SMILE

Johns Hopkins University

Baltimore, MD USA

CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

Penny S. Stevens MBS, MT (ASCP), Document Number Effective Date

Author(s), Name & CLS (NCA)
Title
Sr. International QA/QC Coordinator Pro64-E-04 23 Jan 2009
Review by Amy Rada Review date 04 May 2020
pSMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your
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contact your SMILE representative.

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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. Meningitis, encephalitis, syphilis, or abscess infections.

b. Multiple Sclerosis, Leukemia, and Demylelinating diseases
c. Hemorrhage
d. Brain or spinal cord tumors

II. DEFINITIONS

1. CBC - Complete Blood Count

2. CSF - Cerebrospinal Fluid
3. LIS - Laboratory Information System
4. QC - Quality Control
5. RBC - Red Blood Cell
6. WBC - White Blood Cell

III. 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. Tube #1: To Chemistry for protein and glucose or serology study.

b. Tube #2: To Microbiology for culture and gram stain.
c. Tube #3: To Hematology for cell count and differential
d. 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.
CEREBROSPINAL FLUID (CSF) 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 cannot 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. Neubauer Hemocytometer
b. Neubauer Hemocytometer specific coverslip
c. Capillary pipettes
d. Petri dish containing moist gauze
e. Microscope
f. Gauze
g. Sterile pipettes
h. Microscope slides with etched circles
i. Sterile, disposable cuvettes
j. Shandon Cytospin Cytocentrifuge
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

k. Test tubes

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.

CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

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. Colorless - clear fluid identical to water

ii. Pink
iii. Xanthochromic - yellow color
iv. 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:
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

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.
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.
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

3. Tissue cells are usually large granular cells with

irregular outlines. They should not be included in
either the RBC or the WBC counts.

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 COUNT-

a. 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.
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

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.

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.
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

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.

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) = cells/uL
(Number of squares counted) x (volume of 1 square)

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
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

Total RBC Count

(side 1 count) + (side 2 count) = Average RBC counted

2
3. Diluted Specimens:
Average RBC counted = Total RBC Count (WBC/mm3)
0.9

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 RBC Side 1 Count = 72

WBC Side 2 Count = 26 RBC Side 2 Count = 78

Example Example
Total WBC Count Total RBC Count

22 + 26 = 24; 24 = 27 cells/mm3 72 + 78 = 75; 75 = 83 cells/mm3

2 0.9 2 0.9

b. Diluted 1:10:

WBC Side 1 Count = 99 RBC Side 1 Count = 31

WBC Side 2 Count = 93 RBC Side 2 Count = 39
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

Example Example
Total WBC Count Total RBC Count

99 + 93 = 96; 96 x 10 = 1067 31 + 39 = 35; 35 x 10 = 389 cells/mm3

IX. INTERPRETATIONS AND REPORTING2 RESULTS: 0.9
cells/mm 3

2 0.9
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 a traumatic tap from subarachnoid

hemorrhage:
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

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.

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 Cytocentrifuge
WBC Cell Count Expected Total Cell Recovery
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

0 0 - 40
1-15 20 - 100
6-10 60 - 150
11-20 150 - 250
> 20 >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.

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: 0-5 mm³

< 1 year old: 1-30 mm³
1-4 years old: 0-20/mm³
5-15 years old: 0-10/mm³

1. A great increase of WBCs occurs in acute pyogenic meningitis, the

majority of cells being polymorphonuclear.
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

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.

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
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

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.

Appendix 1
HEMOCYTOMETER COUNTING AREAS
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

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.
CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

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CEREBROSPINAL FLUID (CSF) CELL COUNT AND DIFFERENTIAL

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