your lab focus
science [generalist | hematology | blood banking]
Cellular Sedimentation and Barrier Formation
under Centrifugal Force in Blood Collection Tubes
Fu-Chung Lin, PhD, Richmond Cohen, PhD, Robert Losada, Valerie Bush, PhD
Becton Dickinson, Vacutainer Systems, Preanalytical Solutions
왘 Human blood separation dynamics in
different separator tubes
왘 Cell sedimentation and gel movement
affects serum clarity
왘 Plasma yield, spin times, and plasma
cell interface
BD Vacutainer™ Systems M
introduced the serum separator tubes
(BD SST™) to laboratories
approximately 25 years ago, and they
remain the prevailing standard for blood
collection tubes for clinical diagnostics.
In recent years, laboratories have been
striving for improved efficiencies and
reduced turn-around-time (TAT) of re-
sults. BD Vacutainer™ SST™ tubes pro-
vide a means for improving laboratory
efficiency by using a single tube for
multiple chemistry analyses, improving
sample purity and analyte stability. This
primary tube can be used for instrument
sampling and storage, thereby reducing
the need for aliquot tubes. Frequently,
laboratory professionals centrifuge blood
collection tubes under a variety of condi-
tions to shorten TAT or to preserve tem-
perature sensitive analytes. Associated
with this practice are limitations with
588 respect to specimen quality and product
performance.
In a gel barrier tube, an inert gel
moves to the serum clot, or plasma
packed cell interface under centrifugation
thereby providing a mechanical and
chemical barrier between the supernatant
and the cells. The gel exhibits thixotropic
laboratorymedicine> october 2001> number 10> volume 32
properties (such that it is semi-solid
under static conditions and becomes less
viscous when a force is applied),
enabling it to flow during centrifugation.
Separator gels are designed with a spe-
cific density that falls between those of
the serum or plasma and the cells, thus
determining the location of the interface.
These characteristics make it an ideal
material for separating 2 phases of differ-
ent densities by centrifugation. Despite
the prevalence and utility of separator gel
tubes for blood collection, the
mechanism of gel movement in the tube
during centrifugation is not clearly un-
derstood by many laboratory profession-
als. Forces applied to the gel can be
designated, and initiation of movement
of the gel can be calculated, but the way
the gel moves cannot be calculated. An
understanding of how the gel moves to
the interface and forms a barrier is im-
portant as laboratories seek to improve
efficiency and specimen quality.
Winkelman and Tanasijevic1
recently described RBC movement
through thixotropic gels. However,
some of the terminology used in the
article was confusing.2 It was not clear
if the authors used clotted blood or an-
ticoagulated blood, as the gel
movement differs between the two. Ad-
ditionally, the authors recorded cell
movement at intermittent time intervals
and under high centrifugal forces
(much higher than are normally used).
This made it difficult to translate their
©
observations to common laboratory
practice.
Becton Dickinson has extensively
studied gel movement in clotted and anti-
coagulated blood. The best method of de-
termining gel movement is by visual
inspection with continuous monitoring
during centrifugation. This is accomp-
lished by using a strobe centrifuge and a
video camera that records blood separa-
tion and gel movement during centrifuga-
tion. Our studies have characterized
human blood separation dynamics during
centrifugation of BD Vacutainer tubes
under the manufacturer’s recommended
handling conditions. This article describes
blood separation in tubes with and with-
out gel using clotted and anticoagulated
blood and offers insight as to how labora-
tory efficiency and specimen quality can
be managed.
Methods
Blood was collected from 18
donors into 4 different types of tubes: a
serum tube without gel, a BD SST tube,
a lithium heparin tube without gel, and
a plasma separator tube (BD PST). All
tubes were collected from donors
according to a randomized schedule. All
tubes were 16x100 mm plastic BD
Vacutainer blood collection tubes.
Blood collected in the serum tubes with-
out gel was allowed to clot for 60 min-
utes while blood in BD SST tubes was
allowed to clot for 30 minutes. The
manufacturer recommends centrifugal
 왗your lab focus 왘

A B
free red cell layer

serum

Volume (%)
clot

Time (minutes)

C D
free red cell layer

serum
Volume (%)
gel

clot

Time (minutes)

[F1] (A) Measurements of plain serum tube during centrifugation. Serum volume was calculated from D-C. (B) Percent volume versus time for serum tube
without gel. (C) Measurement of BD SST tube during two stages of centrifugation (before and after the gel has moved to the interface). Serum volume was
calculated from either D-C or D-B; the free RBC layer volume from C-A; and gel volume from (B-A). (D) Percent volume versus time for BD SST tubes.

forces for these tubes of 1000-1300
RCF. Each tube was placed into a hori-
zontal head centrifuge (International
Equipment M) with a strobe attachment
(GenRad 1546 Strobotac). The strobe
light was synchronized with the acceler-
ation (1000 RCF) of the centrifuge to
facilitate video recording and continu-
ous monitoring of blood separation and
gel movement. The positions of cells
and gel at selected time intervals were
measured with a ruler placed within the
view of the video-recording camera.
Column heights were converted to vol-
umes based on known tube dimensions.
The Column heights for the different
phases of blood separation are shown in
F1A, F1C, F2A, and F2C. The time in-
tervals recorded were every 10 seconds
for the first minute, every 30 seconds
from 1-5 minutes, 7.5 minutes and 10
minutes. The percent volumes of the
components were plotted versus time to
display blood separation graphically
[F1B, F1D, F2B, F2D]. The total per-
centages in the gel tubes are greater
than 100% because the volume of the
gel that has not reached the interface is
always included the clot phase [F1D,
F2D]. The total volume percentage in-
cludes all of the gel regardless of its lo-
cation.
Results and Discussion
Our observations of experiments in
the strobe centrifuge indicate that
although cell and gel movement are re-
lated in a clotted specimen and an antico-
agulated specimen, different mechanisms
of gel movement occur. As shown in
F1B, F1D, F2B, and F2D, the centrifugal
force applied to blood tubes has differing
effects on phase movement depending
upon the tube type (gel vs. non-gel) and
the state of the blood (clotted vs. antico-
agulated). These observations indicate
similar cell sedimentation but different
mechanisms of gel movement in a clotted
specimen compared with an anticoagu-
lated specimen. 589
Centrifugation of serum tubes
without gel and BD SST tube
As shown in F1, sedimentation of
cells in both serum tubes without gel
and BD SST tubes begins immediately
after the centrifugal force is applied. In a

© laboratorymedicine> october 2001> number 10> volume 32

 왗your lab focus 왘

A B

plasma

Volume (%)
packed cells

Time (minutes)

C D

plasma
Volume (%)

gel

packed cells

Time (minutes)

[F2] (A) Measurements of plain plasma tube during centrifugation. Plasma volume was calculated from (D-A). (B) Percent volume versus time for plasma
tube without gel. (C) Measurement of BD PST tube after centrifugation. Plasma volume was calculated from D-B, and gel volume from (B-A). (D)
Percent volume versus time for BD PST tubes.

serum tube, the clot begins to compress
followed by the sedimentation of some
free red blood cells (RBCs) not
entrapped in the clot. The free RBCs
settle at a slightly slower rate than the
clot because of relative differences in
density and size. Simultaneous to cell
sedimentation is the expression of serum
with nearly all of the serum recovered in
approximately 2 minutes. Further cen-
trifugation results in relatively small in-
590 creases in serum volume. Although cell
counts were not performed on the super-
natant in these experiments, the data
suggests that centrifugation of
completely clotted serum tubes can yield
relatively clean serum in approximately
2 to 3 minutes. Most manufacturers rec-
ommend spinning serum tubes for 10
minutes to improve barrier formation,
serum clarity and yield.
In BD SST tubes, cells sediment
just as those do in clotted blood without
gel. The clot begins to compress,
followed by sedimentation of free
RBCs within the first minute of
centrifugation. Within 20 seconds of
centrifugation, the free RBCs begin to
settle and are completely settled (1
minute) before the gel first reaches the
interface ( approximately 2 minutes) as
shown in F1D. Therefore, the serum has
clarified before the gel moves to the
interface. The gel does not move imme-
diately to the interface between the
serum and the cells because it cannot
move through the clot. The gel must
move around the clot and along the
wall of the tube in a BD SST tube. On
average, the initiation of gel movement
in the BD SST tube is delayed approxi-
mately 1 to 1.5 minutes after centrifu-
gation begins [F1D]. An entire layer of
gel covers the cells after about 3 to 5
minutes. Because the sedimentation is
completed before an intact gel layer
forms, relatively few cells are entrapped
in the gel of a BD SST tube. Beyond 5
minutes, additional gel on the wall of
the tube moves into position and thick-
ens the barrier, further compressing the
clot. This subsequently increases serum
yield. On average, a complete, thick
barrier is formed in 7 to 8 minutes.
The dynamics of cell and gel move-
ment in the BD SST tube illustrate the
importance of the recommended handling

laboratorymedicine> october 2001> number 10> volume 32 ©


conditions, but also indicate opportuni-
ties for the manufacturer to improve lab
efficiency. Because a thick barrier is not
formed for 7 to 8 minutes when cen-
trifuging at 1000g, it is recommended
to spin for 10 minutes to ensure sample
stability and maximum serum yield.
However, the serum clarifies in less
than 2 minutes. Were the gel to move
completely to the interface 2 minutes
after centrifugation began, free cell sed-
imentation would be concluded, cen-
trifugal spin time could be abbreviated,
and a specimen would be obtained. Al-
though the serum volume might not be
maximized, as seen in F1D, most of the
specimen would be available, and TAT
would be shortened.
Although the inability of the gel to
move completely to the interface at the
ideal time necessitates longer spin
times, the delay does lead to benefits in
terms of serum quality. As mentioned,
visible cell presence within the gel
layer is limited because the gel does not
begin to form a barrier until after cell
sedimentation is virtually complete.
This is significant, as the results of our
experiments on the dynamics of cell
and gel movement in the BD PST tube
will show in the next section.
Centrifugation of plasma tubes and
BD PST tubes
In a plasma tube without gel, cell
sedimentation follows a more linear rela-
tionship with time [F2B] and at a much
slower rate than clotted blood. As
centrifugation proceeds, the cells continue
to settle, and more plasma is expressed. It
takes approximately 7 to 8 minutes for all
of the plasma to be recovered in a
heparinized tube under these centrifuga-
tion conditions. Therefore, while a serum
tube without gel can produce
approximately 40% of the serum in 2
minutes, it takes nearly twice as long for a
heparinized sample to produce the same
volume of plasma. While no cells are vis-
ible in the supernatant of clotted blood,
some low density and smaller cells remain
visible in the plasma after centrifugation
for 10 minutes at 1000g. Because the clot
has a higher density and greater size than
free cells in anticoagulated blood, faster
sedimentation occurs, and fewer cells re-
main in the specimen.
In the BD PST tube, the gel moves to
the plasma: cell interface shortly after
centrifugation begins or as soon as an in-
terface is created (within 30 seconds). As
illustrated in F2D, the gel moves in paral-
lel with cell sedimentation, and the barrier
thickens as it compresses the cells. In-
stead of flowing along the wall of the tube
as in the BD SST tube, the gel moves
through the blood in discrete portions
until it reaches the interface. This results
in faster barrier formation but entrapment
of many more cells above and in the gel
compared to a BD SST tube. A complete
barrier is formed in approximately 1
minute, but it takes approximately 3 min-
utes before the gel begins to clarify. Cells
continue to sediment through the gel with
each successive minute of centrifugation.
Any cells remaining in the barrier after
centrifugation have little clinical signifi-
cance, as there is no driving force to move
them. As mentioned above, low-density
and smaller cells (platelets and some
WBCs) remain in the plasma after cen-
trifugation, yielding a somewhat more
turbid supernatant than serum. The num-
ber and type of cells remaining in the su-
pernatant depend upon the patient’s
hematologic profile. While the plasma
from BD PST tubes contains fewer visible
cells than that from lithium heparin tubes
without gel, reduced counts of low-den-
sity and smaller cells do remain in the
supernatant (data not shown).
Plasma yield increases with longer
spin times until approximately 8 to 10
minutes, when the cells are packed and
final barrier thickness is achieved. Thus,
it takes much longer to obtain the full
volume of plasma in a BD PST tube
compared to serum in a BD SST tube.
The suggested centrifugation conditions
recommended by the manufacturer are
defined for optimal plasma yield and pu-
rity. However, there is clearly an opportu-
nity for the manufacturer of plasma tubes,
as well as serum tubes, to help the labora-
torian with reducing TAT while maintain-
ing specimen quality. A barrier that can
move into position quickly, such as the
current gel, but that allows a pathway for
sedimentation throughout centrifugation,
© laboratorymedicine> october 2001> number 10> volume 32
왗your lab focus 왘
would provide an ideal scenario for effi-
ciency and sample purity.
We have reported on the separation
of blood under recommended handling
and centrifugation conditions. Barrier
formation in gel tubes will vary with
centrifuge types due to the angle of the
tube carrier. Rates of barrier formation
differ according to various ramp-up and
ramp-down accelerations, as well as
centrifugation temperature. Other fac-
tors can also influence sedimentation
rates, eg, serum/plasma viscosity and
cell density.
Summary
We have studied and detailed cell
sedimentation and gel movement in
clotted and anticoagulated blood during
centrifugation of BD Vacutainer™
tubes using recommended handling
conditions. Using a strobe centrifuge
and video camera, we showed that the
dynamics of cell and gel movement are
different for clotted and anticoagulated
blood. In BD SST tubes, the gel must
move around the clot. The gel flows
continuously along the tube wall, delay-
ing its arrival at the interface between
the serum and cells. The benefits of this
delay are that the serum is clarified
prior to barrier formation, and few cells
are entrapped in the gel, allowing for
improved serum quality.
In plasma tubes, cells sediment more
slowly than in serum tubes due to several
factors. In BD PST tubes, the gel can
move readily through the anticoagulated
blood to reach the plasma: cell interface.
Unlike the gel moving in a continuous
flow in BD SST tubes, the gel in BD PST
tubes moves in discrete pieces. Because
the gel reaches the interface faster and
travels through the blood, more cells are
trapped between the gel portions and con-
centrate at the interface. Most of these
cells sediment through the gel during the
remaining centrifugation period. 591
Reducing the overall TAT of report-
ing test results is a common clinical labo-
ratory quality measure. Frequently,
reducing the processing time is a method
to achieve shorter TAT. However, as
demonstrated by our studies, specimen
purity is dependent upon cell separation
 왗your lab focus 왘
and gel movement (in a tube containing
gel). This is particularly true for
plasma samples as cell sedimentation
takes much longer for anticoagulated
blood than clotted blood. Although cell
sedimentation times, and thereby, cen-
trifugation times, can be shortened
with serum tubes, clotting time is criti-
cal to obtaining a cell-free and fibrin-
free serum. Use of lithium heparin
tubes greatly reduces the total TAT, but
shortened centrifugation times may re-
594
laboratorymedicine> october 2001> number 10> volume 32
sult in a supernatant containing some
cells. Some reductions in processing
time of blood collection tubes can be
made without compromising specimen
quality depending on the type of tube
collected.
Gel tubes can significantly improve
serum/plasma purity by separating the
cells from the supernatant. The gel bar-
rier also permits primary tube sampling
without cellular contamination, thus
improving laboratory efficiency.
©
Acknowledgement
We wish to acknowledge Moushmi
Patel for her efforts in collecting the data.
BD, BD Logo, and Vacutainer, SST
and PST trademarks are property of
Becton, Dickinson and Company.
©2001.
1.Winkelman J and Tanasijevic M. How RBCs move
through thixotropic gels. Lab Med 1999;30:476-
477.
2. Cohen R. RBCs on the Move? [Letter] Lab Med
2000; 31:304.