CE U P D A T E -
BODY FLUIDS I
Wright-Giemsa Cytology
of Body Fluids
Techniques for Optimal Cytocentrifuge Slide Preparation
The cytocentrifuge represents a great advance in
the techniques available for making microscopic
slide preparations. The instrument consists of a
centrifuge apparatus that contains a bowl to hold
the slide assemblies. The assembly consists of the
slide, a filter card, and the chamber to hold the
sample, all of which is secured together by a clip.
When the apparatus is in the resting position, the
clip assembly is angled in such a way that the
fluid specimen does not contact the glass slide.
During centrifugation, the assembly tilts, and the
centrifugal force spins the fluid and cells out the
side arm of the chamber onto the slide. The filter
card absorbs the fluid while the cells stay on the
slide. The cells are concentrated approximately
twenty-fold by the cytocentrifugation procedure.
An optimal slide preparation contains an ade-
quate number of evenly distributed cells with
lucid cellular morphology. Some body fluids,
cerebrospinal fluid (CSF) in particular, may be
very hypocellular. Before the advent of the cyto-
centrifuge, CSF samples were concentrated by
ordinary centrifugation, and a "push smear" of
the cell sediment was prepared. The morphology
of these cell-sediment concentrates rarely was
adequate to identify normal leukocyte subtypes,
much less malignant cells. In addition, the cell
yieki on hypocellular CSF samples was scant.
Using the cytocentrifuge, we can obtain a yield of
approximately 35 cells per slide for a CSF sample
that would have a cell count of zero by conven-
tional means.
Although it is tempting to prepare a simple
push smear from a cellular or bloody fluid sam-
ple, such as a pleural, peritoneal, or pericardial
effusion (see "Speaking of Body Fluids"), the
cytocentrifuge preparation yields superior
morphology. Cell distortion is minimized, and
cohesive cell aggregates are uncommon among
benign cells (Figs 1A, IB). Thus, Wright-
Giemsa-stained slides of cytocentrifuged CSF and
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Carol D. Jones, MT(ASCP)SH
P. Joanne Cornbleet, MD, PhD
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ABSTRACT Body fluid analysis can provide important
diagnostic information to the clinician. A key aspect of these
analyses is the white blood cell differential and evaluation of
cellular morphology on slides of specimens prepared with
Wright-Giemsa stains. Despite the clinical imperative for
providing consistently good slides, body fluid specimens pose
special challenges. The cytocentrifuge provides one way to
meet these challenges. We review several techniques for
achieving optimal cytocentrifuge slide preparations.
This is the first article in a three-part series on body fluids. On completion of
the series, participants should be able to prepare high-quality cytocentrifuge
slides from cerebrospinal fluid and other body fluids, identify crystals in
synovial fluid, and distinguish benign from malignant cells.
From Clinical
other body fluid samples show morphologic detail
Laboratories and
similar to that seen in peripheral blood smears, Department of o
enabling the detection of small numbers of malig- e
Pathology, Stanford 3
nant cells. (Calif) University S
Medical Center. £
Consistently excellent slide preparation espe- o
cially is important for body fluid samples. There Reprint requests to 0
may be a delay of one or more days between slide Dr Cornbleet,
Stanford Health
preparation and review of the slide by a patholo- Services, Clinical
gist. After such time, the cells may have deterio- Laboratories, Room
rated so much that adequate morphology cannot H1524, R t 6 , 300
be obtained. In addition, the specimen may not Pasteur Dr, Stanford,
be replaceable; obtaining CSF and other body CA 94305-5272. I
fluid effusions requires uncomfortable or painful
aspiration procedures that can pose a risk to the
Fig 1. A, a "push smear" prepared from a pleural fluid containing adenocarcinoma
(Wright-Giemsa, original magnification, x313) and B, the same sample prepared
by cytocentrifugation (Wright-Giemsa, original magnification, X400). Note the
improved morphology in B, particularly the enhanced nuclear detail.
NOVEMBER 1997 VOLUME 28, NUMBER 11 LABORATORY MEDICINE 713
 * ,
••*
to cytocentrifugation can improve the slide
preparation: washing serous fluids cells, lique-
& # * • i -•••- faction of synovial fluids with hyaluronidase,
and agitation of clots.
•

• • • •
5 •• •

a * ; :
• *

s •
' C • * Washing the Cells of Serous Fluids
C o
Pleural, pericardial, and peritoneal samples often
•
9 s)

q$-\ contain fibrin and other proteins that can clog

* 4, 2 „# 1 " * ?V>* ?S the filter cards, reducing cell yield. These sub-

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stances also can affect the cellular distribution on
-•••v.*
« the final preparation. Two slides made from the
Fig 2. Cytocentrifuge slides from a pleural fluid from a patient with ovarian same pleural fluid sample are compared in Figs
carcinoma (Wright-Giemsa, original magnification: A, x50; B, xlOO). The sample in 2A and 2B. In Fig 2A, the slide was prepared
A was not washed prior to cytocentrifugation. Note the presence of fibrin and the without washing the cells. In Figure 2B, a simple
uneven distribution of cells. In B, the sample was washed before
washing technique was used before cytocentrifu-
cytocentrifugation. Note the improved cell yield and even distribution.
gation. The latter reveals a dramatic improve-
patient. To best serve the patient, the technologist ment in both yield and cellular distribution.
must make the best possible slides the first To wash the cells, place approximately 1 mL of
time—there may not be an opportunity to try serous fluid into a 5-mL conical centrifuge tube,
again. Although the cytocentrifuge is a simple marking the location of the fluid meniscus on the
instrument, slide quality can be enhanced by outside of the tube with an indelible marker.
some simple techniques. Dilute the aliquot with 3 mL to 4 mL of sterile
saline and centrifuge at 400g for 5 minutes.
Specimen-Processing Techniques Decant the supernatant and resuspend the cells in
The best preparations require fresh, unfixed sterile saline to the original volume, as marked on
specimens received directly in the laboratory. the tube. Although the washed sample is not used
Three techniques for processing samples prior to perform the cell count, the approximate origi-
nal concentration of cells is maintained. The
Speaking of Body Fluids... washed-cell suspension now is ready for dilution
and cytocentrifugation. Note that this washing
Cerebrospinal fluid (CSF)—the fluid circulating within the ventricles of procedure should not be used for fluids that have
the brain, and the subarachnoid space of the cranium and spinal canal low cell count or low protein content, such as CSF.

Effusion—an accumulation of fluid, in this case within the pleural, Liquefaction of Synovial Fluids With Hyaluronidase
pericardial, or peritoneal cavities. Effusions sometimes are classified as The normal hyaluronic acid concentration of
transudates (an ultrafiltrate of plasma, usually associated with a synovial fluids renders these specimens highly
systemic disease) or exudates (associated with a localized increase in viscous. The cell count and slide preparation can
capillary permeability). be difficult to perform under these circum-
stances. Viscous synovial fluids can be liquefied
Pericardial cavity—the space between the two membranes that form a by adding a pinch of the enzyme hyaluronidase to
sac enclosing the heart approximately 1 mL of fluid. Incubate at 37°C for
1 to 5 minutes, checking visually for liquefaction.
Peritoneal cavity—the space between the two membranes that line the Washing the cells after liquefaction often
abdominal or pelvic walls and enclose the viscera enhances these slide preparations as well.
Hyaluronidase is available commercially as a
Pleural cavity—the space between the two membranes that line the frozen powder.
thoracic space and enclose the lungs
Agitation of Clots
Serous fluid or effusion—a fluid that is a simple ultrafiltrate of plasma, Although clotted body fluid specimens will yield
without active transport or exclusion of substances. Pleural, peritoneal, inaccurate cell counts, useful information still
and pericardial fluids are serous effusions, while the formation of can be obtained from the microscopic examina-
cerebrospinal fluid is regulated by the cells lining the choroid plexus tion of the cells. Hence, these specimens should
vessels of the ventricles in the brain. not be discarded. If clots are present, they should
be agitated gently with tissue forceps to free
Synovial fluid—the viscous fluid contained within joint cavities trapped cells, improving the sensitivity for the
detection of malignant cells.
LABORATORY MEDICINE VOLUME 28, NUMBER 1 1 NOVEMBER 1997
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Fig 3. A, the components of the sample chamber-clip assembly: from
left, the clip, slide, filter card, and sample chamber.
Cytocentrifuge Techniques
Several techniques can be used while performing
the cytocentrifugation to improve the quality of
the preparation. These include the use of a stan-
dardized dilution protocol, the use of albumin,
and proper handling of the chamber.
Components of the cytocentrifuge apparatus are
shown in Figs 3A and 3B.
A Standardized Dilution Protocol
The tremendous concentrating capability of the
cytocentrifuge method is an important advan-
tage for preparing slides from hypocellular flu-
ids, such as CSF. But it also raises the specter of
overcrowded cells when the fluid specimen is cel-
lular (Figs 4A and 4B). Overcrowded slides may
be difficult to interpret, owing to distortion of
cell morphology or clumping of benign cells.
It is common practice to estimate the appro-
priate dilution by visually examining the turbi-
dity of the sample, adding physiologic saline until
it grossly "looks right." We have found more con-
sistent success by using the WBC and RBC counts
to standardize sample dilution. By using a stan-
dardized scheme for sample dilution prior to
cytocentrifugation, a uniform monolayer of cells
is obtained on every sample.
We dilute the fluid to yield a WBC count of less
than 500 X 106/L (500/uL), and an RBC count of
less than 5,000 X 106/L (5,000/uL). Therefore, if
the reported WBC count is less than 500 X 106/L,
no dilution is performed. If the WBC count is
between 500 X 106/L and 1,000 X 106/L, a 50%
dilution is performed (one drop of specimen and
one drop of sterile, physiologic saline). If the WBC
count is 1,000 X 106/L to 1,500 X 106/L, a 33.3%
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B, the assembled components loaded into the cytocentrifuge bowl
and ready for the addition of the sample before cytocentrifugation.
dilution is performed, and so on. For bloody fluids,
the RBC count also is considered when making the
appropriate dilution. If the RBC count will exceed
5,000 X 106/L, then the sample is diluted further to
bring the RBC under 5,000 X 106/L.
After the specimen has been diluted as just
described, it is ready for cytocentrifugation. In
our laboratory, we place five drops (approxi-
mately 0.25 mL) of this material into each cham-
ber. Samples are cytocentrifuged at 700 rpm for 5
minutes.
For bloody samples with few white cells, one
may speculate that such a large dilution will jeopar-
dize the final yield of the WBCs. We have not
found this to be the case, even when the RBC count
is more than 1 million and the WBC count is only
several hundred. Many of the red cells are drawn
up into the filter card, and the cytocentrifuge
appears to concentrate the white cells preferentially.
Because of this phenomenon, we have not found it I
Fig 4. A peritoneal fluid containing large cell lymphoma. A, the sample was not
properly diluted prior to cytocentrifugation (Wright-Giemsa, original magnification,
x313). The cells are crowded together, distorting the morphology. In contrast, lucid
morphology (B) was attained when the same material was diluted to a WBC of 500
x106/L prior to cytocentrifugation (Wright-Giemsa, original magnification, X313).
NOVEMBER 1997 VOLUME 28, NUMBER 1 1 LABORATORY MEDICINE 718

m
Fig 5. Variation in
cell yield attained necessary to use red-cell lysing reagents or to resort
when the chamber
to push smears for bloody samples.
and filter card are
centered properly
(left), slightly The Use of Albumin
misaligned (center), Add a drop of sterile, 30% albumin to the sample
or grossly chamber before adding the diluted specimen.
misaligned (right).
This not only enhances the adherence of the cells
to the glass slide, but also provides a "cushion"
for the cells as they are spun onto it, reducing
smudge artifact and preserving the morphology.
Proper Handling of the Chamber
There are two things to remember when handling
the chamber and clip assembly. First, when
assembling the clip, filter card, and chamber
together, carefully center the sample chamber
outlet port within the hole in the filter card. As
shown in Fig 5, improper alignment results in a
dramatically decreased cell yield.
Test Your Second, when removing the clip assembly
Knowledge
from the bowl at the end of the cytocentrifuga-
Look for the CE
tion, hold the clip assembly in such a way that
Update exam on
Body Fluids (801) in
any residual fluid in the chamber does not flow
the January 1998 back onto the slide. This can be accomplished by
issue of Laboratory maintaining the clip assembly at the same angle
Medicine. Participants
as when in the resting position in the cytocen-
will earn 3 CMLE trifuge bowl. If fluid does flow back onto the
credit hours.
slide, the cells will absorb the fluid, and have a
shrunken or "rounded-up" appearance, rather
than the excellent morphology seen on a properly
prepared slide.
Spurious Presence of Bacteria
Preparation of a Reagent Blank Slide
Introducing saline and albumin into the process-
ing protocols also introduces the possibility of
716 LABORATORY MEDICINE VOLUME 28, N UMBER 1 1 NOVEMBER 1997
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bacterial contamination. The centrifugation may
bombard the cellular elements with bacteria
when contaminated reagents are used, creating
what appears to be intracellular bacteria. The use
of sterile reagents with meticulous attention to
sources of contamination and proper storage can
minimize this problem. One way to distinguish
spurious from legitimate bacterial presence in a
specimen is to make a "reagent blank" slide,
preparing a cytocentrifuge slide using all the
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reagents except the actual sample. This slide is
evaluated for the presence of bacteria after stain-
ing with Wright-Giemsa. Because our laboratory
processes a large number of samples on all shifts,
we require that a reagent blank slide be checked
as a prerequisite to result reporting whenever
bacteria are found in a sample.
Conclusion
The use of the cytocentrifuge offers an opportunity
to prepare Wright-Giemsa-stained slides with
excellent morphologic detail. This article
describes some techniques for achieving optimal
cytocentrifuge slide preparations. While these
methods may appear cumbersome or time-con-
suming, they can fit smoothly into the work flow
of body fluid analysis. For example, for serous flu-
ids, the technologist can perform the cell count
during the cell wash centrifugation; using the
proper dilution based on this cell count can save
time by eliminating any necessity to remake slides
that are too thick or too thin. Ultimately, the clini-
cal imperative for producing consistently optimal
slide preparations makes the extra time in attend-
ing to these technical details worthwhile.©
Acknowledgment
We are grateful to Nicky Sherwood, MT(ASCP), formerly of
Stanford Hospital, for her role in developing many of these
techniques in our laboratory, and for providing the
photomicrographs.
Recommended Reading
Cytospin3 Operator Guide. Pittsburgh, Pa: Shandon
Lipshaw; 1996.
Keebler C, Somrak T, eds. The Manual of Cytotechnology.
Chicago, 111: ASCP Press; 1993.
Kjeldsberg CR, Knight JA. Body Fluids—Laboratory
Examination of Cerebrospinal, Seminal, Synovial and Serous
Fluids: A Textbook Atlas. Chicago, 111: ASCP Educational
Products Division; 1993.