BODY FLUIDS

Ma. Aristhea Resurreccion, M.D.

Pathology Resident
 OBJECTIVES
 To review on the Anatomy and Physiology of
body fluids
 To have an overview of the collection of the
specimens of body fluids
 To discuss the quantitative and qualitative
examination of body fluids
 BODY FLUIDS
 Cerebrospinal Fluid
 Semen
 Synovial Fluid
 Serous Fluid
Cerebrospinal Fluid
 CSF
 Third major fluid in the body
 Functions:
1. Supply nutrients to the CNS
2. Remove metabolic wastes
3. Produce a mechanical barrier to cushion
brain and spinal cord against trauma
 CSF
Volume:
 20 ml/hr in the choroid plexuses and
reabsorbed by the arachnoid villi
 90-150 ml in adults

 10-60 ml in neonates
 CSF
Collection:
 Lumbar puncture between 3rd and 4th or
4th and 5th lumbar vertebrae
 Note intracranial pressure and prevent
introduction of infection and damage to
neural tissues
 CSF
Collected in 3 containers:
Tube1: Chemical and serologic tests
Tube 2: Microbiological
Tube 3: Cell count
Tube 4: Additional tests for microbiology and
serology
 CSF
Characteristics:
1. Appearance
- crystal clear, hemolyzed/bloody
- cloudy, turbid or miky –
increased protein or lipid
concentration, presence of WBC
- xanthocromic - pink, orange and
yellow
- presence of RBC
degradation products, elevated
bilirubin, carotene, increased protein
concentrations, melanin
pigments
- bloody - indication of intracranial
hemorrhage or traumatic tap
 CSF
Cerebral Hemorrhage Traumatic tap

1. Blood evenly 1. Heaviest

distributed in all 3 concentration of
CSF specimen tubes blood in tube 1
2. No clot 2. Clot formation due to
3. Xanthocromic introduction of
supernatant plasma fibrinogen
into the CSF
3. Clear supernatant
 CSF
2. Cell Count:
- adult - 0-5 WBC/uL
- neonates - 0-30 WBC/uL
(more monocytes)
- specimens with 200 WBC/ml or 400
RBC/uL → clear
CSFcytology ( Lymphocyte : Monocyte ratio 70:30)
 Calculating CSF Cell Counts
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square
 Practice
Calculate the cell count if 3 are found in one WBC
count chamber.
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square

_______3 x (1)______ = 30 cells/uL

(1) X 1 mm3 x 0.1
 Practice
Calculate the cell count if 3 are found in one small
WBC count chamber.
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square

_______3 x 16 x (1)______ = 480 cells/uL

(1) X 1 mm3 x 0.1
 Practice
Calculate the cell count if 3 are found in four
WBC count chamber.
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square

_______3 x (1)______ = 7.5 cells/uL

(4) X 1 sq meters x 0.1
 Practice
Calculate the cell count if 10 are found in four
RBC count chamber.
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square

_______10 x (1)______ = 500 cells/uL

(5) X [0.2 X 0.2 x 0.1]
0.004
 3. Chemistry tests
 Protein
 Glucose
 Lactate
 Glutamine
 3. Chemistry tests
 Protein: 15-45 mg/dL
 Albumin
 Prealbumin
 Alpha globulins:
haptoglobulin and
ceruloplasmin
 Beta globulin:
transferrin
 Gamma globulins:
(IgG, IgM, IgA)
 CSF/Serum albumin index

 IgG index
 3. Chemistry tests
 Glucose: 60-70% of plasma glucose
(drawn 2 hrs prior to LP)
 CSF
 Lactate:
- 25 mg/dl
- elevations can result from any condition that
decreases the flow of O2 to the tissues
- used to monitor severe head injuries

 Glutamine
- 8 to 18 mg/dl
- 35 mg/dl : show disturbance in consciousness
→ coma
 4. Microbiologic
 Gram stain
 Acid Fast stain
 India ink preparation
 Latex agglutination tests
CSF Gram stain : Gram negative diplpcocci characteristic of N.
meningitidis
 5. Serologic
 VDRL
 FTA-ABS
 RPR
 DDx of Meningitis
Bacterial Viral TB Fungal

↑ WBC, ↑ WBC, ↑ WBC, ↑ WBC,

neutrophils lymphocytes lymphocytes and lymphocytes and
monocytes monocytes
Marked ↑ CHON Moderate ↑ CHON Mod to Marked ↑ Mod to Marked ↑
CHON CHON
Marked ↓ CHO Normal CHO ↓ CHO Normal - ↓ CHO

Lactate: >35 Normal Lactate Lactate: >25 Lactate: >25

Pellicle formation Positive india Ink

with C.
neoformans
+ Gram stain and Positive
bact antigen tests immunologic test
for C. neoformans
Seminal Fluid
Analysis
 Functions

 Evaluation of reproductive dysfunction

infertility
 Select donors for therapeutic insemination
 Monitor the success of surgical procedures, such
as varicocelectomy and vasectomy
Physiology
 Sample collection and preparation

 Collected after a minimum of 2 days and a maximum of 7 days of sexual

abstinence
 Obtained by masturbation and ejaculated into a properly labeled, sterile, wide-
mouthed container made of glass or plastic (confirmed to be non-toxic for
spermatozoa)
 Kept at ambient temperature, between 20 °C and 37 °C
 Note if the sample is incomplete, especially if the first, sperm-rich fraction
may be missing a second sample should be collected.
 Semen analysis
 Gross examination  Additional Test
 Appearance  Sperm vitality
 Seminal Fluid Fructose
 Volume
 Anti-sperm antibodies
 Viscosity
 Microbial and chemical testing
 pH  Sperm function tests
 Microscopic
examination
 Sperm count
 Sperm motility
 Sperm morphology
INITIAL MACROSCOPIC
EXAMINATION
 Initial macroscopic examination

LIQUEFACTION
 Within 30-60 minutes at room temperature
 Recognized macroscopically and microscopically
 Immobilized spermatozoa gain ability to move as the semen
liquefies
 Continuous gentle mixing or rotation on a two-dimensional
shaker (room temperature or in an incubator), can produce a
homogeneous sample.
 Initial macroscopic examination
Delayed liquefaction
 additional treatment, mechanical mixing or enzymatic digestion may be
necessary.

1. Addition of an equal volume of physiological medium

(e.g. Dulbecco’s phosphate-buffered saline), followed by
repeated pipetting.
2. Addition of bromelain broad-specificity proteolytic
enzyme
 Initial macroscopic examination

S EMEN p H
 Normal: 7.2-8.0
 Decreased pH
 Increased prostatic fluid
 Ejaculatory duct obstruction
 Poorly developed seminal vesicles

 Increased pH
 Infection
 Initial macroscopic examination
APPEARANCE
 Normal liquefied semen has a homogeneous, grey-
opalescent appearance
 Appear less opaque if the sperm concentration is very low
 Color may also be different:
 red-brown when red blood cells are present, or
 yellow in a man with jaundice or taking certain vitamins or drugs
Initial macroscopic examination

VISCOSITY
 gently aspirating it into plastic disposable
pipette, allowing the semen to drop by gravity
and observing length of any thread
 Normal leaves the pipette in small discrete drops

 Abnormal drop will form a thread >2 cm long

 Initial macroscopic examination

VOLUME
 Range: 2-5 ml Decreased volume
Infertility
 Increased volume Improper functioning of one of the
 Extended periods of abstinence semen producing organs, primarily
seminal vesicles
Inomplete collection
 Sperm count
 Greater than 20-250 million sperm per mL
 Between 10-20 million per mL: borderline
 Sperm numbers

• Left: all nine grids in one chamber of the hemocytometer

• Central: the central grid of 25 large squares (number 5)
• Right: micrograph of part of a filled chamber, showing one of the 25 squares of the central grid bounded
by triple lines and containing 16 smaller squares (the circled square)
 Sperm numbers

Using the haemocytometer

grid
 Count only whole spermatozoa
(with heads and tails).
 A spermatozoon is counted if most
of its head lies between the two
inner lines, but not if most of its
head lies between the two outer
lines
 Practice
Calculate the cell count if 300 are found in five
RBC count chamber.
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square
_____Number of cells counted x dilution____ = cells/uL
Number of squares x volume of 1 square

_______300 x 20______ = 300,000cells/uL

(5) X [0.2 X 0.2 x 0.1]
0.004
 SPERM MOTILITY
 Prepare a wet preparation
 Evaluate at least 200 spermatozoa in a total of at least
five fields in each replicate, calculate the average
percentage
 Assess the motility of all spermatozoa within a defined
area
 Scan and count quickly to avoid overestimating, change
fields often
 SPERM MOTILITY
Categories of sperm movement
The motility of each spermatozoon is graded as follows:
 Progressive motility (PR) spermatozoa moving actively,
either linearly or in a large circle, regardless of speed.
 Non-progressive motility (NP) all other patterns of motility
with an absence of progression, e.g. swimming in small circles,
or when only a flagellar beat can be observed.
 Immotility (IM) no movement.
 Sperm morphology
Determination of sperm morphology comprises the following steps:
 Prepare a smear of semen on a slide
 Air-dry, fix and stain the slide
 Examine the slide with brightfield optics at ×1000 magnification with
oil immersion
 Assess approximately 200 spermatozoa per replicate for the percentage
of normal forms or of normal and abnormal forms
 Compare replicate values
 Sperm morphology

(a) “Feathering” method for undiluted semen.

(b) Pipette method for washed samples. A drop of the sperm suspension (SS) is
spread by pushing the horizontal pipette (P).
 SPERM MORPHOLOGY
 Head
 Acrosome
 Neckpiece
 Midpiece
 Tail
 Sperm morphology
Morphologically “normal” spermatozoa
Common Abnormalities
 Kruger’s strict criteria
 Measuring head, neck and tail size
 Measuring acrosome size

 Evaluating for the presence of vacuoles

 >30% normal forms when using routine criteria

 >14% normal forms when using strict criteria
 Seminal Fluid
Volume: 2-5 ml
Viscosity: pours in droplets
pH: 7.2 – 8.0 (alkaline)
Sperm conc: > 20 million/ml
: 10-20 million/ml borderline
Sperm count: >40 million/ ejaculate
Motility: > 50% with fair quality within 1 hour
Quality: 2.0 or a, b, c
Morphology: 14% normal forms (strict criteria)
30% normal forms (routine criteria)
Round cells: < 1.0 million/mL
Synovial Fluid
 Synovial Fluid
 Joint fluid
 Viscous fluid found in joint cavities
 Ultrafiltrate of plasma across the synovial
membrane
 Composed of mucopolysaccharide containing
hyaluronic acid and protein
 Supplies nutrients to the cartilage and acts as
lubricant to surfaces of moving joints, provides
nutrients to articular cartilage, and lessons shock
during joint compression
 Synovial Fluid
Collection: fine needle aspiration or
arthrocentesis
Volume: < 3.5 ml
Characteristics: clear to pale yellow
: does not clot
Viscosity: >string test 4-6 cm
Cell count: WBC<200 cells/ml
 Synovial Fluid
Neutrophils: <25% of the differential
Crystals: None present
Glucose plasma difference: <10 mg/dL of
blood glucose level
Total protein: < 3g/dL
 Classification of Joint Disorders
Group Classification Pathologic Significance Lab Findings

I. Non-inflammatory Degenerative joint Clear, yellow fluid

disorders, osteoarthritis Good viscosity
WBC < 1,000/ml
Neutro < 30%; No RBC’s
Glucose(blood/SF
difference:mg/dl): 0-10
normal
II. Inflammatory- Immunologic disorders, Transparent to opaque
Immunologic rheumatoid xanthochromic to
white/bloody
arthritis, lupus
Poor viscosity
erythematosus,
WBC 2,000-75,000/ml
scleroderma, polymyositis,
Neutro > 50%; No RBC’s
anklylosing spondylitis, Glucose(bld/SF diff):
rheumatic decreased glucose level
fever, and Lyme arthritis Possible autoantibodies
present
Group Classification Pathologic Significance Lab Findings

II. Immunologic Gout Cloudy or milky fluid

Crystal-induced Pseudogout Poor viscosity
WBC up to 100,000
uL
Neutro <70%
Decreased glucose
Crystals present
Group Classification Pathologic Significance Lab Findings
III. Septic/Infectious Microbial infection Cloudy, yellow-green fluid
Variable viscosity
WBCs 50,000–100,000 L
Neutrophils 75%
Decreased glucose level
Positive culture and Gram
stain

IV. Hemorrhagic Traumatic injury, tumors, Cloudy, red fluid

hemophilia, Low viscosity
other coagulation disorders WBCs equal to blood
Anticoagulant overdose Neutrophils equal to blood
Normal glucose level
Crystals Shape Pathology

Monosodium Needles Gout

urate
Calcium Rods Pseudogout
pyrophosphate Needles
Rhombus
Cholesterol Notched Cholesterolosis
rhombic plates
Hydroxyapatite Small needles Minerals from
cartilage
Corticosteroids Flat plates Drug infections
Monosodium urate crystals under polarized light from a patient with urate
gout
Monosodium urate crystals in synovial fluid
Calcium pyrophosphate dihydrate crystals in synovial fluid from a patient with
pseudogout.
 9

Cholesterol crystals in Synovial fluid. Polarized light.

SEROUS FLUIDS
Serous fluids:

1. Pleural - thoracentesis
2. Pericardial - pericardiocentesis
3. Peritoneal - paracentesis

Collection:
1. EDTA tube: cell count
2. Sterile tube: culture
3. Heparinized tube: chemistry
Effusions - systemic disorder that disrupts
the balance in the regulation of fluid
filtration and reabsorption

1. Transudates results from changes in

hydrostatic pressure
2. Exudates results from an inflammatory
process