CEREBROSPINAL FLUID

o Considered as the third major fluid in the body

o Not an ultrafiltrate of plasma
o VOLUME
 20 mL of CSF is produced every hour
 Adults: 90-150 mL
 Neonates: 10-60 mL
o ORDER OF COLLECTION
 1st tube: Chemistry and serology – frozen
 2nd tube: Microbiology – room temperature
 3rd tube: hematology/ cell count – refrigerated
 4th tube: May be drawn for microbiology to provide better exclusion of skin contamination

TRAUMATIC COLLECTION DIFFERENTIATION

DIFFERENCES INTRACRANIAL HEMORRHAGE TRAUMATIC TAP
Distribution of blood Even in all 3 tubes UNEVEN
Clot formation ABSENT PRESENT
Xanthochromic supernatant COMMON NOT COMMON
Erythrophagocytosis PRESENT ABSENT
D-dimer test POSITIVE NEGATIVE
 CSF PRROTEIN
o Most frequently performed chemical test on CSF
o PROTEINS NORMALLY FOUND IN CSF
 ALBUMIN – major CSF protein
 PREALBUMIN – 2nd most prevalent
 HAPTOGLOBIN AND CERULOPLASMIN
 TRANSFERRIN – major beta-globulin
 “TAU” protein – separate carbohydrate deficient transferring fraction seen only in CSF
 IgG and small amounts of IgA
 MYELIN BASIC PROTEIN
 Presence indicates recent destruction of myelin sheath that protects the axons of
neurons
 Used to monitor course of multiple sclerosis
 Detected by radioimmunodiffusion
 QUALITATIVE TEST

TEST REAGENT POSITIVE RESULT

None-Apelt Ammonium sulfate Cloudy precipitate
Rose Jones Ammonium sulfate White ring
Pandy’s Phenol Bluish white cloud
Nogochi 10% butyric acid Precipitate
Colloidal Gold test Colloidal gold solution Blue = 1+
Purple = 2+
Deep blue = 3+
Pale blue = 4+
Colorless = 5+
  QUANTITATIVE TEST
o TURBIDIMETRIC
 Precipitation of protein using:
1. TRICHLOROACETIC ACID – reagent of choice; precipitates both albumin and globulins
2. SULFOSALICYLIC ACID – precipitates albumin only unless combined with sodium sulfate
o DYE BINDING
 “Protein error of indicator”
 Coomassie brilliant blue
 Ponceau S
o NEPHELOMETRY
 Uses benzalkonium chloride
o ELECTROPHORESIS
 Most frequently performed
 Method of choice when it is necessary to determine if the fluid is CSF
 CSF GLUCOSE
o Blood should be drawn 2 hours prior to spinal tap
o Specimens should be tested immediately because glycolysis occur rapidly in CSF

 LACTATE
o Aids in diagnosis and management of meningitis cases
o Sensitive method for evaluating effectiveness of antibiotic therapy
o Used to monitor severe head injuries
o GLUTAMINE
o Chemical test frequently performed in CSF BUT NOT IN BLOOD
o Used to indirect measure of CSF ammonia

TYPE LACTATE GLUCOSE OTHERS

VIRAL <25 mg/dL Normal
BACTERIAL >35 mg/dL Decreased Gram staining
FUNGAL >25 mg/dL Normal to decreased Presence of “sturburst”
under microscope because
of C. neoformans
TUBERCULAR >25 mg/dL decreased Pellicle formation
o MICROSCOPIC EXAMINATION
o CELL COUNT
 Specimens contain up to 200 WBCs or 400 RBCs may appear clear so it is necessary to examine
all specimens microscopically
 WBC COUNT – ROUTINELY PERFORMED
 Specimens requiring dilution - use 3% glacial acetic acid as diluents to lyse the RBCs
 Methylene blue – added to stain the WBCs

CLARITY DILUTION
Slightly hazy 1:10
Hazy 1:20
Slightly cloudy 1:100
Cloudy 1:200
Bloody or Turbid 1:10,000
 RBC COUNT – done only when there is traumatic tap and correction for leukocytes or proteins is
needed
 TOTAL CELL COUNT – cells are counted in the 4 corner squares and the center square of the
hemocytometer. Use of normal saline solution as diluents
 DIFFERENTIAL COUNT – specimen should be concentrated and stained
- 100 cells should be counted, classified and reported in percentage

SEMINAL FLUID
o To investigate the cause of infertility
o To check the effectiveness of previous vasectomy
o In medico-legal cases, where paternity is being disclaimed on the basis of male sterility
 SPECIMEN COLLECTION, HANDLING AND PRESERVATION
o ABSTINENCE PERIOD – 3-5 days
- Prolonged abstinence will result to higher volume and decreased sperm motility
and increased flavin giving the semen a yellowish color

PORTION SPERM COUNT pH OTHERS

1ST portion is missing Decreased Falsely increased Specimen will not liquefy
Last portion is missing Increased Falsely decreased Specimen will not clot
Semen volume is decreased
 For fertility testing, 2-3 samples must be examined at 2 weeks intervals with 2 abnormal samples considered
significant
 METHODS OF COLLECTION
o Self-production or masturbation – best because it prevents contamination
o Coitus interruptus – withdrawal method
o Vaginal vault aspiration – aspiration of seminal fluid from the vaginal vault after coitus
o Condom method – only nonlubricant-containing rubber or polyurethane condoms should be used
 MACROSCOPIC SEMEN ANALYSIS
o COLOR
 Grayish white to pearly white and translucent – normal
 Rusty red to brown – presence of red blood cells
 Yellowish – urine contamination, antibiotics, prolonged abstinence, pyospermia
 Turbid – infection, increased WBCs
 Clear - infertillity
o VOLUME
 2- 5 mL/ejaculation – normal
 Prolonged abstinence - ↑ volume
 Infertility, improper functioning of semen-producing organs - ↓ volume
o VISCOSITY
 Highly viscous, pours in droplets – normal
 Watery = rated as 0
 Gel-like = rated as 4+
o LIQUEFACTION TIME
 30 mins to 1 hour – normal
 Incomplete liquefaction will impede sperm motility
 Failure of liquefaction should be reported
 o pH
 7.2 to 8.0 – normal
 More basic – possible infection within the reproductive tract
 More acidic – seminal vesicle obstruction, absence of seminal vesicle or increased prostatic fluid
 MICROSCOPIC SEMEN ANALYSIS
o SPERM MOTILITY
 Performed in undiluted specimen under 20 hpf
 Evaluate the speed and direction of motility and grade as follows

GRADE CRITERIA
4.0 a Rapid, straight-line motility
3.0 b Slower speed, some lateral movement
2.0 b Slow forward progression, noticeable lateral movement
1.0 c No forward progression
0 d No movement

o SPERM COUNT AND CONCENTRATION

 2 TYPES OF COUNTING CHAMBER
 MAKLER – undiluted specimens, sperms are immobilized by heating, sperm motility is
assessed in the unheated portion
 Neubauer hemocytometer – counted in 4 corner squares and center square of the large
center square
- Both sides of the hemocytometer are loaded and counted and counts should
agree wihin 10%
- Round cells are immature sperm and WBCs
 DILUTION – 1:20
 DILUTING FLUID
 5% sodium bicarbonate or 1% formalin
 Cold distilled water or tap water
 CLINICAL SIGNIFICANCE
 Azoospermia – complete or total absence of spermatozoa seen
 Necrospermia – presence of sperm cells whether completely dead or immobile
 Oligospermia – deficiency in the number of sperm cells or presence of few motile cells
o SPERM MORPHOLOGY
 Stain and evaluate at least 200 sperm cells under oil immersion
 Evaluate with respect to both head and tail
 HEAD
 Oval-shaped head: approximately 5um long and 3 um wide
 Acrosomal cap – should occupy approximately ½ of head
 MIDDLE PIECE
 Contains mitochondria to provide energy for flagellar motion
 TAIL
 Flagellar, approximately 45 um long
 o OTHER TEST
 FRUCTOSE TEST
 Assesses the function of the seminal vesicles and is used when the sperm concentration
is low
 Specimen are screened using RESORCINOL TEST that produces an orange color when
fructose is present
 Using spectrophotometric methods
 Specimens for fructose levels should be tested within 2 hours or frozen to prevent
fructolysis
 FLORENCE TEST
 Test for choline
 Reagents: potassium iodide and iodine crystals
 (+) brown rhombic crystals examined under the microscope
 BARBIERO’S TEST
 Test for spermine
 Reagent: picric acid and TCA
 (+) yellow leaf-like structures

 SPINBARKEIT TEST
 Test for the tenacity of mucus
 SIMS HUCHNER TEST
 Post-coital test
 Test for the ability of sperm cells to penetrate the cervical mucosa
 SPERM VIABILITY/ BLOOM’S TEST/ EOSIN-NIGROSIN STAIN
 Bluish white – living sperms
 Red – dead sperms

SYNOVIAL FLUID

o Often referred to as the “joint fluid”

o Present within the synovial cavity in free-moving joints to provide lubrication and sole nutrient source for the
joint tissue
o ULTRAFILTRATE OF PLASMA with a very mucoidal substance – hyaluronic acid
o Large amount in the knee cavity
 SPECIMEN COLLECTION AND HANDLING
o Arthrocentesis – needle aspiration in the joints
o When sufficient fluid is collected, it should be distributed into the following tubes based on the required
tests
 A sterile heparinized tube or SPS for gram stain and culture
 A heparin or liquid EDTA tube for cell counts
 Nonanticoagulated tube for other tests
 A sodium fluoride tube for glucose analysis
o COLLECTION TUBE ORDER
 #1 – chemical examination (1-3 mL)
 #2 – microscopic examination (2-5 mL)
 #3 – microbiologial studies (3-10 mL)
 oIf a glucose test is to be performed, the patient should be fasting for at least 6 hours prior to collection
of joint fluid. A 6-hour fast is necessary to establish an equilibrium between plasma and joint glucose
levels
o Synovial diluents
 Normal saline solution, 1% saponin in saline, 0.1 N HCl
 NORMAL VALUES FOR SYNOVIAL FLUID
o VOLUME - <3.5 mL
o COLOR – colorless to pale yellow
o CLARITY - clear
o VISCOSITY – able to form a string 4 to 6 cm long
o LEUKOCYTE COUNT - <200 cells/ul
o NEUTROPHIL - <25% of the differential
o CRYSTALS – none present
o GLUCOSE:PLASMA DIFFERENCE – <10 mg/dL lower than the blood
o TOTAL PROTEIN - <3 g/dL

INCLUSIONS
REITER CELLS Vacuolated macrophages with ingested neutrophils
RA CELLS RAGOCYTE Neutrophils with small, dark, cytoplasmic granules that consist of precipitated rheumatoid
factor
LE CELL Neutrophil containing characteristic ingested “round body”
TART CELL Monocyte that have engulfed nuclear material
SYNOVIAL LINING CELL Similar to macrophage, but may be multinucleated, resembling mesothelial cells
CARTILAGE CELLS Large, multinucleated cells
RICE BODIES Macroscopically resemble polished rice
Microscopically show collagen and fibrin
FAT DROPLETS Refractile intracellular and extracellular globules
Stain with Sudan dyes
HEMOSIDERIN Inclusions within clusters of synovial cells
OCHRONOTIC SHARDS Debris from metal and plastic joint prosthesis
Look like ground pepper
 ROPE TEST/MUCIN CLOT TEST
o Estimation of the integrity of the hyaluronic acid-protein complex (mucin)
o Normal synovial fluid forms a tight ropy clot upon the addition of acetic acid
o Reagent: 2-5 % Acetic acid

CHARACTERISTICS OF SYNOVIAL FLUID CRYSTALS

CRYSTAL SHAPE COMPENSATED POLARIZED LIGHT SIGNIFICANCE
Monosodium urate needles Negative birefringence Gout
Calcium pyrophosphate Rhombic square, rods Positive birefringence Pseudogout
Cholesterol Notched, rhombic plates Negative birefringence Extracellular
Corticosteroid Flat, variable-shaped plates Positive birefringence Injections
Calcium oxalate Envelopes Negative birefringence Renal dialysis
Apatite Small particles No birefringence osteoarthritis
 GROUP CLASSIFICATION LABORATORY FINDINGS
I. NONINFLAMMATORY Clear, yellow fluid
Good viscosity
WBCs <1000/ul
Neutrophils <30%
Normal glucose
II. INFLAMMATORY Cloudy, yellow fluid
IMMUNOLOGIC ORIGIN Poor viscosity
WBCs 2000-75,000/ ul
Neutrophils >50%
Decreased glucose level
Possible auto antibodies
CRYSTAL-INDUCED ORIGIN Cloudy or milky fluid
Low viscosity
WBCs up to 100,000/ ul
Neutrophils <70%
Decreased glucose level
Crystals present
III. SEPTIC Cloudy, yellow green fluid
Variable viscosity
WBCs 50,000-100,000/ ul
Neutrophils >75%
Decreased glucose level
Positive culture and gram stain
IV. HEMORRHAGIC Cloudy, red fluid
Low viscosity
WBCs equal to blood
Neutrophils equal to blood
Normal glucose level
 SEROUS FLUIDS
o Are serum-like fluids formed as ultrafiltrates of plasma which provide lubricantion in the cavities where
they are found
o Serous fluid is produced and reabsorbed at a constant rate
o If an alteration in the hydrostatic and oncotic pressure in the capillaries of the cavities happens, there
will be an increase in fluid volume between the two membranes called effusion

TRANSUDATES – serous effusion that result from disturbance of the fluid production and regulation between serous
membranes
INCREASED HYDROSTATIC PRESSURE DECREASED ONCOTIC PRESSURE
Congestive heart failure Nephrotic syndrome
Salt and fluid retention Hepatic cirrhosis
Malnutrition
Protein-losing enteropathy

EXUDATE – purulent effusions that form in any body cavity as a result of an inflammatory process
INCREASED CAPILLARY PERMEABILITY LYMPHATIC OBSTRUCTION
Microbial infections Malignant tumors
Membrane inflammation Lymphomas
Malignancy Infection and inflammation
Thoracic duct injury
PARAMETERS TRANSUDATE EXUDATE
APPEARANCE Clear Cloudy
FLUID: SERUM PROTEIN RATIO <0.5 >0.5
FLUID: SERUM LD RATIO <0.6 >0.6
WBC COUNT <1,000/ul >1,000/ ul
SPONTANEOUS CLOTTING No Possible
PLEURAL FLUID CHOLESTEROL <45 to 60 mg/dL >45 to 60 mg/dL
PLEURAL FLUID: SERUM CHOLESTEROL RATIO <0.3 >0.3
PLEURAL FLUID: BILIRUBIN RATIO <0.6 >0.6
SERUM-ASCITES ALBUMIN GRADIENT >1.1 <1.1
 PLEURAL FLUID
o APPEARANCE
 Clear/pale yellow – normal
 Turbid – WBCs
 Bloody – malignancy, hemothorax
 Brown – rupture of amoebic liver abscess
 Black – aspergillosis
 Viscous – malignant mesothelioma
 Milky – may be due to chylous or pseudochylous effusions

CHYLOUS PSEUDOCHYLOUS
From thoracic duct leakage Chronic inflammatory conditions
Extractable in ether Not extractable in ether
Stained by Sudan III Not stained by Sudan III
No cholesterol crystal (+) cholesterol crystals
Lymphocytes are predominant Mixed cells
>110 mg/dL TAG <50 mg/dL TAG
Milky white Milky green
 TUMOR MARKERS important in evaluation of effusion of malignant origin:
o CEA (carcinoembryonic antigen)
o CA 125 (metastatic uterine cancer)
o CA 15.3 and CA 549 (breast cancer)
o CYFRA 21-1 (lung cancer)
 PERITONEAL FLUID/ASCITIC FLUID
o PERITONEAL LAVAGE - sensitive test detection of intra-abdominal bleeding in blunt trauma cases
o SERUM-ASCITES ALBUMIN GRADIENT
 Recommended over the fluid to serum total protein and LD ratios to differentiate transudates
from exudates
o PSAMMOMA BODIES
 Contains concentric striations of collagen-like material
 Seen in benign conditions
 Also associated with ovarian and thyroid malignancies
o CA 125 AND CEA – for identifying probable source of tumors producing the exudates
 AMNIOTIC FLUID
o 1ST TRIMESTER – 35 mL of amniotic fluid is derived primarily from maternal circulation
o AFTER THE FIRST TRIMESTER- fetal urine is the major contributor to the amniotic fluid volume
o AMNIOCENTESIS
 Needle aspiration of amniotic fluid
  May be safely done after 14th week of gestation
 16th week of gestation – for assessment of genetic defect/chromosome analysis
 Done in 3rd trimester for assessment of fetal pulmonary maturity or fetal hemolytic disease
o SPECIMEN HANDLING AND PROCESSING
 A maximum of 30 mL of amniotic fluid is collected in sterile syringes
 The first 2 or 3 mL collected can be contaminated by maternal blood, tissue fluid and cells and
are discarded
 Fluid for bilirubin analysis in cases of hemolytic disease of the newborn kust be protected from
light at all times
 Fetal lung maturity test – placed on ice for delivery to lab and ref prior to testing, low speed
centrifuge for no longer than 5 minutes to prevent loss of phospholipids
 Cytogenic studies – maintained at room temperature or body temperature to prolong the life of
cells needed for analysis
 Chemical testing- separate from cellular elements and debris
 TEST FOR FETAL LUNG MATURITY
o L/S (LECITHIN/SPHINGOMYELIN) RATIO
 Reference method
 Up to the 26th week of gestation, the amount of lecithin is less than sphingomyelin
 After 36th week, lecithin increases markedly while sphingomyelin remains constant
o PHOSPHATIDYLGLYCEROL/PHOSPHATIDYLINOSITOL
 Another lung surfactant essential for lung maturity
 Performed by TLC
 May be used in place of L/S ratio
 Production parallels that of lecithin but delayed in diabetic mothers
o AMNIOSTAT-FLM
 Uses antisera specific for phosphatidylglycerol and not affected by specimen contamination with
blood or meconium
o FOAM.SHAKE TEST
 Done by shaking amniotic fluid with 95% ethanol for 15 seconds
 (+) presence of bubbles for 15 minures
o FOAM STABILITY INDEX
 Semiquantitative measure of of amount of surfactants present
 Amniotic fluid is reacted with varying amounts of 95% ethanol
 Value of >47 = fetal lung maturity
o MICROVISCOSITY
 Presence of phospholipids decreased the microviscosity of amniotic fluid
 This change in microviscosity can be measured using fluorescence polarization
 Albumin is used as interna standard
 ≥55 mg/g = fetal lung maturity
o LAMELLAR BODIES AND OPTICAL DENSITY
 Surfactant responsible for fetal lung maturity are produced and secreted by the type II
pneumocytes of the fetal lung in the form of lamellar bodies
 Lamellar bodies enter the alveolar spaces to provide surfactant and also enter the amniotic fluid
 The number of lamellar bodies correlates with amount of phospholipids present in fetal lungs
and they are counted using resistance pulse counting
 TESTS FOR FETAL DISTRESS
o Bilirubin analysis
 For evaluation of HDN
 In the course of normal pregnancy, bilirubin pigment in amniotic fluid decreases
 If there is maternal antibody crossing the placenta and destroying the fetal cells, bilirubin
increases
 LILEY GRAPH
 Zone 1 – non-affected/mildly affected – observe fetus for distress
 Zone 2 – moderately affected – requiring close monitoring and treatment
 Zone 3 – severely affected – interventions is required
o Alpha-fetoprotein
 For detection of neural tube defect
 Major protein produced by the fetal liver during early gestation and found in maternal serum
due to the combined circulation and in amniotic fluid by excretion in fetal urine
 Normal values are based on the week of gestation age, as the fetus produces maximal AFP
between 12 to 15 weeks gestation, after which levels in amniotic fluid begin to decline
o Acetylcholinesterase level
 Elevated in amniotic fluid in neural tube defect because cholinesterase is a component of a
nerve tissue