ANALYSIS OF URINE AND OTHER BODY FLUIDS

_______________________________________, RMT

SPUTUM & BRONCHOALVEOLAR LAVAGE (BAL)

 From upper & lower respiratory
 Tracheobronchial secretion (mixture of plasma, electrolytes, mucin & water) added with cellular
exfoliations, nasal & salivary gland secretions and normal oral flora

Sputum Collection
First morning Most preferred
24 –hour For volume measurement
Throat swab For pediatric patients
Sputum induction For non-cooperative patients
Tracheal aspiration For debilitated patients

Macroscopic Examination
Volume ↓ Bronchial asthma, acute bronchitis, early pneumonia, stage of healing
↑Bronchiectasis, lung abscess, edema, gangrene, tuberculosis,
pulmonary hemorrhage
Odor Odorless Normal
Foul or putrid  lung gangrene, advanced necrotizing tumors
Sweetish  bronchiectasis, tuberculosis
Cheesy  Necrosis, Tumors, Epyema
Fecal  Liver abscess, enteric Gram-negative bacterial infection
Color Colorless or translucent Made up of mucus only
White or yellow ↑ Pus
Gray ↑Pus & epithelial cell
Bright green or greenish ↑ Bile; Pseudomonas
earoginosa infection, lung
abscess
Red or bright red Fresh blood or hemorrhage,
tuberculosis, bronchiectasis
Anchovy sauce or rusty brown Old blood, pneumonia,
gangrene
Prune juice Pneumonia, chronic lung
Olive green or grass green cancer
Cancer
Black Inhalation of dust or dirt,
carbon, charcoal, anthracosis,
smoking
Rusty (with pus) Lobar pneumonia
Rusty ( without pus) Congestive heart failure
Current, jelly-like Klebsiella pneumoniae
infection
Consistency Mucoid  asthama, bronchitis
Serous or frothy  lung edema
Mucopurulent  bronchiectasis, tuberculosis with cavities

Macroscopic Structures Clinical Significant

Dittrich' plugs  Yellow or gray material, size of Bronchitis, Bronchiectasis
a pinhead Bronchial asthma
 Produces foul odor when
crushed
Lung Stone  Hard concretion in a bronchus Histoplasmosis (most
(pneumoliths or  Yellow or white calcified TB common)
broncholiths structures or foreign material Chronic Tuberculosis
Bronchial casts  Branching tree-like casts of the Lobar pneumonia, bronchitis,
bronchi diphtheria
Layer formation  3 layers: Bronchiectasis, lung abscess,
- 1st (top) = frothy mucus gangrene
- 2nd (middle) = opaque, water
material
- 3rd (bottom) = pus, bacteria,
tissues
Foreign bodies  Bronchial calculi (made of Pneumoconiosis

JAN ETHAN V. LOVENDINO, RMT,MSPH

AUBF-Professor (USI)
Page 1
 calcium carbonate & phosphate)
 Asbestos bodies, silica particles
Elastic fibers  Slender fibrils with double Tuberculosis
contour and curled ends
Charcot-layden  Colorless, hexagonal, double Bronchial asthma
crystals pyramid, often needle like; arise
from disintegration of
eosinophils
Pigmented cells  Heart failure cells; hemosiderin- -Congestive heart failure
laden macrophages -Heavy smokers
 Carbon-laden cells: angular
black granules
Curschmann’s  Colorless globules occurring in No clinical significance
spiral myelin variety of sizes and bizarre forms

Myelin Globules  may be mistaken as Biastomyces

Epithelial cells  Creola bodies Bronchial asthma
-cluster of columnar cells
Fungi  Candida albicans, Cryptococcus neoformans, Coccidiodes
immitis, Histoplasma capsulatum, Blastomyces dermatitidis,
Aspergillus fumigatus
Parasites  Migrating Larva: ASH (Heart to lung migration)
-Ascaris lumbricoides
-Strogyloides stercoralis
-Hookworm

 E. histolytica, E. gingivalis, Trichomonas tenax, P.

westermani (egg), E. granulosus, T canis
Others  Neoplastic cells, bacteria, leukocytes

BRONCHOALVEOLAR LAVAGE (BAL)

 A produce for collecting the cellular milieu of the alveoli by use of a bronchoscope through
which saline is instilled into distal bronchi and then withdrawn
 Important diagnostic test for Pneumocytitis carini (pneumocystis jiroveci) in
immunocompromised patients

CELLS SEEN IN BRONCHOALVEOLAR LAVAGE

56-80% Macrophage Most predominant
1-15% Lymphocytes Interstitial disease, pulmonary lymphoma,
nonbacterial infections
<3% Neutrophils Cigarette smokers, bronchopneumonia, toxin
exposure
<1 to 2% Eosinophils Hypersensitivity reactions
4-17% Ciliated columnar bronchial epithelial cells

SWEAT
SWEAT TEST
- Used to diagnose CYSTIC FIBROSIS
 Automated recessive metabolic disorder affecting the mucous secreting glands of
the body
 Associated with pancreatic insufficiency, respiratory distress & intestinal
obstruction

GIBSON AND COOKE PILOCARINE IONTOPHORESIS

- Pilocarpine + mild current = induce sweat production
- Sweat is tested for sodium and chloride
 Sweat sodium =Flame photometry, Ion exchange electrode
 Sweat Chloride = Manual or automated titration

Sweat Na+ and Cl- values:

>70 mEq/L= Diagnostic of CF
40 mEq/L = Borderline for CF

JAN ETHAN V. LOVENDINO, RMT,MSPH

AUBF-Professor (USI)
Page 2
 AMNIOTIC FLUID
Primary functions of amniotic fluid
 Cushion for the fetus
 Allows fetal movement
 Stabilizes temperature
 Proper lung development

Amniotic fluid volume

 From fetal urine and lung fluid
 ________________= Major contributor to the amniotic fluid volume after 1st trimester of
pregnancy.

Quadruple Screening test prior to performing amniocentesis

1. Alpha-fetoprotien (AFP)
2. Human chronic gonadotropin (hCG)
3. Unconjugated estriol (UE3)
4. Inhibin A

Polyhydramnios Oligohydramnios
Increased amniotic fluid volume Decreased amniotic fluid volume
Causes: Causes:
Decreased fetal swallowing of urine Increased fetal swallowing of urine
Neural tube defects Membrane leakage
Urinary tract deformities

Specimen Collection
Method of collection Amniocentesis (up to 30 mL is collected in sterile syringe)
2nd trimester amniocentesis Asses genetic defects
3rd trimester amniocentesis Fetal lung maturity (FLM, fetal hemolytic disease (HDN)

Test for Fetal Lung Maturity
Test for Cytogenetic studies
Test for Hemolytic Disease
of the Newborn (HDN)
Specimen Handling
Place on ICE (delivery)
Refrigerated or Frozen
Filtration-Prevents loss of phospholipids
Room Temperature/ body temperature
Protect from light

AMNIOTIC FLUID vs MATERNAL URINE

ANALYTE AMNIOTIC FLUID MATERNAL URINE
LESS RELIABLE
Protien + -
Glucose + -
MORE RELIABLE
Urea <30 mg/dL
Creatinine <3.5 mg/dL >10 mg/dL

Fern Test
Specimen (Vaginal Fluid)

Slide (Air Dry)

(+) Fern-crystals- AMNIOTIC FLUID

AMNIOTIC FLUID COLOR

COLOR CLINICAL SIGNIFICANCE
Colorless Normal
Blood-streaked Traumatic Tap
Yellow HDN (Bilirubin)
Dark-green Meconium
Dark red-brown Fetal Death

JAN ETHAN V. LOVENDINO, RMT,MSPH

AUBF-Professor (USI)
Page 3
 I. Test for HDN
 A.k.a. O.D. 450 (Optical Density)
 Absorbance of Amniotic Fluid
 NORMAL = High at 365 nm, Low at 550 nm
 HDN = High 450 nm (Bilirubin)
 Results are plotted on Liley graph
 Zone I = Nonaffective/mildly affected fetus
 Zone II = Moderately affected fetus (requires close monitoring)
 Zone III = Severely affected fetus (requires intervention)

II. Test for Neural Tube Defects (NTD)

 Spina bifida
 Anencephaly
 Screening Test = ALPHA FETOPROTIEN
 Increased in – Neural Tube Defects
 Decreased in – Down Syndrome
Confirmatory Test = ACETYL CHOLINESTERASE

III. Test for Fetal Lung Maturity

TEST INFORMATION
Lecithin/ Reference Method
Sphingomyelin (L/S Lecithin = For alveolar stability
Ratio) Sphingomyelin = Serves as a control (due to constant production)

Ration of >2.0 = MATURE FETAL LUNGS

Cannot be done on a specimen contaminated by blood or meconium
Amnoistat- FLM Immunologic test for PHOSPHATIDYL GLYCEROL
Not affected by blood or meconium
Production of PHOSPHATIDYL GLYCEROL is delayed among
diabetic mothers
Foam stability Amniotic fluid + Ethanol Shake for 15 seconds  Stand for 15 mins
(Foam/Shake test) (+) Foam/Bubbles = MATURE FETAL LUNGS
Microviscosity The presence of Phospholipids decreases microviscosity
>55 mg/g Measured by fluorescence polarization
Lamellar body count Lamellar bodies (a.k.a Type II pneumocytes)
 Responsible for production of alveolar surfactants
 >32,000/uL Lamellar body count = Adequate FLM
OD 650 nm High Lamellar bodies- High O.D. (Abrobance
An O.D, of >0.150 is equivalent to:
 L/S ratio of >2.0
 (+) Phosphatidylflycerol

JAN ETHAN V. LOVENDINO, RMT,MSPH

AUBF-Professor (USI)
Page 4
 IV. Test for Fetal Age
 >2.0 mg/dL Creatinine = 36 months (9 months)
TEST FOR FETAL WELL-BEING AND MATURITY
Test Normal Values at Significance
Bilirubin scan A450 > .025 Hemolytic disease of the Newborn
Alpha-fetoprotien <2.0 Multiplies of Median Nueral Tube Disorders
(MoM)
Lecithin-phengomyelin ratio >2.0 Fetal lung maturity
Amniostat-fetal lung maturity Positive Fetal lung maturity/Phosphatidyl
glycerol
Foam Stability Index >47 Fetal lung maturity
Microviscosity (FLM-TDx) >55 mg/g Fetal lung maturity
Optical Density 650 nm >0.150 Fetal lung maturity
Lamellar body count >32,000 Fetal lung maturity

HUMAN CHORIONIC GONADOTROPIN (HCG)

HCG
 Produced by the Cytotrophoblast Cells of the placenta.
 Peaks during 1st trimester of pregnancy (High blood, urine, amniotic fluid)
 Composed of 2 subunits:
 ALPHA = HCG, LH, FSH, TSH
 BETA = unique for HCG

Urine hCG (Preganancy Test)

Specimen = 1st morning urine

HCG Bioassays
Test Animal used Mode of injection Positive Result
Ascheim-Zondek Immature female mice Subcutaneous Formation of
hemorrhagic follicles &
corpora lutea
Friedman Mature virgin female Marginal ear vein Hyperemic uterus &
rabbit corpora hemorrhagica
Hogben -Female toad Lymph sac Oogenesis
-South African clawed
frog
Galli-Mainini -Male frog Subcutaneous Spermatogenesis
-Male toad
Frank-Berman Immature female rats Subcutaneous Ovarian hyperemia
Kupperman Female rats Intraperitoneal Ovarian hyperemia

CEREBROSPINAL FLUID
Cerebrospinal Fluid
 3rd major body fluid
 Functions:
a) Supply nutrients to the nervous system
b) Remove metabolic waste
c) Produce a mechanical barrier to cushion the brain & spinal cord against trauma

JAN ETHAN V. LOVENDINO, RMT,MSPH

AUBF-Professor (USI)
Page 5
Meninges
 Line the brain and spinal cord
 3 Layers: (DAP)
1. DURA MATER (outer layer) = Lines the skull & vertebral canal
2. ARACHNOID MATER (spider-like) = Filaments inner membraine
 SUBARACHNOID SPACE = where CSF flows
3. PIA MATER (innermost layer) = Lines the surface of the brain & spinal cord

CHOROID PLEXUS
 Specific part of the brain that PRODUCES CSF (by selective filtration of plasma portion of
blood)
 20 mL/hr = rate of CSF production

ARACHNOID VILLI/GRANULATIONS
 Reabsorbs CSF = 20 mL/hr

Blood Brain Barrier (BBB)

 Protects the brain from chemicals & other substances circulating in the blood that can harm the
brain tissue
 Disruption of BBB allows WBCs, proteins & other chemicals to enter the CSF (Ex: Meningitis,
Multiple Sclerosis)

CSF total volume:

Adults
 90-150 mL (Strasinger,5th and 6th Ed.)
 140-170 mL (old Strasinger)

Neonates
 10-60 mL

CSF COLLECTION AND HANDLING

 Up to 20 mL CSF can be collected
 Method of collection = LUMBAR PUNCTURE
 Between 3rd, 4th or 5th lumbar vertebrae

JAN ETHAN V. LOVENDINO, RMT,MSPH

AUBF-Professor (USI)
Page 6
3 CSF TUBES
 Tube 1 = Chemistry/Serology (Frozen)
 Tube 2 = Microbiology (Room Temperature)
 Tube 3 = Hematology/ Cell count (Refrigerated)
 (Tube 4) = Microbiology/Serology
If 1CSF tube only
Microbiology  to avoid contamination

Hematology  perform collect agad

Chemistry/Serology  least affected

CSF APPEARANCE
Appearance Clinical Significance
Crystal clear Normal
Hazy/Turbid/Milky/Cloudy High WBCs (>200/uL)
High RBCs (>400/uL)
High Lipids & Protien
(+) Microorganisms
Xanthochromic (Pink/Yellow/Orange) Due to hemoglobin degradation products
Pink = Slight amount of Oxyhemoglobin
Yellow = Oxyhemoglobin to Bilirubin
Orange = Heavy hemolysis
Other causes: High Carotene, High Melanin, High Protein
(>150 mg/dL), Rifampin
Bloody High RBCs (>6,000/uL)
Traumatic tap (puncture of blood vessel
Intracranial hemorrhage (bleeding within the braincase)
Traumatic Tap vs. Intracranial Hemorrhage
Traumatic Tap Intracranial Hemorrhage
Distribution of blood on 3 tubes 1>2>3 Uneven 1=2=3 Even
1 Bloody 3no trace of blood
Clot formation (+) (-)
Due to plasma fibrinogen CSF has no fibrinogen
Supernatant Clear Xanthochromatic
(Rbcs in CSF lyse after 2hr)
Erythrophages (-) (+)
Macrophages with ingested
RBCs
Oily Radiographic Contrast media
Clotted Protien & Clotting Factors
Pellicle (weblike clot on surface) Tubercular meningitis

CSF CELL COUNT

 Any cell count should be performed IMMEDIATELY
 WBCs and RBCs begin to lyse within 1 hour
 40% WBCs disintegrate within 2 hours

Formula for CSF count using Neubauer

WBC ct = # of cells counted x dilution

#of squares counted x depth (0.1)

Sooooo ngayon class!!! Ano ang kailangan natin gamitin na diluting fluid for CSF cell count. Kailangan
natin i- lyse and RBC --- 3% Acetic acid with methylene blue

WBC Count
 Routine performed on CSF
 Normal values:
 Adults = 0-5 WBCs/uL
 Neonates= 0-30 WBCs/uL

CSF DILUTION
Appearnace Dilution
JAN ETHAN V. LOVENDINO, RMT,MSPH
AUBF-Professor (USI)
Page 7
 Clear Undiluted
Slightly Hazy 1:10
Hazy 1:20
Slightly cloudy 1:100
Cloudy/slightly bloody 1:200
Bloody/Turbid 1:10,000

RBC Count
 Done only in cases of traumatic tap
 To correct for WBC count & total protein concentration
 -1WBC for every 700 RBCs seen
 -8 mg/dL Total protein concentration for every 10,000 RBCs/uL (Henry)
 -1 mg/dL Total protein concentration for every 1,200 RBCs/uL (Stasinger)
CSF Differential Count
 Performed on stained smear
 Specimen should be concentrated before smearing by using the following methods:
1) Cytocentrifugation
 Fluid is added to conical chamber
 Cells are forced into a monolayer within a 6mm diameter circle on the slide
 Addition of Albumin
 Increase cell yield/recovery
 Decreases cellular distortion
2) Centrifugation
3) Sedimentation
4) Filtration

Predominant Cells in CSF

 Predominant = Lymphocytes or Monocytes
 Occasional = Neutrophils
 Adults: 70:30 ratio
 70% Lymphocytes
 30% monocytes
 Neonates
 Up to 80% monocytes is considered normal

PLEOCYTOSIS
 Abnormal condition
 Increased number of normal cells in CSF

PREDOMINANT CELLS SEEN IN CEREBROSPINAL FLUID

Type of Cell Major Clinical Significance
Lymphocytes Normal
Monocytes Viral tubercular & fungal meningitis
Multiple sclerosis
Neutrophils Bacterial Meningitis
Early cases of viral, tubercular & fungal meningitis
Cerebral hemorrhage
Macrophages RBCs in spinal fluids
Contrast Media
Blast forms Acute leukemia
Lymphoma cells Disseminated lymphoma
Plasma Cells Multiple sclerosis
Lymphocyte reactions
Ependymal, choroidal, & Diagnostic procedures
spindle-shaped cells
Malignant cells Metastatic carcinomas
Primary CNS carcinom

CSF PROTEIN
Normal Values Adults = 15-45 mg/dL
Infants = 150 mg/dL
Immature = 500 mg/dL
Increased in Damage to the BBB (Most common)
 Meningitis
 Hemorrhage
Production of immunoglobulins within the CNS
 Multiple Sclerosis
Decreased in CSF Leakage
Major CSF Protein ALBUMIN
JAN ETHAN V. LOVENDINO, RMT,MSPH
AUBF-Professor (USI)
Page 8
 2nd Most prevalent Pre-Albumin  Faster migrator
Alpha-globulins Haptoglobulins, Ceruplasmin
Beta-globulins Beta2 transferrin (“tau”)
 Carbohydrate-deficient transferrin
 Found in CSF but not in serum
Gamma-globulins IgG and some IgA
Not found in IgM, Fibrinogen, Lipids ( Beta Lipoproteins) (toooooo Laaaarge)
normal CSf

CSF PROTEIN DETERMINATION

Turbidimetric 1. Tricholoroacetic Acid (TCA)
 Preferred method; precipitates BOTH albumin & globulins
2. Sulfosalicylic Acid (SSA)
 Precipitates albumin only; to precipitate globulins, add sodium
sulfate (Na2SO4)
Dye-binding Coomassie Brilliant Blue (CBB)
 Protein binds to dye ------------ Dye turns from red to blue
 High protein=High blue color

CSF PROTEIN DETERMINATION

CSF/ Serum Albumin Assess the integrity of the blood brain barrier
Index
Normal value = <9
Abnormal = >9
 Correlates the degree of damage
 Index of 100 = complete damage to BBB
IgG index Assess condition with IgG production within the CNS (ex: Multiple
sclerosis)

Normal value = <0.77

Abnormal = >0.77
 Indicative of IgG production within the CNS

ELECTROPHORESIS
 Done in conjunction with serum electrophoresis
 For the detection of oligoclonal bands
 Indicates immunoglobulin production
 The presence of 2 or more oligoclonal bands in CSF but NOT in serum is valuable for the
diagnosis of MULTIPLE SCLEROSIS but not diagnostic
 Other conditions with oligoclonal banding in CSF but not in serum: ENeNG
Encephalitis, Neurosyphilis, Neoplastic disorders, Guillian-Barre syndrome

Multiple Sclerosis
 Demyelinating disorder
 Findings
 (+) anti-myelin sheath autoantibody
 (+) oligoclonal band in CSF but not in serum
 (+) Myelin basic protein (MBP)
 High IgG index

JAN ETHAN V. LOVENDINO, RMT,MSPH

AUBF-Professor (USI)
Page 9
Myelin Basic Protein
 Protein component of the lipid-protein complex that insulate the nerve fibers
 Presence of MBP in CSF indicates destruction of myelin sheath
 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
Normal Values 60-70% of blood glucose
(50-80% mg/dL)
Increased Due to increased plasma glucose
Decreased in Bacterial meningitis
Tubercular meningitis
Fungal meningitis
Normal in Viral meningitis

CSF LACTATE
Notes Inversely proportional to glucose
Normal Value 10-22 mg/dL
Increased in Bacterial meningitis (>35 mg/dL)
Tubercular meningitis (>25 mg/dL)
Fungal meningitis (>25 mg/dL)
Normal in Viral meningitis

CSF GLUTAMINE
Notes Product of ammonia & alpha ketoglutarate
Normal value 8-18 mg/dL
Increased in  Disturbance of consciousness (Coma)
 Reye’s syndrome

CSF ENYMES
1. Lactate Dehydrogenase (LDH)
LDH Isoenzymes in CSF
LD 1 and 2 = Brain tissue
LD 2 and 3 = Lymphocytes
LD 4 and 5 = Neutrophils

Serum LDH:
 Normal LD 2>1>3>4>5
 Flipped pattern (Myocardial Infarction) LD 1>2>3>4>5

CSF LDH
 Normal LD 1>2>3>4>5

JAN ETHAN V. LOVENDINO, RMT,MSPH

AUBF-Professor (USI)
Page 10
  Neurological abnormalities LD 2>1>3>4>5
 Bacterial meningitis LD 5>4>3>2>1

MAJOR LABORATORY RESULTS FOR THE DIFFERENTIAL DIAGNOSIS OF

MENINGITIS
Bacterial Viral Tubercular Fungal
Predominant ↑ Neutrophils ↑ Lymphocytes ↑Lymphocytes, ↑Lymphocyte,
WBC Monocytes Monocytes
Protein ↑ ↑ ↑ ↑
Glucose ↓ (N) ↓ ↓
Lactate ↑ (N) ↑ ↑
Other (+) Gram stain Agents: Agent: Agent:
information (+) Culture ENTEROVIRUSES Mycobacterium Cryptococcus
(+) Limulus Lysate Tuberculosis neofomans
Test Poliovirus
Echovirus (+) AFB (+) Gram stain=
Coxsackievirus (+) Classic starburst
Pellicle/weblike pattern
clot formation after (+) India Ink
12-24 hr -Capsule
refrigeration (Unstained)
-Background
(black)
(+) Immunologic
test for C.
neoformans

LIMULUS LYSATE TEST

 Detects Gram-negative bacterial endotoxin in body fluids & surgical instrument
 Reagents: Blood of horseshoe crab (Hemocyanin (blue) copper)
 Principle:
 In the presence of endotoxin, the amoebocytes (WBCs) will release lysate (Protein)
 (+) Clumping/Clot formation

AGENTS OF BATERIAL MENINGITIS

Age group Causative Agents
Birth to 1 month old Streptococcus agalactiae; Gram (-) rods
1 month to 5 years old Haemophilus influenzae
5 to 29 years old Neisseria maningitidis
>29 years old Streptococcus pneumoniae
Infants, elderly, immunocompromised Listeria monocytogenes

Serological Testing
 Latex agglutination test and ELISA= for detection of bacterial antigens
 VDRL = recommended by CDC for the detection of Syphilis.

JAN ETHAN V. LOVENDINO, RMT,MSPH

AUBF-Professor (USI)
Page 11