CEREBROSPINAL

FLUID
Presented by:
Arrazola, Dioquino, Lizardo, Obra, Sigue
 OUTLINE
Formation and Specimen Collection
Appearance
Physiology & Handling

Traumatic Differential Count

Cell Count
Collection (TAP) on a CSF Specimen

Chemistry Tests Microbiology Tests Serologic Testing

 INTRODUCTION
Cerebrospinal fluid (CSF) is a clear,
colorless, major body fluid of the body found
within the tissue that surrounds the brain and
spinal cord.

Functions:
Provides a physiologic system to supply
nutrients to the nervous tissue
Removes metabolic wastes
Produces a mechanical barrier to cushion
the brain and spinal cord against trauma
 FORMATION AND PHYSIOLOGY

Dura mater “hard mother”

Outer layer that lines the skull and
vertebral canal

Arachnoid mater “spiderweb-like”

Filamentous inner membrane

Pia mater “gentle mother”

The brain and spinal cord are lined by the meninges, Thin membrane lining the surfaces
which consists of three layers: of the brain and spinal cord
The layers of the meninges in the spinal cord
 FORMATION AND PHYSIOLOGY
Choroid plexuses
Location where CSF is produced
20mL/hr of fluid is produced in
adults

Subarachnoid space
Where the fluid flows through
Located between the arachnoid
and pia mater

Arachnoid granulations/villae
Where the circulating fluid is
reabsorbed to maintain volume
The flow of CSF through the brain and
- 90 to 150 mL in adults spinal column
- 10 to 60 mL in neonates
 FORMATION AND PHYSIOLOGY

Blood-brain Barrier
Tight-fitting structure of
Capillary walls throughout the endothelial cells in the
Choroid Plexuses
the body are lined with choroid plexuses.
Capillary networks that
endothelial cells that are
form the CSF from plasma
loosely connected to allow Dis ruption of the blood-
by mechanisms of
passage of soluble brain barrier by diseases
selective filtration under
nutrients and wastes such as meningitis and
hydrostatic pressure and
between the plasma and multiple sclerosis allows
active transport secretion.
tissues. leukocytes, proteins, and
additional chemicals to
enter the CSF.
 CSF is routinely collected by lumbar puncture between the
third, fourth, or fifth lumbar vertebra.

Although this procedure is not complicated, it does

SPECIMEN require certain precautions, including:

COLLECTION
Measurement of intracranial pressure

AND HANDLING
Careful technique to prevent infection or neural tissue
damage

The volume of CSF that can be removed is based on the:

Volume available in the patient (adult vs. neonate)
The opening pressure of the CSF
Specimens are collected in three sterile tubes, which are
labeled 1, 2, and 3 in the order in which they are withdrawn.

Tube 1: Chemical and Serologic tests

Tube 2: Microbiology laboratory
Tube 3: Cell count

Note: A fourth tube may be drawn for the microbiology

laboratory to better exclude skin contamination or for
additional serologic tests.

Excess fluid should not be discarded and should be frozen

until there is no further use for it.
 SPECIMEN COLLECTION
AND HANDLING
Ideally, tests are performed on a STAT basis. If this is not
possible, specimens are maintained in the following manner:

Hematology tubes are refrigerated

Microbiology tubes remain at room temperature
Chemistry and serology tubes are frozen
APPEARANCE
The initial appearance of the
normally crystal-clear CSF can
provide valuable diagnostic
information.

The major technology used to describe

CSF appearance includes crystal-
clear, cloudy or turbid, milky,
xanthochromic, and
hemolyzed/bloody.

Xanthochromia - it is a CSF specimen that is

supernatant that is pink, orange or yellow. One of
the common factor that can cause xanthochromia
is the presence of RBC degradation products. The
xanthochromia that is caused by bilirubin can be
seen in infants due to immature liver function.
TRAUMATIC COLLECTION (TAP)

There are three visual examinations of the collected

specimens can usually determine whether the blood is the
result of hemorrhage or a traumatic tap.

Uneven Blood Distribution

Clot Formation
Xanthochromic Supernatant
 Blood from a cerebral hemorrhage will be
evenly distributed throughout the three CSF
specimen tubes, whereas the traumatic tap
UNEVEN BLOOD will leave the heaviest concentration of

DISTRIBUTION blood in tube 1, and gradually diminishing

amounts in Tubes 2 and 3 since performing
red blood cell (RBC) counts on all three tubes
to measure decreasing constant blood is not
always reliable.
 The fluid collected from a traumatic tap may form
clots. Diseases in which damage to the blood-brain
barrier allows increased filtration of protein and
coagulation factors also cause clot formation.

CLOT FORMATIONThese conditions include meningitis, Froin syndrome,

and blocked CSF circulation through the subarachnoid
space.

NOTE:
A classic web-like pellicle is associated with tubular
meningitis and can be seen after overnight
refrigeration of the fluid.
 RBCs must usually remain in the CSF approx. 2
hours before noticeable hemolysis begins; thus, a
xanthochromic supernatant would be the result of

XANTHOCHROMIC
blood has been present longer than that
introduced by the traumatic tap.

SUPERNATANT NOTE:
To examine a blood fluid for the presence of
xanthochromia, the fluid should be centrifuged in a
microhematocrit tube and the supernatant
examined against a white background.
CELL COUNT
The cell count that is routinely performed on CSF
specimens is the WBC count.

RBC counts are usually determined only when a

traumatic tap has occurred and a correction for
leukocytes or protein is desired.

Cell Count should be performed immediately because

WBC and RBCs begin to lyse within 1 hour.
 METHODOLOGY

Normal adult CSF = 0-5 WBCs/uL

Children = 30 mononuclear cells/uL (considered normal in
newborns)

Specimens that contain up to 200 WBC or 400 RBC/uL

may appear clear, so it is necessary to examine all
specimens microscopically.
 CALCULATING CSF CELL
COUNTS

The standard Neubauer calculation formula used for

blood cell counts is also applied to CSF cell counts to
determine the number of cells per microliter.
 TOTAL CELL COUNT
Dilutions for total cell counts are made with normal
saline, mixed by inversion, and loaded into the
hemocytometer with a Pasteur pipette. Cells are
counted in the four corner squares and the center square
on both sides of the hemocytometer.
 QUALITY CONTROL OF CSF AND OTHER BODY
FLUID CELL COUNTS
All diluents should be checked biweekly for contamination by
examining them in a counting chamber under 400×
magnification.
Contaminated diluents should be discarded and new solutions
prepared.
The speed of the cytocentrifuge should be checked monthly
with a tachometer, and the timing should be checked with a
stopwatch.
If non-disposable counting chambers are used, they must be
soaked in a bactericidal solution for at least 15 minutes and
then thoroughly rinsed with water and cleaned with isopropyl
alcohol after each use.
Differential Count on a CSF
Specimen
The differential count should be performed on a
stained smear and not from the cells in the counting
chamber.
 METHODS AVAILABLE FOR
SPECIMEN
CONCENTRATION:

1. Sedimentation
2. Filtration
3. Centrifugation
4. Cytocentrifugation
Cytocentrifugation
Fluid is added to the conical
chamber, and as the specimen is
centrifuged, cells present in the
fluid are forced into a monolayer
within a 6-mm diameter circle on
the slide. Fluid is absorbed by the
filter paper blotter, produc- ing a
more concentrated area of cells. As
little as 0.1 mL of CSF combined
with one drop of 30% albumin
produces an ade- quate cell yield
when processed with the
cytocentrifuge.
 CSF Cellular Contents

The cells found in normal CSF are primarily

lymphocytes and monocytes. Adults usually have
a pre- dominance of lymphocytes to monocytes
(70:30), whereas the ratio is essentially reversed
in children. Improved concentration methods are
also showing occasional neutrophils in normal
CSF. The presence of increased numbers of these
normal cells (termed pleocytosis) is considered
abnormal, as is the finding of immature
leukocytes, eosinophils, plasma cells,
macrophages, increased tissue cells, and
malignant cells.
 When pleocytosis involving neutrophils,
lymphocytes, or monocytes is present, the CSF
differential count is most frequently associated with
its role in providing diagnostic information about the
type of microorganism that is causing an infection of
the meninges (meningitis). A high CSF WBC count of
which the majority of the cells are neutrophils is
considered indicative of bacterial meningitis.
Likewise, a moderately elevated CSF WBC count with
a high percentage of lymphocytes and monocytes
suggests meningitis of viral, tubercular, fungal, or
parasitic origin.
 CSF Cellular Contents

As seen in Table 9–3, many pathologic

conditions other than meningitis can be
associated with abnormal cells in the
CSF. Therefore, because laboratory
personnel become so accustomed to
finding neutrophils, lymphocytes, and
monocytes, they should be careful not
to overlook other types of cells. Cell
forms differing from those found in
blood include macrophages, choroid
plexus and ependymal cells, spindle-
shaped cells, and malignant cells.
 Type of predominant
cells seen in CSF
Neutrophils

In addition to bacterial meningitis, increased neutrophils also are seen in the early stages (1 to 2 days) of viral,
fungal, tubercular, and parasitic meningitis. Neutrophils may also contain cytoplasmic vacuoles following
cytocentrifugation (Fig. 9–9). Granules are also lost more rapidly in CSF. Neu- trophils associated with bacterial
meningitis may contain phagocytized bacteria (Figs. 9–10 and 9–11). Although of little clinical significance,
neutrophils may be increased fol- lowing central nervous system (CNS) hemorrhage, repeated lumbar punctures,
and injection of medications or radi- ographic dye.
 cont…

Neutrophils with pyknotic nuclei indicate degenerating cells. They may resemble nucleated red blood cells
(NRBCs) but usually have multiple nuclei. When a single nucleus is present they can appear similar to NRBCs (Fig.
9–12). NRBCs are seen as a result of bone marrow contamination during the spinal tap (Figs. 9–13 and 9–14). This is
found in approximately 1% of specimens. Capillary structures and endothelial cells may be seen following a
traumatic tap (Fig. 9–15).
 Type of predominant
cells seen in CSF
Eosinophils

Increased eosinophils are seen in the CSF in association with

parasitic infections, fungal infections (primarily Coccidioides
immitis), and introduction of foreign material, including
medications and shunts, into the CNS (Fig. 9–17).
 Type of predominant
cells seen in CSF
Macrophages

The purpose of macrophages in the CSF is to remove cellular debris and

foreign objects such as RBCs. Macrophages appear within 2 to 4 hours after
RBCs enter the CSF and are frequently seen following repeated taps. They
tend to have more cytoplasm than monocytes in the peripheral blood (PB)
(Fig. 9–18).
The finding of increased macrophages indicates a
previous hemorrhage (Fig. 9–19). Further degradation of
the phagocytized RBCs results in the appearance of dark
blue or black iron- containing hemosiderin granules (Figs.
9–20 through 9–23). Yellow hematoidin crystals
represent further degeneration. They are iron-free,
consisting of hemoglobin and unconjugated bilirubin
(Figs. 9–24 and 9–25).
cont…
 Malignant Cells of Hematologic and
Nonhematologic Origin

Lymphoblasts, myeloblasts, and monoblasts (Figs. Metastatic carcinoma cells of nonhematologic

9–29 to 9–31) in the CSF are frequently seen as origin are pri- marily from lung, breast, renal, and
a serious complication of acute leukemias. gastrointestinal malignan- cies. Cells from
Nucleoli are often more prominent than in blood primary CNS tumors include astrocytomas,
smears. retinoblastomas, and medulloblastomas (Fig. 9–
Lymphoma cells are also seen in the CSF and 35). They usually appear in clusters and must be
indicate dis- semination from the lymphoid tissue. distinguished from normal clusters of ependymal,
They resemble large and small lymphocytes and choroid plexus, lymphoma, and leukemia cells.
usually appear in clusters of large, small, or Fusing of cell walls and nuclear irregularities and
mixed cells based on the classification of the lym- hyperchromatic nucleoli are seen in clusters of
phoma. Nuclei may appear cleaved, and malignant cells. Slides containing abnormal cells
prominent nucleoli are present (Figs. 9–32 to 9– must be referred to pathology.
34).
CHEMISTRY TESTS
CSF is formed by filtration of the plasma.

Abnormal values result from alterations in the

permeability of the blood–brain barrier or increased
production or metabolism by the neural cells in response
to a pathologic condition.
 CEREBROSPINAL PROTEIN
The most frequently performed chemical test on CSF is
the protein determination.

Reference values for total CSF protein = 15 to 45 mg/dL

CSF contains protein fractions similar to those found in

serum.

Albumin makes up most of CSF protein. But in contrast to

serum, prealbumin is the second most prevalent fraction
in CSF.
 CEREBROSPINAL PROTEIN
Alpha globulins include primarily haptoglobin and
ceruloplasmin. Transferrin is the major beta globulin present.

“tau” a separate carbohydrate-deficient transferrin fraction

seen in CSF and not in serum.

CSF gamma globulin is primarily immunoglobulin G (IgG), with

only a small amount of immunoglobulin A (IgA).

Immunoglobulin M (IgM), fibrinogen, and beta lipoprotein are

not found in normal CSF.
CLINICAL SIGNIFICANCE OF ELEVATED PROTEIN
VALUES
Elevated total protein values = pathologic conditions.
Abnormally low values = fluid is leaking from the CNS.

The causes of elevated CSF protein:

damage to the blood–brain barrier
immunoglobulin production within the CNS
decreased normal protein clearance from the fluid
neural tissue degeneration.

The most common causes of elevated CSF protein are meningitis

and hemorrhage conditions that damage the blood–brain
barrier.
 METHODOLOGY

The two most routinely used techniques for

measuring total CSF protein use the principles of
turbidity production or dye-binding ability.

The turbidity method has been adapted to

automated instrumentation in the form of
nephelometry.
 PROTEIN FRACTIONS
Routine CSF protein procedures are designed to measure total
protein concentration.

Protein that appears in the CSF as a result of damage to the

integrity of the blood–brain barrier contains fractions
proportional to those in plasma, with albumin present in the
highest concentration.

To accurately determine whether IgG is increased because it

is being produced within the CNS or is elevated as the result
of a defect in the blood–brain barrier, comparisons between
serum and CSF levels of albumin and IgG must be made.
Normal IgG index values vary slightly among laboratories;
however, in general, values greater than 0.70 indicate IgG
production within the CNS.
 ELECTROPHORESIS AND IMMUNOPHORETIC
TECHNIQUES
The primary purpose for performing CSF protein
electrophoresis is to detect oligoclonal bands.

To ensure that the oligoclonal bands are present as the result

of neurologic inflammation, simultaneous serum electrophoresis
must be performed.

Disorders such as leukemia, lymphoma, and viral infections

may produce serum banding.

Banding representing both systemic and neurologic

involvement is seen in the serum and CSF with HIV infection.
The presence of two or more oligoclonal bands in the CSF that are
not present in the serum can be a valuable tool in diagnosing
multiple sclerosis, particularly when accompanied by an increased
IgG index.

Other neurologic disorders including encephalitis, neurosyphilis,

Guillain-Barré syndrome, and neoplastic disorders also produce
oligoclonal banding that may not be present in the serum.

Low protein levels in the CSF make concentration of the fluid

before performing electrophoresis essential for most
electrophoretic techniques.

CSF immunofixation electrophoresis (IFE) and isoelectric focusing

(IEF) followed by silver staining.
 MYELIN BASIC PROTEIN

The presence of myelin basic protein (MBP) in the

CSF indicates recent destruction of the myelin
sheath that protects the axons of the neurons
(demyelination).

Immunoassay techniques are used for measurement.

 CSF GLUCOSE
Glucose enters the CSF by selective transport across the
blood–brain barrier, which results in a reference value that is
approximately 60% to 70% that of the plasma glucose.

If the plasma glucose is 100 mg/dL, then a reference CSF

glucose would be approximately 65 mg/dL.

CSF glucose is analyzed using the same procedures employed

for blood glucose. Specimens should be tested immediately
because glycolysis occurs rapidly in CSF.

The diagnostic significance of CSF glucose is confined to

finding values that are decreased relative to plasma values.
 CSF GLUCOSE
Elevated CSF glucose values are always a result of plasma
elevations.

Low CSF glucose values can be of considerable diagnostic

value in determining the causative agents in meningitis.

Decreased CSF glucose values are caused primarily by

alterations in the mechanisms of glucose transport across the
blood–brain barrier and by increased use of glucose by the
brain cells.
 CSF LACTATE
In bacterial, tubercular, and fungal meningitis, CSF lactate
levels greater than 25 mg/dL occurs much more consistently
than does decreased glucose.

Levels greater than 35 mg/dL are frequently seen with

bacterial meningitis, whereas in viral meningitis, lactate levels
remain lower than 25 mg/dL.

Tissue destruction within the CNS owing to oxygen deprivation

(hypoxia) increases CSF lactic acid levels.

CSF lactate levels are frequently used to monitor severe head

injuries.
 CSF GLUTAMINE
Glutamine is produced from ammonia and a -ketoglutarate by
the brain cells. This process serves to remove the toxic
metabolic waste product ammonia from the CNS.

normal concentration of glutamine in the CSF is 8 to 18 mg/dL.

Elevated levels are associated with liver disorders that result

in increased blood and CSF ammonia.

Several methods of assaying glutamine are available and are

based on the measurement of ammonia liberated from the
glutamine.
 CSF GLUTAMINE

As the concentration of ammonia in the CSF increases, the

supply of a -ketoglutarate becomes depleted.

Some disturbance of consciousness is almost always seen

when glutamine levels are more than 35 mg/dL.

CSF glutamine test is frequently requested for patients with

coma of unknown origin. Approximately 75% of children with
Reye syndrome have elevated CSF glutamine levels.
Microbiology Test
to identify causative agent in meningitis
CONFIRMATORY TEST:
CSF culture

PRELIMINARY DIAGNOSIS:
Gram stain
Acid-fast stain
India ink preparation
Latex agglutination test
 CSF CULTURE

Microorganism must be
recovered from the CSF fluid
culture to signify positive
identification.

Culture time: 24 hrs (bacterial

meningitis) to
6 weeks (tubercular meningitis)
 GRAM STAIN

Detects bacteria and fungal organisms, and is routinely performed on

CSF from all suspected cases of meningitis. Cytocentrifuge provides a
highly concentrated specimen for gram stains. Blood cultures should be
taken because causative organisms are often present in both CSF and
blood.

Centrifuge: 1500 g for 15 minutes

 GRAM STAIN

Streptococcus pneumoniae (gram-positive cocci)

Haemophilus influenzae (pleomorphic gram-negative rods)
Escherichia coli (gram-negative rods)
Neisseria meningitidis (gram-negative cocci)
Bacteria encountered in newborns:
Gram-positive cocci (Streptococcus agalactiae)
Gram-positive rods (Listeria monocytogenes)
 ACID FAST STAIN

Not routinely performed on specimens

unless tubular meningitis is suspected.
INDIA INK PREPARATION

performed to detect the presence of

thickly encapsulated Cryptococcus
neoformans. Particular attention
should be paid to the Gram stain for
the classic straburst pattern produced
by Cryptococcus.
LATEX AGGLUTINATION TEST

performed to detect the presence of

Cryptococcus neoformans in serum
and CSF.
ENZYME IMMUNOASSAY TECHNIQUES

Lateral flow assay (LAF)

Enzyme-linked Immunosorbent
Assay (ELISA)
Bacterial Antigen Test (BAT)
Serologic Testing
performed to detect the presence of neurosyphilis.
 Serologic Testing

Venereal Disease Research Laboratories (VDRL)

Flourescent Treponemal Antibody-absorption (FTA-ABS)
Rapid Plasma Regain (RPR)
thank you
 SYNOVIAL
FLUID
Bantegui, Edma, Lim,Lovenaria, Ojerio,
SYNOVIAL FLUID

referred to as “joint fluid”

viscous liquid found in the cavities of
the movable joints (diarthroses) or
synovial joints.
provides nutrients to the articular
cartilage and lessens the shock of joint
compression that occurs during
activities (walking, jogging).
formed as an ultrafiltrate of plasma
across the synovial membrane.

(e.g. shoulder, hip, elbow and knee)

Bones in the synovial joints are lined with smooth articular
cartilage and separated by a cavity containing the synovial fluid.
Joint is enclosed in a fibrous joint capsule lined by the synovial
membrane.
Smooth articular cartilage and synovial fluid together reduce
friction between the bones during joint movement.

Filtration is nonselective except for the exclusion of high-

molecular weight proteins.
Most of the chemical constituents, although seldom of clinical
significance, have concentrations similar to plasma values and
they provide nutrients for the vascular-deficient cartilage.
Synoviocytes or Synovial intimal cells

a synovial membrane contains specialized cells.

secrete a mucopolysaccharide containing hyaluronic acid and a
small amount of protein (approximately ¼ of the plasma
concentration) into the fluid. Large hyaluronate molecules
contribute the noticeable viscosity to the synovial fluid.

Arthritis
a damage to the articular membranes produces pain
and stiffness in the joints.
Laboratory Results of Beneficial tests most frequently
Synovial Fluid Analysis performed on synovial fluid are:

White blood cell count

used to determine the
Differential
pathologic origin of
Gram stain
arthritis.
Culture
Crystal examination.
Reference Values
Variety of conditions associated
with arthritis:

infection
inflammation
metabolic disorders
trauma
physical stress
advanced age
The patient’s clinical history must also be
considered when assigning a category.
 SPECIMEN COLLECTION AND
HANDLING

·Arthrocentesis: collected by needle aspiration, fluid is often collected in a syringe that

has been moistened with heparin
·The amount of fluid present varies with the size of the joint and the extent of fluid
buildup in the joint.
Fluid in Adult knee cavity: NORMAL- < 3.5 mL
INFLAMMATION- increase - >25 mL
·Only a few drops of fluid are obtained, and used for microscopic analysis or culturing.
·The vol. of fluid collected should be recorded
·NORMAL synovial fluid DOES NOT CLOT
·Synovial fluid from a DISEASED JOINT may contain FIBRINOGEN and will CLOT.
-When sufficient fluid is collected, it should be distributed into specific tubes based on
the required tests.
SPECIMEN COLLECTION AND
HANDLING
 SPECIMEN COLLECTION AND
HANDLING

Powdered anticoagulants should not be used because they may produce

artifacts that interfere with crystal analysis.
Nonanticoagulated tubes for other tests must be centrifuged and separated
to prevent cellular elements from interfering with chemical and serologic
analyses.
 COLOR AND CLARITY

A report of the gross appearance is an

essential part of the synovial fluid analysis.
The word “synovial” comes from the Latin
word for egg, ovum.
Normal viscous synovial fluid resembles egg
white.
Colorless to pale yellow- normal synovial
fluid
Deeper yellow- the presence of
noninflammatory and inflammatory
effusions
 COLOR AND CLARITY

Greenish tinge- bacterial infection.

Reddish- the presence of blood from a
hemorrhagic arthritis must be
distinguished from blood from a traumatic
aspiration. This is accomplished primarily
by observing the uneven distribution of
blood or even a single blood streak in the
specimens obtained from a traumatic
aspiration.
 COLOR AND CLARITY

CLARITY/TURBIDITY
-frequently associated with
the presence of:
WBCs
Synovial cell debris
Fibrin
Crystals- the fluid may appear
milky
 VISCOSITY
Synovial fluid viscosity comes from polymerization of
the hyaluronic acid and is essential for the proper joints
lubrication.
Arthritis affects both the production of hyaluronate and
its ability to polymerize = fluid viscosity.

Several methods are available to measure the synovial fluid

viscosity, the simplest being to observe the fluid’s ability to
form a string from the tip of a syringe, a test that easily can
be done at the bedside
String measuring 4-6 cm = Normal
 VISCOSITY
Hyaluronate polymerization can be measured using a Ropes, or mucin clot test.
When added to a solution of 2-5% acetic acid, normal synovial fluid forms a solid
clot surrounded by clear fluid.
As the ability of the hyaluronate to polymerize decreases, the clot becomes less
firm, and the surrounding fluid increases in turbidity.
The mucin clot test is reported in terms of :
- good (solid clot)
- fair (soft clot)
- low (friable clot)
- poor (no clot)

Mucin clot test is not routinely performed, because all forms of arthritis decrease viscosity and little
diagnostic information is obtained.
Formation of a mucin clot after adding acetic acid can be used to identify a questionable fluid as synovial
fluid
 CELL COUNTS

Total leukocyte count is the most frequently performed cell count on synovial fluid. To prevent cellular
disintegration, counts should be performed as soon as possible or the specimen should be refrigerated. Very
viscous fluid may need to be pretreated by adding one drop of 0.05% hyaluronidase in phosphate buffer per
milliliter of fluid and incubate at 37°C for 5 minutes.

Manual counts on thoroughly mixed specimens are done using the Neubauer counting chamber.
Clear fluids can usually be counted undiluted, but dilutions are necessary when fluids are turbid or
bloody.
Normal saline can be used as a diluent. If it is necessary to lyse the RBCs, hypotonic saline (0.3%) or
saline that contains saponin is a suitable diluent. Methylene blue added to the normal saline stains
the WBC nuclei, permitting separation of the RBCs and WBCs during counts performed on mixed
specimens.
The recommended technique is to line a petri dish with moist paper and place the hemocytometer
on two small sticks to elevate it above the moist paper.
Counting procedure:

For counts less than 200 WBCs/ L, count all 9 large squares.
For counts greater than 200 WBCs / L in the above count, count the 4 corner squares.
For counts greater than 200 WBCs / L in the above count, count the 5 small squares used for a RBC .

WBC counts less than 200 cells/ L are considered normal and may reach 100,000 cells/ L or higher in
severe infections.
 DIFFERENTIAL COUNT

-Should be performed on cytocentrifuged preparations or on thinly smeared slides. Fluid should be incubated with
hyaluronidase prior to slide preparation. Mononuclear cells, including monocytes, macrophages, and synovial tissue cells, are
the primary cells seen in normal synovial fluid.

Neutrophils should account for less than 25% of the differential count and lymphocytes less than 15%.
Increased neutrophils indicate a septic condition, whereas an elevated cell count with a predominance of
lymphocytes suggests a nonseptic inflammation.
In both normal and abnormal specimens, cells may appear more vacuolated than they do on a blood smear.

increased numbers of these usually normal cells, other cell abnormalities include the presence of eosinophils, LE
cells, Reiter cells (or neu- trophages, vacuolated macrophages with ingested neu- trophils), and RA cells (or
ragocytes, neutrophils with small, dark cytoplasmic granules consisting precipitated rheuma- toid factor). Lipid
droplets may be present after crush injuries, and hemosiderin granules are seen in cases of pigmented
villonodular synovitis.
DIFFERENTIAL COUNT
 Crystal Identification in
SYNOVIAL FLUID utilizes
Microscopic Method
important in evaluating arthritis

Crystal Formation may result to..

acute, painful inflammation
could become a chronic condition

Causes of Crystal formation could be...

metabolic disorders
decreased renal excretion [produces elevated
blood levels of crystallized chemicals]
CRYSTAL degeneration of bone and cartilage
IDENTIFICATION Injection of medication on the joint.
[Corticosterroids]
Primary Crystals in
SYNOVIAL FLUID

Monosodium Urate (MSU) -

cases of Gout
Calcium Pyrophosphate
Dihydrate (CPPD) - cases of
Pseudogout.
COMMON CRYSTALS:
OVERVIEW
Frequent Causes of GOUT
Increased Serum Uric Acid (UA)
Impaired purine metabolism
Increased consumption of
foods with high-purine
content, alcohol and fructose.
Chemotherapy (Leukemia)
Decreased renal excretion of
Uric Acid (UA).
 The PSEUDOGOUT
associated with degenerative
arthritis
produces cartilage calcification
produces endocrine disorders
(elevates Serum Calcium levels
Hydroxyapatite | Basic
Calcium Phosphate
associated with Calcified
Cartilage Degeneration
Cholesterol Crystals
associated with chronic
inflammation
ADDITIONAL
CRYSTALS
Corticosteroids
associated with join injection
medications

Calcium Oxalate
visible in renal dialysis
patients

ADDITIONAL
CRYSTALS
 Scratches from slides

Starch Dust
(from gloves)

ARTIFACTS Precipitated
Talcum Powder Anti-coagulants
CHARACTERISTICS OF
SYNOVIAL FLUID
CRYSTALS
Name of Crystal
Monosodium Urate
Shape
Needles
Compensated Polarized Light
Negative Birefringence
Significance
Gout
APPEARANCE
 Name of Crystal
Calcium Pyrophosphate
Shape
Rhomboid Square, rods
Compensated Polarized Light
Positive Birefringence
Significance
Pseudogout
APPEARANCE
Name of Crystal
Cholesterol
Shape
Notched, Rhomboid Plates
Compensated Polarized Light
Negative Birefringence
Significance
Extracellular
APPEARANCE
 Name of Crystal
Corticosteroid
Shape
Flat, Variable-shaped plates
Compensated Polarized Light
Positive and Negative Birefringence
Significance
Injections
APPEARANCE
Name of Crystal
Calcium Oxalate
Shape
Envelopes
Compensated Polarized Light
Negative Birefringence
Significance
Renal Dialysis
APPEARANCE
 Name of Crystal
Apatite | Calcium Phosphate
Shape
Small Particles (Electron
Microscopy Required)
Compensated Polarized Light
No Birefringence
Significance
APPEARANCE Osteoarthritis
 UNSTAINED WET PREP OF MSU CRYSTALS. (X400) NOTICE
CHARACTERISTIC YELLOW-BROWN OF THE URATE CRYSTALS
WRIGHT’S STAINED NEUTROPHILS CONTAINING CPPD CRYSTALS
(X1000)
 EXTRACELLULAR MSU CRYSTALS UNDER COMPENSATED POLARIZED
LIGHT. NOTICE CHANGE IN COLOR WITH CRYSTAL ALIGNMENT (X100)
STRONGLY BIREFRINGENT MSU CRYSTALS UNDER POLARIZED
LIGHT
WEAKLY BIREFRINGENT CPPD CRYSTALS UNDER POLARIZED LIGHT
(X1000)
 MSU CRYSTALS UNDER COMPENSATED POLARIZED LIGHT. THE
YELLOW CRYSTAL IS ALIGNED WITH THE SLOW VIBRATION (X500)
CPPD CRYSTALS UNDER COMPENSATED POLARIZED LIGHT. THE
BLUE CRYSTAL IS ALIGNED WITH THE SLOW VIBRATION (X1000)
 Synovial Fluid
chemically, an ultrafiltrate of
CHEMISTRY plasma.

TESTS
Chemistry test values are
approximately same as
Serum values
GLUCOSE DETERMINATION Protein and Uric Acid Test
decreased glucose values, CHEMISTRY Increased protein levels,
indicates inflammatory
TESTS present in inflammatory
(G2) or septic (G3) and hemorrhagic
disorders. disorders.
Same process to serum
Fasting for 8 hours is protein determination.
needed.

Blood Glucose and Synovial Fluid Samples Elevated Synovial Uric acid levels confrims
should be collected simultaneously. GOUT diagnosis when crystals can’t be
demonstrated in the fluid.
Normal Value:
Normal Value: 10mg/dL 3 g/dL: Protein
Fluid Lactate or Acid Phosphatase
Could be requested for monitoring
or Rheumatoid Artritis severity and CHEMISTRY
prognosis
TESTS

IMPORTANT NOTICE:
Specimens for Crystal Analysis
shouldn’t be refrigerated. Additional
crystals interferes with the ID of
significant crystals.
 MICROBIOLOGY TESTS

Purpose of Testing:
Identify causative organisms in synovial fluid.
Diagnostic Tools:
Gram Stain: Reveals bacterial characteristics.
Culture: Grows and identifies microorganisms.
Enrichment medium (Chocolate agar)
Common organisms that causes infection in synovial fluid:
fastidious Haemophilus spp.
Neisseria gonorrhoeae
Common Infections:
Bacterial (most frequently found), fungal, viral infections can be
detected.
 SEROLOGIC TESTS

Role of Serologic Testing:

Integral in diagnosing joint disorders associated with the immune system and inflammation.
Primary Testing Location:
Most tests are conducted on serum (blood).
Synovial Fluid Analysis:
Serves as a confirmatory measure, particularly in challenging diagnoses.
Autoimmune Diseases:
Rheumatoid arthritis and systemic lupus erythematosus diagnosed by demonstrating specific
autoantibodies in serum.
Synovial Fluid Confirmation:
Autoantibodies can also be found in synovial fluid, providing additional confirmation.
Lyme Disease and Arthritis:
Arthritis in Lyme disease can be confirmed by demonstrating antibodies to Borrelia burgdorferi
in the patient's serum.
THANK YOU FOR
LISTENING!<3
Manila Adventist College
1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture

Serous Fluid
Other type of Body Fluids

BELARMINO. ENRIQUEZ. MACALALAD. PICART. TORRES

Introduction
Serous fluid: the fluid between the parietal
membrane and visceral membrane.

Function: provide lubrication between

membranes, necessary to prevent the friction
that occur as a result of movement of the
enclosed organs

Production and reabsoprtion: constant rate

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Introduction
Pleural Fluid: obtained from the pleural cavity,
located between the parietal pleural
membrane lining of the chest and visceral
pleural membrane covering the lungs

Pericardial fluid: found between the

pericardial serous membranes

Peritoneal fluid: a liquids that acts as a

lubricant in the abdominal cavity

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Formation of Serous fluids
Formed as ultrafiltrates of plasma; no
additional material is contributed by the
mesothelial cell that lines the
membranes.

Production and reabsorption are subject

to hydrostatic pressure and colloidal
pressure (oncotic pressure) from the
capillaries

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Formation of Serous fluids
Effusion: increase in fluid between the
membranes due to the disruption of the
mechanisms of serous fluid formation and
reabsorption.

Primary causes of effusions:

increased hydrostatic pressure
decreased oncotic pressure
increased capillary permeability
lymphatic obstruction

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Specimen Collection and
Handling
Fluids for laboratory examination are collected by needle aspiration form the
respective cavities:

Thoracentesis (pleural)
Pericardiocentesis (pericardial)
Paracentesis (peritonial)

Volume: >100 mL

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Specimen Collection and
Handling
EDTA tube (cell counts and differential)
Sterile heparinized or sodium polyanethol sulfonate (SPS) evacuated tubes
(Microbiology and cytology)
Centrifugation: used for concentration of large amount of fluids for better
recovery of microorganism and abnormal cells
Chemistry tests: clotted specimens in plain tubes or in heparin tubes
Specimens for pH: maintained anaerobically in ice

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
General Classification of the
cause of an effusion
Transudate Exudate
Effusions that form because of a Produced by conditions that directly
systematic disorder that disrupts the involve the membranes of the
balance in the regulation of fluid particular cavity, including infections
filtration and reabsorption. and malignancies.

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Laboratory tests to differentiate between
transudates and exudates:
Appearance
Total protein
Lactic dehydrogenase
Cell counts
Spontaneous clotting

Most reliable differentiation: determining the

fluid: blood ratios for protein and lactic
dehydrogenase

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
General Laboratory Procedures
Serous fluid examination
Classification as transudate or exudate
Chemistry
Microbiology
Cytology

All procedures are perfomed in the same manner on all serious fluids.
However, the significance of the test results and the need for specialized test
vary among fluids.

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
General Laboratory Procedures
Test that are usually performed on all serous fluids:
Evaluation of the appearance
Differentiation between a transudate and an exudate

Effusions of exudative origin are then examined: presence microbiologic and

cytologic abnormalities
Additional tests (based on specific clinical symptoms)

RBC and WBC counts are not routinely performed: provide little diagnostic
information

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
General Laboratory Procedures
WBC and RBC Count >1000/uL: Exudate
Performed manually by using a Neubauer counting chamber or by electronic cell
counters
Frequently include a count of all nucleated cells
Inclusion of tissue cells and debris (electronic cell counter)

Differential cell count: routinely performed, Wright’s stained, cytocentrifuged

specimens
Smears mus be examined WBCs, normal and malignant tissue cells
Any suspicious cells seen are referred to the cytology lab or the pathologist

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
Pleural Fluid
Located between the parietal pleural
membrane lining the chest wall and the
visceral pleural membrane covering the lungs

Pleural effusion: either transudative or

exudative

Two additional procedures:

pleural fluid cholesterol and fluid: serum
cholesterol ratio
pleural fluid: serum total bilirubin ratio

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
Pleural Fluid: Appearance

Sudan III staining is strongly positive

with chylous material

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Pleural Fluid: Appearance

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 PF: Hematology Tests
Differential cell count: most diagnostically significant hematology
test performed on serous fluids

Primary cells associated with pleural fluids:

Macrophages (64%-80%)
Neutrophils (1%-2%)
Lymphocytes (18%-30%)
Eosinophils (>10%)
Mesothelial cells
Plasma cells
Malignant cells

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 PF: Hematology Tests
Increased in pleural fluid neutrophils
bacterial infection, such as pneumonia
effusion resulting from pancreatitis and pulmonary infarction

Lymphocytes
normally noticeably present in both transudates and exudates in
variety of forms (small, large, and reactive)
More prominent nucleoli and cleaved nuclei may be present

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 PF: Hematology Tests
Elevated lymphocyte counts (>18%
to 30%)
effusion resulting from
tuberculosis, viral infections,
malignancy and autoimmune
disorders
rheumatoid arthritis
systemic lupus erythematous

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 PF: Hematology Tests
Increased in eosinophil levels (>10%)
trauma resulting in the presence of air or blood (pneumothorax
and hemothorax)
allergic reactions and parasitic infections

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 PF: Hematology Tests
Mesothelial cells
Pleomorphic; resemble lymphocytes,
plasma cells and malignant cells
frequently making identification
difficult
Often appear as single small or large
round cells with abundance blue
cytoplasm (normal mesothelial cells)

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 PF: Hematology Tests
Increased mesothelial cells is not a
diagnostically significant finding, but they
mabe increased in pneumonia and
malignancy

Noticeable lack of mesothelial cells

associated with tuberculosis is of more
significance
result from exudate covering with
pleural membranes

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Characteristics of Malignant Cells
Increased nucleus:cytoplasm (N:C) ratio. The higher the ratio, the more poorly
differentiated are the cells.
Irregularly distributed nuclear chromatin
Variation in size and shape of nuclei
Increased number and size of nucleoli
Hyperchromatic nucleoli
Giant cells and multinucleation
Nuclear molding
Cytoplasmic molding (community borders)
Vacuolated cytoplasm, mucin production
Cellular crowding, phagocytosis

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
Characteristics of Malignant Cells

Special staining techniques and flow cytometry may be used for positive
identification of tumor cells.

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Chemistry Tests
Most common chemical test performed
on pleural fluid:
Glucose (<60 mg/dl)
pH (<7.3)
Adenosine deaminase (ADA) (higher
than 40 U/L)
Amylase (often elevated first)

Triglyceride levels may also be measured

to confirm the presence of a chylous
effusion

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Chemistry Tests
Decreased glucose level: >60mg/dL

Pleural fluid pH lower than 7.2: need for chest-tube drainage, in addition to
administration of antibiotics in cases of pneumonia

Acidosis: pleural fluid pH should be compared with the blood pH

pH at least 0.30 degrees lower than the blood pH is considered significant

pH value as low as 6.0: esophageal rupture that is allowing the influx of gastric
fluid

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Chemistry Tests
ADA levels higher than 40 U/L: tuberculosis
frequently elevated with malignancy

Serum, elevated amylase levels: pancreatitis

amylase is often elevated first in the pleural fluid
elevated salivary amylase: esophageal rupture and malignancy

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Microbiologic Tests
Microorganisms primarily associated with pleural effusions include:
Staphylococcus aureus
Enterobacteriaceae
Anaerobes
Mycobacterium tuberculosis (PCR)

Gram stains, cultures (both aerobic and anerobic), acid fast stains, and
mycobacteria cultures are performed (clinically indicated)

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Serologic Tests
Used to differentiate effusions of immunologic origin from noninflammatory
processes.

Most frequently performed:

Antiunclear antibody (ANA)
Rheumatoid factor (RF)

Detection of tumor markers

Carcinoembryonic antigen (CEA)
CA 125 (metastatic uterine cancer)
CA 15.3 and CA 549 (breast cancer)
CYFRA 21-1 (lung cancer)

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
Manila Adventist College
1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Pericardial Fluid
10 to 50 mL of fluid is found between the pericardial
serous membranes

Primarily the result of changes in the membrane

permeability due to:
infection (pericarditis)
malignancy
trauma-producing exudates

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Pericardial Fluid
Metabolic disorders that are primarily
causes of transudates:
uremia
hypothyroidism
autoimmune disorders

Effusion is suspected when cardiac

compression (tamponade) is noted during
the physician’s examination

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Laboratory Tests
Determine whether fluid is transudate or an exudate

Fluid:serum protein and lactic dehydrogenase (LD) ratios

>100 WBCs/uL with a high percentage of neutrophils: bacterial endocarditis

Cytologic examination of pericardial exudates for the presence of malignant

cells is an important part of the fluid analysis.

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Laboratory Tests
Bacterial cultures and Gram stains are performed on concentrated fluids when
endocarditis is suspected.
Infections are frequently caused by previous respiratory infection including:
Streptococcus, Staphylococcus, adenovirus and coxsackievirus

Effusions of tubercular origin are increasing as a result of AIDS

Acid-fast stains and chemical test for adenosine deaminase are often
requested

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Peritoneal Fluid
Ascites: accumulation of fluid between the peritoneal membrane
Ascitic fluid: accumulated fluid

Heptic disorders (e.g. cirrhosis) are frequently causes of ascitic transudates

Bacterial infections (peritonitis) - often result of intestinal perforation or a

ruptured appendix - and malignancy are the most common causes of
exudative fluids

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Peritoneal Fluid

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Peritoneal Fluid
Normal saline: introduced into peritoneal cavity as a lavage to detect
abdominal injuries that have not yet resulted in fluid accumulation

Peritoneal lavage: sensitive test to detect intra-abdominal bleeding in blunt

trauma cases, and results of the RBC count can be used along with
radiographic procedures to aid in determining the need for surgery.

RBC counts greater than 100,000/mL: blunt trauma injuries.

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Transudates vs. Exudates
The serum-ascites albumin gradient (SAAG)
recommended over the fluid: serum total protein and LD ratioes to detect
transudates of hepatic origin

Fluid and serum albumin levels are measured concurrently, and the fluid albumin
level is then subtracted from the serum albumin level

A difference (gradient) of 1.1 or greater suggest a transudate effusion of hepatic

origin, and lower gradients are associated with exudative effusions

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 Transudates vs. Exudates

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 LT: Chemical Testing
Glucose: bacterial and tubercular peritonitis and malignancy
Amylase: determined on ascitic fluid to ascertain cases of pancreatitis
Gastrointestinal perforations
Alkaline phophatase: intestinal perforation
BUN and creatinine: ruptured or accidental puncture of bladder during the
paracentesis
Bilirubin: leakage of bile
Amylase or Lipase: pancreatitis or damage to the pancrease

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 LT: Microbiology Testing
Gram stains and bacterial culture:
bacterial peritonitis
Inoculation of fluid into blood culture
bottles: increases the recovery of
anaerobic organisms
Acid-fast chains, adenosine deaminase
and cultures: tuberculosis

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
 LT: Serologic Tests
Tumor markers CEA & CA: primary source
of tumors producing ascitic exudates

CA 125 Antigen w/ negative CEA: source is

from the ovaries, fallopian tubes, or
endometrium

Manila Adventist College

1st Semester S.Y. 2023-2024
MLS 108 | AUBF - Lecture
Presented by Calundre, Evangelista, Malic, Ramirez, Vigal
Analysis in Urine and Body Fluids: Part III Chapter XV

Amniotic
Fluid
9th Group
Learning Objectives:
* State the functions of amniotic fluid.
* Describe formation and composition of amniotic fluid.
* Differentiate maternal urine from amniotic fluid.
* State indications for performing an amniocentesis.
* Describe the specimen-handling and processing procedures for testing
amniotic fluid for bilirubin, fetal lung maturity (FLM), and cytogenetic analysis.
* Discuss the principle of spectrophotometric analysis for evaluation of
hemolytic disease of the newborn.
Learning Objectives:
* Interpret a Liley graph.
* Describe the analysis of amniotic fluid for the detection of neural tube
disorders.
* Explain the physiological significance of the lecithinsphingomyelin (L/S) ratio.
* State the relationship of phosphatidyl glycerol to FLM.
* Define lamellar bodies and describe their significance to FLM.
* Discuss the principle of and sources of error for the L/S ratio, Amniostat-FLM,
lamellar body count, and Foam Stability Index for FLM
Physiology
Amnion: a membranous sac that
surrounds the fetus

Amniotic fluid
Provides protective cushion for
fetus
Allows fetal movement
Stabilizes fetal temperature
exposure
Permits proper lung development
Exchanges water and chemicals
among the fluid, fetus, and
maternal circulation
Fetus in Amniotic Sac
Physiology
Volume
Production: fetal urine, lung fluid, and
maternal circulation
During the first trimester, the
approximately 35 mL of amniotic fluid is
derived primarily from the maternal
circulation
Increased amniotic fluid peak at 800 to
1200 mL in the third trimester is the result
of fetal urine
Increased urine is regulated by increased
fetal swallowing
Lung fluid adds lung surfactants to
amniotic fluid; used as a measure of lung
maturity
Physiology
Polyhydramnios
Excess amniotic fluid from failure
of fetus to swallow >1200 mL
Neural tube disorders,
structural/chromosomal
abnormalities, cardiac
arrhythmias, infections
Oligohydramnios
Decreased amniotic fluid from
increased fetal swallowing,
membrane leakage <800 mL
Umbilical cord compression
Chemical Composition
Composition similar to maternal plasma with
sloughed fetal cells
Cells are used for cytogenetic analysis
The fluid also contains biochemical substances
that are produced by the fetus, such as bilirubin,
lipids, enzymes, electrolytes, urea, creatinine, uric
acid, proteins, and hormones
Fetal urine increases creatinine, urea, and uric
acid
Fetal age can be estimated by creatinine
<36 weeks = 1.5 to 2.0 mg/dL
>36 weeks = >2.0
Maternal Urine versus
Amniotic Fluid
Needed to determine premature membrane
rupture or accidental puncture of maternal
bladder from amniocentesis
Measure creatinine, glucose, protein, and urea
Amniotic fluid has <3.5 mg/dL creatinine and
<30 mg/dL urea
Values as high as 10 mg/dL for creatinine and
300 mg/dL for urea may be found in urine
The presence of glucose, protein, or both is
associated more closely with amniotic fluid
Maternal Urine versus
Amniotic Fluid
Fern test: specimen air dries on glass slide;
examine microscopically for “fern-like” amniotic
fluid crystals
Indications for Amniocentesis
Collection of fetal epithelial cells in amniotic fluid indicate the
genetic material of the fetus
Examined for chromosome abnormalities by
Karyotyping
Fluorescence in situ hybridization (FISH)
Fluorescent mapping spectral karyotyping
DNA testing
Biochemical substances that the fetus has produced
Analyzed by thin-layer chromatography
 Specimen Color &
Collection
Handling Appearance
Amniocentesis: needle aspiration
Perform all handling procedures
of fluid from amniotic sac Normal amniotic fluid is
immediately, and deliver to
Transabdominal colorless, with slight to moderate
laboratory promptly
amniocentesis turbidity from cells
Amber tubes to protect bilirubin
Maximum of 30 mL collected in integrity
Blood streaked: traumatic tap,
sterile syringes. Deliver fetal lung maturity (FLM) tests abdominal trauma, intra-amniotic
Discard first 2 to 3 mL for on ice; refrigerate or freeze up to 72 hemorrhage
contamination. hours if needed Fetal versus maternal blood:
Cytogenetic specimens kept at room use Kleihauer-Betke
Protect specimens from light for
temperature or 37°C to prolong cell Bilirubin: bright yellow
bilirubin analysis for HDN at all
life
times: amber tubes or black Meconium (1st bowel movement):
Centrifuge or filter fluid for chemical
plastic tube covers. dark green
tests to remove debris; filter only for
Fetal death: dark red-brown
FLM tests
 Specimen Color &
Collection
Handling Appearance
Amniocentesis: needle aspiration
Perform all handling procedures
of fluid from amniotic sac Normal amniotic fluid is
immediately, and deliver to
Transabdominal colorless, with slight to moderate
laboratory promptly
amniocentesis turbidity from cells
Amber tubes to protect bilirubin
Maximum of 30 mL collected in integrity
Blood streaked: traumatic tap,
sterile syringes. Deliver fetal lung maturity (FLM) tests abdominal trauma, intra-amniotic
Discard first 2 to 3 mL for on ice; refrigerate or freeze up to 72 hemorrhage
contamination. hours if needed Fetal versus maternal blood:
Cytogenetic specimens kept at room use Kleihauer-Betke
Protect specimens from light for
temperature or 37°C to prolong cell Bilirubin: bright yellow
bilirubin analysis for HDN at all
life
times: amber tubes or black Meconium (1st bowel movement):
Centrifuge or filter fluid for chemical
plastic tube covers. dark green
tests to remove debris; filter only for
Fetal death: dark red-brown
FLM tests
Tests for
Fetal Distress
Hemolytic Disease of the Newborn
(HDN)
Most commonly Rh-negative mothers
Other red blood cell (RBC) antigens
can also produce HDN
Fetal cells with antigens enter
maternal circulation and cause
production of maternal antibodies
Maternal antibodies cross the
placenta and destroy fetal cells with
the corresponding antigen
Tests for
Fetal Distress
Bilirubin from RBC destruction appears in the
amniotic fluid
The amount of unconjugated bilirubin present
correlates with the amount of RBC destruction
Spectrophotometric analysis of fluid optical
density (OD) between 365 and 550 nm is
plotted on semilog paper
Bilirubin causes OD rise at its maximum
absorbance level of 450 nm; difference
between baseline and 450 nm peak is the
ΔA450
Difference is plotted on a Liley graph
Tests for
Fetal Distress
Liley graph
Plots ΔA450 against gestational
age
Consists of three zones based on
hemolytic severity
Zone I: mildly affected fetus
Zone II: requires careful monitoring
Zone III: severely affected fetus,
may require induction of labor or
intrauterine exchange transfusion
Tests for
Fetal Distress
Neural tube defects
Alpha-fetoprotein (AFP) produced by
the fetal liver prior to 18 weeks’
gestation
Increased levels in maternal blood or
amniotic fluid indicate possible
anencephaly or spinal bifida
Increased levels are found when skin
fails to close over neural tissue
Measure maternal blood first, then
amniotic fluid
Tests for
Fetal Distress
Normal values based on weeks of
gestation (maximum AFP 12 to 15 weeks)
Report multiples of the median (MoM)
Median is laboratory’s reference level for
a given week of gestation
More than two times the median (MOM)
is abnormal
Follow with fluid amniotic
acetylcholinesterase (AChE): more
specific for neural disorders
Do not perform on a bloody specimen
Fetal Lung
Maturity (FLM)
Most common complication of early delivery is
respiratory distress syndrome (RDS)
Lack of lung surfactant, which keeps the alveoli
open during inhaling and exhaling
Surfactant decreases the surface tension on the
alveoli so they can inflate more easily
Many laboratory tests are available for FLM
Lecithin-Sphingomyelin (L/S) Ratio
Considered the reference method
Lecithin is the primary component of the lung surfactants; increased production
occurs after the 35th week
Sphingomyelin is produced at a constant rate after the 26th week and serves as a
control for the rise in lecithin
L/S ratio is 1.6 prior to week 35 and rises to 2.0 or greater for alveolar stability
after week 35
Therefore, preterm delivery is considered safe with an L/S ratio of 2.0 or higher
Test is performed using thin-layer chromatography
Many laboratories have replaced the L/S ratio with the quantitative phosphatidyl
glycerol immunoassays and lamellar body density procedures
Phosphatidyl Glycerol
Lung surface lipid phosphatidyl glycerol is also needed for
lung maturity
Normally parallels lecithin, except in diabetics, so must be
included in L/S ratio
Amniostat-FLM is an immunologic agglutination test for PG
using antibody specific for phosphatidyl glycerol that can
replace the L/S ratio (no special equipment needed)
Blood and meconium do not interfere with the test
 Foam Stability
Performed at the bedside
Amniotic fluid is mixed with 95% ethanol, shaken for 15 seconds, and
allowed to sit undisturbed for 15 minutes
A continuous line of bubbles around the outside edge indicates the
presence of a sufficient amount of surfactant to maintain alveolar
stability (alcohol is an antifoaming agent, and fluid can overcome this)
 Foam Stability Index
0.5 mL amniotic fluid added to increasing amounts of 95% ethanol
Provides ethanol:fluid ratios of 0.42 mL to 0.55 mL in 0.01 mL
increments, giving semiquantitative measure of surfactant amount
A value >47 indicates FLM
Correlates well with L/S ratio and tests for phosphatidyl glycerol
Lamellar Bodies
Lamellar bodies: storage form of surfactant
Approximately 90% phospholipid and 10%
protein
Secreted by the type II pneumocytes of the fetal
lung to the aveolar space at about 24 weeks of
gestation
Increase in amniotic concentration from 50,000
to 200,000/mL by the end of the third trimester
OD of 150 at 650 nm correlates with L/S ratio
of 2.0 and the presence of PG
Lamellar Body Count (LBC)
Lamellar body diameter ranges in size from 1.7 to 7.3 fL or 1 to 5 µm
LBCs can be obtained using the platelet channel of automated
hematology analyzers
Advantages of LBC
Rapid turnaround time
Low reagent cost
Wide availability
Low degree of technical difficulty
Low volume of amniotic fluid required
Excellent clinical performance
Specimens contaminated with meconium or mucous cannot be used
Protocol for Performing LBC
1. Mix the amniotic fluid sample by inverting the capped sample
container five times.
2. Transfer the fluid to a clear test tube to allow for visual
inspection.
3. Visually inspect the specimen. Fluids containing obvious mucus,
whole blood, or meconium should not be processed for an LBC.
4. Cap the tube and mix the sample by gentle inversion or by
placing the test tube on a tube rocker for 2 minutes.
Protocol for Performing LBC
5. Flush the platelet channel; analyze the instrument’s diluents
buffer until a background count deemed acceptable by the
laboratory is obtained in two consecutive analyses.
6. Process the specimen through the cell counter and record the
platelet channel as the LBC.
Presented by Group 9

Thank you
so much for
listening!
Amniotic Fluid
 Semen
Aquidado, Digma, Limbanganon, Mugisha,
Sigua
Introduction
Advances in the field of andrology
Assisted reproductive technology (ART)
Increased concern over fertility (by couples)

Patients with abnormal results on the routine semen

analysis performed in the clinical laboratory often are
referred to Specialized Andrology for further testing to
determine the need for in vitro fertilization (IVF)

Four Fractions
1. Testes
2. Epididymis
3. Seminal vesicles
4. Prostate gland
5. Bulbourethral glands

Mixing of all four fractions during ejaculation is essential for the production of a normal semen specimen
The testes are paired glands in the scrotum that contain the
seminiferous tubules for the secretion of sperm

The external location of the scrotum contributes to a

lower scrotum temperature that is optimal for sperm
development.

Germ cells
production of spermatozoa
located in the epithelial cells of the seminiferous
tubules

Specialized Sertoli cells

“nurse cell”
provides support and nutrients for the germ cells as
they undergo mitosis and meiosis (spermatogenesis)

Spermatogenesis → Sperm (non motile) will enter the

epididymis → Sperm mature and develop flagella
90 days

The sperm remain stored in the epididymis until ejaculation, at which time they are propelled through the
ductus deferens (vas deferens) to the ejaculatory ducts.
Ejaculatory ducts receive both the sperm from the ductus deferens and fluid from the seminal vesicles.

Seminal Fluid
60% to 70%
transport medium for the sperm
contains a high concentration of fructose and flavin

Spermatozoa metabolize the fructose for the energy needed for the flagella to propel them through the
female reproductive tract.

Fructose - motility of the sperm

Flavin - gray appearance of the sperm

Muscular prostate gland

located just below the bladder
surrounds the upper urethra
aids in propelling the sperm through the urethra by contractions during ejaculation.

Prostate Fluid
20% to 30% (acidic)
milky acidic fluid

Acid phosphatase, citric acid, zinc, and proteolytic enzymes

responsible for both the coagulation and liquefaction of the semen following ejaculation
Bulbourethral glands
5% - form of a thick, alkaline mucus that helps to neutralize acidity from the prostate secretions and
the vagina
located below the prostate

It is important for semen to be alkaline to neutralize the vaginal acidity present as a result of normal
bacterial vaginal flora.

Without this neutralization, sperm motility would be diminished.

Specimen Collection
The laboratory should provide the patient with warm sterile
glass or plastic containers
The specimen is collected in a room provided by the
laboratory if not appropriate the specimen should be kept at
room temperature and delivered to the laboratory within 1
hour of collection
Specimens awaiting analysis should be kept at 37°C.
Specimens should be collected by masturbation.
Only nonlubricant-containing rubber or polyurethane
condoms should be used.
Ordinary condoms are not acceptable because they contain
spermicides

Laboratory personnel must record

patient’s name
birth date
period of sexual abstinence
completeness of the sample
difficulties with collection
the times of specimen collection and specimen receipt
Specimen Collection
Most of the sperm are contained in the first portion of the
ejaculate
making complete collection essential for accurate testing
of both fertility and post vasectomy specimens.

When a part of the first portion of the ejaculate is missing

the sperm count will be decreased
the pH falsely increased
the specimen will not liquefy

When part of the last portion of ejaculate is missing

the semen volume is decreased
the sperm count is falsely increased
the pH is falsely decreased
the specimen will not clot
Specimen Collection
Specimens are collected following a period of sexual
abstinence of at least 2 days to not more than 7 days

Specimens collected following prolonged abstinence

tend to have higher volumes
decreased motility

When performing fertility testing (WHO) recommend 2 or 3

samples be collected not less than 7d or more than 3w
apart with two abnormal samples considered significant
Specimen Handling
and Analysis
All semen specimens are potential reservoirs for HIV and
hepatitis viruses, and standard precautions must be
observed at all times during analysis.
Specimens are discarded as biohazardous waste.
Sterile materials and techniques must be used when
semen culture is to be performed or when the specimen is
to be processed for bioassay, intra-uterine insemination
(IUI), or in vitro fertilization (IVF).

Semen analysis for fertility evaluation

macroscopic and microscopic examination.

Parameters reported include appearance, volume, viscosity,

pH, sperm concentration and count, motility, and
morphology.
 Macroscopic
Examination

Appearance Volume
Normal; Gray-white, translucent ( musty Normal = 2-5 mL
odor) Increased in = Abstinence
Increased white turbidity = Infection Decreased in = Infertility, incomplete
(WBC) collection
Yellow coloration = Urine contamination,
Medications
 Macroscopic
Examination

Viscosity Liquefaction
Normal = Pour in droplets Fresh Semen Specimen = 30-60 mins
Abmormal = 2 cm long threads Failure to liquefaction within 60 mins =
Reporting; Deficiency in Prostatic enzyme
0 = Watery
4= gel like
May also be reported as low, normal or high
pH
Indicates the balance between the pH values
from acidic prostatic secretion and alkaline
seminal vesicle secretion.
within 1 hour of ejaculation, the pH should
be measured, due to the loss of CO2
Sperm Concentration
Valid Measurement for fertility
-the actual number of sperm present in a semen specimen.
Reference Values for Sperm Concentration:
>20 to 250 million sperm per milliliter
Border Line: Between 10 and 20 million per milliliter

more than one semen specimen should be evaluated for infertility studies

Factors that affect the Sperm Concentration

days of sexual abstinence before collection
infection
stress
Sperm Concentration
Total sperm count= Sperm Concentration x specimen volume

-Neubauer counting chamber

the same manner in counting cerebrospinal fluid cell count.
The amount of dilution and the number of squares counted
varies among the laboratory.
1:20 dilution- most commonly used prepared using a
mechanical(positive-displacement)
-Immobilizes the sperm before counting.
Sodium Bicarbonate and formalin
-A traditional diluting fluid that preserves the cells
Saline and Distilled Water
-can achieve good results
 Sperm Count
Neubauer counting chamber
similar to manual RBC count, Four corner and center squares of
the large center square
Allow to settle for 3 to 5 minutes after charging both sides of the
hematocytometer.
Both counts should agree within the 10 % if the counts do not
agree, both the dilution and the counts are repeated.
Phase or Bright-field microscopy
Crystal violet
-additional of stain to the diluting fluids aids in visualization when
using bright-field microscopy.
Sperm Count
Immature sperm and WBCs, often
referred to as “round “ cells must NOT
be included, their presence can be
significant and needs to be identified
separately.

Immature sperm cells(spermatids) and

leukocytes are counted in the same
manner as the mature sperm.

Leukocytes = > 1 million leukocytes > 1 million spermatids per milliliter

per milliliter indicates spermatogenesis caused
associated with inflammation or by viral infections, exposure to
infection of reproductive organs toxic chemicals, and genetic
that may lead to infertility. disorders.
Sperm Concentration and Sperm Count

Sperm Count= # of cells counted x DF

# of squares counted x 0.1

2 WBC squares
# of sperm counted x 1000= sperm in M/mL
5 RBC squares
# of sperm counted x 1,000,000 = sperm in M/mL
Sperm Motility
capable of forward, progressive movement is critical for fertility,
because once presented to the cervix, the sperm must propel
themselves through the cervical mucosa to the uterus, fallopian
tubes, and ovum.

Traditionally, clinical laboratory reporting

subjective evaluation performed by examining an undiluted
specimen and determining the percentage of motile sperm and the
quality of the motility.
Sperm Motility
capable of forward, progressive movement is critical for fertility,
because once presented to the cervix, the sperm must propel
themselves through the cervical mucosa to the uterus, fallopian
tubes, and ovum.

Traditionally, clinical laboratory reporting

subjective evaluation performed by examining an undiluted
specimen and determining the percentage of motile sperm and the
quality of the motility.
 Sperm Motility Assestment
well-mixed, liquefied semen specimen
within 1 hour of specimen collection
Amount of semen on a slide
10 μL under a 22 × 22 mm cover slip
calibrated positive-displacement pipette
allow it to settle for 1 minute
Sperm Motility Assestment
evaluating approximately 20
high-power fields
examine 200 sperm per slide
and count the percentages of
the different motile categories
using a manual cell counter
evaluated by both speed and
direction
Sperm Motility Assestment
Computer-assisted semen analysis (CASA)

provides objective determination of both sperm

velocity and trajectory (direction of motion),sperm
concentration and morphology
primarily in laboratories that specialize in andrology
and perform a high volume of semen analysis.
Sperm Morphology

oval-shaped head approximately 5 μm

long and 3 μm wide and a long

flagellar tail approximately 45 μm long

Sperm Morphology
Acrosomal cap

Critical to ovum penetration

Enzyme-containing located at the tip of
the head
Approximately half of the head
cover approximately two thirds of the
sperm nucleus
Sperm Morphology
Neckpiece

attaches the head to the tail and the

midpiece

Midpiece
7.0 μm long
Thickest part
surrounded by a mitochondrial sheath
-produces the energy required by the tail for motility.
Evaluation of Sperm
Morphology
thinly smeared, stained slide under oil
immersion
Stains
Wright’s, Giemsa, Shorr, or Papanicolaou stain
Air-dried slides
stable for 24 hours
Routine Criteria
identified abnormalities in
head structure include:
-double heads
-giant heads
-amorphous heads
-pinheads
-tapered heads
constricted heads
abnormally long neckpiece
may cause the sperm head
to bend backward and
interfere with motility
Kruger’s strict criteria Normal Values
Routine Criteria
greater than 30%
measuring head, neck, and tail size normal forms

measuring acrosome size Strict Criteria

greater than 14%
presence of vacuoles normal forms

use of a stage micrometer or morphometry

not routinely performed in the clinical laboratory but
is recommended by the WHO.
Calculating Round Cells
used when counting cannot be
performed during the
hemocytometer count and to
verify counts performed by N= number of spermatids or
hemocytometer. neutrophils counter per 100
mature cells
> 1 million WBCs per milliliter S= sperm concentration in
per ejaculate millions per milliliter

inflammatory condition associated with infection and poor sperm

quality and may impair sperm motility and DNA integrity
ADDITIONAL TESTING

For abnormalities discovered in

any of the routine parameters.

Sperm Vitality
Used to differentiate live (unstained) and dead
(stained).

DECREASED sperm vitality when a specimen

has a normal sperm concentration with
markedly decreased motility.

Should be assessed within 1 hour of

ejaculation.

For normal vitality = 50% of more living living

cells, should correspond to previously
evaluated motility
Procedure
Mix specimen with an Eosin-nigrosin stain

Preparation of smear

Counting number of dead cells in 100 sperm

using BRIGHTFIELD or PHASE-CONTRAST
microscope.

NOTE: Living cells are not infiltrated by dye and

remain bluish-white, whereas dead cells are
stained red against a purple background.
Possible Problems
If large group is VITAL
but IMMOBILE =
Defective Flagellum

If IMMOTILE and
NONVIABLE and High
number = Epididymal
Pathology
 SEMINAL FLUID FRUCTOSE

Low sperm concentration - caused by low fructose

levels in semen

Low fructose levels - caused by abnormalities in the

seminal vesicles, bilateral congenital absence of the
vas deferens, obstruction of the ejaculatory duct,
partial retrograde ejaculation, androgen deficiency
 Resorcinol Test
procedure:
1. Prepare 150mg resorcinol in 23mL HCL
diluted in water
2. Mix 1mL of semen with 9mL of reagent
3. Boil
4. Observe for orange-red color

NOTE:
Resorcinol test produces orange color when
fructose is present
Normal quantitative level of fructose is equal to or
greater than 13um per ejaculate
Test within 2 hrs of collection to prevent fructolysis
ANTISPERM ANTIBODIES

Can be present in both males

and females (more in males)
detected in semen, cervical
mucosa or serum
can possibly cause infertility
 NORMAL CONDITIONS OF
ANTISPERM ANTIBODIES
Blood-testes barrier separates sperm from male immune
system. When barrier is disrupted through trauma,
infection or vasectomy reversal (vasovasostomy), the
antigens on the sperm reproduce an immune response
that damages the sperm. Damaged sperm may cause the
production of antibodies in the female partner.
HOW TO DETECT:
IN MALES:
Antibodies are suspected when clumps of sperm are
observed due to sperm agglutination antibodies.
head-to-head, heat-to-tail, tail-to-tail pattern
Graded as: few, moderate or many

IN FEMALES:
Presence of antisperm antibodies results in a normal semen
analysis accompanied by continued fertility.
Demonstrated by mixing the semen with the female cervical
mucosa or serum and observing for agglutination.
 2 FREQUENTLY USED TESTS TO DETECT
THE PRESENCE OF ANTI-BODY-
COATED SPERM
1. Mixed Agglutination Reaction (MAR) Test
- screening procedure used to detect the presence of
immunoglobulin G (IgG) antibodies.
semen sample containing motile sperm is incubated with IgG
AHG and suspension of latex particles or treated RBCs coated
with IgG.
The end results will form clumps
Less than 10% of the motile sperm attached to the particles
are considered “normal”
 2 FREQUENTLY USED TESTS TO DETECT
THE PRESENCE OF ANTI-BODY-
COATED SPERM
2. Immunobead Test
- can be used to detect the presence of IgG, IgM, and IgA
antibodies
shows what part of the sperm is being affected by
autoantibodies
Head-directed antibodies - can interfere with penetration into
cervical mucosa or ovum
Tail-directed antibodies - can affect movement through
cervical mucosa
Microbial and
Chemical Testing
·The presence of more than 1 million
leukocytes per millimeter indicates infection
within the reproductive system, frequently the
prostate.

·Routine aerobic and anaerobic cultures and

tests for Chlamydia trachomatis, Mycoplasma
hominis, and Ureaplasma urealyticum are
most frequently performed.
 Analytes

1. Neutral α –glucosidase
lack of seminal fluid, decreased neutral α - 1. Would cause decreased fructose level
glucosidase
2. Glycerophosphocholine, and L-carnitine 2. suggests a disorder of the epididymis.
3. Decreased zinc, citric acid, glutamyl
transpeptidase, and acid phosphatase 3. Indicates the lack of prostatic fluid
Spectrophotometric methods
used to quantitate citric acid and zinc

Also, laboratory tests may be used to determine the presence of semen in a specimen.
In terms of rape cases, the specimen is enhanced using xylene and is examined under a
phase microscope.

Motile sperm can be detected for Non-motile sperm can persist for 3
up to 24 hours after intercourse days
Cont.
Only the heads remain and may be present and could still be detected for 7 days
after intercourse.
Seminal fluid contains a high concentration of prostatic acid phosphatase, an
enzyme that can be detected and aid in determining the presence of semen in a
specimen.
Prostatic Specific Antigen [PSA], A more specific method is the detection of
seminal glycoprotein p30 for the presence or the absence of sperm.
Further information can often be obtained by performing ABO blood grouping
and DNA analysis on the specimen.
Post vasectomy
Semen Analysis
·A much less involved procedure when compared with infertility analysis
because the only concern is the presence or absence of spermatozoa.

The length of time required for complete sterilization can vary greatly among
patients and depends on both time and number of ejaculations.
Therefore, finding viable sperm in a post-vasectomy patient is not
uncommon, and care should be taken not to overlook even a single sperm.

·Specimens are routinely tested at monthly intervals, beginning at 2nd month

post vasectomy and continuing until two consecutive monthly specimens
show no spermatozoa.
Wet Preparation
Help assess the appearance, motility, and dilution required to optimally
assess the number of spermatozoa. Wet preparations use phase microscopy
for the presence of motile and non-motile sperm.

A negative wet preparation is followed by specimen centrifugation for 10

minutes and examination of the sediment.

Sperm Function Test

·The tests are most commonly performed in specialized andrology
laboratories and include the hamster egg penetration assay, cervical mucus
penetration test, hypo-osmotic swelling test, and the in vitro acrosome
reaction.
Semen Analysis QC
The routine semen analysis has been subjective to very little quality
control.
The analysis resulted from a lack of appropriate control materials and the
subjectivity of the motility and morphology analyses.
Increased interest in fertility testing has promoted the development of
quality control materials and in-depth training programs.
The standardized procedures developed by the WHO (World Health
Organization) have provided a basis for laboratory testing and reporting.
The use of CASA (Computer Assisted Semen Analysis) has aided in
reducing the subjectivity of the analysis.
Laboratories can now participate in proficiency testing programs that
include sperm concentration, vitality, and morphology. Commercial
quality control materials and training aids are available and should be
incorporated into laboratory protocols.