SMILE
Johns Hopkins University
Baltimore, MD USA
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
Author(s), Name &
Title
Penny S. Stevens MBS, MT (ASCP),
CLS (NCA)
Document Number
Effective Date
Sr. International QA/QC Coordinator
Pro64-E-04
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.
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CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
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CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
I.
PRINCIPLE:
1. Cerebrospinal Fluid, also called CSF, is the product of the secretory activity
of the choroid plexus. It is the third major fluid of the body and supplies
nutrients to the nervous tissue, removes metabolic waste, and protects the
brain and spinal cord from trauma.
2. CSF examination is requested when the physician suspects:
a.
b.
c.
d.
II.
DEFINITIONS
1.
2.
3.
4.
5.
6.
III.
Meningitis, encephalitis, syphilis, or abscess infections.
Multiple Sclerosis, Leukemia, and Demylelinating diseases
Hemorrhage
Brain or spinal cord tumors
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.
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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.
ii. Hematology - cell count and differential.
iii. Chemistry
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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.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
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Neubauer Hemocytometer
Neubauer Hemocytometer specific coverslip
Capillary pipettes
Petri dish containing moist gauze
Microscope
Gauze
Sterile pipettes
Microscope slides with etched circles
Sterile, disposable cuvettes
Shandon Cytospin Cytocentrifuge
Test tubes
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2. Reagents:
a. Quality Control Material - Level I and II
b. Methylene Blue - Maintained in flammable cabinet in Hematology.
Store at room temp.
c. Diff Quick Stain
d. Saline
V.
QUALITY CONTROL:
1. Two levels of quality control will be tested at least once each day that a
CSF Cell Count is performed.
2. Both levels of QC will be performed on the first shift during which patient
testing is ordered. If day shift performed a cell count, the next shift to
perform patient testing must repeat and record results for quality control
level II.
3. The quality controls come ready to use. No further preparation is
necessary.
4. When a new vial of control is opened, label with the date and initials of
tech placing reagent in use.
5. Store the controls tightly capped at 2-8°C when not in use. Stored at this
temperature, the controls are stable until expiration date. After opening,
the controls are stable for six months when refrigerated.
6. Discard the controls if there is any evidence of microbial contamination.
The level 2 control may appear slightly turbid after mixing.
7. Procedure for cell count controls:
a. Remove CSF Controls from the refrigerator, and allow the controls
to remain at room temperature for 15 minutes before mixing.
b. Mix the controls thoroughly by inverting the vials several times and
by squeezing the bulb in the cap at least 10 times but AVOID
FOAMING to minimize cell lysis.
c. Using the glass dropper provided, charge both sides of the
hemocytometer chamber. Do not over or under fill.
d. Immediately recap the controls and return them to the refrigerator.
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e. Count all nine squares in the hemocytometer chamber. All RBC
and WBC counts will be performed as stated in the procedure and
calculation sections. Controls must be processed as undiluted and
unstained CSF.
f. Perform the controls in the same manner as patient samples.
Record all results on the CSF Quality Control Worksheet (appendix
2) and in the LIS.
g. The controls must be within the expected ranges posted on the
Body Fluid Quality Control Worksheet. (appendix 3)
h. If all results are within limits, proceed with patient testing.
i.
Procedure for out of range control:
i. Review all reagents for expiration dates.
ii. Repeat the procedure with a new vial of control(s).
iii. If results are still outside of the expected ranges, notify the
Hematology supervisor immediately. Corrective action must
be taken before reporting patient results.
iv. Out of range controls will be recorded in the Quality Control
log and the hematology corrective actions log along with the
corrective action taken.
VI.
CALIBRATION: NOT APPLICABLE
VII.
PROCEDURE:
1.
MACROSCOPIC EXAMINATION:
a. Immediately after the samples have been received, complete the CSF
Worksheet with the total volume of fluid.
b. Evaluate for color: Gently invert CSF tube #3 and hold both the
uncentrifuged sample and the Hematology water standard against a
white background. Report the color as follows:
i.
ii.
iii.
iv.
Colorless - clear fluid identical to water
Pink
Xanthochromic - yellow color
Brown
c. Evaluate for clarity: Gently invert tube #3 and hold this uncentrifuged
sample together with the water standard against the 12-font print
standard. Report appearance as follow:
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i. Clear - crystal clear fluid identical to water.
ii. Hazy - turbidity present but print standard can be read easily
through the tube.
iii. Cloudy - print standard cannot be read through the tube.
2. MICROSCOPIC EXAMINATION:
a. UNSTAINED CELL COUNT
i. Prepare the hemocytometer - clean the coverslip and counting
area with distilled water and follow with an alcohol wipe. Allow
it to dry thoroughly.
ii. Prepare the humidity chamber 1. Place 1-2 layers of gauze on the bottom of a Petri dish.
It must provide a level resting area or the fluid in the
hemocytometer will pool resulting in an inaccurate
count.
2. Wet the gauze slightly with distilled water.
3. Place two wooden sticks or straws on top of the gauze.
This will provide a resting area for the hemocytometer
and keep it above the wet gauze which makes removal
easier.
4. Put the hemocytometer on top of the sticks (or straws)
in the Petri dish and place the clean, dry coverslip on
the hemocytometer.
iii. Mix the specimen and estimate (based on turbidity) if the
specimen can be counted diluted or undiluted.
iv. If the specimen is clear, colorless, or there is a very small
volume of CSF, the specimen should not be diluted. Charge
the hemocytometer and count all nine squares on both sides.
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CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
v. To charge the hemocytometer:
1. Draw up well mixed specimen using a rubber capillary
tube bulb and capillary tube or a 15 µL pipette.
2. Place the end of the capillary tube against the
hemocytometer and charge both sides with the fluid.
Very little pressure is needed - the hemocytometer
should fill by capillary action. Be careful not over or
underfill and do not bump the coverslip or the count will
be inaccurate.
3. Cover the petri dish with the lid and let the chamber sit
undisturbed for five (5) minutes before counting.
vi. If the cells are too numerous to count, the fluid must be diluted
according to the number of cells present. See below for dilution
steps.
NOTE: The most accurate count is achieved by counting as
many squares as possible (all 9 on both sides of the
hemocytometer).
Unmaneageable counts result in inaccurate results - do not
attempt to perform an unmanageable count. If the cell count
yields more than 100 cells per 9 squares (one side of the
hemocytometer), perform a dilution.
vii. Cells in the hemocytometer appear as follows:
1. RBC’s have a distinct outline with halos and clear
centers. If crenated, they have many fine-pointed
projections.
2. WBC’s are granular.
3. Tissue cells are usually large granular cells with
irregular outlines. They should not be included in either
the RBC or the WBC counts.
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CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
viii. Count the stained WBCs on both sides of the chamber (all
eighteen squares - nine on each side). Record results on the
Body Fluid Worksheet - appendix 2.
ix. Count the RBC’s on both sides of the chamber (all eighteen
square). Record results on the CSF Worksheet.
x. Proceed with calculations.
3. STAINED CELL COUNTa. Staining is optional and may be used at the discretion of testing
personnel. In this procedure, Methylene Blue is used to stain WBC’s.
This helps differentiate them from RBC’s and can improve count
accuracy.
b. There is no dilutional effect with this procedure.
c. Methylene Blue is stored in the hematology flammable chemical
cabinet. Dispense a small working solution into a 1 mL specimen cup.
d. Place a rubber capillary tube bulb on a clean capillary tube. Depress
the bulb and hold. Place the other end of the capillary tube in the
methylene blue and release the bulb drawing the stain up and fill the
capillary tube at least 1/2 full.
e. Depress the bulb again and dispense all of the stain onto a disposable
gauze pad. Discard the gauze. The capillary tube should be coated
but not filled with stain.
f. With a sterile pipette, transfer a small portion of well mixed
uncentrifuged CSF sample into a 1-mL specimen cup to prevent
contamination in the original patient specimen tube.
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g. Using the stain coated capillary tube, draw up the CSF sample to at
least 3/4 full. Carefully mix the capillary tube by gently depressing
and releasing the capillary tube bulb. Allow the tube to sit undisturbed
for approximately two (2) minutes.
h. Alternatively, use a calibrated 15 µL pipette and sterile tip. Draw
methylene blue into the pipette and then discard all of the fluid. A
small residue will remain in the pipette tip, which is a sufficient volume
for staining. Proceed with specimen collection from the patient aliquot.
i.
Using a clean hemocytometer, charge both sides of the chamber with
the stain coated specimen and allow the chamber to sit in a moist petri
dish for five (5) minutes.
j.
Count the stained WBCs on both sides of the chamber (all eighteen
squares). Record results on the Body Fluid Worksheet - appendix 2.
k. Count the RBC’s on both sides of the chamber (all eighteen square).
Record results on the Body Fluid Worksheet - appendix 2.
l.
Proceed with calculations.
m. NOTE: If there is a 10% or greater difference between the counts
from each chamber of the hemocytometer, the difference must be
investigated for both unstained and stained cell counts.
4. DILUTIONS:
a. Obtain 2 mL’s of 0.85% fresh uncontaminated saline from the blood
bank department.
b. Charge a hemocytometer with undiluted specimen and review
microscopically to estimate the best dilution. Remember the ideal cell
count is less than 100 cells per 9 squares (one side).
c. If the specimen is excessively bloody or turbid, it may be necessary to
perform counts on WBC’s and RBC’s using different dilutions.
d. Dilutions must be prepared in sterile specimen tubes and labeled
accordingly. The most commonly used dilutions are prepared as
follows:
i. 1:10 - 0.1 mL sample to 0.9 mL of Saline.
ii. 1:100 - 0.1 mL of the 1:10 dilution to 0.9 mL of Saline.
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NOTE: If a different dilution is needed and you are uncertain of what
specimen volume to use, contact the Hematology supervisor. Use only
calibrated pipettes to perform dilutions.
e. Perform the cell count using either the unstained or stained
procedures listed above.
f. Record the dilution and all results on the CSF worksheet.
5. SMEAR PREPARATION AND DIFFERENTIAL COUNT:
a. Prepare a smear by a cytocentrifuge technique for the differential
count (cytospin). Refer to the Shandon or Wescor Cytocentrifuge
SOP’s for step by step instruction.
b. Prepare at least two (2) cytospin slides REGARDLESS OF THE WBC
COUNT OBTAINED.
c. A differential cell count must be performed if 1 or more
WBC/mm3 are found during the cell count..
d. The Supervisor and the Pathologist must review the cytocentrifuge
slide and the CSF Worksheet during the next regular duty shift.
e. Enter all differential results on the Body Fluid Worksheet - appendix 2
and in the LIS.
VIII.
CALCULATIONS:
1. Standard formula:
(Number of cells counted) x (dilution factor)
(Number of squares counted) x (volume of 1 square)
= cells/uL
2. QC and Undiluted Specimens:
Note: The following calculations assume a counting area of 9 large squares
at 0.1 μL volume per square. See the CSF worksheet and appendix 1 for a
diagram of square volumes and alternate counting options.
Total WBC Count
(side 1 count) + (side 2 count) = Average WBC counted
2
Average WBC counted
= Total WBC Count (WBC/mm3)
0.9
Total RBC Count
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(side 1 count) + (side 2 count) = Average RBC counted
2
Average RBC counted
= Total RBC Count (WBC/mm3)
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CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
3. Diluted Specimens:
Total WBC Count
(Side 1 count) + (Side 2 count) = Average WBC counted
2
(Average WBC counted) x (Dilution Factor) = Total WBC Count (WBC/mm3)
0.9
Total RBC Count
(Side 1 count) + (Side 2 count) = Average RBC counted
2
(Average WBC counted) x (Dilution Factor) = Total RBC Count (RBC/mm3)
0.9
4. Calculation Examples:
a. Undiluted:
WBC Side 1 Count = 22
WBC Side 2 Count = 26
Example
Total WBC Count
22 + 26 = 24;
2
RBC Side 1 Count = 72
RBC Side 2 Count = 78
Example
Total RBC Count
24 = 27 cells/mm3
0.9
72 + 78 = 75;
2
75 = 83 cells/mm3
0.9
b. Diluted 1:10:
WBC Side 1 Count = 99
WBC Side 2 Count = 93
RBC Side 1 Count = 31
RBC Side 2 Count = 39
Example
Total WBC Count
Example
Total RBC Count
99 + 93 = 96; 96 x 10 = 1067 cells/mm3
2
0.9
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31 + 39 = 35;
2
35 x 10 = 389 cells/mm3
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CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
IX.
INTERPRETATIONS AND REPORTING RESULTS:
1. REPORTING RESULTS
a. Record all results, color, clarity, manual cell count, and differential
results on the Body Fluid Worksheet - appendix 2 during testing. Do
not record any results on scrap paper.
b. Any notes or comments should also be added to the worksheet. (i.e.,
specimen dilution, stained or unstained count, precipitate presence,
etc.)
c. 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.
d. After the manual differential is completed a pathology review must be
ordered for the specimen in the LIS.
e. 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.
f. 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.
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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.
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:
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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
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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.
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 and in the LIS.
X.
REFERENCE RANGES:
RBC Total Cell Count
All Ages:
0 mm³
WBC Total Cell Count:
Adults:
< 1 year old:
1-4 years old:
5-15 years old:
0-5 mm³
1-30 mm³
0-20/mm³
0-10/mm³
1. A great increase of WBCs occurs in acute pyogenic meningitis, the majority
of cells being polymorphonuclear.
2. A slight to moderate increase of polys occurs in meningitis accompanying
brain abscess. This also occurs in the early stages of tuberculosis, syphilitic
meningitis, and poliomyelitis. After which the mononuclear cells are
predominant.
3. The cell count is usually normal in multiple sclerosis, epilepsy, brain tumor,
and cerebral arteriosclerosis.
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CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
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. Cell
counts must be performed promptly.
3. 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.
XII.
APPENDICES:
1. Hemocytometer counting areas
2. Body Fluid Worksheet
3. Body Fluid Quality Control Worksheet
XIII.
REFERENCES:
1. Manufacturer’s Package Insert; Spinal Fluid Cell Controls; Quantimetrix
Corporation; 2001.
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.
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Appendix 1
HEMOCYTOMETER COUNTING AREAS
FIGURE 1 Represents one-side of a hemocytometer chamber. Tech must count
both sides. W = White Blood Cell counting areas and R = Red Blood Cell
counting areas.
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CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
SOP VALIDATION
SOP NAME:
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL
Clear and specific title and principle:
Comments:
yes / no
All necessary supplies, equipment, and materials are listed:
Comments:
yes / no
SOP is sufficiently detailed to be understood but not overly complex:
Comments:
SOP text adequately describes process/procedure:
Comments:
SOP accomplishes purpose:
Comments:
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yes / no
Reviewed by: (Name & Title)
Signature: __________________
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Date: __________________
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