CE update [cytology | generalist]
Principles of Cytocentrifugation
Barry O. Stokes, PhD
Wescor, Logan, UT
DOI: 10.1309/FTT59GWKDWH69FB0
After reading this article, the reader should be able to identify the principles involved in cytocentrifugation and understand
the potential of the method.
Cytology exam 30401 questions and the corresponding answer form are located after the “Your Lab Focus” section
on p. 441.
왘 The cytocentrifuge can be used to
transfer any sedimentable particles
from liquid suspension onto a
microscope slide, but is primarily
used to transfer biological cells.
왘 It is widely used in clinical medicine
and biological research.
왘 The literature of clinical applications
is extensive and includes such
specimens as cerebrospinal fluid,
synovial fluid, urine, fine needle
aspirates, and a variety of body
fluid and lavage samples.
Prior to the introduction of cyto-
centrifugation nearly 40 years ago,1-4
the main methods for transferring cells
were: making direct smears of cell sed-
iments collected by conventional cen-
trifugation, gravity sedimentation onto
microscope slides, and filter collection
techniques. Comparisons to these
methods indicate that cytocentrifuga-
tion is a suitable method for cell prepa-
ration, although it is not always
superior.5-13 The principle concerns in
such comparisons are that cells are lost
during the cytocentrifugation process,
and that the loss can be preferential for
small cells.5,14,15 In addition, the forces
of cytocentrifugation can produce arti-
factual morphology in the cells of in-
434 terest, but the effects are not usually
severe, and some can even be benefi-
cial.16,17 Recently, automated thin-layer
cell transfer systems have challenged
the role of cytocentrifugation as the
standard method for cell transfer in
cytology.18-21 They offer high-quality
specimens without the need for exten-
sive experience, but at a high cost rela-
tive to cytocentrifugation. These
laboratorymedicine> july 2004> number 7> volume 35
systems were primarily developed for
the Pap smear market, but have also
been applied to the preparation of body
fluids. As a result of their success in
the Pap smear market, cytocentrifuga-
tion is, in turn, being explored as a
low-cost alternative thin-layer method
for gynecological analysis.22-24
Cytocentrifugation is being
increasingly employed in
microbiology25,26 and hematology27
laboratories. The use of direct smears
is considered by the College of Ameri-
can Pathologists (CAP) to produce sub-
optimal specimens and is being
formally discouraged, with cytocen-
trifugation recommended as the
replacement method for hematology.28
While cytocentrifugation remains a
widely used laboratory method, the
loss of cells and the production of aber-
rant cell morphology are important
concerns for technologists.
Centrifugation Principles
The principles of centrifugation
are well-known and are available at the
textbook level.29 Simply stated, the
principles involve particles of density
(dp), sedimenting at speed (dx/dt)
through the fluid medium of density
(dm) when a centrifugal force (F) is
applied to the particle mass (density x
volume (V)). It is useful to this discus-
sion to consider the simplified expres-
sion:
dx/dt = (dp-dm)VF
K the flow of the suspension fluid away
The buoyancy of the medium
opposes the forward centrifugal force
by lowering the effective particle
mass. The term (dp-dm) accounts for
the buoyant action of the medium.
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Frictional forces, included in the term
K, also oppose the centrifugal force.
These are a function of the characteris-
tics of both the particle and the
medium. The relative centrifugal force
term (RCF), where the radius (r) is in
millimeters, is conveniently expressed
in gravities as the familiar expression
involving rotor revolutions per minute
(RPM):
[ ]
RCF (gravities) = 1.118r RPM 2
1000
Simply stated, particle sedimenta-
tion speed increases with the rotational
speed of the centrifuge, and it is faster
for large or dense particles and slower
for small or light particles.
Cytocentrifugation
Commercially available cytocen-
trifuges fall into 2 categories as illus-
trated in F1 (ie, those which remove
the suspension fluid during cell sedi-
mentation and those which retain it). In
the latter case, the principles are basi-
cally the same as those for centrifuga-
tion. The cell sediment in response to
the applied centrifugal force (B) and
the fluid is basically static. Practical
protocols are widespread for centrifug-
ing cells in aqueous media, primarily
expressed in time and g force. Such
protocols are applicable to
cytocentrifugation with fluid retention.
When fluid removal and sedimen-
tation are simultaneous, hydraulic
forces (A) on the cells are caused by
from the sample area, usually into an
absorbent medium. The hydraulic
forces are complex. They are greatest at
the edge of the sample area and least at
its center. They also vary throughout the
©
run and between runs. These hydraulic
forces dramatically affect the sedimen-
tation of cells and render centrifugation
principles inadequate to predict the re-
sults of cytocentrifugation.
Cell Loss Considerations
Where the fluid is retained during
sedimentation, it must be removed be-
fore the slide can be further processed.
Cell loss can occur during manual or
automated fluid removal processes.
Cells collected without fluid removal
experience less force pressing them
against the microscope slide at a given
rotor speed than those with the fluid
removed. This is due to the relative
buoyancy of the surrounding medium,
[ie, effective cell density ≅ (1.05-1.0)
for fluid versus (1.05-0) for air]. This
produces less flattening against the
slide and therefore less adhesion,
which increases the potential for cell
loss during fluid removal and subse-
quent fixation and staining operations.
These losses can be important.30 In
principle, cells collected in this manner
should be given greater centrifugal
force than those collected with simulta-
neous fluid removal to promote contact
with the slide. Manual fluid removal
should be carefully done, and fixation
and staining may need special consid-
eration.
When fluid removal and sedimen-
tation are simultaneous, hydraulic
forces on the cells can cause the
process to fail or be suboptimal. When
the velocity of the fluid being removed
is much greater than the sedimentation
velocity of the cells, most of the cells
will be lost with the fluid. Obtaining
high cell recovery depends on balanc-
ing the centrifugal and hydraulic
forces during the run. This is accom-
plished by providing an appropriate
centrifugal force while controlling the
velocity of the fluid removal at a slow
enough rate to allow the cells to sedi-
ment to the microscope slide. It should
also be noted that the cell collection
process is complete once the suspen-
sion fluid has been removed. Running
the cytocentrifuge beyond the point of
fluid removal cannot improve the cell
recovery and is probably detrimental
to cell morphology. Little has been
© laboratorymedicine> july 2004> number 7> volume 35
Cytocentrifugation Chambers
Simultaneous Fluid Removal Fluid Retention
Chamber Absorbent Chamber
Tunnel Medium Slide Tunnel Slide Seal
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Sample
Fluid Sample
Fluid
Seal
[F1] The above figure illustrates cross sections of cytocentrifuge chambers. In the case of
simultaneous sedimentation and fluid removal, it shows a sample which is in contact with a
microscope slide and an absorbent medium. The arrow A represents hydraulic force on the
cells that tends to move the cells into the absorbent medium and arrow B represents the
centrifugal force that causes sedimentation of the cells to the microscope slide. Where fluid is
retained, the only force exerted on the cells is the centrifugal force. The fluid must be removed
after the sedimentation process is completed.
published on fluid flow rates during principle can also account for the
cytocentrifugation. Fluid flow is pri- preferential loss of small cells since
marily controlled by equipment design they sediment at a slower rate than
and fluid/sample characteristics, but larger cells. Eliminating preferential
the fluid removal time also depends on loss of small cells requires nearly
the rotor speed and sample volume.31 quantitative recovery of the cell popu-
The fluid removal can be observed lation, and this demands attention to
with a strobe light, but little attention the fluid removal time. Observation
is generally paid to this variable. The that the least preferential loss of small
primary indication of fluid removal cells occurs in the center of the sample
time for most operators is the presence area14 is consistent with the fact that
of residual fluid at the end of a run. If the hydraulic forces are least there.
no residual is ever observed, the fluid Start-up losses can also occur
removal time is unknown and may be where cells are lost into an absorbent
short enough to cause cell loss. As the material prior to establishing the cen-
fluid removal time goes to 0, the re- trifugal force. This is especially prob-
covery also goes to 0. As it increases, lematic with very small sample
the recovery increases asymptotically volumes32 and is minimized by
to 100%. The available data31 indicate increasing the sample volume to 0.1 to
that an absorption (fluid removal) time 0.3 mL and/or prewetting the
greater than 3 minutes is necessary to absorbent material prior to the run. It
maintain a high recovery of leukocytes should also be noted that using a slow
in a 0.3 mL sample volume at 1,000 rotor acceleration prolongs the period
RPM (110 g). Recovery of bacteria, of of absorption of sample prior to estab- 435
course, will be more difficult due to lishing the centrifugal force and can
their small size. Higher speeds and cause minor cell loss at start up.
longer fluid removal times will be re- Prewetting the absorbent material is a
quired for high recovery of smaller useful precaution when using a slow
cells. Failure to balance the magnitude rotor acceleration.
of the hydraulic and sedimentation
forces probably accounts for most of Morphological Considerations
the reports of low recovery due to Perhaps the first consideration
simultaneous fluid removal. This here is the health of the cells. Once
 cells are removed from the body, they
begin to degrade and eventually
become worthless in any procedure.
The best morphology will be obtained
with fresh cells maintained and run in a
protective environment. The presence
of nutrient solutions (balanced saline or
tissue culture media) and proteins
(BSA or HSA) will prolong viability
and protect the cells during the cyto-
centrifugation process.
The force applied to the cells is an
important factor in the morphology of
the preparation, but the force experi-
enced by the cells is more important
than the force developed by the rotor.
This distinction is due to the buoyant
force of the suspension fluid which
counteracts the force developed by the
spinning rotor. When the suspension
fluid has been removed, the cells are
essentially suspended in air which has
near 0 density. This change in buoy-
ancy increases the force experienced by
the cells about 20 fold, [ie, (1.05-
0)/(1.05-1.0) ≅ 20]. In normal practice
with healthy cells, this force is useful
to flatten the cells and promote adhe-
sion to the slide, and it produces only
minor cell distortion. The force experi-
enced after the fluid is removed
depends on the g force and hence the
rotor speed. Decreasing the speed will
soften the forces but can adversely af-
fect the cell recovery by slowing the
particle sedimentation rate. Prolonging
the run time much beyond the point of
fluid removal is not useful and can be
detrimental to morphology. The gen-
tlest procedure for fragile cells is to
process them in equipment without
fluid removal or to extend the fluid re-
moval time by adding protein to the
suspension fluid and interrupt the run
prior to removing all of the fluid. At
the end of the run, the operator should
manually remove the fluid carefully to
436 avoid dislodging the cells from the mi-
croscope slide.
Cytocentrifugation
Procedures
The instrument variables in cyto-
centrifugation are fairly simple. They
are speed, time, and equipment design.
The main complexity lies in the nature
of the sample, and success is largely
laboratorymedicine> july 2004> number 7> volume 35
determined by the extent of sample
characterization and pretreatment. Pos-
sible pretreatments include:
• Adjusting cell concentration
• Liquifying or diluting viscous
samples
• Removing precipitates or debris
• Lysing erythrocytes
• Adding nutrients and/or proteins
• Adding preservatives
In general, one needs a fresh (or
well-preserved) sample of healthy cells
at a suitable concentration, an absence
of interfering materials, and a suspen-
sion fluid that allows proper cell sedi-
mentation and fluid absorption.
Achieving these objectives is not al-
ways simple and may require more
than 1 pretreatment procedure. The
combination of a suitably treated sam-
ple and appropriate instrument parame-
ter selections determines the final
quality of the cytocentrifuged speci-
men.
Perhaps the most important single
factor in specimen preparation is to
obtain an appropriate number of cells
in the sample applied to the instrument.
If too many cells are applied, the re-
sulting specimen will be thick, layered,
and difficult to read. It may even
slough off during fixation and staining
and be lost. Ideally, one obtains a cell
count and alters the sample concentra-
tion to provide the required cell con-
centration. The count need not be
highly accurate, and a rather wide
range will produce acceptable results.
Experienced operators can often visu-
ally judge the sample concentration
and get acceptable results. The number
of cells required is primarily a function
of the equipment selected and its asso-
ciated cell collection area on the micro-
scope slide.
Another crucial factor is the qual-
ity of the specimen. Samples should be
processed within a few hours of collec-
tion or well preserved immediately
upon collection. Since fixation hardens
the cells and prevents flattening against
the microscope slide, fresh samples are
preferred. Unfortunately, fresh samples
are not always available, and cytocen-
trifuged specimens are often prepared
from samples fixed in Saccomano’s or
other fixatives. In unfixed samples, a
number of factors affect cell quality
and stability, including: osmolality,
available nutrients, and microbial con-
tamination. The presence of proteins in
the suspending fluid can also protect
the cells against degradation during the
process of cytocentrifugation. Refriger-
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ation is useful in preventing cell degra-
dation for short periods when
processing needs to be delayed. Best
results are usually obtained when fresh
cells are suspended in a nutrient-con-
taining medium with small amounts of
protein and processed immediately
after collection. A number of less im-
portant decisions are necessary to pro-
duce acceptable cytocentrifuge
specimens, including: equipment selec-
tion, sample volume, rotor speed, and
processing time. The question of equip-
ment selection depends on the objec-
tives of the operator. The major
determining factors are the desired
specimen area on the microscope slide
and the desired sample size to be
processed. Cost, design features, con-
venience, and personal preferences also
enter into the equipment selection
process. The speed and time selections
depend on the equipment selected and
the sample characteristics. A wide
range of choices will yield useable
samples for many purposes, even
though cell collection may not be
quantitative. In much of clinical prac-
tice, optimization of the specimen is
less important than cost-effective pro-
duction of specimens.
Summary and Conclusions
Cytocentrifugation has been a
mainstay in clinical and research labo-
ratories for decades as a means of
preparing microscopic specimens from
cell suspensions. Significant concern
exists regarding the recovery of cells in
cytocentrifugation. The role of the hy-
draulic forces in the process has been
neglected. When the fluid is removed
during cell collection and the fluid flow
becomes too fast (short fluid removal
time), cells are swept away from the
sample area before they are able to sedi-
ment to the microscope slide. Quantita-
tive cell recovery in cytocentrifugation
is only possible if the speed of fluid
removal is controlled at a rate that is
©
slow enough to allow the cells to sedi-
ment to the microscope slide. The fluid
removal rate depends both on the char-
acteristics of the sample and the instru-
ment settings. Where the equipment
does not remove the fluid during sedi-
mentation, care must be taken to pro-
vide enough centrifugal force to flatten
the cells and to carefully remove the
fluid once the run is completed. Fortu-
nately, quantitative cell recovery is not
always required, and very acceptable
results may be obtained with routine
procedures. Morphological considera-
tions are also important in the process
and require attention to cell characteris-
tics and the forces placed on them dur-
ing cytocentrifugation.
Acknowledgements: The author
wishes to thank Patti Nelson for assis-
tance in developing the article; Susan
Udy for preparing the manuscript; and
Wayne Barlow, Kent Thomas, and
Dennis Briscoe for their comments and
suggestions.
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