CHAPTER 14

Cell counts on CSF and

other body fluids
Objectives
At the end of this chapter, students will be able to:
 Define terms related to body fluids
 Identify different types of body fluids
 Define CSF
 Define Serous fluids: pleural, pericardial, peritoneal
(Ascitic), and Synovial fluid
 Explain the analysis of CSF
 Discuss semen analysis that can be done in hematology
 Perform cell counts on Serous fluids: pleural, pericardial,
peritoneal (Ascitic)
 Perform cell count on synovial fluid
 Apply QC measures in body fluid examination
14.1. introduction to Body Fluids
 Are ultrafiltrates of plasma
 Fluids serve as lubricants as membranes move against
each other
 Body fluids commonly analyzed in hematology lab include:
 CSF

 Serous fluid
 Synovial fluid (fluid from the joints)
 Semen
Introduction cont’d
Serous fluids:
 Are fluids from closed body cavities such as pleural,
pericardial, peritoneal/ascitic cavities:
 Pleural fluid from the pleural cavity of lungs
 Pericardial from around the heart
 Peritoneal from around the abdominal and pelvic organs
Definition of terms
 Effusion: an increase in volume of any serous fluid
 Transudates: effusion as a result of a mechanical
disorder affecting movement of fluid across a memebrane
 Exudates: are effusions resulting from inflammatory
responses that directly affect the serous cavity (includes
infections and malignancies)
Characteristics of Serous Effusions:
Transudate versus Exudate
Observation / Test Transudate Exudate

Appearance Watery, clear, pale Cloudy, turbid, purulent, or bloody;

yellow, does not clot may clot (fibrinogen present)

WBC count Low, < 1,000/µL with 500-1,000 cells/µL or more, with
> 50% mononuclear increased PMNs, increased
cells lymphocytes with TB or rheumatoid
arthritis
Red cell count Low, unless from a > 100,000/µL, especially with a
traumatic tap malignancy
Total protein Low >3g/dl (or > than half the serum
level)
Lactate Varies with serum Increased (>60% of serum level
dehydrogenase level because of cellular debris)
Glucose Not applicable Lower than serum level with some
infections and high cell counts
14.2. Cerebrospinal fluid (CSF) analysis

i. About CSF
 Is a fluid in the space called sub-arachnoid space
between the arachnoid mater and pia mater
 Protects the underlying tissues of the central nervous
system (CNS)
 Serve as mechanical interface to:
 prevent trauma

 regulate the volume of intracranial pressure

 circulate nutrients

 remove metabolic waste products from the CNS

 Act as lubricant

 Has composition similar to plasma except that it has

less protein, less glucose and more chloride ion
CSF cont’d
 Maximum volume of CSF
 Adults 150 mL
 Neonates 60 mL
 Rate of formation in adult is 450-750 mL per day or 20 ml
per hour
 reabsorbed at the same rate to maintain constant
volume
 Collection by lumbar puncture/tap done by experienced
medical personnel
 About 1-2ml of CSF is collected for examination
 lumbar puncture is made from the space between the 4th
and 5th lumbar vertebrae under sterile conditions.
 Collecting a CSF specimen

 Collected in three
sequentially labeled tubes
 Tube 1 for chemical and
immunologic tests
 Tube 2 for Microbiology
 Tube 3 for Hematology
(gross examination, total Location of CSF
WBC & Diff)
 This is the list likely to
contain cells
introduced by the
puncture procedure
 CSF ont’d

ii. Clinical Significance

 Diagnosis of meningitis of bacterial, fungal,
mycobacterial and amoebic origin or differential
diagnosis of other infectious diseases
 subarachnoid hemorrhage or intracerebral hemorrhage

iii. Principle of CSF analysis

 CSF specimen examined visually and microscopically
and total number of cells can be counted and identified
 CSF ont’d
iv. Specimen: the third tube in the sequentially collected
tubes*
 must be counted within 1 hour of collection (cells
disintegrate rapidly). If delay is unavoidable store at 2-8oC.
 All specimens should be handled as biologically hazardous
 Leukocyte count on CSF
v. Equipment and Reagents: same as for WBC
counting on whole blood

vi. Method
Gross appearance
 Is visual assessment of CSF for turbidity, color and
viscosity
 Normal CSF is crystal clear in appearance with
viscosity comparable to water
 Abnormal CSF may appear
 cloudy, smoky, hazy, opalescent, turbid or grossly
bloody
 Method: Gross appearance cont’d

Turbidity may be graded from 0 to 4+ as follows:

0 = crystal clear fluid
1+ = faintly cloudy, smoky or hazy with slight (barely visible)
turbidity
2+ = turbidity clearly present but news print easily read
through tube.
3+ = news print not easily read through tube
4+ = grossly turbid, news print cannot be seen through tube.
Note:
 Slight haziness indicates WBC count of 200-500/uL
 Turbidity indicates WBC count of > 500/uL
 Turbidity in general could result from large number of
leukocytes or bacteria, or increase in proteins or lipids
 Gross appearance cont’d

1. Bloody CSF specimens

 Can result from a traumatic a spinal tap (often occur in
children)
 Grossly blood specimen: may indicate subarachnoid
hemorrhage or intracerebral hemorrhage
 If the specimen is bloody:
 There is a need to differentiate between a traumatic tap
and a patient’s clinical condition
 If the specimen in the 1st tube is bloody and is clear in the last
tube, it indicates traumatic tap
 If the specimen has the same bloody color in all the three
tubes, it indicates clinical condition
 Gross appearance cont’d

2. Color
 Any color should be reported (N.B. normal CSF is crystal
clear)
 Xanthochromia: is yellow coloration of CSF
 yellow color could be due to:
 Result of release of hemoglobin from lysed red
blood cells increase in bile pigments
 Specimen collected 2 hours post arachnoid
hemorrhage
 Gross appearance cont’d
3. Viscosity
 Normal CSF has viscosity comparable to that of water
 Clotting may occur
 from increased fibrinogen

 resulting from a traumatic tap

 or rarely may be associated with meningitis or

subarachnoid block
 Microscopic cellular enumeration
 Cell count is performed by manual method
 Electronic methods should be used with care
 RBC counts are of limited value
 WBC counts are useful for differential diagnosis

Normal Range:
 0-5 WBC/µL or 0-5 x 106/L

 Neonates have higher value of 0-30 cells/µL

 Low WBC with turbidity could indicate high

concentration of bacteria
 WBC between 100-10,000 x 106/L could indicate acute
untreated bacterial meningitis
 WBC >50,000 x 106/L are unusual and suggest
intraventricular rupture of a brain abscess
 Microscopic examination cont’d
 Differential count is performed when WBC >30 cells/ µL
 Smear is prepared from centrifuged CSF settlement

Total Leucocyte Count on CSF

 If CSF is clear
 Mix well the undiluted CSF and properly charge the
improved Neubauer counting chamber
 count the cells in 9mm2 area
 Multiply the number by 10/9 to get the number of
WBC/mm3

 Total Leucocyte Count on CSF cont’d
 If CSF is slightly turbid
 prepare a 1:10 dilution with 2% acetic acid (1 drop CSF
and 9 drops 2% acetic acid)
 count the cells in 9mm2 area in the improved Neubauer
counting chamber
 Multiply counted cells by 100/9
 If CSF is purulent, proceed with the dilution and counting
as for a blood sample i.e prepare 1:20 dilution, count the
cells on 4mm2 area and multiply counted cells by 50.
 Technique for Counting Mixture of WBC and RBC in
bloody CSF
 This is done to find the true WBC count when the CSF is
bloody due to
 traumatic tap
 Perform the WBC and RBC counts on the patient’s blood
and CSF.
 Multiply the ratio of RBC count on CSF to RBC count on
blood by the blood leucocyte count and subtract this
product from the WBC count of CSF.
RBCCSF x WBCblood= WBCADDED
RBCBLOOD

Corrected WBCCSF = WBCCSF – WBCADDED

Excercise

 Example:
RBCBLOOD = 5 x 106/mm3
RBCCSF = 2,500/mm3
WBCBLOOD = 12 x 103/mm3
WBCCSF = 70/mm3

WBCADDED = 2,500 x 12,000

5 x 106
= 60/mm3

Corrected WBCCSF = 70 – 60 = 10/mm3

Differential Leukocyte Count on CSF
 Centrifuge the CSF at 2500 rpm for 10 min
 Remove supernatant (can be saved for other analysis)
 Re-suspend the sediment
 Prepare a smear from the re-suspended sediment
 Stain using Wright stain
 Wash off stain with water and air-dry
 Identify the types of leucocytes (PMNs or mononuclear
cells) and their number may be expressed as
percentage of the total count
 Count at least 100 cells using the oil immersion objective
 Artifacts due to distortion of cells can lead to
misidentification
CSF cont’d

vii. Quality control:

 count both sides of hemocytometer (18 mm2 area) for the
total WBC
 Increasing the number of cells to be counted for
differential count (instead of 100 cells count 200 cells)
 Check staining quality (e.g. staining time, pH, filtering)
 Proper centrifugation (Speed and time)
 Properly follow SOP
 viii. Sources of errors
 General sources of error in dilution, charging, counting ,
calculating etc that were discussed for WBC count also
apply here
 Delay in analysis
 Centrifugation time and speed during sediment preparation
for Diff count
 Staining time
 Improper handling of sample
Differential Leukocyte Count on CSF
 Cells in the spinal fluid may include:
 granulocytes
 mature or reactive lymphocytes,
 momonuclear phagocytes,
 plasma cells, blast cells and
 malignant cells (indicating primary tumors of brain and
spinal cord)
 Others like nucleated red cells, and intracellular
bacteria.
ix. Interpretation of CSF diff count

 An increase in the number of WBC in CSF is called

Pleocytosis. Counts above 500/mm3 usually arise from a
purulent infection and the predominant cells are
neutrophils.
i. Infectious/inflammatory causes
 Bacterial meningitis
 Early viral meningitis
 Early tuberculosis
 Amoebic encephalomyelitis
ii . Noninfectious causes
 Reaction to CNS hemorrhage
 Reaction to lumbar puncture
 Chronic granulocytic leukemia involving the CNS
 Lumbar puncture with needle that is contaminated with detergent
14.3. Other body fluids
 Pleural fluid, pericardial fluid, peritoneal/ascitic fluid,
synovial fluid, semen
 Same protocol followed as with CSF
 Macroscopic examination for:
 Turbidity
 Color
 Viscosity
 Microscopic
 total white cell count
 Differential
 Gram stain and culture is done in bacteriology laboratory
Review Questions
1. What is the function of body fluids?
2. What is the role of CSF in our body?
3. Mention at least three different types of body fluids
4. What is the difference between transudates and exudates
5. Define serous fluids
6. Define CSF
7. How do you perform the total leukocyte count on a:
a) clear CSF b) slightly turbid CSF c) purulent CSF
8) How do you correct the total leukocyte count to a true
value when the count is performed on a sample of CSF
that is slightly turbid due to traumatic tap?
9) How is the differential leukocyte count performed on a
sample of CSF?