SMILE
Johns Hopkins University
Baltimore, MD USA
CYTOCENTRIFUGE TECHNIQUE FOR SMEAR PREPARATION
Author(s), Name &
Title
Penny S. Stevens MBS, MT (ASCP),
CLS (NCA)
Document Number
Effective Date
Sr. International QA/QC Coordinator
Pro6.4-E-05
23 Jan 2009
SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your
lab’s specific processes and/or specific protocol requirements. Users are directed to countercheck facts when
considering their use in other applications. If you have any questions contact SMILE.
Name, Title
Signature
Date
Name, Title
Signature
Date
Approved By
SOP Annual
Review
Version # [0.0]
Revision
History
Name (or location)
Revision Date
[dd/mm/yy]
Description (notes)
# of copies
Name (or location)
# of copies
Distributed
Copies to
106739189
Version 1.0
Page 1 of 12
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
I acknowledge that I have read, understand and agree to follow this SOP.
Name (print)
106739189
Signature
Version 1.0
Page 2 of 12
Date
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
I.
PRINCIPLE:
1. The cytocentrifuge method is used as a means to concentrate cells within a
defined area to enable morphological identification and differential counting.
It provides more cells than that which would be present in a wedge smear
preparation on the same sample and therefore, provides for greater precision
in counting.
2. The principle of all cytocentrifugation techniques is that the cell is denser
than the suspending fluid and under an applied force the cell will have
greater momentum than the fluid. The cellular movement occurs after
passing through the sample chamber. The cells are projected towards the
microscope slide with sufficient momentum to cross the exit port formed by
placing a filter card between the chamber sample and the glass slide
resulting in cell to slide adhesion.
II.
DEFINITIONS
1.
2.
3.
4.
5.
6.
III.
CBC - Complete Blood Count
CSF - Cerebrospinal Fluid
LIS - Laboratory Information System
QC - Quality Control
RBC - Red Blood Cell
WBC - White Blood Cell
SPECIMENS:
1. Cerebrospinal Fluid:
a. A physician obtains CSF by lumbar puncture and always under
aseptic conditions.
b. It is a routine practice to collect three (3) sterile tubes of CSF (1-5 ml
per tube) for analysis.
2. Tubes should be collected and labeled sequentially by the physician at the
time of collection. All tubes must be labeled properly and delivered
immediately to the following sections:
a.
b.
c.
d.
106739189
Tube #1: To Chemistry for protein and glucose or serology study.
Tube #2: To Microbiology for culture and gram stain.
Tube #3: To Hematology for cell count and differential
If only one tube is collected, perform testing in the following order to
preserve specimen and avoid contamination:
i. Microbiology - culture and gram stain.
Version 1.0
Page 3 of 12
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
ii. Hematology - cell count and differential.
iii. Chemistry
3. Cell lysis can begin within one (1) hour of collection so prompt delivery to the
laboratory is critical.
4. Specimens must be handled as STAT. If possible, notify laboratory
personnel before specimen collection to ensure staff is ready for testing
immediately after collection.
5. Always deliver specimens to laboratory personnel by hand - never drop
specimens off or leave unattended.
6. Clotted specimens are not satisfactory for testing.
a. If the specimen is clotted, the cell count can not be performed. Notify
the physician immediately.
b. Prepare a Cytocentrifuge smear and review for malignant cells. Do
not perform or report a manual differential.
c. Document actions taken and all notification in the LIS and on the CSF
worksheet.
7. Never run a body fluid through the CBC automated counting instrument. Cell
counts are performed manually using a hemocytometer.
8. Hematology specimens are retained for 7 days at 2-8°C in the hematology
refrigerator in the container marked "Fluids". If specimens are transferred for
additional testing, they will be stored in the transfer departments as required
by department procedure.
IV.
EQUIPMENT & REAGENTS
1. Equipment:
a. Cytocentrifuge –Wescor or Thermo Shandon Cytocentrifuge
b. Sterile pipettes
c. Pre-cleaned etched glass cytospin slides
d. Wescor Cytofunnel sample chamber
e. Wescor absorbant filter pads, pre-punched
f. Shandon Cytofunnel sample chamber with filter
g. Shandon Cytoclip
h. LIS label
2. Reagents:
a. 22% Bovine Albumin Solution (protein concentration)
106739189
Version 1.0
Page 4 of 12
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
b. Diff Quick or Wright’s Stain
V.
QUALITY CONTROL - NOT APPLICABLE
VI.
CALIBRATION: Centrifuge speed verifications are performed semi-annually
by the medical maintenance department. Document accordingly on the Body
Fluid QC and Cytocentrifuge Maintenance Worksheet. Appendix 3 in the CSF
Cell Count SOP
VII.
PROCEDURE:
a. Determine the amount of fluid to be used depending on the appearance.
Varied amounts may be used.
i. Clear fluids: 2 drops of well mixed patient specimen
ii. CSF fluid: Add 2 drops 22% Albumin Solution. This is a protein
concentration that will aid in cell to slide adhesion and decrease
cellular distortion due to the centrifugal force.
iii. The cytofunnels are capable of holding 0.1mL to 0.5 mL total
volume.
b. Cloudy or bloody fluids: Perform a dilution using physiological saline.
Albumin solution will not be required. Prepare 2 slides using the same
dilution as used during the hemacytometer count and prepare an undiluted
slide. Annotate the dilution factors on the slides.
c. Label the slides with an LIS label or pencil. NOTE - the labels should be
placed on the opposite side of the etched circle. The circle is visible through
the glass and should not be on the same side as the patient specimen.
d. The cytofunnels are specific to the centrifuge in use. Use only Wescor
Cytopro funnels with tan or white absorption pads with the Wescor
Cytocentrifuge and Thermo Shandon Cytofunnels with the Thermo Shandon
Cytocentrifuge.
e. The slides are also specific to the centrifuge in use. Ensure that the funnel
opening aligns directly with the etched circle on the cytospin slide and use
only Wescor Cytopro cytocentrifuge precleaned etched slides with the
Wescor Cytocentrifuge and Shandon cytospin slides with the Thermo
Shandon Cytocentrifuge.
f. WESCOR CYTOCENTRIFUGE:
i.
106739189
Version 1.0
Turn the instrument on. The power switch is located on the back,
bottom, right of the instrument.
Page 5 of 12
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
ii.
Lift the lid and remove the rotor assembly.
iii.
Remove the sealed rotor assembly lid by pulling up on the silver
button. Lift the lid off.
iv.
Insert the slides with the etched glass facing forward and the
printing legible.
v.
Select the appropriate Cytopro funnel as follows:
1. Tan (slow): Clear colorless CSF or urine samples (thin
samples)
2. White (fast): Synovial, sputum and turbid body fluids (thick
samples)
3. Tan & White: Other fluids as necessary based on sample
cellularity, turbidity and viscosity.
vi.
Press down on the spring arm and insert the funnel. Add patient
sample as indicated previously (under section VII.a-b.) and cap the
funnel.
vii. Place the lid on the rotor assembly and seal it by pressing the
silver button. Insert the assembly into the cytocentrifuge and close
the lid.
viii. Press [STOP] and select the [CYTOCENT] key. Press the number
1 for program cycle 1 and press [RUN]. All hematology body fluid
samples will use cycle 1. This program is set at 1000 RPM for 4
minutes.
ix.
Set new programs as follows:
1. Select [CYTOCENT] and then enter the number of the program
i.e., [1], [2], [3], etc. to be programmed.
2. Select [PROG] and then enter the desired speed, time and
acceleration. The acceleration options are 1 = Low, 2 =
Medium or 3 = High.
x.
The display will revert back to the cytocentrifuge main menu
automatically once the acceleration has been entered
g. THERMO SHANDON CYTOCENTRIFUGE:
i.
106739189
Version 1.0
Place the labeled glass slide in the cytospin clip. Place a
disposable Shandon cytofunnel sample chamber with attached
filter facing the labeled slide.
Page 6 of 12
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
ii.
Close the cytospin clip.
iii.
Add patient sample as indicated previously (under section VII.a-b.)
and cap the funnel.
iv.
Press the ON/OFF switch on the front of the instrument to turn
power on.
v.
Make sure that the bench or support surface is solid immovable
and flat.
vi.
If the ‘BAL’ and ‘!’ alarm lights flash lift the front of the instrument
about ½ inch from the bench. Place the centrifuge back to normal
positions. This will reset the movement detection system.
vii. Press [OPEN LID] and at the same time lift the see through safety
cover.
viii. If it won’t open, the emergency release is located on the upper left
side of the upper housing. Remove the cap and insert a pencil
into the aperature.
1. Remove the Sealed Head assembly and pull up the center
button of the lid until it clicks, and lift off the lid.
2. If the lid sticks, lift the centrifuge and touch the ball bearings
located under the assembly. This releases the vacuum.
3. Place the cytoclip with labeled slide into the sealed head
assembly. Make sure to balance the cytoclip in the centrifuge.
4. Close the sealed head by pushing the center button to secure.
Carefully place assembly into centrifuge and close lid.
5. The [LID LOCK] alarm light will indicate that the safety cover is
not closed correctly. Press [OPEN LID] and carefully close
again.
6. Select the desired program by pressing [LOAD].
a. 1-BODY FLUIDS, 1000 rpm, 10 min.
b. 2-CSF, 800 rpm, 6 min. 5-Platelet Smear 2000 rpm, 10
min.
c. Press [ENTER] and [START].
106739189
Version 1.0
Page 7 of 12
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
7. In order to create a loaded program, select [LOAD], choose a
number 1-9 using the numeric keys, press [ENTER].
a. Select [SET TIME], choose a time from 1 to 99 minutes,
press [ENTER].
b. Select [SET SPEED], choose a speed between 200 and
2000, press [ENTER].
c. Select [SAVE], choose the number that was just loaded,
press [ENTER]. The information will be retained in
memory for future use.
8. Press the [OPEN LID] key and remove the sealed head
assembly. Open the sealed head assembly by pulling the
center button. Remove cytospin clips and funnels.
9. To unload the cytofunnel from the cytoclip: Hold the clip firmly.
Push the spring against the slide to release it from the hooks.
Allow the cytofunnel to pop out of the clip.
h. Dispose of all used cytofunnels. Do not reuse.
i.
VIII.
Dry slides horizontally before staining with wrights or Diff Quick stain
CALCULATIONS:
a. Differential results are reported as percentages not total number of cells
counted. If cell count is performed using anything other than 100 cells,
convert the cells to percentage as follows:
i. [(number of cell type counted)/(total number of cells counted)] x 100
ii. If 50 total cells are counted: 15 Lymphocytes and 35 Neutrophils:
1. 15 lymphocytes/50 total cells x 100 = 30% lymphocytes
2. 35 Neutrophils/50 total cells x 100 = 70% neutrophils
3. Your calculations must total 100% when added.
b. Insert the following comment: “X number of total cells counted during
differential cell count and converted to percentage.”
IX.
REPORTING RESULTS
a. Refer to the CSF Cell Count Procedure for additional information.
b. Record all results, color, clarity, manual cell count, and differential
results on the Body Fluid Worksheet (appendix 2 of the CSF Cell
Count SOP) during testing. Do not record any results on scrap paper.
106739189
Version 1.0
Page 8 of 12
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
c. Any notes or comments should also be added to the worksheet. (i.e.,
specimen dilution, stained or unstained count, precipitate presence,
etc.)
d. All results, cell count data and differential, will be reported in the LIS
and results released to the physician within the one hour of receipt in
the laboratory.
e. After the manual differential is completed a pathology review must be
ordered for the specimen in the LIS.
f. The results from microbiology and chemistry must also be printed
from the LIS and attached to the hematology results. All section
results must be available for the pathologist at the time of their review.
g. Enter all pathologist comments in the LIS and reported to the
physician. Upon completion, the finalized CSF worksheet will be
maintained in the PATHOLOGY REVIEW book for one month.
2. INTERPRETATION OF RESULTS:
a. CSF is normally clear, colorless, and hypocellular. Any turbidity or
color presence is abnormal.
i. To differentiate
hemorrhage:
a
traumatic
tap
from
subarachnoid
1. Traumatic tap - staining of the (3) tubes of CSF is
uneven, being greatest in the first tube, and least in the
last tube. After centrifugation, the supernatant is
colorless and the specimen tends to clot.
2. Subarachnoid hemorrhage - the blood is evenly mixed,
the supernatant becomes yellowish within a few hours
after the hemorrhage, and the fluid will not clot.
ii. Pink color - indicates RBC lysis and hemoglobin release. It
can be seen 4 to 10 hours after a subarachnoid hemorrhage.
iii. Yellow or xanthochromic - indicates pathologic bleeding
resulting from hemoglobin breakdown to bilirubin in the
subarachnoid space. Xanthochromia persists for 2 to 3 weeks
after hemorrhage. It is also caused by a very high protein
concentration in the CSF or by liver disease.
106739189
Version 1.0
Page 9 of 12
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
iv. Brown - indicates the presence of methemoglobin, which forms
after a subdural or intracerebral hematoma.
b. The CSF normally contains small numbers of lymphocytes and
monocytes.
c. Ventricular lining cells, ependymal cells or choroid plexus cells may
occasionally be seen in normal or abnormal CSF.
d. Additional cell types that may be found in normal CSF include bone
marrow cells, chondrocytes (cartilage cells), squamous epithelial cells,
fibrous tissue, and adipose tissue.
e. Abnormal cells that may be seen in CSF are plasma cells, monocytes
(together with neutrophils and lymphocytes), lipophages (foamy
macrophages), and malignant cells.
f. Others terms used to describe monocytes are "reticulomonocytes",
"histiocytes", and "macrophages".
3. CORRELATION OF RESULTS
a. Compare the cytospin manual differential and total cell count results.
They should correlate as follows:
Hemocytometer
WBC Cell Count
Cytocentrifuge
Expected Total Cell Recovery
0
1-15
6-10
11-20
> 20
0 - 40
20 - 100
60 - 150
150 - 250
>250
i. If they do not correlate:
1. Verify the cytospin was prepared using the same
dilution as the hemocytometer cell count.
2. Verify calculations
3. Repeat the hemocytometer cell count.
4. Prepare a new cytospin specimen and repeat the
manual differential cell count.
106739189
Version 1.0
Page 10 of 12
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
b. Other section correlation - Before results are released, compare
results with microbiology and chemistry. If discrepancies are
detected between results, testing must be repeated if sample
volume permits.
c. If the problem can not be resolved, notify the attending physician
and document all actions taken on the Body Fluid Worksheet appendix 2 in the CSF Cell Count Procedure and in the LIS.
X.
REFERENCE RANGES:
Leukocyte Percent Differential:
Adults:
Lymphocytes…………… 60% ± 20%
Monocytes……………… 30% ± 15%
Neutrophils ……….…….. 2% ± 4%
Neonates:
Lymphocytes…………… 20% ±15%
Monocytes………….……70% ± 20%
Neutrophils ……………….4% ± 4%
XI.
PROCEDURAL NOTES:
1. Specimens must be well mixed. Failure to mix the specimen can cause
erroneous results.
2. Leukocytes may begin to lyse within one (1) hour after collection. Testing
must be performed promptly.
3. Differential cell counts cannot be performed on clotted CSF specimens.
Notify the physician immediately. If the physician requests that the fluid
be tested despite the clot, add the following comment in the LIS and on
the CSF worksheet: Clotted Specimen - results are questionable. Testing
performed at Dr. [Name]’s request.
4. Variable cellular distortion may be seen with the cytocentrifuge method.
a. The cells near the center of the circle are often smaller, have less
cytoplasm, and denser nuclear chromatin than those in the
periphery.
106739189
Version 1.0
Page 11 of 12
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
b. Considerable nuclear distortion such as clefting or lobulation may
be seen
c. Other artifacts frequently encountered are peripheral localization of
nuclear lobes in polymorphonuclear leukocytes, peripheral
cytoplasmic vacuolation, localization of cytoplasmic granules, and
holes in the nuclei.
d. Benign cells, such as mesothelial cells and lymphocytes, often
clump together.
5. When CSF or serous fluid is received from patients with diagnosed or
suspected leukemia or any other malignancy, prepare 6 extra Cytospin
slides for possible histological testing. Label the slides in pencil and do not
stain.
XII.
APPENDICES- Refer to the CSF Cell count procedure for the following
appendices:
1. Body Fluid Worksheet
2. Body Fluid Quality Control Worksheet
XIII.
REFERENCES:
1. CSF Cell Count Procedure
2. King-Strasinger, Susan; Urinalysis and Body Fluids; Fourth Edition; F.A.
Davis Book Publisher; 2001; Pages 150 to 164.
3. Kjeldsberg, Carl; Body Fluids; American Society of Clinical Pathologist
Book Publisher; 1993; Pages 71 to 75 and 321 to 323.
4. Wescor Cytocentrifuge Methods Manual, M2365-4 Rev 4; Logan, UT;
2003
5. Shandon Cytospin 3, Cell Preparation System, Life Sciences International
Europe, 3/1997
106739189
Version 1.0
Page 12 of 12