N 98) (N 43) types of WBC ltered components.
Mean (SD) Mean (SD)
The results for PCs from the standard bags are
Filtered RCC presented in Table 1. A statistically signicant
Hb (g/unit) 53.3 (7.8) 59.2 (7.8)
Hb loss (%) 13.0 (7.6) 10.4 (4.4)
dierence (p < 0:003) has been observed between
WBC Log depletion 4.36 (0.6) 4.14 (0.3) the automatic procedure compared to manual
procedure, with a remarkable increment of the
PCs
Plts (10e11) 0.61 (0.2) 0.71 (0.3)
platelet count, roughly 20%, in the PCs. Table 3
Recovery (%) 73.5 (15.1) 82.0 (24) presents the results obtained for each of several
dierent manufacturers. The automatic prepara-
tion method was found to yield excellent and
comparable platelet counts with both the Terumo
Table 3 and Fresenius blood bag systems, with a recovery
Comparison of the results produced by Compomat G4 of red greater than 92.9% and 95.0%, respectively. This is
cell concentrates using bag systems from three dierent manu- a statistically (p < 0:001) signicant dierence, as
facturers compared to the results with the Maco Pharma
Results with Terumo Fresenius Maco Pharma bags, which had a recovery of 62.1%.
dierent bag (N 20) (N 14) (N 8) Similarly, the automated method proved supe-
system Mean (SD) Mean (SD) Mean (SD)
rior to the manual method for the preparation
manufacturers
of PCs from blood bag systems with lters, with
RCC
Volume (ml) 340.0 (12.4) 370.4 (25.1) 365.4 (18)
a mean recovery per unit of 0.71 1011 versus
Hb (g/unit) 62.6 (3.2) 68.4 (6.5) 57.2 (3.9) 0.61 1011 (Table 2). Table 4 presents the results
Ht (%) 52.8 (2.1) 53.8 (2.6) 52.8 (2.2) obtained for the two dierent bag manufacturers.
PCs
No statistically signicant dierences were found
Plts (10e11) 0.82 (0.16) 0.85 (0.3) 0.60 (0.33) in the results obtained with the Fresenius and
Recovery (%) 92.9 (11.8) 95.0 (14.4) 67.1 (10.2) Baxter blood bag systems with in-line lters.
The average processing time using the Compo-
mat was 2 min and 15 s for each bag. The total
soft-housing. The priming of the rst, before the time for the procedure using automatic equipment
passive ltration, was performed automatically by was 4 min and 50 s, while the total time for the
use of the top press, while for the second both manual operation was 6 min and 36 s.
 D. Pasqualetti et al. / Transfusion and Apheresis Science 30 (2004) 2328
There were no problems with the equipment,
and no breakdowns during the study. The only
unexpected events during the study were two
leaking seals, failures traced to incorrect posi-
tioning of the tubing in the welding clamp.
4. Discussion
It is well known that, for transfusion therapy
to be eective, there must be quality and consis-
tent blood components. This study provides data
on the performance of an available automated
instrument, Compomat G4 (Fresenius Hemo-
Care), for separating blood components. Perfor-
mance, the accuracy of the separation process, and
the reliability of the device using dierent bag
systems, were evaluated and compared with the
manual method previously used in our Blood
Center.
Performance, accuracy and reproducibility of
the automatic method were assessed in two ways,
both on ability to meet the standards in the
European guidelines, and on the extent of varia-
tion in the results. The results obtained for the
components produced were all well above the
standards of the European guidelines, and the
standard deviation of the parameters measured
were either the same or lower for the automatic
procedure than for the manual procedure.
Our results demonstrate that when this device
was used to prepare red cell concentrates (RCC)
following a low-speed centrifugation to prepared
PRP, the level of hemoglobin in the RCC was
higher than for the manual method. When this
device was used in conjunction with in-line lters,
there was less loss of hemoglobin during prepara-
tion of the ltered RCC than for the manual
method. In Europe, it is required [1] that RCC and
ltered RCC units contain more than 43 and 40 g
hemoglobin/unit, respectively. This study suggests
that, in the light of the ongoing standardization of
these methods, higher values for the expected lev-
els of hemoglobin could be set.
Similarly, our results demonstrate that when
this device is used to separate PPP to yield PC,
there is a marked improvement in the platelet
content of the component. Indeed, the level of
27
recovery was high enough to allow us to reduce the
number of units used for the preparation of pooled
PCs. This reduces the risk of exposure to infectious
agents and to allogeneic stimuli as well as reducing
the costs of this product. In our facility, where the
increased request for PCs does not allow storage of
the PCs for more than one or at most two days, we
have established that it is best to prepare platelets
by this two-step method, using the automated
device Compomat G4.
Usage of pools of PC made by the two-step
centrifugation method has, in the past, been
questioned. Since the beginning of the 1990s in the
United States, in order to reduce risks of alloim-
munization, there has been an increase in the use
of apheresis-derived platelet concentrates (APCs),
rather than single unit PCs, for treatment of
thrombocytopenic patients. In 1994, more than
half of all transfused platelets were performed with
APCs [13], with markedly increased costs. How-
ever, the TRAP Study group [14] demonstrated
that, when blood products are leucodepleted, there
is no advantage of APCs over PCs with regard to
alloimmunization rates. It also was suggested, by
several studies in the past, that the quality of BC-
PCs was better than PRP-PCs [15,16]. However,
recent studies demonstrate that there are no dif-
ferences in low morphological score and recovery
between ltered pooled BC-PCs and PRP-PCs [17]
and that, in vitro, the dierences of activation,
brinogen and glycoproteins were not signicant
after one day of storage [7].
Our Center processes approximately 20,000
blood units/year, and for each product uses a
specic kind of bag, with the aim of improving the
quality process, so we evaluated the universal
applicability of the instrument, using the same
separation process with bag systems from several
dierent manufacturers. The results conrm that
this device can be used successfully with bags from
numerous sources.
In conclusion, given the improvement in the
production and content of the blood components
produced, the compliance with quality require-
ments and the easy, rapid and reproducible tech-
nology, we would recommended the use of the
Compomat/Compomaster system to process blood
into its components.
28 D. Pasqualetti et al. / Transfusion and Apheresis Science 30 (2004) 2328
Acknowledgements
The authors express their appreciation to Mrs
M.L. Salvagnini and Mrs G. Alimenti for the
technical work and for help with the preparation
of the samples tested in this study. The authors are
also grateful to Dr. Chiara Franciosi for helpful
discussions.
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