This document summarizes guidelines for examining various body fluids, including cerebrospinal fluid, pleural fluid, peritoneal fluid, and semen. For cerebrospinal fluid, normal volumes and cell counts are provided. Guidelines are given for collection into tubes and microscopic examination, including normal cell differentials. Similar information is provided for examining pleural fluid, peritoneal fluid, and semen, including gross appearance, cell counts, differentials, and factors that affect analysis.

1. EXAMINATION OF BODY FLUIDS
Dr Utkarsh Sharma
Moderated by: Dr Navita Gupta

2. EXAMINATION OF CSF
• In adults, approximately 500 mL of
cerebrospinal fluid (CSF) is produced each day
(0.3 to 0.4 mL/min). The total adult volume
varies from 90 to 150 mL.
• In neonates, the volume varies from 10 to 60
mL.
• the total CSF volume is replaced every 5 to 7
hours

3. • The normal opening adult pressure is 90 to 180
mm of water in the lateral decubitus position
with the legs and neck in a neutral position.
• Opening pressures above 250 mm H2O are
diagnostic of intracranial hypertension.
• If the opening pressure is greater than 200 mm
H2O in a relaxed patient, no more than 2.0 mL
should be withdrawn.

4. • The CSF specimen is usually divided into three
serially collected sterile tubes:
Tube 1 for chemistry and immunology studies;
Tube 2 for microbiological examination
Tube 3 for cell count and differential.
 An additional tube may be inserted in the No. 3
position for cytology if a malignancy is suspected.
• Note: Tube 1 should never be used for
microbiology because it may be contaminated
with skin bacteria.

5. • Glass tubes should be avoided because cell
adhesion to glass affects the cell count and
differential.
• Specimens should be delivered to the laboratory
and processed quickly to minimize cellular
degradation, which begins within 1 hour of
collection.
• Refrigeration is contraindicated for samples in
which flow cytometry is likely to be needed for
detection of leukemia or lymphoma cells.

6. GROSS EXAMINATION
• Normal CSF is crystal clear and colorless and has
a viscosity similar to that of water.
• Clot formation may be present in patients with
traumatic taps, complete spinal block (Froin’s
syndrome), or suppurative or tuberculous
meningitis.
• It is not usually seen in patients with
subarachnoid hemorrhage.
• Clots may interfere with cell count accuracy by
entrapping inflammatory cells and/or by
interfering with automated instrument counting

7. • Viscous CSF may be encountered in patients
with
 Metastatic mucin producing adenocarcinomas.
 Cryptococcal meningitis due to capsular
polysaccharide
Liquid nucleus pulposus resulting from needle
injury to the annulus fibrosus.

8. • Pink-red CSF usually indicates the presence of
blood and is grossly bloody when the RBC
count exceeds 6000/μL. It may originate from
a subarachnoid hemorrhage, intracerebral
hemorrhage, or cerebral infarct or from a
traumatic spinal tap.
• Xanthochromia commonly refers to a pale
pink to yellow color in the supernatant of
centrifuged CSF.

10. Visible CSF xanthochromia may also be due to the
following:
• (1) oxyhemoglobin resulting from artifactual red blood
cell lysis caused by detergent contamination of the needle
or collecting tube, or a delay of longer than 1 hour
without refrigeration before examination;
• (2) bilirubin (bilirhachia) in jaundiced patients;
• (3) CSF protein levels over 150 mg/dL,
• (4) disinfectant contamination;
• (5) carotenoids (orange) in people with dietary
hypercarotenemia (i.e., hypervitaminosis A);
• (6) melanin (brownish) from meningeal metastatic
melanoma; and
• (7) rifampin therapy (red-orange).

11. Differential Diagnosis of Bloody CSF
• A traumatic tap occurs in about 20% of lumbar
punctures. Distinction of a traumatic puncture from a
pathologic hemorrhage is, therefore, of vital importance.
• In a traumatic tap, the hemorrhagic fluid usually clears
between the first and third collected tubes but remains
relatively uniform in subarachnoid hemorrhage.
• Xanthochromia, microscopic evidence of
erythrophagocytosis, or hemosiderin-laden macrophages
indicate a subarachnoid bleed in the absence of a prior
traumatic tap. RBC lysis begins as early as 1 to 2 hours
after a traumatic tap. Thus, rapid evaluation is necessary
to avoid false-positive results.

12. MICROSCOPIC EXAMINATION
Total Cell Count
• The normal leukocyte cell count in adults is 0 to 5
cells/μL.
• It is higher in neonates, ranging from 0 to 30
cells/μL, with the upper limit of normal
decreasing to adult values by adolescence.
• No RBCs should be present in normal CSF. If
numerous (except with a traumatic tap), a
pathologic process is probable (e.g., trauma,
malignancy, infarct, hemorrhage).

13. Differential Cell Count
• A differential performed in a counting chamber is
unsatisfactory because the low cell numbers give
rise to poor precision, and identifying the cell
type beyond granulocytes and “mononuclears” is
difficult in a wet preparation.
• Direct smears of the centrifuged CSF sediment
are also subject to significant error from cellular
distortion and fragmentation.
• The cytocentrifuge method is rapid, requires
minimal training, and allows Wright’s staining of
air-dried cytospins. It is the recommended method
for differential cell counts in all body fluids.

14. • In adults, normal CSF contains small numbers of
lymphocytes and monocytes in an approximate 70 : 30
ratio.
• A higher proportion of monocytes is present in young
children, in whom up to 80% may be normal.
• No general consensus regarding an upper limit of normal
for PMNs has been established. Many laboratories
accept up to 7% neutrophils with a normal WBC count.
• The number of PMNs may be decreased by as much as
68% within the first 2 hours after lumbar puncture owing
to cell lysis .
• A total PMN count of over 1180 cells/μL (or more than
2000 WBCs/μL) has a 99% predictive value for bacterial
meningitis.

16. Cerebrospinal fluid cytology (lymphocyte to monocyte
distribution ratio 70 : 30).

17. Choroid plexus cells in cerebrospinal fluid.

18. Cluster of blastlike cells in cerebrospinal fluid
from a premature newborn.

22. Hemosiderin-laden macrophages (siderophages) from the
cerebrospinal fluid of a patient with subarachnoid
hemorrhage. Hemosiderin crystals (golden-yellow) are also
present.

23. Acute lymphoblastic leukemia in cerebrospinal fluid. Note
uniformity of the blast cells.

24. Burkitt’s lymphoma in cerebrospinal fluid. The cells
are characterized by blue cytoplasm with vacuoles
and a slightly clumped chromatin pattern.

25. CSF protein levels of 15 to 45 mg/
dL have long been accepted as the “normal”
reference range.

27. EXAMINATION OF PLEURAL FLUID
• The pleural cavity is a potential space lined by
mesothelium of the visceral and parietal pleurae.
• The pleural cavity normally contains a small amount of
fluid that facilitates movement of the two membranes
against each other.
• An accumulation of fluid, called an effusion, results from
an imbalance of fluid production and reabsorption.
• Except for an EDTA tube for total and differential cell
counts, the specimen should be collected in heparinized
tubes to avoid clotting.

28. • Because serous effusions are more forgiving than
CSF in maintaining cellular integrity, fresh specimens
for cytology may be stored for up to 48 hours in the
refrigerator with satisfactory results
• Initial classification of a pleural fluid as a transudate
or an exudate greatly simplifies the process of
arriving at a correct final diagnosis.
• Transudates generally require no further workup.

31. GROSS EXAMINATION
• Transudates are typically clear, pale yellow to straw-
colored, and odorless, and do not clot.
• Approximately 15% of transudates are blood tinged. A
bloody pleural effusion (hematocrit >1%) suggests
trauma, malignancy, or pulmonary infarction .
• A traumatic tap is suggested by uneven blood
distribution, fluid clearing with continued aspiration, or
formation of small blood clots.
• A pleural fluid hematocrit greater than 50% of the
blood hematocrit is good evidence for a hemothorax.

32. • Exudates may grossly resemble transudates, but most
show variable degrees of cloudiness or turbidity, and
they often clot if not heparinized.
• Turbid, milky or bloody specimens should be
centrifuged and the supernatant examined.
• If the supernatant is clear, the turbidity is most likely
due to cellular elements or debris.
• If the turbidity persists after centrifugation, a chylous
or pseudochylous effusion is likely.

34. Cell Counts
• Leukocyte counts have limited utility in separating
transudates (<1000/μL) from exudates (>1000/μL).
• Although RBC counts above 100,000/μL are highly
suggestive of malignancy, trauma, or pulmonary
infarction, they are not specific for these conditions.
• Mesothelial cells are common in pleural fluids from
inflammatory processes, however, conspicuously scarce
in patients with tuberculous pleurisy and rheumatoid
pleuritis, and in patients who have had pleurodesis.
• Nucleoli and nuclear cleaving are more prominent in
lymphocytes of effusions than in the peripheral blood.

35. Mesothelial cells in pleural fluid

37. Small cell carcinoma of the lung showing typical
molding of nuclei.

38. EXAMINATION OF PERITONEAL FLUID
• Ascites is the pathologic accumulation of excess fluid
in the peritoneal cavity.
• Up to 50 mL of fluid is normally present in this
mesothelial-lined space.
• Diagnostic paracentesis is performed in most patients
with new ascites, or if there is a change in the clinical
picture of a patient with ascites, such as rapid fluid
accumulation or fever development.
• A minimum of 30 mL is needed for complete
evaluation.
• If possible, at least 100 mL should be provided for
cytologic examination.

39. GROSS EXAMINATION
• Transudates are generally pale yellow and clear.
• Exudates are cloudy or turbid because of the presence of
leukocytes, tumor cells, or increased protein levels.
• The presence of food particles, foreign material, or green-
yellow bile staining in a DPL specimen suggests
perforation of the gastrointestinal or biliary tract.
• Acute pancreatitis and cholecystitis may also cause
greenish discoloration.
• As little as 15 mL of blood per liter of fluid produces a
bright red opaque color.
• Bloody ascites is also seen in malignancy and
tuberculosis.

40. MICROSCOPIC EXAMINATION
• The total leukocyte count is useful in distinguishing
ascites due to uncomplicated cirrhosis from
spontaneous bacterial peritonitis (SBP), which is
caused by migration of bacteria from the intestine
into the ascitic fluid.
• Approximately 90% of patients with SBP will have
leukocyte counts greater than 500/μL, more than 50%
of which are neutrophils.
• Eosinophilia (>10%) is most commonly associated
with the chronic inflammatory process associated
with chronic peritoneal dialysis, lymphoma, and
ruptured hydatid cyst.

41. Semen Analysis
• In addition to its use in the evaluation of reproductive
dysfunction, particularly infertility, semen analysis is used
to select donors for therapeutic insemination and to
monitor the success of surgical procedures, such as
varicocelectomy and vasectomy.
• The patient should be instructed to collect semen after 2
to 5 days of sexual abstinence.
• Longer periods of abstinence usually result in a higher
semen volume but reduced sperm motility.
• In such a case, a second semen specimen may be
collected 2 hours after the first sample is collected.

42. • The semen specimen should be delivered to the
laboratory within 1 hour of collection and kept warm
during transportation.
• Two specimens collected within 2- to 3-week intervals
should be used for evaluation, and if they are markedly
different, additional specimens should be collected.
• Semen should be thoroughly mixed before
examination, and its viscosity recorded.
• The appearance of a yellow hue in a semen specimen is
associated with pyospermia, and a rust color is due to
small bleedings in the seminal vesicle.

44. • A hemocytometer or microchamber should be used for the
sperm count.
• Total sperm count is then calculated by multiplying the
dilution factor (normal concentration range, 15-50
million/mL) by its volume (normal range, 2-5 mL).
• Progressive motility (normal range 32% or above) is
expressed as the percentage of sperm that move. In addition,
forward movement is graded.
• Sperm that move rapidly in a straight line with little yaw
and lateral movement are grade 4
• if they move more slowly, grade 3.
• Grade 2 sperm move even more slowly and with substantial
yaw.
• Grade 1 sperm have no forward progression.
• Zero progression denotes absence of any motility.

45. • If motility is less than 50%, a viability stain of eosin
Y with nigrosin as a counterstain is done. In bright-
field microscopy, dead sperm will stain red, whereas
live sperm will exclude the dye and appear unstained.
• In samples with no visible sperm, such as
postvasectomy semen, the entire sample should be
centrifuged and the pellet examined for intact or
damaged sperm fragments. The analysis should be
repeated in 4 to 6 months.

46. • Agglutination occurs when motile sperm stick to one
another in an orientation that is reproducible within a
given specimen, such as head to head, tail to tail,
midpiece to midpiece, or mixed ways, depending on the
specificity of sperm antibodies.
• Agglutination suggests an immunologic cause of
infertility, and a description of the type of agglutination
should be recorded.
• This can usually be distinguished from clumping due to
bacterial infection or tissue debris, which typically
involves nonspecific orientation of the sperm.

47. • Round cells should be differentiated into two classes:
immature germ cells with a single or double highly
condensed nucleus with a relatively large area of
cytoplasm, and polymorphonuclear leukocytes, which
are smaller than the germ cells and have a lower
nuclear/cytoplasmic ratio.
• Peroxidase staining specifically identifies the
polymorphonuclear leukocytes in the presence of
lymphocytes and other cells that normally occur in
semen.
• Bacterial contamination should be noted, as should the
presence or absence of epithelial cells.

48. • Typically, more than 4% of the sperm in a semen
specimen should exhibit normal morphology.
• A strict morphology score of greater than 4% of normal
indicates excellent fertilizing capacity. Scores between
0% and 3% predict probable inability to fertilize.
• Wide variability in the size of the acrosomal cap is the
most obvious characteristic of abnormal sperm. An
acrosomal cap less than one third of the head surface is
considered abnormal, as are retention of a cytoplasmic
droplet greater than one-half of the head size and a tail
less than 45 μm long.
• Of particular note is the direct relationship between
acrosome size and the frequency of fertilization or
pregnancy.