SMDNG
CSF (CEREBROSPINAL FLUID)
• CSF flows through the subarachnoid
space
• 70% - derived by ultrafiltration and
secretion through the choroid plexus
• 30% - ependymal cells of the
ventricles and subarachnoid space
• Arachnoid villi, reabsorbed the CSF
back to the circulation
BLOOD BRAIN BARRIER
2 components
1. Capillary Endothelium - held
together by intracellular tight
junctions
2. Choroid plexus - single layer of
specialised choroidal ependymal
cells connected by tight
junctions
CSF COLLECTION AND HANDLING
SPECIMEN COLLECTION
- Up to 20 mL CSF can be collected
Method of collection = Lumbar puncture
3 CSF TUBES
(between 3rd, 4th or 5th lumbar
vertebrae)
1. Chemistry/Serology freezing
- least affected by blood or
bacteria introduced by spinal
tap
2. Microbiology RT
- usually designated for
microbiology
3. Hematology Ref Temp
-least likely contain cells introduced by
the spinal tap
4.Microbiology/Serology!
- better exclusion of skin
contamination or for additional
tests
Transparency
Normal: Crystal clear
Variations/Turbidity
- usually caused by the following:
1. Cellular Elements:
● Turbidity or cloudiness begins to
appear
● WBC counts - >200 Cells / UL
● RBC counts. - 7400 cells. 1uL
● grossly bloody - 76,000
cells/uL
2. microorganisms like bacteria, fungi
and amoeba
3. contrast media
Specific Gravity: 1.006 - 1.008
pH: - pH7.3. 7.45
Pressure- 90-180 mm. H20 ( 500-200
mmH20)
 SMDNG

Clot and Pellicle formation - Intracranial hemorrhage

(bleeding within the braincase)
- overnight
Normal: no clot due to absence of Oily
fibrinogen web-like clots - TB
meningitis - Radiographic contrast media

Clotted
CSF TOTAL VOLUME
- Protein & clotting factors; meningitis,
Adults 90-50m Froin syndrome, blockage of CSF
140-10 ml circulation

Neonates 10-60 ml
I
CSF APPEARANCE

Crystal clear

- Normal Pellicle- Tuberular meningitis

Hazy/Turbid/Milky/Cloudy TRAUMATIC TAP VS. INTRACRANIAL

⬆️WBCs > 200/uL

HEMORRHAGE
TRAUMATIC INTRACRANIAL
RBCs > 400/uL) TAP HEMORRHAGE
lipids & protein
microorganisms Distribution of Uneven even
blood on 3 1>2>3 1=2=3
Xanthochromic tubes
(Pink/Yellow/Orange)
Clot formation Due to (-) CSF has
Plasma no fibrinogen
- Due to hemoglobin degradation fibrinogen
products
Supernatant Clear xanthochromi
Pink - slight amount of c
oxyhemoglobin (RBC in CSF
Yellow - oxyhemoglobin lyse after 2
hrs)
Orange - heavy hemolysis
Erythrophages absent present

⬆️
Other causes: (Macrophages
w/ Ingested

⬆️
Carotene RBCs)

⬆️
Melanin
Protein (≥ 150 mg/dL) D-dimer negative positive
Rifampin
CSF CELL COUNT
Bloody - Any cell count should be

- ⬆️RBCs (> 6,000/uL) -

performed IMMEDIATELY
WBCs and RBCs begin to lyse
- Traumatic tap (puncture of blood
within 1 hour 40% WBCs
vessel)
disintegrate within 2 hours
 SMDNG

PREDOMINANT CELLS IN CSF

WBC Count
- Routinely performed on CSF ● Predominant = Lymphocytes
Normal values (70%) and Monocytes (30%)
Adults 0-5 WBCS/ uL ● Оссsional =Neutrophils
Neonates 0-30 WBCs/uL
Adults: (70:30 ratio)
WBC Diluting Fluid: Neonates (Inversed ratio) Up to 80%
Acetic acid with methylene blue monocytes

Formula for CSF WBC Count: CYTOCENTRIFUGE

- Fluid is added to conical chamber
- Cells are forced into a monolayer
CSF DILUTION
w/in a 6mm diameter circle on
Clear undiluted the slide

Slightly hazy 1:10

⬆️
Addition of albumin

⬇️
- cell yield / recovery
Hazy 1:20
- cellular distortion
Slightly cloudy 1:100
PLEOCYTOSIS
Cloudy/Slightly bloody 2:00 - Abnormal condition
- Increased no. of normal cells in
Bloody/Turbid 1:10,000
CSF

RBC Count
- Done only in cases of traumatic tap
- To correct for WBC count & total
protein concentration

-1 : WBC for every 700 RBCs seen

-8 mg/dL : Total protein

concentration for every 10,000
BBCs/uL (Henry)

-1 mg/dL : Total protein concentration

for every1 ,200 RBCs/uL (Strasinger)
PREDOMINANT CELLS SEEN IN
CEREBROSPINAL FLUID
CSF Differential Count
- stained smear Lymphocytes & Monocytes
- Specimen should be concentrated
- Normal
before smearing by using the - Microscopic Findings
following methods: - Viral, tubercular & fungal
1. Cytocentrifugation meningitis
2. Centrifugation - Multiple sclerosis
3. Sedimentation
- All stages
4. Filtration
 SMDNG

- Monocytes mixed with CNS carcinoma

lymphocytes
- Seen in clusters with fusing of
Neutrophils cell borders & nuclei

- Bacterial meningitis
- Early cases of viral, tubercular & CSF PROTEIN
fungal meningitis
- Cerebral hemorrhage Normal Values

- Granules less prominent in blood Adults = 15 - 45 mg/dL (<1% or 1/200

- Cells disintegrate rapidly that of serum protein)
Infants= 150 mg/dL
Macrophages Immature = 500 mg/dL

- RBCs in spinal fluid, contrast Increased in

media
- May contain phagocytized RBCs
appearing as empty vacuoles or
ghost cells, hemosiderin
granules and hematoidin crystals
Blast forms
- Acute leukemia
- Lymphoblasts, myeloblasts, or
monoblasts
Lymphoma cells
- Disseminated lymphoma
- Resemble lymphocytes with cleft
nuclei
1. Damage to the BBB (most common)
● meningitis
● Hemorrhage
2. Production of immunoglobulins within
the CNS multiple adlerrs
3. Decreased normal protein clearance
from the fluid
4. Neural tissue degeneration
Decreased in
1. CSF leakage/ trauma
2. Recent puncture
3. Rapid CSF production
4. Water intoxication
Albumin
- Major CSE Protein

Plasma cells Pre-Albumin

- multiple sclerosis, lymphocyte - 2nd Most Prevalent

reactions
Haptoglobin, ceruloplasmin
- Traditional and classic forms
seen - Alpha-globulins

Ependymal, choroidal, & spindle Beta-globulins

shaped cells
- Beta 2 - transferrin “tau protein”
- Diagnostic procedures - Carbohydrate-deficient
transferrin
- Seen in clusters with distinct - Found in CSF but not in serum
nuclei and distinct cell walls
Gamma globulins
Malignant cells
- IgG and some IgA
- Metastatic carcinomas, primary
 SMDNG

IgM, Fibrinogen, Lipids

- NOT found in normal CSF

CSF ELECTROPHORESIS
CSE PROTEIN DETERMINATION
Total Protein
Trichloro acetic Acid / TCA
albumin & globulin
- Preferred method; precipitates
BOTH albumin & globulins
Dye-binding
- For the detection of oligoclonal
bands (in the v-region)
- Indicates immunoglobulin
production
- Done in conjunction w/ serum
electrophoresis to ensure that
banding is due to neurologic
inflamm.
- The presence of 2 or more
oligoclonal bands in CSF but
NOT in serum is valuable for the

➡️
Coomassie Brilliant Blue diagnosis of mutiple sclerosis
-Protein binds to dye Dye turns from
red to blue Other conditions with oligoclonal banding
- ↑ Protein = ↑ Blue color in CSF but not in serum:
1. Neurosyphilis
PROTEIN FRACTIONS
2. encephalitis
CSF /Serum Albumin Index 3. neoplastic disorders
4. Guillain-Barre syndrome
- assess the integrity of the blood
brain barrier Oligoclonal bands in serum but not in
NV: <9 CSF = leukemia, lymphoma, viral
Abnormal : >9 infections

9-14 = slight impairment Oligoclonal bands in serum and CSF

15-30 =moderate impairment = HIV
>30 =severe impairment
100 ＝complete damage to BBB MULTIPLE SCLEROSIS
- Demyelinating disorder
CSF/Serum Albumin Index
= CSF Albumin (mg/dL) Findings:
Serum Albumin (g/dL) (+) Anti-myelin sheath autoantibody
(+) Oligoclonal band in CSF but not in
IgG Index
serum

⬆️
Assess conditions with IgG production (+) Myelin basic protein (MBP)
within the CNS (ex: Multiple sclerosis) IgG index

NV:<.077 (Graffs Textbook: 0.30 to MYELIN BASIC PROTEIN (MBP)

0.70) - Protein component of the
Abnormal : >.077 lipid-protein complex that insulate
the nerve fibers
Indicative of IgG production within the - Presence of MBP in CSF indicates
CNS (Ex. Multiple sclerosis) destruction of myelin sheath
 SMDNG

- Used to monitor the

course of multiple sclerosis
CSF GLUCOSE
Determination:
Done in conjunction with blood glucose
Specimen for blood glucose should be
drawn 2 hours prior to spinal tap (to
allow time for equilibration between
CSF and plasma glucose)
Normal values : 60-70% of blood
glucose (50-80 mg/dL)
CSF GLUTAMINE
Notes:
Product of ammonia &
alpha-ketoglutarate
Indirect test for the presence of
excess ammonia in the CSF
Normal values : 8-18 mg/dl
Increased: Disturbance of
consciousness (coma, Reye's
syndrome)

Increased: Due to increased plasma

glucose CSF ENZYMES

Decreased in: Bacterial tubercular and LACTATE DEHYDROGENASE (LDH)

fungal meningitis
Serum LDH:
Normal in: Viral meningitis ● Normal 2 >1>3>4>5
● Flipped pattern (AMI) 1>2
Example:
Blood glucose = 100 mg/dL CSF LDH
CSF glucose = 65 mg/dL ● Normal LD 1>2>3>4>5
● Neurological abnormalities 2>1
CSF LACTATE ● Bacterial meningitis
S>4>3>2>1
Notes:
Inversely proportional to glucose CREATINE KINASE (CK)

Normal values : 10-22 mg/dL =⬆️ in:

● stroke
Increased: Bacterial meningitis (>35 ● multiple sclerosis degenerative
mg/dL) disorders
tubercular & fungal meningitis. > 25 ● brain tumors
mg/dL ● viral & bacterial meningitis
● epileptic seizure
Decreased in: Bacterial tubercular and
fungal meningitis ASPARTATE AMINOTRANSFERASE
(AST)
Normal in: Viral meningitis

Example:
⬆️:
● intracerebral
Blood glucose = 100 mg/dL
● subarachnoid hemorrhage
CSF glucose = 65 mg/dL
● bacterial meningitis

CSF LDH Isoenzymes
● LD 1 and 2 (Brain tissues)
● LD 2 and 3 (Lymphocytes)
● LD 4 and 5 (Neutrophils)
DIFFERENTIAL DIAGNOSIS OF
MENINGITIS
Bacterial Meningitis
⬆️
Predominant WBC Neutrophils
⬇️
Protein
⬆️
Glucose
Lactate
Other Information (+) Gram stain
(+) Culture
(+) Limulus lysate test
LIMULUS LYSATE TEST
- Detects Gram-negative
endotoxin in body fluids &
surgical instrument
Reagent: Blood of horseshoe crab
Principle: Limulus Polyphemus
- In the presence of endotoxin,
the amoebocytes (WBCs) will
release lysate (protein)
(+) Clumping/Clot formation
SEMINAL FLUID
MALE REPRODUCTIVE ORGANS
AND THEIR ROLE IN SEMEN
PRODUCTION
● Seminiferous tubules of testes -
site of spermatogenesis
● Interstitial cells of Leydig of
testes - produces and secretes
testosterone
● Epididymis - sperm maturation
and storage until ejaculation
● Vas/ductus deferens - propels
sperm to ejaculatory duct
SMDNG
● Seminal vesicles - provides
nutrients (fructose) for sperm
● Prostate gland - provides
enzymes and proteins for
coagulation and liquefaction
● Bulbourethral gland - add
alkaline mucus to neutralize
prostatic acid and vaginal acidity
COMPOSITION
• Sperm cells
- 5% of the semen
- Produced by the SEMINIFEROUS
TUBULES of the Testes
• Secretions from:
● Testes - 5%
● Seminal Vesicles - 60%
● Prostate Glands. - 20 %
● Epididymis, Vas Deferens,
Bulbourethral Glands & Urethral
Glands. - 10 - 15 %
Chemical constituents:
● Acid phosphatases -
distinguishes semen from other
fluids
● Zinc - a decreased amount of this
in seminal fluid has been
associated with disorders of the
prostate gland
● Fructose - major nutrient of
spermatozoa
● Potassium, Citric Acid, Ascorbic
Acid
● Proteolytic enzymes- controls the
liquefaction and coagulation of
seminal fluid
● Spermine and Choline - from the
prostate glands
 SMDNG

● More acidic - seminal vesicle

NORMAL VALUES FOR SEMINAL
obstruction, absence of seminal
FLUID
vesicle or increase prostatic fluid
● Color - Grayish white to pearly
white and translucent MICROSCOPIC
● Volume - 2-5 ml/ejaculation Sperm Motility
● Odor - Performed in undiluted specimen
- Fishy under 20 HPF
- Distinct - Evaluate the speed and direction
- Chlorox-like of motility and grade as follows:
- Musty
● Viscosity - Highly viscous, Pours
Grade Criteria
in droplets
● Liquefaction time - 30mins to 1 hr 4.0 A Rapid, straight-line
● pH - 7.2 to 8.0 motility

NORMAL VALUES FOR SEMINAL 3.0 B Slower Speed,

Some lateral
FLUID
movement
VARIATIONS
2.0 B Slow forward
progression,
Color
noticeable lateral
movement
• Rusty Red to Brown - presence of
red cells (bleeding) 1.00 No forward
• Yellowish - urine contamination, progression
antibiotics, prolonged abstinence,
pyospermia OD No movement
• Turbid - infection, increased wbc
• Clear - infertility MORPHOLOGY

Volume
1. Increased in prolonged abstinence
2. Decreased in infertility

Viscosity
• Rated as:
0=watery
4+=gel like

Liquefaction
● Incomplete liquefaction will
impede sperm motility
● Failure of liquefaction should be
reported
pH
● More basic - possible infection
within the reproductive tract

Head
• Oval shaped head - approx 5um long
and 3um wide (5x3x45)
• Acrosomal cap - should occupy approx.
½ of head
• Critical to ovum penetration
Middle Piece - mitochondria
Tail - approx. 45um long
Stains:
- Papanicolau's - Provides objective
- Giemsa - determination of both
- Wrights
CASA:
- Provides objective determination of
both sperm velocity and trajectory
- Sperm Concentration
SPERM COUNT AND CONCENTRATION
Types of Counting Chambers
1. Makler
2. Neubauer counting chamber
• Dilution: 1:20
Diluting Fluids
- 5% NaHCO3, 1% Formalin
- Cold distilled water or tap water
Clinical Significance
● Azoospermia - complete or total
absence of spermatozoa
● Necroospermia - presence of
sperm cells whether completely
dead or immobile
SMDNG
● Oligospermia - def. in number of
sperm cell
Formula
• Sperm concentration
- Short cut method for using 5 RBC
squares
NUMBER OF SPERM COUNTED × 1
MILLION = SPERMS IN MILLION/mL
- Short cut method for using 2 WBC
squares
NUMBER OF SPERM COUNTED x
100,000 = SPERMS IN MILLION/mL
- Using the Neubauer Counting Chamber
Sperms/ UL=
# of sperms counted x Dilution 1:20
area of squares counted × volume of
square
• Formula of Sperm count
- To convert sperms/uL to
sperms/ml, multiply it by 1,000
Sperm Count
• Sperm Motility - > 50% with grade of
2.0 within 1 hr
• Sperm Concentration - 20-160
million/ml or > 20million/ml
• Sperm Count - >40 million/ejaculate
Other TEST
Fructose Test
- Specimens are screened using
Resorcinol Test
- Normal Fructose level is >13
umol/ejaculate
- Specimens for fructose level
should be tested within 2hrs or
frozen to prevent Fructolysis
Florence Test
- Test for Choline
- (+) brown rhombic crystals
 SMDNG

Barbieros Test
- Test for Spermine
- Reagent: Picric acid & TCA
- (+) YELLOW LEAF LIKE
STRUCTURES

Spinbarkeit test
- Test for tenacity of mucus

Sperm viability test/Blooms

test/Eosin-Nigrosin Stain
- Bluish white = living sperms
- Red = dead sperms
- Normal Value = 75% viable/living sperms
(25% dead sperms)

Two test to detect the presence of

Ab-coated sperm cells
1. MAR (Mixed agglutination reaction)
2. Immunobead test

Sperm Function Test

1. Hamster Egg penetration
2. Cervical mucus penetration
3. Hypo osmotic swelling
4. In vitro Acrosome reaction