MFG*ILMT MLS IV-B

SEMINAR (BODY FLUIDS)

SEMINAL FLUID

Seminal fluid or semen complex body fluid used to transport sperm or spermatozoa
- analyzed routinely to evaluate fertility and to follow up after a vasectomy to ensure its
effectiveness
- evaluation of quality for donation purposes and forensic application
- composed primarily of secretions from the testes, epididymis, seminal vesicles, and
prostate gland, with a small amount derived from bulbourethral glands
- sperm production is regulated by Sertoli cells (in seminiferous tubules) which
functions as a barrier

Testes exocrine function (secretion of sperm)
- endocrine function (secretion of testosterone)

Epididymis tubular network wherein the luminal fluid from sertoli cells are carried for the maturation of the
immotile and immature sperm
- serves in the concentration of the sperm by absorption of lumen fluid and their storage until
ejaculation

Seminal vesicles and prostate gland serves as accessory gland of the male reproductive system
- produce and store fluids that provide the principal transport medium for
sperm

Composition of semen
Spermatozoa 5%
Seminal fluid 65-70%
Prostate fluid 25-30%
Bulbourethral glands 5%

Specimen collection
It should be collected in a plastic or glass container , or in a special type of condom known as a collection
condom or through masturbation. It should be performed after a 48- 72hr absence of sexual activity so it
contains an accurate account of sperm count and viability.
The specimen should arrive at the laboratory as soon after collection as possible so that an accurate
Liquefaction time can be reported.
During transportation it should be kept near body temperature.
The patient should be asked if any of the sample is lost since the highest concentration of sperm is in the
first part of ejaculation.


MFG*ILMT MLS IV-B

Apperance of semen
Normal semen: grayish white and opalescent
If brown or red presence of blood
If yellow associated with certain drugs
If turbid with less translucent there is an increase in large numbers of leukocytes
Odor: musty

Volume of semen
Analysis of seminal fluid should take place immediately following liquefaction not more than 60 minutes
after collection.
A normal, complete ejaculate collection recovers 2 to 5mL of seminal fluid.
Viscosity of semen
After complete liquefaction, the viscosity of the semen is evaluated using Pasteur pipette and observing
the droplets that form when the fluid is allowed to fall by gravity
A normal specimen is watery and forms into discrete droplets. Abnormal viscosity or fluid thickness is
indicated by the formation of a string or thread greater than 2cm length.
Motility of semen
Laboratory evaluation of sperm motility is performed by examining the undiluted specimen
microscopically and determining the percentage of sperm showing active motility.
Approximately 25 high-power fields should be examined.
- A minimum motility of 50-60 percent with a quality of fair (2.0) is considered normal for specimens
tested within the 3-hour time period.
Motility Grading Criteria
0 immotile
1 motile, without forward progression
2 motile, with slow nonlinear or meandering progression
3 motile, with moderate linear (forward) progression
4 motile, with strong linear (forward) progression

Concentration and sperm count
Normal concentration: 20-250 million per mL of sperm
- determined by using a hemacytometer after preparing an appropriate dilution of 1:20
Sperm count= sperm concentratio n(sperm/mL) x Volume of ejaculate (mL)

Morphology
Normal sperm: oval head measuring 3x5 m and a long tapering tail
Abnormality:
associated with poor ovum penetration
include double heads, giant and morphous heads, pin heads, tapering heads, and constricted heads
MFG*ILMT MLS IV-B

double or coiled tails impeded motility
immature sperm (spermatids) may be present and must be differentiated from WBC

**Sperm morphology should be reported from a stained specimen examined under oil immersion, the
recommended stain is the hematoxylin, crystal violet, or Giemsa stains.
**Additional tests: sperm viability, seminal fluid fructose level and sperm agglutinins

Normal semen analysis
Semen volume: 2ml or more
Semen pH: 7.2-8.0
Liquefaction time: 20-30 mins after collection
Sperm collection: 20 million spermatozoa per ejaculate or more
Sperm count: 40 million spermatozoa per ejaculate or more
Sperm morphology: more than 30% are normal
Sperm motility: 50% show progressive movement; 25% or more with rapid progressive movement
Sperm vitality: 75% or more live
WBC: fewer than 1 million WBC/ml

Semen Biochemistry
Acid phosphatase: marker for prostatic function
Citric acid: can indicate prostatic function low levels may indicate dysfunction or a prostatic duct
obstruction
Zinc: marker for prostatic function colorimetric assay (WHO)
Fructose: marker for seminal vesicle function, and is a substrate for sperm metabolism
spectrophotometric assay (WHO)
-Glucosidase: secreted exclusively by the epididymis and so is a marker for epididymal function
spectrophotometric assay (WHO


SYNOVIAL FLUID

Synovial fluid - normally thick, straw-colored liquid in small amounts in joints, bursae (fluid-filled sacs in bones)
and tendon sheaths
- lubricates the joint and allows for ease movement
- ultrafiltrate or dialysate of plasma and contains levels of glucose and uric acid

Specimen collection:
After the joint area is cleaned, the health care provider inserts a sterile needle through the skin and into
the joint space. Fluid is then drawn through the needle into a sterile syringe.
The fluid sample is sent to the laboratory.
The laboratory technichian:
MFG*ILMT MLS IV-B

o checks the sample's color and clarity
o places the sample under a microscope, counts the number of red and white blood cells, and looks
for crystals (in the case of gout) or bacteria
o measures glucose, proteins, uric acid, and lactic dehydrogenase (LDH)
o cultures the fluid to see if any bacteria grow
Normal synovial fluid: will not clot
Abnormal synovial fluid: fluid from diseased joint may contain fibrinogen and form a clot
**therefore, both anticoagulated and non-anticoagulated specimens should be collected

Recommended specimen containers:
1. EDTA tube (Lavender top tube) for cell counts, differentials and viscosity.
2. Heparanized tube (dark green top tube) for chemistry and immunologic III-47 tests
3. Plain sterile tube or syringe (with needle removed) for microbiologic testing and crystal examination

Appearance
Color:
should be evaluated in a clear glass tube against a white background
colorless but is often pale yellow because of diapedesis of a few RBCs associated with even mild trauma
straw to yellow colored (xanthochromia) seen in noninflammatory and inflammatory disorders

Clarity:
relates to the number and types of particles within the synovia
normal synovial fluid: transparent; newsprint is easily read through
translucent fluid: obscures details but black and white areas can be distinguished
opaque fluid: completely obscures background
leukocytes are most commonly responsible for changes in clarity, however, very large numbers of crystals
may produce an opaque, milky fluid without leukocytes
shimmering, oil-appearing specimen abundance of cholesterol crystals which may grossly resemble pus
increased turbidity less often due to concentration of fibrin, free-floating rice bodies, metal and plastic
particles from patients with joint prostheses, or cartilage fragments in osteoarthritis

Viscosity:
normal synovial fluid: viscid and does not clot
difference from other fluids derived from plasma: high content of hyaluronic acid (mucin)


Total cell counts
total leukocyte counts should be performed promptly
o degenerative loss begins as soon as one hour following arthrocentesis
cell counts are usually performed in a standard hemocytometer
MFG*ILMT MLS IV-B

leukocyte counts: > 10,000/UI
o > 50,000/UI
crystal induces arthritis
chronic inflammatory arthritis
septic arthritis
o < 10,000/UI
osteoarthritis
osteochondritis dissicans
trauma
synovioma
upper reference level for leukocytes: 150-200/uL

Differential leukocyte counts
Neutrophil: 20%
Lymphocytes: 15%
Monocytes and macrophages: 65%

Recommended tests
Routine Tests
Gross examination (color,clarity)
Total and differential leukocyte counts
Gram stain and bacterial culture (aerobic and anaerobic)
Crystal examination with polarizing microscope and compensator
Crystal identification
Types of Crystals
Gout and pseudogout most common crystalline arthropathies
- caused by deposition of monosodium urate (MSU) and calcium pyrophosphate
dehydrate (CPPD) crystals
normal joint fluid is negative for any crystals

Monosodium urate (MSU)
- thin, needle-like crystals
- polarize light and negatively birefringent

Calcium pyrophosphate dehydrate (CPPD)
- present in pseudogout
- smaller and rodlike or rhomboid
- polarize light but are positively birefringent

Corticosteroid crystals
- needle-shaped
MFG*ILMT MLS IV-B

- maybe seen following intraarticular injections
- iatrogenic
- strongly variably birefringent
- polymorphic clumps, rods and rhomboids
- may cause joint pain for several hours

Cholesterol crystals
- may be present in chronic effusions from patients with osteoarthritis or RS
- very large
- flat and rectangular shaped
- notched corners
- varying birefringence
- rheumatoid arthritis

Calcium phosphate (apatite) crystals
- small chunky rods
- seen in calcific periarthritis, osteoarthritis and inflammatory arthritis

Slide preparation
A small amount of hyaluronidase should be added to synovial fluid prior to performing cell counts or
preparing cytocentrifuge slides to liquefy the fluid

Mucin Clot Test
- Ropes Test
- estimation of the integrity of the hyaluronic acid-protein complex (mucin)
- normal fluid forms a tight ropy clot upon the addition of acetic acid
- good mucin clot: indicates good integrity of the hyaluronate
- poor mucin clot: one that breaks up easily; associated with destruction or dilution of hyaluronate

Chemical Tests
Glucosetypically a bit lower than blood glucose levels; may be significantly lower with joint
inflammation and infection
Proteinincreased with bacterial infection
Lactate dehydrogenaseincreased LD (LDH) level may be seen in rheumatoid arthritis, infectious arthritis,
or gout


Microbiologic Tests
Normal synovial fluid: has small numbers of WBCs and RBCs but no microorganisms or crystals present
Culture and susceptibility testing determine what type of microorganisms present

Infectious Disease Test
MFG*ILMT MLS IV-B

- in addition to chemistry tests, other tests may be performed to look for microorganisms if infection is
suspected

Serologic Tests
Rheumatoid Factor (RF) antibody to immunoglobulins
- present in the serum of patients with RS
- false positive RF can result from other chronic inflammatory diseases


AMNIOTIC FLUID

Amniotic fluid found around the developing fetus, inside a membranous sac, called amnion
- formed in the placenta
- volume increases from:
o 30ml at 10 weeks,
o 450ml at 20 weeks
o up to 800-1000 ml at 37 weeks
- serves as cushion, protection and serves as the key role in the exchange of water and molecules
between the fetus and the maternal circulation
- the fluid absorbs jolts, prevent adherence of the embryo to the amnion and allows fetal
movement

Hydramnios- increase in amniotic fluid volume (1,500-2,000ml)
- caused by maternal diabetes, congenital malformations and gastrointestinal defects that prevents
FETAL SWALLOWING.

Oligohydramnios- decreased amount of amniotic fluid (less than 400ml)
- occur with premature rupture of the membranes and with congenital malformations.

The amniotic fluid uses to assess the status of the fetus
Tests to diagnose genetic and congenital disorders before birth
Test to detect fetal distress from HEMOLYTIC DISEASE OF THE NEWBORN or from infection
Test to assess FETAL LUNG MATURITY
To assess the ability of the fetus to survive early delivery

Amniocentesis
obtained by needle aspiration
15-18 weeks of gestation for genetic studies
10-20ml (maximum 30ml)
dispensed into sterile plastic specimen containers
cell culture and chromosomal studies stored at body or room temp
MFG*ILMT MLS IV-B

phospholipid analysis transported on ice and centrifuged at 500g

Appearance
Normal amniotic fluid: colorless to pale yellow
Slightly turbid due to fetal cells, vernix and hair
Dark yellow or amber color associated with bilirubin
Green color indicates meconium; newborn first fecal bowel movements
Very dark red brown associated with fetal death

Difference of amniotic fluid to maternal urine
Urine: higher levels of creatinine and urea
Amniotic fluid: higher levels of glucose and protein

Testing for neural tube defects- alpha fetoprotein and acetylcholine esterase
anencephaly
spina bifida
high levels of AFP
AChE is more specific than AFP testing

Fetal distress testing
1. HDN or Erythroblastosis Fetalis when a mother develops an antibody to an antigen of fetal
erythrocytes
2. Infection vaginal vaginosis and trichomoniasis have been linked to preterm delivery and spontaneous
abortion
3. Respiratory distress syndrome most common death in premature and newborn
immature fetal lungs, lack of surfactant (allows alveoli to function normally during
inhalation and exhalation)

Fetal lung maturity test
1. Lecithin: Sphingomyelin Ration and Phosphatidylglycerol
- levels of lecithin and sphingomyelin are relatively equal
- after 34 weeks, levels of sphingomyelin decreases, while levels of lecithin increases
- (L/S) ration of 2.0 or greater is associated with fetal pulmonary system maturity
- Phosphatidylglycerol detectable only 35 week of gestation (not affected by blood and
meconium)
2. AMNIOSTAT-FLM - uses antibodies against phosphatidyl glycerol to detect fetal lung surfactant.
3. FOAM STABILITY - screening test
4. MICROVISCOSITY FLOURESCENCE POLARIZATION ASSAY - this assay provides flourescence polarization
surfactant: albumin ratio
5. LAMELLAR BODIES - provide reliable estimate of fetal lung maturity

MFG*ILMT MLS IV-B


CEREBROSPINAL FLUID

Formation
- ventricular choroid plexuses (70%)
- ependymal lining cells (30%)

Blood-brain barrier
- tight junction between capillary endothelial cells and epithelial cells in the choroid
- prevent some substances to enter CSF
- small molecules and lipid soluble substances pass through easily

Function:
cushion of the brain and spinal column
serves as a nutrient and metabolic waste exchange fluid
adjust its volume in response to changes in cerebral vessel changes

Specimen collection
Lumbar puncture:
Adults intervertebral space between L3 and L4
Small children and infants intervertebral space between L4 and L5

Physical characteristics
clear
colorless
viscosity similar to water

Traumatic Tap
Blood distribution: there is significant difference in the amount of blood present between first and last tube
Clot formation: may form clot with bloody fluid

Hemorrhage
Blood distribution: homogenous amount of blood present in all tubes
Clot formation: may form clot but with the absence of bloody fluid

Cell Count
Specimen used: well-mixed, undiluted specimen
Counted in Neubauer counting chamber
Cells normally found
o PMNs
o Monocytes
MFG*ILMT MLS IV-B

o Lymphocytes
No RBCs are present

Differential count
Specimen used: cytocentrifuged preparations stained with Wrights stain
Lymphocytes 28-96%
Monocytes 16-56%
PMNs 0-7%
Eosinophils, ependymal cells, histiocytes rarely seen

Pleocytosis increased amount of RBC

Neutrophilic pleocytosis:
Bacterial meningitis
Cerebral abscess
Subdural empyema
CNS hemorrhage
Intrathecal treatments
Postseizure

Lymphocytic pleocytosis
Later stage of viral, tubercular, fungal, syphilitic meningitis
Increased number of leukocytes in Guillian-Barr syndrome

Plasmacytes
Multiple sclerosis

Eosinophils
Parasitic and fungal infections
Allergic reactions

Mixed pleocytosis
Chronic bacterial meningitis
Meningitis of fungal or tuberculin origin
Rupture of cerebral abscess


Other cells in normal CSF:
Ependymal cells
Choroidal cells
PAM cells
MFG*ILMT MLS IV-B


Chemical analysis
1. Protein
- Total protein: 15-45 mg/dL
- comprised by low molecular protein
Methods:
dye-binding
immunochemistry
modified biuret methods
turbidimetric methods
- increased in endocrine disorders, traumatic tap and infections
- decreased in hyperthyroidism
o Albumin derived from transport across the blood-brain barrier
o Protein electrophoresis presence of oligoclonal bands may establish diagnosis
2. Glucose
- 60-70% of plasma in normal adults
- normal range: 50-80 mg/dL
- increased in hyperglycemia and traumatic tap
- decreased in CNS infections, hypoglycemia, impaired glucose transport and metastatic carcinoma
3. CSF Lactate
- present in CSF due to CNS anaerobic metablosim
- levels are independent from plasma lactate levels
- used to differentiate viral meningitis (30mg/dL) from other forms of meningitis (>35 mg/dL)
- increased levels usually reflect CNS tissue hypoxia
4. CSF Glutamine
- produced from ammonia and -ketoglutarate by the brain cells
- serves to remove the toxic metabolic waste product ammonia from CNS
- elevated levels are associated with liver disorders (Reye Syndrome)

Microbiology procedures
meningitis is the most serious diagnosis
the most sterile tube collected is used

Stains
Gram Stain- demonstrates 60-90% sensitivity
Wright Stain
Ziehl-Neelsen Stain- stains Mycobacteria tuberculosis
Fluorescent Rhodamine Stain- stains M. tuberculosis
India Ink- to detected Cryptococcus neoformans (25-50% sensitivity)
Cultures
MFG*ILMT MLS IV-B

Sediment of centrifuged CSF is inoculated into thioglycolate broth and plates of blood agar, chocolate, and
MacConkey agar
If Haemophilus is suspected, Strips of X-V may be applied to the blood agar plate
If fungal meningitis is suspected, Sabouraud dextrose agar should be inoculated
If Mycobacteria is suspected, inoculate Middlebrook broth and agar
NOTE:
It is important to note that if antibiotic therapy was administered prior to the collection of CSF for
culture, the recovery of microorganisms may be significantly reduced.

SEROUS FLUID
Serous fluid fluid between the membranes which provides lubrication as the surfaces move against each other

Formed under the influence of
Hydrostatic pressure
Osmotic pressure
Capillary permeability

Pleural Fluid
- fluid in the lung cavity
- it is about 3- 20 ml under normal conditions.
- drained by the lymphatic system
- normally its a clear or pale yellow fluid.
Turbidity: white blood cells and microorganisms
Blood: traumatic injury, malignancy, and traumatic tap
Milky: chylous or pseudochylous material
Neutrophils: bacterial infection
Lymphocytes: tuberculosis and malignancy
Low glucose: tuberculosis, rheumatoid inflammation and malignancy
Low pH: tuberculosis, malignancy and esophageal rupture
Elevated amylase: pancreatitis

Pleural Effusion
- an abnormal amount of fluid around the lung.
- most pleural effusions are not serious by themselves, but some require treatment to avoid problems
- causes:
o Congestive heart failure
o Pneumonia
MFG*ILMT MLS IV-B

o Liver disease (cirrhosis)
o End-stage renal disease
o Nephrotic syndrome
o Cancer
o Pulmonary embolism
o Lupus and other autoimmune conditions
- symptoms:
o Shortness of breath
o Chest pain, especially on breathing in deeply (pleurisy, or pleuritic pain)
o Fever
o Cough
- diagnosis:
o Chest X-ray film
o Computed tomography (CT scan)
o Ultrasound
Pericardial Fluid
- fluid surrounding the heart
- clear and pale yellow
- volume 10- 15 ml
Milky: lymphatic drainage
Tirbidity: infection and malignancy
Blood: tuberculosis, tumor and cardiac puncture
Neutrophils: bacterial endocarditis
Low glucose: bacterial infection and malignancy

Pericardial Effusion
- the presence of excessive pericardial fluid within the potential space of pericardium.
- rapid accumulation of pericardial fluid may cause elevated intrapericardial pressures with as little as 80 mL
of fluid, while slowly progressing effusions can grow to 2 L without symptoms

Cardiac tamponade
- occurs when the heart is squeezed by fluid that collects inside the sac that surrounds it
- three principal features
o Elevation of intracardiac pressures
o Limitation of ventricular filling
o Reduction of cardiac output
- causes:
o any condition lead to pericarditis can lead to pericardial effusion .
o the most common cause are:
Neoplastic disease
MFG*ILMT MLS IV-B

Idiopathic pericarditis
Uremia
Following cardiac operation
Trauma
- signs and symptoms:
o shortness of breath
o weakness and fatigue
o anxiety
o tachycardia
o jugular vein engorged
o cyanosis
- diagnosis:
o physical examination
o electrocardiogram (ECG)
o chest X-ray film

Peritoneal Fluid
- fluid in the abdominal cavity
- Clear or pale yellow
- > 50 ml
Turbidity: peritonitis and cirrhosis
Blood: trauma
Neutrophils: peritonitis
Low glucose: tubercular peritonitis and malignancy
Elevated amylase: pancreatitis, gastrointestinal perforation
Elevated alkaline phosphatase: intestinal perforation
Elevated urea or creatinine: ruptures bladder



Peritoneal Effusion
- an accumulation of fluid in the peritoneal cavity and is also known as ascites.
- other name: hydroperitoneum and abdominal dropsy

Transudate and exudate
Transudate
- produced as a result of disruption of fluid production and regulation between the serous membranes
Exudate
- caused by conditions producing damage to the serous membranes

MFG*ILMT MLS IV-B

Ascites
Common causes:
Transudate
1. Cirrhosis
2. Congestive heart failure
3. Hypoalbuminemia
Exudate
Metastatic ovarian cancer and infective peritonitis

Signs and symptoms:
abdomen related
everted umbilicus
flank fullness
lank dullness( if absent this means that there is < 10% chance of having Ascites) there is at least 1.5 liters
of Ascites if dullness is present], shifting dullness
fluid thrill

Diagnosis:
USG : confirm the diagnosis of minimal amount of ascites
Paracentesis

General Laboratory Procedures
Routine fluid examination including:
- classification as a transudate or exudate
- Appearance
- cell count (differential)
o cell counts are usually performed manually using the Neubauer counting chamber
o differential counts are performed on (Wright stained smears)
o any suspicious cells been on the differential should be referred to the cytology laboratory or the
pathologist
- chemistry
- microbiology procedures