ANALYSIS OF URINE AND OTHER BODY FLUIDS

WEEK 11
CEREBROSPINAL FLUID AND SEMINAL FLUID

CEREBROSPINAL FLUID FORMATION AND PHYSIOLOGY

• CSF
o Formed in choroid plexus located in the 2 lateral
ventricles, third ventricle, and fourth ventricle
o 500 mL/day
o Total volume: 90-150 mL (for adults)
o There is constant production and absorption for
homeostasis
• CSF-CHOROID PLEXUS

o Has a cellular and vascular component

a. Cellular- single layer of tall columnar
epithelial cells called ependymal cells CIRCULATION OF CSF
• Ependymal cells have a lot of
mitochondria providing energy for the
active secretion of small molecules from
ependymal cells to the ventricular space

b. Vascular component- fenestrated capillaries;

has pores on the endothelial wall that allow
passage of small molecules like ions, water
from blood/plasma to ventricular space by
hydrostatic pressure (pressure of blood flow
exerted against endothelial cells) • Formation at lateral ventricles of the brain→flow into 3rd
o CSF Components come from plasma particularly, ventricle→4th ventricle→subarachnoid space→circulate to
small molecules brain and spinal cord until it reaches the top of the brain
o MAIN GOAL OF CSF FORMATION: transfer called superior sagittal sinus
metabolites from blood to CSF to supply brain • Subarachnoid space- located in between the arachnoid
and spinal cord tissue and pia mater
 ANALYSIS OF URINE AND OTHER BODY FLUIDS

CSF REABSORPTION

• From the subarachnoid space, CSF returns to the • Lateral decubitus/fetal position OR in minor cases, in
systemic blood circulation a sitting position
• To maintain homeostasis
• Occurs at the top outer surface of the brain that houses
the arachnoid villi—finger-like projections of the arachnoid
membrane which serves as a one-way valve that allow
CSF to return to bloodstream
• If there is enough hydrostatic pressure by the buildup of
CSF→opens arachnoid villi/granulations→allow CSF
reabsorption→CSF returns to systemic circulation
• In hydrocephalus, there is buildup of CSF in ventricles

FUNCTIONS OF THE CSF

1. Mechanical barrier- cushion brain and spinal cord
against trauma—shock absorber
2. Supply nutrients to nervous tissue- to neurons and
glial cells
3. Remove metabolic wastes- via CSF reabsorption
4. Provides support to brain
TRIVIA: An adult brain weights ~1,500 g however due to CSF
the effective weight of the brain is ~50 g thus without CSF, the
brain will become heavy—our brain floats

THE BLOOD BRAIN BARRIER (BBB)

• Plays a role in the composition of the CSF • Locate puncture site- lumbar interspace between L3
• Has 2 components: cellular and vascular and L4 OR between L4 and L5
a. Cellular- made up of astrocytes specifically, the • Do not collect at a higher interspace since the spine of
podocytes/foot processes an adult usually ends at the level of the 1st or the 2nd
b. Vascular- endothelium with tight junctions lumbar vertebrae as to not puncture the spine
o Tight junctions prevent the paracellular • Practice aseptic technique (iodine then local
movement of small molecules anesthetic)
• BBB modifies composition of CSF—BBB is restrictive • NEEDLE: Gauge 20; Length 90 mm; no barrel just
• Substances with high lipid solubility may cross the BBB by needle
simple diffusion (oxygen) due to the lipid bilayer • “Pop” sound heard as it penetrates the dura
• Allows essential metabolites (glucose, vitamins and ions, mater→then advance a little further→clear fluid flows
amino acids) through spinal needle
o Carrier mediated transport • Measure CSF opening pressure using monometer
o Glucose transport protein • Collected in plastic tubes so that cells will not adhere
o Leucine-preferring transport proteins which will lead to decreased cell counts
• CSF is NOT an ultrafiltrate of plasma • BOTTLE 1 contains first stream, BOTTLE 2 contains
o Na, Cl, and Mg: ↑ in CSF than plasma second stream etc.
o K and Total Ca: ↓ in CSF than plasma • 1st tube: chemistry and serology
o Ultrafiltrate of plasma must have the same o Most likely contaminated with microbes,
composition as plasma tissue fluid and blood cells
• 2nd tube: microbiology
SPECIMEN COLLECTION AND HANDLING o May contain some blood cell contaminants
I. LUMBAR TAP/PUNCTURE • 3rd tube: hematology/cell count
• Routine method for CSF collection performed by o Least cellular or debris contamination
physician • 4th tube: back up for microbiology
• LOW VOLUME SPECIMENS: collected in ONE tube
 ANALYSIS OF URINE AND OTHER BODY FLUIDS

o cOrder of testing: Differences Intracranial Traumatic tap

MICROBIOLOGY→HEMATOLOGY→CHEM hemorrhage
ISTRY/SEROLOGY Blood distribution EVEN in all 3 UNEVEN—tube
o SPECIMEN HANDLING: processed within tubes 1 has highest
30 mins to 1 hr. from time of collection blood
o Delay: false ↓ cell counts (lysis of WBCs); concentration
false ↑ lactate levels (anaerobic glycolysis); Clot Formation ABSENT (not PRESENT
problems with recovering viable microbial enough (plasma
organisms fibrinogen to fibrinogen)
• CSF PRESERVATION clot)
o 1st tube: frozen (-15 to -30 Degrees) Xanthochromic COMMON NOT COMMON
o 2nd tube: placed in incubator (37 Degrees)— supernatant
most CSF pathogens are very fastidious Erythrophagocytosis PRESENT ABSENT
o 3rd tube: refrigerated ( to 8 Degrees) (hemosiderin-laden
II. VENTRICULAR PUNCTURE macrophages)
D-dimer test POSITIVE NEGATIVE
III. CISTERNAL PUNCTURE (formation of
fibrin at
EXAMINATION OF CSF hemorrhage site)

I. PHYSICAL EXAMINATION/APPREARANCE OF II. MICROSCOPIC EXAMINATION

CSF • Cell counts: WBC, RBC, total cell counts
Color Colorless • CSF differential counts
Clarity Crystal clear • Must be done immediately
Viscosity Same with water • 40% of granulocytes: lyse after 2 hrs.
Specific gravity 1.006 to 1.008 • Refrigerate samples
pH 7.30 to 7.45 • In SOME cases, clear samples: ↑concentration of RBCs
Pressure 50 to 180 mmHh and WBCs
TOTAL CELL COUNT
Description Possible Causes • Count RBC and WBC
Slightly hazy 200-500 WBC/uL • Clear: may be counted undiluted (no overlap)
Cloudy >500 WBC/uL • Counting area: ALL 9 Squares
Turbid/Milky WBC, RBC (>400/uL), • Turbid: dilution is required
proteins, increased lipid o Diluting fluid: NSS
concentration, o Counting area: 4 WBC squares, RBC square
microorganisms, aspirated RBC COUNT
epidural fats, radiographic
• Done only in traumatic tap
contrast media
• Correction for WBC count or protein is needed
Clotted Froin’s disease (increased
• Computed: RBC count= Total cell count – WBC count
proteins and clotting factors)
WBC COUNT
Bloody Nonpathologic: traumatic tap
Pathologic: intracranial or • Normal CSF WBC Counts
subarachnoid hemorrhage o Adult: 0-5 WBC/uL
Viscous Cryptococcal meningitis o Children: values are higher than adults
Oily Radiographic contrast media o Neonates: 0-30 mononuclear cells/uL
Pellicle formation Tubercular meningitis (web- • Routinely performed
like pellicle after overnight • Diluting fluid: 3% glacial acetic acid to lyse RBCs
refrigeration of CSF) Clarity Dilution
Xanthochromia (yellow Presence of RBC Slightly Hazy 1:10
discoloration of CSF but also degradation product Hazy 1:20
includes other colors) Pink: very slight amount of Slightly Cloudy 1:100
oxyhemoglobin Slightly bloody/Cloudy 1:200
Orange: heavy hemolysis Bloody or Turbid 1:10,000
Yellow: conversion of CELL COUNT: METHOD FOR LOW WHITE CELL COUNT:
oxyhemoglobin to CLEAR CSF SAMPLE
unconjugated bilirubin • Fill in WBC pipette with undiluted CSF
• Charge and count number of cells in all 9 large squares
*elevated serum bilirubin • Total number of cells counted x VCF x DF
levels • VCF= 1/ (no. of squares counted x volume of 1 square)
*elevated carotene levels • DF= (total volume – 1)/ initial volume
(dietary hypercarotenemia)
• CSF cell count/uL
*protein concentration >150
CELL COUNT: METHOD FOR MODERATE WHITE CELL
mg/dL
COUNT: CSF APPEARS HAZY OR SLIGHTLY TURBID
*normal neonate (immaturity
of BBB) • Fill in WBC pipette with undiluted CSF up to 1st mark
Brown Methemoglobin, melanin • Dilute specimen up to 11th mark
(meningeal melanosarcoma) • Discard, charge and count on 4 WBC squares sand
central square
 ANALYSIS OF URINE AND OTHER BODY FLUIDS

CELL COUNT: METHOD FOR HIGH WHITE CELL COUNT: Prealbumin (Transthyretin)- Fibrinogen
CSF IS PURULENT OR VERY TURBID 2nd most prevalent
• Fill in WBC pipette up to 0.5 mark Haptoglobins and b-lipoproteins
• Dilute to 11th mark Ceruloplasmin- a-globulins
• Discard, charge, count on 4 corner large squares (WBC Transferrin- major b-globulin
squares) “TAU” protein- ONLY in CSF
CORRECTIONS FOR CONTAMINATION IgG and small amounts of
• Correct for introduction of WBCs or proteins because of IgA
traumatic tap
• WBC added= [WBC (blood) x RBC (CSF)] / RBC (blood) ELEVATED IN DECREASED IN
• Corrected CSF WBC count= actual count – added WBCs Meningitis and hemorrhage Leakage of fluid from CNS
CSF DIFFERENTIAL COUNT (most common causes) (otorrhea/rhinorrhea)
Damage to BBB Recent puncture
• Performed on stained smear
Production of Increased reabsorption due
• Specimen should be concentrated before smearing:
immunoglobulins within the to increased cranial
sedimentation, filtration, centrifugation, cytocentrifugation
CNS (multiple sclerosis) pressure
• Add 1 drop of 30% albumin to 0.1 mL of CSF→place in
Decreased clearance of Water intoxication (dilute
cytocentrifuge
normal protein from CSF CSF proteins)
• 30% albumin- increase yield of WBCs and decreases cell
Degeneration of neural
distortion
tissue
NORMAL CSF DIFFERENTIAL COUNT
Endocrine/metabolic
Age Lymphocytes Monocytes Neutrophils
disorders
Neonates 5-35% 50-90% 0-8%
(0-2 months)
CORRECTION FOR CONTAMINATION FROM TRAUMATIC
Children ( 2 NOT YET ESTABLISHED TAP
mo. – 18 yr)
• If blood hematocrit and serum protein values are
Adults (>18 40-80% 15-45% 0-6%
yr.) NORMAL: SUBTRACT 1mg/dL protein for every 1,200
RBCs
• Pleocytosis- any increase in cellularity
• TOTAL PROTEIN ADDED=
Pleocytosis Description 𝑡𝑜𝑡𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 (𝑠𝑒𝑟𝑢𝑚) 𝑥 (1 − 𝐻𝑐𝑡) 𝑥 𝑅𝐵𝐶 (𝐶𝑆𝐹)
Neutrophilic Bacterial meningitis; 90% neutrophils
Early viral, fungal, tubercular, and 𝑅𝐵𝐶 (𝑏𝑙𝑜𝑜𝑑)
parasitic meningitis MEASUREMENT OF TOTAL CSF PROTEIN
Noninfectious causes: subarachnoid or
intracerebral hemorrhage TURBIDIMETRIC METHOD
Lymphocytic Viral meningitis • Addition of precipitating agents that will bind to CSF
Tubercular, fungal, syphilitic meningitis protein which will increase turbidity that will be read by the
Later stages: lymphocytes spectrophotometer
predominates Sulfosalicyclic Acid (SSA) Albumin produce more
Plasma cell Acute viral and chronic inflammatory turbidity than globulin
conditions
Multiple sclerosis Add sodium sulfate so that
Eosinophlic 10% or greater are associated with globulin can be equally
parasitic and fungal infections measure
(Coccidiodes immitis) infections Trichloroacetic Acid (TCA) Reagent of choice
Allergic reaction to malfunctioning
intracranial shunts Precipitates both albumin
Idiopathic eosinophilic meningitis and globulins
Macrophage Subarachnoid or intracerebral
hemorrhage (erythrophagocytosis) DYE-BINDING TECHNIQUES
Nonpathologically (most frequent) diagnostic procedures • Coomasie Brilliant Blue G250
significant cells like pneumoencephalography and in o Protein binds to dye: color change from red to
fluid from ventricular taps or during blue
neurosurgery o Concentration of protein is directly proportional to
Malignant cells Hematologic origin: lymphoblasts, the intensity of blue color
myeloblasts, and monoblasts; acute • Ponceau S
leukemias; lymphoma PROTEIN FRACTIONS
Nonhematologic origin: astrocytomas, • Albumin
retinoblastomas, medulloblastomas • IgG
1. CSF/SERUM ALBUMIN INDEX
𝑚𝑔
𝐶𝑆𝐹 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 ( )
III. CHEMICAL EXAMINATION 𝑑𝐿
𝑔
• CSF Protein 𝑠𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 ( )
𝑑𝐿
o Most frequently performed <9 Normal (intact BBB)
o NORMAL VALUE: 15-45 mg/dL 9-14 Minimal impairment of the BBB
Proteins normally found in Proteins NOT normally 15-100 Moderate to severe impairment
CSF found in CSF >100 Complete breakdown of BBB
Albumin- major CSF protein IgM
 ANALYSIS OF URINE AND OTHER BODY FLUIDS

2. IgG INDEX be converted to urea→ammonia which is lipid-

𝑚𝑔 𝑔 soluble will build up in blood
𝐶𝑆𝐹 𝑖𝑔𝐺 / 𝑠𝑒𝑟𝑢𝑚 𝐼𝑔𝐺 ( )
𝑑𝐿 𝑑𝐿 circulation→ammonia traverses
𝑚𝑔 𝑔
𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 ( )/ 𝑠𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 ( ) BBB→ENCEPHALOPATHY
𝑑𝐿 𝑑𝐿
• REFERENCE INTERVAL: 0.3-0.70 • Reye’s syndrome common in children taking
• >0.70: indicate IgG production within CNS aspirin
• <0.3: compromised BBB 7. LD ISOENZYMES
SAMPLE PRACTICE: • Lactate dehydrogenase enzymes
➢ Serum albumin: 4.2 g/dL; Serum IgG: 12 g/dL; CSF • Differentiate types of meningitis
albuin: 35 mg/dL; CSF IgG: 150 mg/dL INCREASED LD CONDITION
➢ CSF/SERUM ALBUMIN INDEX= ISOENZYMES
8.33→NORMAL/INTACT BBB LD1 and LD2 Brain tissue destruction
➢ IgG INDEX= 1.50= IgG PRODUCTION WITHIN CNS LD2 and LD3 Viral meningitis
3. ELECTROPHORESIS LD4 and LD5 Bacterial meningitis
• Method of choice when it is necessary to
determine whether a fluid id CSG by “Tau” IV. MICROBIOLOGIC EXAMINATION
identification
• Detection of oligoclonal bands for clinical
impression of multiple sclerosis
• “Regarding laboratory findings, MS is
characterized by the presence of oligoclonal IgG
bands (OCB) ONLY in CSF and NOT in
corresponding serum, reflecting an intrathecal
synthesis of IgG sustained by few clones of
antibody-secreting B cells sequestrated into
the CNS
• OCB in CSF and not in serum→ MULTIPLE
SCLEROSIS Gram positive cocci in pairs and in chains (S. pneumoniae)
4. CSF GLUCOSE
• NORMAL VALUE: 60-70% plasma glucose (of
patient)
• Differentiate types of meningitis
Type of meningitis Glucose Others
concentration
Bacterial ↓ce ↑WBC
(neutrophils)
Tubercular ↓ ↑WBC
(lymphocytes and
monocytes)
Viral NORMAL ↑WBC
S. pneumoniae in culture
(lymphocytes)
• Uses the 2nd tube of CSF
Fungal Normal to ↓ (+) India Ink
(stains capsule of • Commonly performed are Gram’s stain and culture
C. neoformans) • S. pneumoniae- common cause of bacterial meningitis
5. CSF LACTATE exp. In adult population
• Increased when there is increase in anaerobic
SEMINAL FLUID PHYSIOLOGY
glycolysis when there is bacterial infection of
meninges CELLULAR COMPONENT FLUID COMPONENT
NORMAL VALUE 10-22 mg/dL Sperm cells (5%) SEMINAL PLASMA:
i. Seminal vesicle:
VIRAL MENINGITIS <25 mg/dL
60-70%
TUBERCULAR AND >25 mg/dL
ii. Prostate gland: 20-
FUNGAL MENINGITIS
30%
BACTERIAL MENINGITIS >35 mg/dL iii. Bulbourethral
6. CSF GLUTAMINE gland: 5%
• NORMAL VALUE: 8-18 mg/dL
• SEMINAL VESICLE SECRETIONS: Flavin, Fructose,
• Indirect measure of CSF ammonia which may Fibrinogen like protein and Semenogelin I and II,
lead to encephalopathy Prostaglandins, K, HCO3, Mg, Prolactin,
• Produced by brain cells from ammonia and Phosphorylcholine, ergothionine, ascorbic acid
alpha-ketoglutarate • Seminal fluid has a yellow tinge color due to FLAVIN (from
• As CSF ammonia increases, alpha-ketoglutarate seminal vesicle)
decreases→COMA • PROSTATE GLAND SECRETIONS: Acid phosphatase,
• Elevated in liver disorder that result in increased Fibrinogenase, Fibrinolysin and Prostate Specific Antigen,
blood ammonia and CSF ammonia AND Reye’s Zinc, Citric Acid, Spermine
syndrome (75% of children with this disease
have increased ---)
• Liver is the organ responsible for detoxification of
metabolic waste products thus, ammonia cannot
 ANALYSIS OF URINE AND OTHER BODY FLUIDS

o Limit exposure of semen to fluctuations in

temperature
o Control the time between collection and analysis
• COLLECTION AT HOME
o Inability to produce a sample by masturbation in
the clinic
o Lack of adequate facilities near the laboratory
o Exact time of collection should be noted
o Delivery/transport of sample to the lab: WITHIN 1
HOUR and should be kept between 20-37
Degrees
• INFORMATION NEEDED ON REPORT FORM
o Patient’s complete name
o Birthdate
Spermine Crystals o Personal code number
• Spermine crystals determine whether fluid is semen or not o Period of abstinence
• BULBOURETHRAL GLAND SECRETIONS (“Cowper’s o Date and time of collection
Gland secretions): Mucus, Alkaline fluid o Completeness of sample
o Any difficulties in producing the sample
o Interval between collection and start of semen
analysis
METHODS OF COLLECTION
1. MASTURBATION
• Preferred method: complete and uncontaminated semen
sample
• CONSIDERATIONS:
o Glans penis: cleaned with wet towel (avoid use of
soap)
o Oil lubricants should be AVOIDED
o Collect entire ejaculate into receptacle/container
• If done at HOME:
o Delivery/transport of sample to the lab: WITHIN 1
HOUR
o Sample should be kept at 20-37 Degrees
SPECIMEN COLLECTION
• PREPARATIONS 2. COLLECTION OF SEMEN BY CONDOM
o Collected after 2-7 days of sexual abstinence • Inability to produce sample by masturbation
o Clear oral and written instructions: EMPHASIZE • Only special non-toxic condoms should be used
semen sample needs to be complete and that o Ordinary latex: agents interfere with sperm
ANY LOSS should be reported motility and spermicidal
o CONTAINER: clean and sterile, wide mouth, kept • Record time of collection
at room temperature or body temperature • If done at HOME:
• ONLY COMPLETE COLLECTIONS ARE ACCEPTABLE o Delivery/transport of sample to the lab: WITHIN 1
First/Initial Ejaculate Last portion of Ejaculate HOUR
NOT COLLECTED NOT COLLECTED o Sample should be kept at 20-37 Degrees
Sperm count is falsely Semen volume will be 3. COITUS INTERRUPTUS
decreased decreased • Not reliable
pH is falsely increased Sperm count falsely • DISADVANTAGES:
(more basic) increased o First portion of ejaculate may be lost (contains
Coagulum will fail to liquefy pH falsely decreased highest number of spermatozoa)
(prostatic secretions are o Cellular and biological contamination
concentrated) o Low pH of vaginal fluid affects the sperm motility
Happens because the Coagulum will not form UNIVERSAL PRECAUTIONS
initial portion contains the • SEMEN samples are BIOHAZARD—may contain HIV,
majority of the sperm hepatitis virus, and Herpes Simplex Virus
concentration, prostatic SEMEN ANALYSIS
secretions (responsible 1. PHYSICAL PROPERTIES
for acidity of semen), and • Appearance
responsible of liquefaction NORMAL SEMEN Gray-white, light yellow,
of semen opaque
Last portion contains the OTHER APPEARANCES
majority of seminal vesicle Brown/reddish hue Hemospermia/hematospermia
secretions (responsible Yellow Jaundice, urine
for coagulum formation) contamination, prolonged
abstinence, certain
• COLLECTED in a PRIVATE ROOM NEAR THE medications
LABORATORY
 ANALYSIS OF URINE AND OTHER BODY FLUIDS

Clear Sperm concentration is very

low
Increased white turbidity ↑ WBCs

• Liquefaction Rate/Time
o Complete liquefaction: WITHIN 30 MINS.
o Rarely: up to 60 MINS
o ABNORMAL: > 60 MINS.
o Normal liquified semen samples may contain
jelly-like granules gelatinous bodies) which do not
liquefy
o Assessment: Macroscopically (homogenous and
Eye reticle
watery) or Microscopically (1 drop on
slide→sperm cells are freely moving) • Eye reticle- assess easier for sperm motility
• Viscosity • Apply petroleum jelly on all sides of the cover slip to avoid
o Performed after complete liquefaction of sample drying of semen sample
o Using a PASTEUR PIPETTE • To avoid interferences of dried samples, assess sperm
▪ Normal: small discrete drops motility 5mm away from all edges of the coverslip
▪ Abnormal: string/thread formation >2cm
→ High mucus content
→ Production of anti-sperm
antibodies
→ Oligoasthenospermia
(↓concentration and motility of
sperm)
• Volume
o NORMAL: 2-5 mL (some reference: 1.5- 5 mL)
o Best measured by weighing the sample in the
vessel
o Measuring by aspiration using pipette or syringe
or by decantation IS NOT RECOMMENDED due
to residues that may not be measured
o Collect the sample in a pre-weighed
container→weight vessel with semen→subtract
weight of container→calculate volume from
sample weight (DENSITY OF SEMEN: 1g/mL)
• pH
• NORMAL: 7.2-7.8
• Assessed within 1 HOUR
• pH paper in the range of 6-10 SHOULD BE USED
o mix semen sample • EXAMPLE 1: Sperm motility estimates replicate counts of
o spread drop of semen evenly onto paper 200 spermatozoa are: progressive, 30% and 50%; non-
o wait for color change to be uniform (appropriate progressive, 5% and 15%; immotile, 65% and 35%
read time) o The most common category: IM (Immotile)
o compare color to comparator o With an average of: 50% (65+35/2)
2. SPERM MOTILITY o And a difference of: 30% (65-35)
o Basing from EXAMPLE 1, assessment is
• Mix semen→remove aliquot (label as R1) immediately
INVALID→CAN NOT RELEASE RESULTS
after mixing→remix semen sample and remove another
aliquot (label as R2)→for each replicate, prepare a wet • EXAMPLE 2: Sperm motility estimates replicate counts of
preparation 200 spermatozoa are: progressive, 37% and 28%; non-
progressive, 3% and 6%; immotile, 60% and 66%
• Consistent amount should be used covered with coverslip
o The most common category: IM (Immotile)
• ASSESS ~200 spermatozoa PER REPLICATE (AT
o With an average of: 63% (60+66/2)
LEAST 200 but can go beyond 200)
o And a difference of: 6% (66-60)
• 2 replicates to validate results
o Basing from EXAMPLE 2, assessment is
• After preparation of 2 replicates, let is settle for 1 MIN VALID→ RELEASE RESULTS
before assessing in the microscope o PROGRESSIVE: 32.5% (37+28/2)
• GRADE o TOTAL MOTILITY: 37% [(37+28/2) + (3+6/2)]
Progressive Sperm moves in a o Normal PR but decreased Total motility
linear/straight fashion OR in • SPERM MOTILITY REPORTING
large circles (whether fast or • Total Motility: PR (progressive) + NP (nonprogressive)
slow in movement)
• NORMAL VALUES
Non-progressive There is just a flagellar beat,
Progressive Motility At least 32%
there is flagellar and
Total motility At least 40%
movement of the head but
stuck on its place, OR
moves in small circles
Immotile NO movement
 ANALYSIS OF URINE AND OTHER BODY FLUIDS

Step 2: Check the acceptability for the most common category

SAMPLE PROBLEMS o Most common category: IM
A. Sperm motility estimates in replicate counts of 200 o With an average of: 63%
spermatozoa are: o And a difference of: 6% (66-60)
Replicate 1 Replicate 2 Step 3: Check for its acceptability using the acceptability table
PR 65 94
NP 13 31
IM 122 75
TOTAL 200 200
Step 1: Calculate the average percentage per category (to
check for the most common category)
Replicate 1 Replicate 2 Average
PR 32.5% 47% 39.75%
(65/200) *100 (94/200) *100 (32.5+47/2)
NP 6.5% 15.5% 11%
(13/200) *100 (31/200) *100 (6.5+15.5/2)
IM 61% 37.5% 49.25%
(122/200) *100 (75/200) *100 (61+37.5/2)
TOTAL 100% 100% 100%
Step 2: Check the acceptability for the most common category
o Most common category: IM
o With an average of: 49.25%
o And a difference of: 23.5% (61-37.5)
Step 3: Check for its acceptability using the acceptability table

o For an average on 63%, a difference of up to 10%

would be expected to occur by chance alone
o Conclusion: VALID→PROCEED TO REPORT
RESULTS
Step 4: Report Results
o Progressive: 32.5%
o Total Motility (PR+NP): 37% (32.5+4.5)
o Progressive motility is within the normal limit but the
total motility is less than the normal limit

3. SPERM COUNT
• PROCEDURE: fill WBC pipette up to 0.5 mark with
liquefied and well mixed seminal fluid sample→dilute
specimen with chilled distilled water up to the 11th
mark→shake the preparation and discard the first 3-4
drops→charge BOTH sides of the counting chamber→let
cells settle for at least 4 mins in a wet house
preparation→focus under LPO then HPO→locate the
central grid
o For an average on 49.25%, a difference of up to 10% • Chilled distilled water is used to immobilize the sperm for
would be expected to occur by chance alone accurate counts
o Conclusion: INVALID→DO NOT REPORT RESULTS

B. Sperm motility estimates in replicate counts of 200

spermatozoa are:
Replicate 1 Replicate 2
PR 74 56
NP 6 12
IM 120 132
TOTAL 200 200
Step 1: Calculate the average percentage per category (to
check for the most common category)
Replicate 1 Replicate 2 Average • 1, 3, 7 and 9 are the WBC squares
PR 37% 28% 32.5% • 5 is the RBC square (CENTRAL GRID)- 5x5
(74/200) *100 (56/200) *100 (37+28/2) • Sperm counts are done systematically row per row
NP 3% 6% 4.5% • Count ONLY WHOLE spermatozoa (with head and tail)
(6/200) *100 (12/200) *100 (3+6/2) • The MIDDLE of the 3 lines defines the square’s boundary
IM 60% 66% 63% • ALL cells within the boundary line are counted
(120/200) *100 (132/200) *100 (60+66/2) • Sperm head that touches the middle:
TOTAL 100% 100% 100%
 ANALYSIS OF URINE AND OTHER BODY FLUIDS

o Cells are counted when most of the head lies onn • With 1:20 Dilution
the LOWER or LEFT MIDDLE line— “L” Rule Dilution Factor=
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒−1
o Cells are NOT counted if it lies on the UPPER 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
11−1
or RIGHT BOUNDARY LINE = = 20
0.5
• Cells are NOT counted if it lines between the 2 outer lines
(does not fall under the L RULE) o Replicate 1- 232 in 3 rows
• COUNT at least 200 spermatozoa in EACH replicate o Replicate 2- 235 in 3 rows
o SUM: 467 (232+235)
o DIFFERENCE: 3 (235-232)
o ACCEPTABILITY: VALID→PROCEED TO
COMPUTATION AND RELEASE OF RESULTS
SUM ACCEPTABLE DIFFERENCE
144-156 24
157-169 25
170-182 26
183-196 27
197-211 28
212-226 29
227-242 30
243-258 31
259-274 32
275-292 33
Middle line boundary
293-309 34
310-328 35
329-346 36
347-366 37
367-385 38
386-406 39
407-426 40
427-448 41
449-470 42
471-492 43
493-515 44
516-538 45
539-562 46
563-587 47

• CONCENTRATION OF SPERMATOZOA IN SEMEN:

𝑵 𝟏
𝑪 = ( ) 𝒙 ( ) 𝒙 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝒇𝒂𝒄𝒕𝒐𝒓
𝒏 𝟐𝟎
o C= concentration of spermatozoa in semen
L Rule-red arrows are sperms (arrows are heads) o N= total number of sperm cells counted
• EXAMPLE 1 o n= total number of rows
Replicate 1
NOTE: No
15 17 14 17 16 =79 • Basing from EXAMPLE 1:
need to count
15 15 16 15 14 =75 4th and 5th 467 1
15 18 16 17 12 =78 232 𝐶=( ) 𝑥 ( ) 𝑥 20
chamber 6 20
19 17 15 19 19 since at least 𝑠𝑝𝑒𝑟𝑚𝑎𝑡𝑜𝑧𝑜𝑎
𝐶 = 77.83
14 15 17 18 9 200 𝑛𝐿
spermatozoa 𝐶 = 77.83 𝑥 106 /𝑚𝐿
Replicate 2 count was **NOTE: 1 nL= 1𝑥 106mL
19 16 18 11 14 =78 reached on
14 17 15 18 15 =79 the 3rd row
15 15 15 16 17 =78 235 • SPERM/ EJACULATE
10 18 14 9 10 𝑺𝒑𝒆𝒓𝒎 𝒔𝒑𝒆𝒓𝒎 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏
= 𝒙 𝒔𝒆𝒎𝒆𝒏 𝒗𝒐𝒍𝒖𝒎𝒆
9 13 12 9 8 𝒆𝒋𝒂𝒄𝒖𝒍𝒂𝒕𝒆 𝒎𝑳

o FOR EXAMPLE: If you have finished your 200

sperm count on the 3rd box of the 3rd row, we are • Basing from EXAMPLE 1:
not allowed to stop at the middle of the 𝑠𝑝𝑒𝑟𝑚 77.83𝑥106
row→FINISH counting the WHOLE row = 𝑥 2.5 𝑚𝐿
𝑒𝑗𝑎𝑐𝑢𝑙𝑎𝑡𝑒 𝑚𝐿
o If you had counted up until the 3rd row of the 𝑠𝑝𝑒𝑟
= 194.58𝑥106
first counting chamber, you MUST count 3 𝑒𝑗𝑎𝑐𝑢𝑙𝑎𝑡𝑒
rows as well in the 2nd counting chamber • NORMAL VALUES
EVEN IF IT DOES NOT REACH 200 Sperm concentration 15 𝑥 106 spermatozoa per mL
spermatozoa—NUMBER OF ROWS MUST BE Total sperm number per 39𝑥 106 spermatozoa per
THE SAME ejaculate ejaculate
 ANALYSIS OF URINE AND OTHER BODY FLUIDS

• EXAMPLE 2
Replicate 1 NOTE: No need
13 20 21 19 12 =85 to count 4th and
25 12 16 18 16 =87 5th chamber since
at least 200
15 21 11 9 8 =64 236 spermatozoa
21 18 19 14 9 count was
21 16 18 18 23 reached on the 3rd
row. No need to
reach 200 on
Replicate 2 the 2nd chamber
13 10 14 12 16 =65 since the
10- 9 15 12 13 =59 counted
16 16 11 13 14 =70 194 number of rows
10 18 14 9 10 MUST BE THE
SAME
9 13 12 9 8
• After drying, dip into fixative solution→dip in EOSIN (first
• With 1:20 Dilution stain)→blot→dip in METHYLENE BLUE (second
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒−1
Dilution Factor= stain)→wash→let dry→LPO then HPO then OIO
𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
11−1
= = 20
0.5

o Replicate 1- 236 in 3 rows

o Replicate 2- 194 in 3 rows
o SUM: 430 (236+194)
o DIFFERENCE: 42 (236-194)
o ACCEPTABILITY: INVALID→ DO NOT
PROCEED TO COMPUTATION AND RELEASE
OF RESULTS
SUM ACCEPTABLE DIFFERENCE
144-156 24
157-169 25
170-182 26
183-196 27
197-211 28
212-226 29
227-242 30
243-258 31
259-274 32
275-292 33
293-309 34
310-328 35
329-346 36 • After staining, count spermatozoa and categorize it
347-366 37 whether normal or abnormal (and type abnormality/defect)
367-385 38 • COUNT AT LEAST 200
386-406 39 • REPORT in terms of PERCENTAGES
407-426 40
427-448 41
449-470 42
471-492 43
493-515 44
516-538 45
539-562 46
563-587 47

4. SPERM MORPHOLOGY

• Make smear like doing PBS→let dry